IMMUNITY 
 
 AND 
 
 SPECIFIC THERAPY 
 
 BY 
 
 W. D'ESTE EMERY, M.D., B.Sc. LOND. 
 
 CLINICAL PATHOLOGIST TO KING'S COLLEGE HOSPITAL AND PATHOLOGIST TO THE CHILDREN'S 
 * HOSPITAL, PADDINGTON GREEN ; FORMERLY ASSISTANT BACTERIOLOGIST TO THE ROYAL 
 COLLEGES OF PHYSICIANS AND SURGEONS, AND SOMETIME LECTURER ON PATHOLOGY 
 AND BACTERIOLOGY IN THE UNIVERSITY OF BIRMINGHAM 
 
 WITH ILLUSTRATIONS 
 
 OF THE 
 
 UNIVERSITY 
 
 OF 
 
 PAUL B. HOEBER 
 
 69, EAST 59T.H STREET 
 
 NEW YORK 
 
 1909 
 
PRINTED IN ENGLAND 
 
PREFACE 
 
 IN writing this book I have attempted to give a connected and 
 symmetrical outline of the chief facts definitely known with regard 
 to the method in which the body protects itself against infections, 
 and of their applications in the diagnosis, prevention, and treat- 
 ment of disease. It is not written in support of the views of any 
 particular school of thought, and, when dealing with subjects still 
 under discussion, I have tried to give a fair and impartial, though 
 necessarily succinct, account of each of the rival theories. The 
 factors in many of the problems of immunity are so complex, 
 and our knowledge of the subject grows and alters so rapidly, 
 that it is quite impossible to deal with it dogmatically at the 
 present time. I have kept in view, as far as possible, the re- 
 quirements of the physician and surgeon who may require an 
 epitome of the theoretical basis of the modern methods of diagnosis 
 and treatment, now assuming so much importance, and of the 
 student who desires a general survey of the subject before com- 
 mencing more advanced studies. 
 
 My best thanks are due to Mr. H. K. Lewis for the ready and 
 courteous way in which he has acceded to all my suggestions and 
 requirements ; to Drs. Whitfield and Briscoe, from whom I have 
 received some valuable suggestions; and to Professor Herbert 
 Jackson, of King's College, for kindly reading the sections dealing 
 with the more purely chemical and physical questions and for 
 much useful information connected therewith. I have also to 
 thank Sir Almroth Wright and Drs. R. W. Allen, Eyre, and 
 Bolduan; Messrs. Macmillan and Co., Kegan Paul and Co., and 
 the proprietors of the Lavcet, British Medical Journal, and the St. 
 Bartholomew's Hospital Journal for permission to use illustrations 
 from their publications. 
 
CONTENTS 
 
 CHAPTER PACE 
 
 GLOSSARY . . ' . . ix 
 
 I. INTRODUCTORY AND GENERAL . ... I 
 
 II. ON THE NATURE OF TOXINS . . . -37 
 
 III. THE PHENOMENA OF ANTITOXIN FORMATION . . 60 
 
 IV. INTERREACTIONS OF TOXIN AND ANTITOXIN . . 69 
 
 V. THE ORIGIN OF ANTITOXIN THE SIDE-CHAIN THEORY. Q2 
 
 VI. IMMUNITY TO TOXINS . . . . IT 5 
 
 VII. BACTERIOLYSIS AND ALLIED PHENOMENA . . 139 
 
 VIII. THE AGGLUTININS . . . . 204 
 
 IX. THE PRECIPITINS .... 226 
 
 X. PHAGOCYTOSIS . . . . -. . 238 
 
 xi. "REACTIONS" AND SIMILAR PHENOMENA . . 300 
 
 XII. COLLOIDAL THEORY OF ANTIBODIES . . . 319 
 
 XIII. ON IMMUNITY TO BACTERIA .... 33! 
 
 XIV. PRACTICAL APPLICATIONS .... 358 
 
 BIBLIOGRAPHY . . . . . .421 
 
 LIST OF AUTHORITIES . . . . . 439 
 
 INDEX . . . . . . . 443 
 
ERRATA 
 
 Page 9, line 5 from bottom, omit " to " after " -cytes." 
 14, line 13, for " rather of " read " than with." 
 36, line 22, for " of read "to." 
 ,, 45, line 29, for " antitoxin " read " toxin." 
 
 48, line 14. for "became" read "become"; line 25, for " united 
 
 with" read "injured." 
 
 ,, 52, line 10. for " supernatural " read " supernatant. " 
 ,, 55, line 4 from bottom, for " properties " read " effects." 
 
 57, line i, for "is" read " are " ; bottom line, insert " upon " after 
 
 " toxins," and for " defends " read " depends." 
 ,, 70. line 6. for " haemoglobin " read " haemolysin." 
 ,, 78, lines 17 and 19, for " c.c. " read "parts"; and line 18, for 
 
 " 16-6 c.c. " read " i6'6 parts." 
 79, top line, for "antitoxin " read " toxin." 
 90, line 22, for " toxin " read " antitoxin." 
 ,, 91, line 18, for " toxic " read " neutral" ; line 26, omit " as. " 
 
 93, line 22, for "injection" read "infection." 
 ,, 106, line 26, for "it" read " the toxin " 
 
 ,, 107, line 21, for " to tetanus antitoxin " read "of tetanus toxin." 
 ,, no, line 9, for " antitoxin" read " toxin." 
 122, line 3, for " toxin " read " antitoxin." 
 
 128, line 20, for " leucocytes " read " bacteria ingested." 
 
 129, line 3 from bottom, for " toxin" read " antitoxin." 
 152, line 7, for "joined " read " formed." 
 
 171, line 10, for " rabbit" read " goat." 
 
 192, line 30. for " nephrotoxin " read " hepatotoxin." 
 
 220, line 7 from bottom, for " they " read "it." 
 
 252, line 12, for "leucocytes " read "bacteria." 
 
 256, line 15, for "complement" read "amboceptor." 
 
 284, line 13, for " opsonix " read " opsonic." 
 
 287, line 8, for "which in" read "in which." 
 
 290, line 19, for " bacteria can " read " leucocytes can ' 
 
 296, line 8, for " bacteria " read " leucocytes." 
 
 307, line 12. for " local " read ' general." 
 
 310, line 6 from bottom, for " y " read "a." 
 
 323, line i, for "or" read "on"; line 28, for " complement " read 
 
 " amboceptor." 
 3-15 line 13, should reaJ, "those which hava no defensive layer, or 
 
 which have numerous receptors " etc. 
 348, line 14, for " bacteria" read " leucocytes." 
 351, line 14, for "installations " read "instillations." 
 355, last line, for "research " read "defence." 
 360, line 3, for " able " read ' unable." 
 365, line 29. for '" -stable ' ' read " labile. ' ' 
 378, line 8 from bottom, read "are accompanied by but the slightest," 
 
 etc. 
 
 386, line 21. for 'benefits " read '"benefit." 
 407, line 10 from bottom, for ' rise " read <; use." 
 419, line 25, for "heated " read "beaten." 
 
GLOSSARY 
 
 Active immunity. Immunity due to an active struggle against some infective 
 material, vaccine, or toxin. 
 
 Addiment. See Alexin or Complement. 
 
 Agglutinin. A specific antibody which brings about agglutination i.e., 
 causes the bacteria, cells, etc., for which it is specific, to collect into 
 clumps. Non-specific substances (acids, etc.) have a similar action, but 
 are not properly termed agglutinins. 
 
 Agglutinogen. The antigen of agglutinin i.e., the substance which, when 
 injected into a suitable animal, leads to the formation of agglutinin. 
 
 Agglutinoid, A modification of agglutinin which has retained the power of 
 uniting with the specific bacteria, etc., but has lost that of causing them 
 to clump. 
 
 Aggressin (aggredior, I attack). A substance secreted by bacteria and possess- 
 ing the power of inhibiting phagocytosis of the organism producing it. 
 
 Alexin (dX&jw, I ward off). A defensive substance having an injurious effect 
 on bacteria, and occurring in the serum of normal and immune animals. 
 It is analogous in many respects to the bacterial toxins, and, like them, 
 easily destroyed by heat, chemical agents, etc. It is probably identical 
 with complement, q.v. (For other synonyms, see p 143.) 
 
 Amboceptor (ambo, both, and capio, I take). A specific antibody produced 
 by the injection of bacteria, red corpuscles, cells, etc., and exerting, with 
 the help of alexin or complement, a solvent action on these substances. 
 The term is Ehrlich's, and its use should involve the acceptance of his 
 theory of its action. (For synonyms, see p. 142.) 
 
 Anaphylaxis (a or dm, privative, and 0uXa<r(rw, 1 guard). The opposite of 
 prophylaxis i.e., a condition in which the susceptibility of the animal 
 (especially to toxins, serums, etc.) is abnormally increased. Practically 
 identical with hypersensitiveness. 
 
 Antibody. A substance formed by the injection into an animal of a substance 
 (its antigen) not normally found in the juices of that animal (and probably 
 in all cases of proteid constitution, or closely allied thereto), which unites 
 with its antigen and modifies it in some way. 
 
 Antiferment, or antienzyme. An antibody to a ferment or enzyme. 
 
 Antigen (dvTi, against, and yiyvu. I produce). A substance which, when 
 injected into a suitable animal, has the power of leading to the produc- 
 tion of an antibody. In most, if not in all, cases it is proteid in nature. 
 
 Antitoxin. An antibody to a toxin i.e., a specific substance formed by an 
 animal in consequence of the presence in its tissues or juices of a given 
 toxin, which the antitoxin thus produced has the power of neutralizing. 
 
 Arthus' phenomenon. A form of hypersensitiveness to serum, where in a 
 sensitized animal local lesions (gargrene, abscesses, etc.) develop in a 
 region where serum is injected, and the animal may become cachectic 
 and die. 
 
 Atrepsy (d, privative, and rptyw, I nourish). A condition in which an infective 
 agent or collection of malignant cells dies in the animal body owing to 
 its being unable to obtain suitable nourishment : a conception intro- 
 duced by Ehrlich to explain certain forms of immunity e.g., to malignant 
 growths. 
 
 Attenuation. The change which an organism undergoes whereby it becomes 
 less virulent. 
 
X GLOSSARY 
 
 Autohsemolysin. A substance which has the power of dissolving the red cor- 
 puscles of the individual which produces it ; an autochthonous ambo- 
 ceptor or immune body to an animal's own red corpuscles. 
 
 Bacteriolysis. The phenomenon of solution of bacteria, more especially by 
 the action of specific antibodies, aided by alexin or complement. 
 
 Bacteriotropin, or bacteriotropic substance (Tptiru, I turn). A substance 
 (usually a specific antibody) which has the property of uniting with 
 bacteria, and in someway altering their properties, usually rendering them 
 more suitable for phagocytosis. 
 
 Bordet's phenomenon. The absorption of alexin or complement which is 
 brought about by means of a cell or bacterium combined with amboceptor 
 or immune body, apart altogether from the alexin which is necessary for 
 the action ; the complete removal of all alexin from a fluid by means of 
 a cell-immune-body compound. 
 
 Chemotaxis (rdfa, an arrangement). The attraction or repulsion of leucocytes, 
 bacteria, etc., by substances in solution in the fluid containing the cells 
 in question. 
 
 Complement (compleo, I fill up). A synonym for alexin, q.v. The term was 
 introduced by Ehrlich, since on his theory this substance unites with the 
 amboceptor which has already united with the bacterium, etc., and thus 
 completes the conditions necessary for solution. 
 
 Complementoid. A modification of complement which possesses the combin- 
 ing powers of that substance, but which has lost its active solvent 
 properties. 
 
 Cytase. Metchnikoffs term for the digestive (proteolytic) enzyme secreted 
 by leucocytes ; a synonym for alexin or complement. 
 
 Cytolysin. An antibody (immune body or amboceptor) to a cell, having the 
 power of sensitizing that cell so that it is completely or partially dissolved 
 on the addition of alexin. 
 
 Cytolysis. The solution usually partial of cells by means of an antibody 
 (immune body or amboceptor) and alexin. 
 
 Cytotoxin. A substance acting as a cellular toxin, especially an antibody and 
 alexin ; practically identical with a cytolysin. 
 
 Danysz effect. The decrease in the neutralizing effect of antitoxin which is 
 manifested when the toxin is added in portions, with an interval between 
 each, rather than all at once. 
 
 Desmon (5e<r/<i6s, a bond). A synonym for immune body or amboceptor. 
 
 Deviation of complement. The phenomenon in which the solvent effect of 
 immune body on cells or bacteria (in presence of alexin) diminishes as an 
 excess of the antibody is added ; also known as the Neisser-Wechsberg 
 phenomenon. 
 
 Dominant complement. Where (on Ehrlich's theory) two or more different 
 complements unite with a complex molecule of amboceptor which has 
 united with a bacterium, corpuscle, or cell, that which has the more 
 potent action is known as the dominant. Its effect may be produced 
 without the action of the other complements, or the necessary amount 
 may be smaller. 
 
 Ehrlich's phenomenon. The fact that the difference between the amount of 
 toxin exactly neutralized by one unit of antitoxin and the amount which 
 (added to one unit of antitoxin) just leaves one lethal unit free is greater 
 than one lethal dose of simple toxin. 
 
 Endocomplement. A complement contained within a red corpuscle, probably 
 in all cases lecithin or an allied substance. 
 
 Endotoxin. A bacterial toxin contained within the substance of the bticterium, 
 and not liberated except when the cell is destroyed. 
 
 Ergophore group (tpyov, work, and 0^/>ar, I bear). The part of a molecule of 
 antigen or antibody on which the specific properties of the substance 
 depends (toxophore, zymophore, agglutinophore, etc.), in distinction from 
 the haptophore or combining part of the molecule. 
 
GLOSSARY XI 
 
 Exotoxin. A soluble bacterial toxin which is excreted by the bacterium into 
 the surrounding fluid during the life of the organism. 
 
 Fixation of complement. A synonym for Bordet's phenomenon, q.v. 
 Fixator. A synonym for immune body or amboceptor. 
 
 Gastrotoxin. A cytotoxin or cytolysin acting on the cells of the mucous 
 
 membrane of the stomach. 
 Gengou's reaction. The removal of all alexin or complement from a fluid by 
 
 means of a compound of a precipitin and its antigen ; analogous with 
 
 Bordet's phenomenon, except that in this case the reacting antigen is a 
 
 soluble substance. The two are often grouped together as the Bordet- 
 
 Gengou reaction. 
 Group reaction. A reaction with an antibody (usually an agglutinin) which 
 
 is common to several species of bacteria, forming a well-defined group 
 
 e.g. , the coli group, or the pasteurelloses. 
 
 Haemagglutinin. A substance which agglutinates red corpuscles. 
 
 Hsemolysin. A substance which dissolves red blood-corpuscles, or at least 
 releases the haemoglobin which they contain. The term is used mainly 
 for an antibody having, in conjunction with alexin, a solvent action of 
 this nature. 
 
 Haptin. A portion of a molecule of protoplasm having combining affinities 
 for food molecules, and forming an antibody when shed (v. Receptor). 
 
 Haptophore group, or Radicle (CCTTTW, I fasten). That portion of a substance 
 (whether antigen or antibody) which has the power of entering into com- 
 bination with its appropriate antibody or antigen, as the case may be. 
 Thus a molecule of toxin is supposed to contain a group of atoms which 
 can combine with a cell or molecule of antitoxin, and a second which can 
 then exert a toxic action. The former is known as the haptophore 
 group. 
 
 Immune body (immunis, exempt from public service). A specific antibody, 
 produced by the injection of bacteria or other cells, and having the 
 power of altering these substances in such a way as to render them com- 
 pletely or partially soluble on the addition of alexin. It is the same as 
 amboceptor, but the term implies no theory and is generally preferable. 
 Synonyms : substance sensibilatrice, desmon, preparator, copula, etc. 
 
 Incitor constituent of serum. A substance which aids phagocytosis, espe- 
 cially thermostable opsonin. 
 
 Isoagglutinins. An agglutinin which, ' occurring in the serum of a certain 
 animal, will agglutinate the red corpuscles of other animals of that 
 species, but not those of the individual which produces it. 
 
 Isohsemolysin. An immune body or amboceptor which, occurring in the 
 serum of a certain animal, dissolves (in conjunction with alexin) the red 
 corpuscles of other animals of that species, but not those of the individual 
 which produces it. 
 
 Koch's phenomenon. The tuberculin reaction, or rise of temperature and 
 sudden exacerbation of the local lesions occurring in a tuberculous animal 
 after injection of a culture of tubercle bacilli, living or dead, tuberculin, 
 or other specific tuberculous material. 
 
 Lactoserum. A serum containing a precipitin for milk proteids. 
 
 Leucotoxin. An antibody (immune body or amboceptor) which, in conjunc- 
 tion with alexin, exerts a toxic influence on leucocytes. 
 
 Lysis (Xims, a loosening). The solution of cells, bacteria, etc., mostly by 
 means of antibodies or other protective substances. 
 
 LO dose of toxin. The amount which is exactly neutralized by one unit of 
 antitoxin. 
 
 L+ dose of toxin. The amount which, added to one unit of antitoxin, behaves 
 just like one lethal dose of toxin, bringing about a fatal result in test 
 animals within the time-limit fixed. The fact that the L_|_ dose -the L 
 dose is greater than one lethal dose constitutes the Ehrlich phenomenon. 
 
Xll GLOSSARY 
 
 Macrocytase. In Metchnikoff's phraseology, the digestive enzyme secreted by 
 the large mononuclear leucocytes, and having a special action on cells 
 rather than on bacteria ; really a synonym for alexin, especially for one 
 acting on cells or red corpuscles. 
 
 Macrophage. Metchnikoff's term for a large phagocyte which, according to 
 him, is especially adapted to the ingestion of cells or corpuscles rather 
 than of bacteria They may be large lymphocytes, large hyaline cells, 
 endothelial or other tissue cells. 
 
 Microcytase. The digestive enzyme of Metchnikoff's microcytes or poly- 
 nuclear leucocytes ; supposed to have a special action on bacteria. 
 Practically identical with alexin. 
 
 Microphage. A small leucocyte supposed by Metchnikoff to be specially 
 active against bacteria, and to have little or no phagocytic action on cells 
 or corpuscles. They are polynuclear leucocytes. 
 
 Negative phase. The sudden diminution in the amount of an antibody (and 
 possibly of other defensive substances) in the blood which follows 
 immediately on the injection of an antigen. 
 
 Neisser-Wechsberg phenomenon. Deviation of the complement, q.v. 
 
 Nephrotoxin. A cytotoxin specific for renal cells. 
 
 -ogen. A suffix usually employed to denote an antigen in relation to its anti- 
 body e.g., agglutinogen, the substance which on injection into an 
 animal leads to the production of agglutinin. Also used for a preliminary 
 non-active form of an active substance e.g., opsoninogen, a substance 
 which under certain conditions becomes opsonin. 
 
 -oid (eldos, a figure or appearance). A suffix denoting a secondary modifica- 
 tion of an active substance in which it appears to retain its power of 
 entering into combination with its antibody or antigen, but has lost its 
 specific activity ; a molecule of antigen or antibody which has lost its 
 ergophore, but retained its toxophore, group e.g., complementoid or 
 toxoid, q.v. 
 
 Opsonin (opsono I cater for, I prepare for food. Derived from &\}/ov, cooked 
 meat, a sauce or relish). A substance or combination of substances of 
 whatever nature which has the power of combining with a bacterium, 
 cell, or other substance, and rendering it more easily ingested by a 
 leucocyte or other phagocyte. 
 
 Passive immunity. Immunity due to the injection of serum from an animal 
 which has acquired immunity to a toxin or infective agent. 
 
 Pfeiffer's phenomenon. The classical Pfeiffer's phenomenon consists in the 
 globular transformation, loss of staining reaction, and finally complete 
 disappearance of cholera vibrios, when introduced into the peritoneal 
 cavity of an immunized guinea-pig, or into that of a normal one if 
 immune serum be also injected. Also applied to the similar, but usually 
 less complete, destruction of other bacteria under similar conditions, or 
 to bacteriolysis in general. 
 
 Phytotoxin. A poisonous substance formed by one of the higher plants, but 
 otherwise closely resembling a bacterial toxin, more especially in its 
 power to give rise to the production of an antitoxin on injection e.g., 
 ricin, abrin. 
 
 Polyceptor. Amboceptor which possesses several haptophore groups capable 
 of anchoring several molecules of different sorts of complement, the 
 most important of which is termed the dominant (Ehrlich). 
 
 Polyvalent serum. A serum containing antibodies against several strains of 
 the same species of bacteria e.g., streptococci. 
 
 Polyvalent vaccine. A vaccine composed of the dead bodies of several strains 
 of the same bacterial species. A vaccine composed of more than one 
 species of organism is termed a mixed vaccine. 
 
 Positive phase. The period during which the amount of antibody or other 
 protective body in the serum is increased owing to the injection of an 
 antigen. In general terms it corresponds to the period of exalted im- 
 
GLOSSARY Xlll 
 
 munity due to vaccination, injection of toxin, etc., and is very variable 
 in duration. 
 
 Precipitin. An antibody to a soluble form of proteid, having the power of 
 precipitating or coagulating that proteid by a process of clumping its 
 molecules. 
 
 Precipitogen. The antigen to a given precipitin. Thus when a serum is 
 injected into an animal numberless substances are introduced, a certain 
 number of which only give rise to the formation of precipitin, and are 
 called precipitogens. Also called precipitable substance. 
 
 Precipitogenoid. Heated precipitable substance, which has retained its 
 power of combining with precipitin, but no longer forms a precipitate 
 after doing so. 
 
 Precipitoid. Precipitin which has lost its active or ergophore, but retained its 
 combining or haptophore, group ; the latter has also increased in 
 affinity for precipitable substance. The name is also applied to pre- 
 cipitogenoid. 
 
 Predisposition. The opposite of immunity ; the state of an animal, in virtue 
 of which it is readily infected with a given agent. 
 
 Preparator. Metchnikorf's term for immune body or amboceptor. 
 
 Prophylaxis. Any process by which the vulnerability of an animal by an 
 infective agent or toxin is diminished or removed ; a process for the 
 induction of immunity, more especially in its practical application to the 
 prevention of disease. 
 
 Prostatotoxin. A x cytolysin for the cells of the prostate. 
 
 Pro-zone. In constructing a curve indicating the action of an antibody at 
 different dilutions, it sometimes happens that stronger solutions have 
 less effect than more dilute ones. The region of the curve in which this 
 inhibition of the action is brought about by an excess of the active sub- 
 stance is termed the pro-zone. It occurs with substances other than 
 antibodies. Also called zone of inhibition. 
 
 Receptor. In Ehrlich's side-chain theory a part of a living molecule of pro- 
 toplasm which has the power of attracting and combining with a molecule 
 of food proteid (or of toxin, etc.) from the fluid with which it is bathed, 
 and of building it up into the whole molecule, and thus utilizing it as 
 nourishment, to aid which process it may also seize one or more 
 molecules of complement. When shed into the blood these receptors 
 constitute antibodies. 
 
 1. Simple (e.g., those constituting antitoxin). In the antibodies formed 
 by this group we can only distinguish one group o'f atoms a haptophore 
 group having the power of combining with the specific antigen (e.g., 
 toxin), and preventing its subsequent union with a living cell, thus render- 
 ing it inert. 
 
 2. Complex (e.g., agglutinin), in which we can recognize two separate 
 properties, presumably situate in different groups of atoms : (a) a hapto- 
 phore, combining group, as above ; and (b) an ergophore group, on 
 which the activity depends, and which may be destroyed whilst (a) 
 remains intact. 
 
 3. Compound (e.g., amboceptor, on Ehrlich's theory). In them there 
 are t-uv or more haptophore groups, one of which combines with the 
 antigen, the others with one or more molecules of complement. 
 
 Sensitization of bacteria, corpuscles, etc. The addition of immune body, so 
 that the objects are prepared or sensitized to the action of alexin. 
 
 Side-chain theory. The theory (Ehrlich's) which accounts for the develop- 
 ment of antibodies by supposing that the receptors (q.v.) which combine 
 with the specific antigen may, under certain circumstances, be produced 
 in excess and cast off into the surrounding fluid ; these receptors, retain- 
 ing their power of combining with antigen, constitute the antibodies in 
 question. A brilliant conception, which has been the cause of enormous 
 advance in our knowledge of problems connected with immunity. 
 
XIV GLOSSARY 
 
 Smith's (Theobald) phenomenon. The acquisition of hypersensitiveness to 
 serum and other proteid substances (normally inert) which occurs in 
 some animals as a result of minute doses of these substances, and leads 
 to rapid death, with acute symptoms, when a second injection is given. 
 
 Specificity (species, an image). A direct relation of cause and effect between 
 two substances (such as diphtheria toxin and its antitoxin, the latter being 
 only produced by, and acting only on, the former), or between a substance 
 and a phenomenon (such as the tuberculin reaction, produced only by 
 tuberculous products in a tuberculous animal). The specific products of 
 a micro-organism are those produced only by that organism, so that their 
 recognition is proof of its presence. In the same way a specific disease 
 is one produced only by a certain bacterium (such as diphtheria or 
 anthrax), and not by several organisms (such as suppuration or actino- 
 mycosis) . 
 
 Spermotoxin. A cytolysin to spermatozoa. 
 
 Stimulin. A substance having the power of stimulating the action of the 
 leucocytes (more especially in regard to phagocytosis) by a direct action 
 on the leucocyte itself. The existence of these substances is doubtful, 
 most of the phenomena supposed to be caused by them being due (a) to 
 the action of opsonins, and (b) to substances which have a positive 
 chemotactic action, attracting leucocytes to the region. 
 
 Syncytiotoxin. A cytolysin acting on the cells of the placenta. 
 
 Thermolabile. Easily destroyed by heat. In general thermolabile substances 
 are destroyed, completely or partially, by an exposure to 55 C. for half 
 an hour or to 60 C. for 10 minutes. 
 
 Thermostable. The opposite to thermolabile, q.v. 
 
 Thyrotoxin. A cytolysin acting on the cells of the thyroid gland. 
 
 Toxin (ro&Kbv <j>dpiu.a.Koi> , the drug with which poisoned arrows were anointed. 
 TO^OV, a bow). The specific poison on which the pathogenic activity of a 
 micro-organism depends. The fact of its being specific excludes simple 
 chemical substances which may also exert a toxic action. 
 
 Toxoid. A secondary modification of a toxin which has lost its power of 
 producing toxic symptoms, but retained that of combining with antitoxin 
 or susceptible cells ; or, in Ehrlich's terminology, one that has lost its 
 toxophore, but retained its haptophore, group. 
 
 Toxone. A specific substance of feeble toxicity and slight affinity for anti- 
 toxin which is supposed to be produced by certain bacteria, notably that 
 of diphtheria, in which case it is believed to be the cause of paralysis. 
 Unlike toxoid, it is a primary product. Its existence is doubted, and the 
 effects attributed to minute amounts of toxin by some authors. 
 
 Toxophore group. The portion of a molecule of toxin on which the toxic 
 activity depends, the destruction of which converts the molecule into one 
 of toxoid. 
 
 Trichotoxin. A specific cytotoxin for ciliated epithelium. 
 
 Vaccination. The production of active immunity by some process less 
 severe than the induction of an ordinary attack of the disease in 
 question. 
 
 Vaccine. A substance (usually a dead culture or living culture of mitigated 
 virulence) the injection of which leads to the production of active im- 
 munity with less risk than that which accompanies an ordinary attack of 
 the disease. 
 
 Virulence (virus, a poison). The property or properties of an organism in 
 virtue of which it is able to give rise to disease in animals or to produce 
 a powerful toxin. 
 
 Zootoxin. A poisonous substance of animal origin which resembles in other 
 respects (and especially in that it can give rise to the production of an 
 antitoxin) the bacterial toxins e.g., snake venom, eel serum. 
 
 Zymophore group (&M, leaven). The portion of an enzyme or enzyme-like 
 substance on which the specific properties depend, in contradistinction to 
 the combining or haptophore portion. 
 
OF THE 
 
 UNIVERSITY 
 
 OF 
 
 >R^ 
 
 IMMUNITY AND SPECIFIC 
 THERAPY 
 
 CHAPTER I 
 INTRODUCTORY AND GENERAL 
 
 IMMUNITY is the power which certain living organisms possess of 
 resisting influences which are deleterious to others. In its widest 
 form it includes the power of resisting poisons, adverse physical 
 influences, and diseases of all kinds. Thus, many men can and 
 do acquire some degree of immunity against nicotine, alcohol, and 
 other poisons ; some bacteria are immune to temperatures which 
 are quickly fatal to others ; and some individuals and races have 
 a very real immunity to gout and other metabolic diseases to 
 which their less fortunate brethren are more prone. In any 
 complete discussion of the subject these forms of immunity would 
 require some consideration, but in what follows we shall, in the 
 main, limit ourselves to the investigation of immunity against 
 the diseases of bacterial origin. In doing so we must not be 
 thought to consider the other diseases metabolic and what not 
 as being unimportant. The very reverse is the case, and the 
 subject which calls most urgently for research at the present day 
 is the nature and mechanism of immunity against malignant 
 tumours, and of this we have recently acquired a little know- 
 ledge. But the diseases other than those of bacterial origin will 
 not be dealt with, for the simple reason that our knowledge of 
 their intimate causes is still unknown, and until they are dis- 
 covered, and until the physiological disturbances of the economy 
 which occur in these diseases are more fully known, the nature 
 of the corresponding immunity is obviously extremely difficult 
 of study. The bacterial diseases are quite different, for here 
 
2 INTRODUCTION 
 
 the causes are fully known ; the diseases themselves can be 
 reproduced (in most cases) at pleasure, and the physiological dis- 
 turbances which take place are fairly well investigated. There 
 are, of course, gaps, and those not inconsiderable, in our know- 
 ledge ; but, on the whole, the nature of these diseases is nearly as 
 well ascertained as the present state of normal physiology will 
 allow. Further, we can not only reproduce the diseases, but we 
 can reproduce in most cases any degree of immunity to them 
 which we may require for purposes of protection or research, and 
 we can investigate the differences between the cells and fluids of 
 the immunized person or animal and the corresponding parts of a 
 normal organism, and we can attempt to correlate them with the 
 production of the immune state. We have, therefore, a very 
 large amount of information on the subject, and although this 
 information is at present incomplete, we have already obtained 
 results of the highest practical and theoretical importance ; and 
 the value of these results leads us to believe with confidence that 
 our methods are right, that we are on the right track, and that a 
 solution of the problems that have at present baffled research will 
 come in the near future. 
 
 As denned above, immunity is a function of all living material, 
 and one of the highest importance. Biologists have compiled 
 lists of the essential properties of living protoplasm nutrition, 
 reproduction, and the like but have not realized that immunity 
 to bacterial action is the first necessity for continued life. 
 Consider for a moment a small water animal say a hydra 
 occurring in water which naturally contains saprophytic bacteria. 
 Whilst the animal lives these organisms do not affect its proto- 
 plasm in any way, the latter being immune to their action ; but 
 on the animal's death rapid putrefaction occurs, and in a few 
 hours its protoplasm is broken down by bacterial action: the 
 immunity has ceased. Immunity to putrefactive bacteria is 
 therefore a condition of life in the lower animals. But the same 
 is true in every respect for those of a higher grade, man included. 
 From the moment of birth we are surrounded with air containing 
 bacteria which are not pathogenic in the ordinary sense, but 
 which only fail to be so because of the inherent power of immunity 
 to saprophytic bacteria, which is a fundamental property of all 
 living material. Apart from this, the organisms present in the 
 air, alimentary canal, skin, etc., would flourish as rapidly as they 
 do in a corpse, and life would only be possible for a few hours, 
 
INTRODUCTORY AND GENERAL 3 
 
 or perhaps minutes. Readers of one of Mr. Wells's ingenious 
 romances may perhaps remember how the strange beasts from 
 Mars which invaded this planet died rapidly, being evolved in a 
 region in which there were no bacteria, and in which this power 
 of resisting their action had not been developed. The example is 
 a striking one, and is strictly scientific, though we may wonder 
 how the rotation of nitrogen, in which bacteria play so essential a 
 part, is brought about in Mars ; for this process of the breaking 
 down of dead proteids by bacterial action, and the preparation of 
 its nitrogen for use in plants, is essential for continued life on the 
 planet. Without decomposition all the combined nitrogen of the 
 world would soon become locked up in the dead bodies of animals ; 
 plants would starve and die, and animals (which are all dependent, 
 directly or indirectly, on plant nitrogen) would likewise become ex- 
 tinct. It is a most marvellous natural phenomenon that these putre- 
 factive bacteria should be found wherever life occurs, and wherever 
 their aid may be required to deal with the protoplasm when dead, 
 and that this same protoplasm should have acquired such potency 
 in resisting their attacks whilst still alive. Absence of bacteria 
 or absence of immunity are alike incompatible with animal life. 
 
 Considerations of this nature lead us to a short discussion of 
 the difference between the pathogenic and non-pathogenic bacteria, 
 and we find that there is, theoretically, none. Any bacterium 
 will produce disease if it grows in the tissues of the living body, 
 and all bacteria 1 will do so if the necessary degree and form of 
 immunity is not present. A pathogenic organism is one which 
 can grow in the living tissues, and it can do so only because those 
 mechanisms of immunity which are sufficient in the case of the 
 saprophytic bacteria are powerless to resist it ; but in most cases, 
 as we shall show, a higher degree of immunity can be produced 
 artificially, and the microbe in question then becomes non-patho- 
 genic to that particular animal. So, too, with the bacteria 
 ordinarily regarded as non-pathogenic. Under certain circum- 
 stances, some of which are known and some still unknown, the 
 resistance of the body or of a part of it may be broken down to 
 such an extent that these organisms may gain access, flourish, 
 and give rise to disease. Thus, B. proteus may give rise to 
 phlebitis, growing in the thrombosed vein, and giving off toxins 
 which have an injurious action on the tissues. 
 
 1 Bacteria growing only at very high or very low temperatures, or on media 
 very poor in nitrogen, perhaps excepted. 
 
 I 2 
 
4 INTRODUCTION PATHOGENICITY 
 
 As a matter of high theory, therefore, there is no fundamental 
 distinction between pathogenic and non-pathogenic bacteria, and 
 we can imagine circumstances in which the tissues are vulnerable 
 to attack by almost any microbic species. Practically, however, 
 we shall consider an organism as pathogenic when the immunity 
 of the animal which it attacks is not so perfectly developed that 
 its presence in the tissues is but transient and unaccompanied by 
 any noticeable ill-effects, but in which there is a balanced contest 
 of longer or shorter duration between the injurious powers of the 
 microbe and the defensive mechanism of the host, accompanied 
 by more or less injury to the tissues and disturbances of the 
 physiological economy of the latter, and resulting either in the 
 death of the invader or of the patient. All grades occur. In 
 most staphylococcic infections the chances are enormously on 
 the side of the host, and the immunity is sufficiently high to 
 localize the process before it has gone far. In typhoid fever the 
 natural immunity and the pathogenic power of the organisms are 
 more nicely matched ; the contest between them is of long duration 
 and doubtful issue. And in some forms of human disease, but 
 more especially in artificial infections of animals with highly 
 virulent cultures, the power of immunity seems almost nothing, 
 the bacterium growing apparently unchecked and death occurring 
 within a few hours. We say that these organisms have different 
 degrees of pathogenicity, but it would be equally correct to say 
 that there are different degrees of resistance against them, since an 
 organism that is highly virulent towards one animal species may 
 be quite harmless to another, so that pathogenicity is not an 
 inherent property of certain bacteria. 
 
 Thus far we have considered the resistance of the host as if it 
 were fixed and definite, but this is not the case. It has been 
 known from time immemorial that certain diseases especially 
 those due to infection are followed by a greater or smaller degree 
 of immunity, so that a second attack is unlikely at any rate, for 
 some time. Smallpox, scarlet fever, and measles are amongst the 
 most striking examples, and in them the protection given by the 
 disease is in most instances absolute and lifelong. This is known 
 as acquired immunity, and we shall enunciate it as a law that all 
 recovery from infective disease is due to, and followed by, some 
 degree of acquired immunity, though this may be slight, transient, 
 and perhaps local. 
 
 Take, for example, a case of pneumonia, a disease which may 
 
INTRODUCTORY AND GENERAL 5 
 
 occur repeatedly and at short intervals in the same person. 
 Pneumococci are widely distributed, and are almost universally 
 present in the mouth ; the necessary exciting cause, therefore, is 
 always at hand. Under ordinary circumstances the power of 
 resistance is sufficient to ward off the infection, but when this 
 barrier of immunity is broken down by certain adverse circum- 
 stances by excessive fatigue or starvation, by cold, or by an over- 
 dose of alcohol or other poison the pneumococcus gains access 
 to the tissues, and infection 1 occurs. The balanced contest 
 spoken of above then takes place. The pneumococcus grows in 
 ' the lungs and blood and produces a toxin, which tends to reduce 
 the general health and the resistance of the body still further; and 
 looking at the problem only from this side, it would appear that 
 the process would go on until all the immunity was broken down, 
 and the pneumococcus could flourish unchecked. This, indeed, 
 might perhaps happen did not death supervene and bring with it 
 conditions unfavourable for the growth of this organism. But all 
 this time the tissues of the host have been reacting, and (in non- 
 fatal cases) sooner or later a condition is brought about in which 
 the noxious power of the coccus and the immunity of the patient 
 are exactly level, so that the disease neither advances nor retro- 
 cedes ; and the process goes still farther, and the patient develops 
 such a degree of resistance as will not only render him immune to 
 the spread of the infection, but will suffice to sterilize his tissues 
 of the pneumococci which have already gained access. In other 
 words, there has been an acquisition of immunity; the patient has 
 become immune to the pneumococcus, and it is this, and this 
 only, which has brought about the cure of the disease. 
 
 This process may be represented very diagrammatically, as 
 shown on p. 6. 
 
 The line ag represents the degree of immunity to the organism 
 in question, the pneumococcus. At b some event takes place 
 (e.g., exposure to cold) by which the resistance is lowered to such 
 a degree that infection can occur. This takes place at c, with the 
 result that the immunity falls still farther. At this time the 
 bacteria begin to flourish in the tissues in increasing numbers. 
 This is represented by the ascending line i. The immunity falls 
 and bacterial action increases until a certain point is reached, 
 
 1 I have elsewhere defined infection as the access of living, virulent, 
 pathogenic bacteria to a region whence their toxins may act on the tissues of 
 the body (Rose and Carless's " Surgery," sixth edition et seq., chap. i.). 
 
6 RECOVERY FROM DISEASE 
 
 when the reserve forces of the patient have been brought into 
 action, with the result that the immunity rises (from d to e). 
 Somewhere during this rise (not necessarily or probably at its 
 commencement) the contest turns in favour of the host ; the bacteria 
 are rapidly destroyed, and the disease is cured. Usually, but not 
 necessarily, there is a rise to a level higher than the previous 
 normal one (e to/), of longer or shorter duration, and then a rever- 
 sion to the normal g. If exposure to cold again takes place, a 
 fresh infection may now occur. 
 
 Now it must be emphasized that natural recovery from disease 
 only takes place in virtue of an acquisition of immunity to the 
 infecting agent, and in no other way ; and, further, that, except in 
 a few instances, medical treatment simply aims in aiding this 
 phenomenon. If we exclude the various sera and vaccines, there 
 
 k 
 
 FIG. i. 
 
 are but two therapeutic agents which have a direct curative effect 
 mercury in syphilis and quinine in malaria. 1 In these diseases 
 the physician can apply a direct remedy, but in other cases the 
 aim and object of treatment is to support the patient's strength 
 until the natural development of acquired immunity takes place, 
 and in some cases to aid this development by certain empirical 
 means. It is found that all agents which tend to improve the 
 general vitality and facilitate the performance of the normal physio- 
 logical processes have this action ; hence the importance of suitable 
 food in amounts and at intervals suited to the patient's com- 
 plaint, of fresh air at a proper temperature, of the removal of 
 pain, and other symptoms which tend to impair the patient's 
 
 1 Arsenic and some other drugs in the treatment of various protozoal 
 infections (trypanosomiasis, etc.) may also be included. It is interesting to 
 notice that all the diseases directly combated by simple means are protozoal 
 in origin. 
 
INTRODUCTORY AND GENERAL 7 
 
 strength. These agents are all-important in medical treatment 9 
 but in themselves they are useless, and they only act by hastening 
 the evolution of the immunity, without which the disease must 
 necessarily progress to a fatal issue. This is well seen in the few 
 diseases in which the development of immunity, in face of a natural 
 infection, is but slight, or perhaps altogether absent, such as 
 leprosy or hydrophobia. Here ordinary medical treatment is 
 powerless, and all our hopes for the future are concerned with the 
 discovery of a direct specific remedy. 
 
 It is this connection between immunity and recovery that 
 renders the subject so important to the physician, and the neglect 
 with which its study is treated by the general members of the pro- 
 fession a matter of such profound regret. In our medical educa- 
 tion at the present day we pay, and rightly, much attention to the 
 study of physiology, for without a knowledge of the processes of 
 the healthy body we can hardly hope to diagnose and treat its 
 derangements when diseased; and our physicians are in many 
 cases competent physiologists. But it is equally important to 
 understand the method in which the diseased body combats and 
 cures an infection ; and, although our knowledge of this is as yet 
 imperfect, it is increasing day by day, and results of the greatest 
 interest to the practising physician have already been obtained. 
 And I, for one, think that an intelligent appreciation of what is 
 actually taking place in the body, of the conservative and adverse 
 forces, and of the conditions necessary for cure, will always be of 
 value to the therapist, although it may not give any definite 
 information as to what drug is to be prescribed. 
 
 Let us revert to the subject of NATURAL IMMUNITY. We may 
 define it roughly as the immunity possessed by a certain individual 
 in virtue of its belonging to a given animal species ; it is inherent 
 to a greater or less extent in all members of that species, and is not 
 dependent on any event taking place during the life of the animal 
 in question. In most cases it is present at birth, though this is 
 not absolutely essential. 
 
 Examples are numerous. The lower animals are immune to 
 the gonococcus, and, with few exceptions (the higher apes), to 
 syphilis also. On the other hand, most of the diseases of the 
 lower animals do not affect man fowl cholera, canine distemper, 
 and rinderpest are a few of many examples. In some cases all 
 animals, with a few exceptions, are immune : this is the case with 
 the venereal diseases, and in some of the protozoal infections of 
 
8 NATURAL IMMUNITY 
 
 the lower animals. In others different types of the infecting 
 organism occur, and a given species is susceptible to one, immune 
 to others ; for example, there are three, and perhaps more, varieties 
 of tubercle bacillus, which resemble one another in many points, 
 and which attack respectively man, cattle, and birds, and each 
 animal species is more or less immune to bacilli from animals far 
 removed in the scale. 
 
 In general terms, the immunity or susceptibility of different 
 animals depends to some extent on their zoological affinities. 
 Thus man is pre-eminently susceptible to the Spirochata pallida, 
 the anthropoid apes less so, but still not immune, and the lower 
 animals entirely refractory. Rinderpest affects cattle, sheep, 
 goats, and other ruminants, and South African horse-sickness 
 horses, asses, and mules. But to this rule there are numerous 
 exceptions : thus, almost all warm-blooded animals are susceptible 
 to anthrax, but the Algerian sheep and white rat are relatively 
 immune, the wild rat being susceptible. And of the domestic 
 animals we find cattle to be highly susceptible to tubercle, whereas 
 goats, though closely allied zoologically, are almost immune. 
 
 Natural immunity does not exist to an equal degree in all 
 individuals of a species. This is well seen in man during an 
 epidemic, where, of a certain number of persons who are exposed 
 to an infection (and, as far as we know, receive the same dose of 
 the materies morbi), some escape the disease altogether, some have 
 a slight, and others a severe, attack, whilst yet others die rapidly. 
 Sex has some influence here, but it is usually difficult to trace, 
 since the males and females of a community are in most cases 
 exposed to an infection in varying degree. 
 
 Age is of more importance, and, in quite general terms, we may 
 say that the younger the infant the less its immunity. Certain 
 diseases, such as measles, scarlet fever, and whooping-cough, are 
 rarely seen except in infants, and this is not altogether due to 
 acquired immunity preventing a second attack in later life. 
 Epidemic diarrhoea due to bacilli of the dysentery group is 
 rarely seen in this country, at least except in the early years of 
 life, and the same is true of cerebro-spinal meningitis and some 
 other diseases. It is also interesting to notice that the variation 
 in immunity may take a qualitative rather than a quantitative 
 form. The best example is in the case of the pneumococcus. 
 This organism is the chief cause of suppurative processes of 
 whatever region in infants, whereas in adults it is (except in 
 
INTRODUCTORY AND GENERAL 9 
 
 certain regions) a decidedly rare cause of abscesses and other 
 pyogenic processes. It is evident that the form of immunity 
 which prevents the pneumococcus from gaining access to the 
 tissues and giving rise to abscess formation is in abeyance in the 
 young and well developed in the adult ; yet the two are more 
 nearly equal in their resistance to this organism in its role of a 
 producer of pneumonia. There are also very marked differences 
 in regard to local immunity in the two ages, but of those we shall 
 speak subsequently. 
 
 Natural immunity must not be regarded as a fixed and definite 
 quantity, since all individuals vary enormously in their resisting 
 powers against various diseases at different times and under 
 different conditions. The factors which tend to break down the 
 immunity against any or all infections may be referred to as the 
 banal causes of the diseases in question. They are not in them- 
 selves sufficient to lead to these diseases, but when they come 
 into action and an infecting agent is present the disease will arise. 
 Hence they are often referred to as predisposing causes of disease, 
 and to the lay public they are the actual causes, since they are 
 usually open and obvious, and the real infecting agent is, of 
 course, unknown They are of the utmost importance in pre- 
 ventive medicine, and wherever the probability of an infection is 
 apprehended, a study of the patient's surroundings and habits 
 may often lead to the giving of advice by which these banal 
 causes of infection may be avoided and the disease warded off. 
 In general these predisposing causes are a study for the physician 
 rather than for the pathologist, and in some cases we are quite in 
 the dark as to the method in which they act. Their study cannot 
 be conveniently undertaken here before the mechanisms and pro- 
 cesses of immunity have been described, but it will be useful to 
 enumerate some of the more important. 
 
 Of these cold and wet, especially in combination, are unquestion- 
 ably the most important. The exact way in which they act is 
 not definitely known, but there are materials for a number of 
 suggestions. Thus, as we shall have abundant opportunity of 
 seeing, immunity is to a very large extent a function of the leuco- 
 cytes, to which are specialized cells to which the defence of the 
 body is entrusted. Now the functions (movement and phagocy- 
 tosis) which can be easily investigated are found to be dependent 
 in a very high degree on temperature, acting best at the tempera- 
 ture of the body, or slightly above ; and it is highly probable that 
 
10 COLD, WET, AND FATIGUE 
 
 the more subtle functions of the leucocytes may be similarly 
 depressed by a low temperature. The exposure of the skin to 
 cold, especially if the animal heat be abstracted more quickly 
 by evaporation of moisture on the surface, will lead to a cooling 
 of the blood which circulates through it, and hence to a slight, 
 though appreciable, cooling of the whole blood. This, it is true, 
 is soon compensated for, and no great amount of cooling of the 
 whole body occurs; but even so, it is quite possible that the 
 periodical chilling of the leucocytes during their repeated passages 
 through the cold skin may be sufficient to diminish greatly their 
 functional activity, and to lower the resistance to a point at which 
 infection can occur, and when once pathogenic bacteria have 
 gained a foothold, the resistance will for a time tend to decrease. 
 There is also some evidence going to show that exposure to cold 
 may lessen the production of the defensive substances which occur 
 in the blood (alexin, antibodies, etc.), though this is not fully 
 proved. It is worthy of note that the loss of immunity due to the 
 action of cold and wet on one part of the body (such as the feet) 
 is a general one, and may result in a nasal catarrh, an attack of 
 pneumonia, acute rheumatism, etc., according to the nature of the 
 infection at hand. It is not necessarily a local infection of the 
 chilled region. This is very well shown experimentally. Fowls 
 are immune to anthrax, but are rendered susceptible if they are 
 kept for some time standing in cold water ; and this acquired 
 susceptibility is then a general one, and not merely of the feet. 
 
 Cold and wet, as is well known, have less action when accom- 
 panied by energetic muscular exercise, so long as this does not 
 reach the extent of undue fatigue. This is not because less heat 
 is lost during exercise. The reverse is the case. The suggested 
 explanation is that the muscular metabolism leads to an increased 
 production of heat, and at the same time the cutaneous capillaries 
 are dilated and the heart accelerated, or that the circulation of 
 blood through the skin occurs quickly ; further, the internal 
 temperature of the body may actually be raised several degrees. 
 The result is that the temperature of any given leucocyte never 
 falls much below normal, if at all, since it comes from the internal 
 regions where the temperature is raised, passes rapidly through 
 the skin, and returns again to the interior of the body. 
 
 The effect of fatigue, either alone or in conjunction with cold and 
 wet, is also well known, and is one reason for the excessive mor- 
 tality from disease of armies in the field. It is less explicable, 
 
INTRODUCTORY AND GENERAL II 
 
 but may probably be connected in some way with the presence in 
 the blood of katabolic products of muscular activity, which have 
 an injurious action on the cells of the tissues in general and on the 
 leucocytes in particular. Further, the metabolic products formed 
 during the action of the muscles are acid in reaction, and it is 
 found that some at least of the protective substances which occur 
 in the blood (alexins and opsonins) act best in an alkaline medium. 
 This diminution of immunity after muscular fatigue is manifested 
 in animals as well as in man. White rats which have been made 
 to work in a revolving cage are more susceptible to anthrax than 
 normal white rats, the pre-existing immunity being broken down. 
 
 Insufficient or unsuitable food is a factor of importance, especially, 
 perhaps, in the aetiology of tuberculosis. It is, however, rarely 
 seen alone in this country, at any rate and in the poorer classes 
 its effects are usually complicated by insufficient clothing, un- 
 cleanly habits, and by insufficient ventilation of their houses. For 
 this reason we may perhaps be led to exaggerate its importance ; 
 and whilst it is, of course, true that semi-starvation, in common 
 with other weakening influences, does pave the way for infective 
 processes, we do not find that a supply of food restricted enough 
 to cause a marked reduction of the bodily strength and some 
 degree of anaemia is necessarily associated with any infective 
 disease, though the patient may live under conditions in which 
 infective material is present in abundance. This is well seen in 
 fasting men, in hysterical anorexia, and in patients with imperme- 
 able cesophageal strictures. The blood, it may be pointed out, is 
 not one of the tissues that suffers first in starvation, and its im- 
 portance to the body in many ways is so great that it is kept in 
 good functional activity whilst other regions waste quickly. 
 
 It is probable that insufficient food lowers the resistance of the 
 body in certain directions rather than in others. In the East 
 plague follows famine with some regularity, but there is little or 
 no connection between famine and cholera. But in these latitudes 
 at the present time the disease most commonly due to bad or in- 
 sufficient food is tuberculosis. Formerly it was relapsing fever, or, 
 as it was sometimes called, famine fever, a disease which is now 
 almost extinct as a result of the general cheapening of foodstuffs. 
 
 It is worthy of note that the number of leucocytes per cubic 
 centimetre diminishes in starvation, and is generally lower in the 
 badly-nourished than in the well-fed ; and these cells, as we shall 
 see, are pre-eminently concerned in immunity, and this in a great 
 
12 EFFECTS OF A VITIATED ATMOSPHERE 
 
 many ways. It was recognized long ago that post-mortem wounds 
 are much more dangerous when received whilst fasting than during 
 the process of digestion, and it is possible that this may be due to 
 some extent to the increased number of leucocytes which occur in 
 the blood during the process. 
 
 Exposure to a vitiated atmosphere, if of long duration, is a most 
 potent cause of the breaking down of immunity, and when con- 
 sidered on a large scale, and in view of its effect on the general 
 death and disease rate, is probably of greater importance than all 
 other causes combined. It is especially important in connection 
 with tuberculosis, and nothing is more striking than to notice its 
 effect on the peasantry of some regions, in which, in spite of 
 exposure to abundant fresh air during the daytime, and a supply 
 of food which certainly does not fall below the physiological 
 minimum, and is usually more abundant, phthisis and other 
 tuberculous diseases are rife. These affections are in general 
 common in cold and windy climates, and less prevalent in warmer 
 countries, and there is little doubt that the main reason for this is 
 the habit which dwellers in cold countries frequently contract of 
 hermetically sealing all entrances to their rooms to keep out the 
 cold. But this is frequently seen in warmer regions, and even 
 throughout the South of England there is an almost universal 
 opinion amongst the lower classes that night air is injurious. 
 This is probably a survival from the time when malaria was 
 indigenous in this country. 
 
 Apart from tubercle, the effect of bad air is especially mani- 
 fested in the causation of diseases of the lungs, nose, throat, etc., 
 and its effect is probably partly general and partly local. The 
 effect of irritating vapours is, of course, local. Thus exposure to 
 nitrous fumes is often followed by the rapid development of 
 pneumonia, and this is, or may be, due to the pneumococcus, 
 which is able to invade the injured lung. 
 
 We do not know the mechanism by which ordinary vitiated air 
 acts on the general immunity. 
 
 Prolonged anesthesia is probably a cause of considerable 
 importance, though one not easy to estimate. The prevalence of 
 ether-pneumonia is not yet ascertained, and has been hotly 
 debated. It falls, of course, into the same category as the 
 pneumonia due to irritating vapours, as described above. Apart 
 from this, however, there is reason to believe that prolonged 
 anaesthesia has some effect in lowering the general resisting 
 
INTRODUCTORY AND GENERAL 13 
 
 power of the body to the common pyogenic bacteria, and that the 
 mere length of an operation should be an indication for the most 
 scrupulous care in antiseptic precautions. It is perhaps con- 
 ceivable that the anaesthetic drug present in the blood may be 
 sufficient to paralyze the leucocytes for a sufficient time to allow 
 bacteria to gain a foothold in the body. 
 
 Certain drugs, of which the most important is alcohol, have an 
 important action in this respect. The liability of alcoholic 
 subjects to pneumonia and some other infective diseases is well 
 known, and in them the prognosis is more than usually unfavour- 
 able. We have but little knowledge of the action of alcohol in 
 this respect. It may be that it acts as a direct inhibitant of the 
 activity of the leucocytes, and it is known to destroy certain 
 delicate defensive substances (alexins and opsonins) which play 
 some part in the defence of the body against microbic invasion, 
 but it is not known whether these effects are actually manifested 
 in the circulating blood. It is also possible that alcohol tends to 
 inhibit the formation of these defensive substances. 
 
 Alcohol tends to lower the temperature of the body by increas- 
 ing the amount of heat lost. It dilates the superficial vessels and 
 accelerates the heart's' action in a way somewhat similar to 
 muscular exercise, but does not, like it, raise the temperature of 
 the interior of the body. Hence the effect of alcohol in conjunc- 
 tion with cold and wet is to increase their ill-effects. More blood 
 is forced through the chilled skin and more heat is lost. The 
 injurious effect of alcohol during exposure to cold is well known. 
 The results, however, are different when alcohol is taken after 
 exposure, and when the sufferer has reached warmth and shelter. 
 There the increased flow in the cutaneous capillaries leads to a 
 warming of the skin and consequent cessation of the chilling of 
 the blood, although the loss of heat may go on. 
 
 Diseases the most important of which are Bright's disease and 
 diabetes lead to a general lowering of the level of immunity, and 
 a consequent predisposition to other diseases. We have no 
 knowledge of the way in which they act. 
 
 There are many causes which act locally, and cause a local 
 lowering of the resistance. Some of these have been hinted at 
 above, but their consideration will be deferred for the present. 
 
 In considering the nature, severity, and prognosis of any disease, 
 two factors have to be recognized : (i) the immunity of the patient, 
 
14 VIRULENCE OF BACTERIA 
 
 and (2) the virulence of the infecting bacterium. A third the 
 number of bacteria which gain access is also of importance, 
 especially under experimental conditions, for it is found that, 
 within limits, lack of virulence can be compensated for by an 
 increase in the dose given. It is, however, one which we can 
 rarely estimate in natural disease ; besides which the growth of 
 bacteria is so rapid that, if not checked by the resisting power of 
 the body, a single organism would multiply in a very few hours 
 to an enormous extent, and render it a matter of but little impor- 
 tance whether one or a hundred bacteria had gained access at 
 first. The number of bacteria is probably of more importance in 
 connection with the occurrence or non- occurrence of infection, 
 rather of the severity of the disease when once infection has 
 occurred. Thus we find in epidemics of typhoid fever due to 
 water or milk that the disease is most prevalent in those who 
 take a large amount of the infective material, but it is not neces- 
 sarily more severe in them than in the patients who have appa- 
 rently become infected with a small dose. This is, however, not 
 the case with artificial infection of animals, for there the severity 
 of the disease (in animals as similar as possible in age, weight, 
 etc.) is fairly proportional to the dose given. But the conditions 
 are somewhat different in the two cases, and in the artificial injec- 
 tion of animals we eliminate altogether the steps by which, e.g., 
 the typhoid bacillus passes the natural barriers, and gains access 
 to the tissues. 
 
 The question of virulence is of much greater importance, and 
 is one which must be more fully discussed subsequently, after we 
 have seen the methods in which the host immunizes itself against 
 the bacterium. Some general points must be mentioned here. 
 
 Cultures of the same organism, identical in all respects in 
 morphological, cultural, and chemical characters, may differ 
 enormously in this respect : thus a culture of streptococci may be 
 entirely devoid of virulence to rabbits, or may be so potent that a 
 minimal dose, containing probably but a single coccus or short 
 chain, may be inevitably fatal. Similar facts hold for pneumo- 
 cocci. According to Eyre, a virulent culture may kill when 20 to 
 200 cocci are injected, whereas an avirulent one may fail to do 
 so in massive doses. In most organisms there is, perhaps, not 
 such a marked difference, but all pathogenic bacteria vary greatly 
 in this respect, and cultures from different sources show marked 
 variations in pathogenicity. 
 
INTRODUCTORY AND GENERAL 15 
 
 Further, the same culture can be made to undergo variation, 
 its virulence being either exalted or diminished, and this is a 
 subject of the utmost importance. An increase in virulence is the 
 more difficult to secure, and can practically only be procured by 
 passage through animals, or by other closely allied process. 
 
 Passage is carried out thus : the avirulent culture is made to 
 infect animals either by the administration of massive doses, or 
 by the simultaneous injection of some substance which lowers the 
 local or general resistance (lactic acid, alcohol, the toxins of 
 B. prodigiosus, etc.). In any case, the organism is made to cause 
 an infection which may or may not be allowed to progress to a 
 fatal issue. From the animal thus infected a second culture is 
 made, and the material used to inoculate a second animal, and 
 the organism will be found to have undergone a noticeable access 
 of virulence. The process is repeated as often as is necessary, 
 and ultimately the virulence of the culture will be brought to 
 its highest possible pitch. The simplest method, where available, 
 is to give the injections into the peritoneum, and to make the 
 cultures by withdrawing some of the peritoneal fluid in a sterile 
 pipette, and incubating it as it is, or after the addition of broth. 
 
 This method was introduced by Pasteur, and is of especial 
 value in preparing the vaccine used against rabies. The organism 
 of this disease is unknown, but the virus occurs in the brain, and 
 emulsions of this substance are used for inoculation. It is found 
 that the virus occurring naturally in rabid dogs (the " virus of 
 the streets ") is comparatively avirulent to rabbits. This is 
 shown by the long incubation period fifteen to eighteen days 
 after intracerebral injection. After about fifty passages through 
 rabbits, the virus becomes so exalted that the incubation period 
 is shortened to six days, and the process cannot be carried further. 
 This virus is called the " fixed virus," and its potency is main- 
 tained unaltered, no matter how many more passages are made. 
 
 Passage does not necessarily raise the virulence of the culture 
 to all animals ; it may do so only for the species used for the pro- 
 cess, the action on other species remaining unaltered or even 
 falling. Nor is passage necessarily followed by an increased 
 degree of virulence the virus of rabies diminishes in this respect 
 when passed through apes. 
 
 Phenomena suggesting a process akin to passage occur under 
 natural conditions. Pneumococci are frequently found in the 
 mouths of healthy persons, and are, as a rule, of feeble virulence, 
 
l6 INCREASED VIRULENCE 
 
 whilst those which are isolated from the lungs in pneumonia, or 
 from pneumococcic lesions in general, are usually far more virulent. 
 Other explanations are possible, but it seems likely that the 
 sequence of events is as follows : The avirulent pneumococci gain 
 access to the body owing to a temporary loss of immunity, due to 
 one or other of the causes enumerated above, and then these are 
 transmitted to a process in all respects like passage, the result 
 being that they undergo a gradual increase in virulence. The 
 struggle of the conservative forces will then be increasingly difficult, 
 and the patient may succumb to an infection with an organism 
 which was at first but slightly virulent. This adaptation of an 
 organism to its environment during the course of a disease may 
 probably be found in the future to be of great importance, as indi- 
 cating a necessity for successive changes in the vaccine or serum 
 used in the treatment of a chronic infection. 
 
 An example worthy of notice has recently been given by 
 Ehrlich. It is not exactly on the same lines, since it deals with 
 an alteration in the body of the power possessed by the parasite of 
 resisting chemical agents of relatively simple composition, rather 
 than in the power of resisting the natural forces of the body, an 
 increase in which constitutes an increase in virulence. Ehrlich 
 investigated the preventive and curative action of atoxyl and of 
 various aniline dye-stuffs, such as fuchsin and trypanroth, on mice 
 infected with trypanosomiasis. He found in a certain number of 
 cases a cure might be obtained e.g., by feeding infected mice with 
 fuchsin or by the injection of atoxyl and that when this occurred 
 the trypanosomes were not entirely destroyed, but remained latent 
 in the body. This is a phenomenon of fairly frequent occurrence, 
 and is called by Ehrlich, " immunitas non sterilisans." After a 
 time a relapse occurred, and was cured by a fresh dose of the drug, 
 but after several of these recurrences this beneficial effect ceased. 
 It was then found that the trypanosomes had been immunized or 
 acclimatized to the agent in question say, to fuchsin and pos- 
 sessed the power of infecting mice previously treated with fuchsin 
 and immune to ordinary trypanosomes ; but the organism had 
 not altered in its susceptibility to other dye-stuffs or to atoxyl, 
 and mice infected with it could be cured by these agents, and not 
 by fuchsin. Further, it was found possible to create a race of 
 trypanosomes resistant to two or more of these agents, and these 
 acquired characters were made permanent after several passages. 
 If we substitute for the drugs used by Ehrlich the substances 
 
INTRODUCTORY AND GENERAL 17 
 
 which are developed in the body as defensive agents during an 
 attack of disease, and imagine the same process to go on, we 
 shall have an exact reproduction of the rise in virulence occurring 
 during an attack of disease. If we defended ourselves against 
 trypanosomes by the development of fuchsin, Ehrlich's fuchsin- 
 resistant race would be extremely virulent for us. 
 
 The development of epidemics of diseases is probably due in 
 some cases to a spontaneous rise in virulence of the infecting agent, 
 but we have no knowledge of the causes by which this is produced. 
 
 The second method of increasing the virulence of a culture is 
 less general, and of greater theoretical than practical interest. It 
 consists in the cultivation of the organism for several generations 
 in the blood-serum of an animal which has been immuned to the 
 bacterium in question. It was discovered by Walker in the case 
 of B. typhosus, and is found in the case of some other organisms. 
 It is referred to subsequently, and we need only say here that it is 
 allied to passage ; the organism is immunized to the fluids of the 
 resistant animal in vitro instead of in vivo. And the virulence of a 
 culture is in general best sustained by a close approximation to 
 the conditions of the body. Thus it is more rapidly lost at the 
 temperature of the room than at that of the body, and the most 
 suitable culture medium is usually one containing body fluids 
 unaltered by heat. Thus Marmorek cultivates his virulent strepto- 
 cocci in broth to which one-third of its volume of ascitic fluid has 
 been added. In the case of diphtheria bacilli the virulence (as 
 estimated by its power of forming toxin) is best maintained by 
 daily transplantations into broth previously raised to the body 
 temperature, and when treated in this way shows little or no 
 change for years. 
 
 Diminution in virulence occurs, as a rule, when the organism is 
 submitted to conditions quite unlike those of the animal body, and 
 is usually the more rapid the greater the divergence. At the same 
 time, the growth under these conditions gradually becomes (in 
 most cases) more abundant. The organism gradually adapts itself 
 to a saprophytic habitat, losing in so doing its distinctive chemical 
 properties which made it virulent as a parasite. Old laboratory 
 cultures of bacteria which have been grown on artificial media for 
 many generations are usually almost devoid of virulence, though 
 here there are great variations, some species becoming inert far 
 quicker than others. 
 
 The subject is important, since cultures of diminished (" miti- 
 
 2 
 
l8 DIMINUTION IN VIRULENCE 
 
 gated ") virulence are frequently employed as vaccines in the pro- 
 duction of artificial immunity. The following are some of the 
 chief methods employed : 
 
 1. By prolonged culture in artificial media, as described above. 
 This method was introduced by Pasteur in the case of fowl 
 cholera. The loss of virulence is a progressive one, and cultures 
 ten months old are devoid of virulence. 
 
 2. By cultivating the organism at a temperature above the 
 optimum for saprophytic growth. This was also introduced by 
 Pasteur, and is used in preparing the vaccines to anthrax. The 
 organism is cultivated at a temperature of 42-5 C., and all 
 virulence is destroyed in about six weeks, though the cultures 
 retain their power of growth unaltered. The first vaccine is prepared 
 by allowing growth to continue at this temperature for twenty- 
 four days. In appearance the bacilli are unaltered, but they have 
 lost the power of killing rabbits and guinea-pigs, though they 
 are still fatal to mice. The second vaccine is cultivated at a high 
 temperature for a fortnight only; it is virulent to mice and 
 guinea-pigs, but not to rabbits. 
 
 The process may also be carried out by a short exposure to a 
 higher temperature. Chauveau's vaccine consists of blood con- 
 taining anthrax bacilli, heated to a temperature of 50 to 55 C. for 
 ten or fifteen minutes. The bacilli remain alive, but are mitigated 
 in virulence. 
 
 3. In some cases the addition of various chemical antiseptics in 
 minute amounts to the culture medium has a similar effect. This 
 is the case with anthrax also. Addition of chemical substances is 
 also made with the idea of destroying toxins, but this is a different 
 phenomenon. 
 
 4. The virulence may be destroyed by drying. This method 
 was introduced by Pasteur for the preparation of a vaccine against 
 rabies. We have already described the method by which he 
 obtained the fixed virus and its action on rabbits. He found that 
 by suspending the spinal cords of rabbits dead of this fixed virus 
 over caustic potash at a temperature of 23 C. the virulence was 
 entirely removed in fifteen days. Drying for a shorter time 
 diminished the virulence, but did not remove it entirely. 1 
 
 1 The more modern idea is that the process of drying kills off a large 
 number of the pathogenic organisms, and that the use of the dried cord is 
 merely another method of giving very minute doses of virus of normal 
 strength. 
 
INTRODUCTORY AND GENERAL IQ 
 
 5. In some cases (as has been noted above) passage through 
 animals diminishes the virulence. In some cases this can be 
 exalted by passage through a series of animals of one species and 
 diminished by the use of another. Pasteur showed this to be the 
 case in swine erysipelas, the potency of which (as tested on pigs) 
 is increased by passage through pigeons and decreased by passage 
 through rabbits. Cultures thus attenuated are used as vaccines. 
 
 The term ACQUIRED IMMUNITY is one that is used to denote an 
 increased resistance to an organism dependent on some modifica- 
 tion in the animal's constitution due to some definite process to 
 which it is subjected, but not including the modifications due to 
 improvements in the general health due to betterment of the 
 environment. For example, a person living in insanitary sur- 
 roundings will undoubtedly acquire a higher degree of resistance 
 to the tubercle bacillus on being moved to more healthy ones, but 
 we do not speak of that as acquired immunity. The distinction 
 is this: The elevation of the natural resisting powers due to 
 improvement in the general vitality is a more or less general one, 
 and affects the immunity to most or all bacteria almost equally ; 
 whereas in acquired immunity in the narrower sense, to which 
 the use of the term is restricted by pathologists, the alteration is 
 in the powers of resistance to one bacterium only. For example, 
 a debilitated person removed to a more healthy environment, 
 given better food, tonics, etc., would become more resistant to 
 the attacks of smallpox, and to other diseases as well ; we should 
 speak of that as an augmentation of the natural immunity. But 
 after an attack of smallpox, or after vaccination, his immunity 
 to smallpox is enormously increased, whereas his resistance to 
 other organisms is unaltered ; this is acquired immunity. 
 
 This is expressed by the use of the word specific, embodying an 
 idea difficult to define, but implying a direct relationship of cause 
 and effect, and, moreover, that a certain effect is only produced 
 by a certain definite cause. Thus the toxin of diphtheria is 
 specific for the diphtheria bacillus in the sense that it is produced 
 by that organism, and by no other ; diphtheria antitoxin is specific 
 for diphtheria toxin, since it is produced only as a result of the 
 injection of that substance ; the reaction caused by the injection 
 of tuberculin into a tuberculous animal is specific, etc. 
 
 In most instances there is another difference between a rise in 
 natural immunity and the development of acquired immunity, in 
 that the latter is much stronger. Thus, the power of resistance 
 
20 ACQUIRED IMMUNITY 
 
 to smallpox of a perfectly healthy person is probably not great, 
 whereas that produced by an attack of the disease or by vacci- 
 nation is for a time almost absolute. Yet all degrees of acquired 
 immunity exist, from the very slight amount which is developed 
 during an attack of pneumonia, and which is probably only just 
 sufficient to cut short the disease, to the enormous degree that 
 can be obtained in animals hyperimmunized to diphtheria or 
 tetanus toxin or hypervaccinated to B. typhosus. Perhaps our 
 conception of immunity in the past has been influenced too 
 strongly by a study of these latter conditions, which are readily 
 induced in the laboratory, but rarely if ever seen in the actual 
 practice of medicine. They represent in an extreme form the 
 changes which follow disease of natural origin, and possess the 
 theoretical interest which attaches to all extreme cases. 
 
 ACQUIRED IMMUNITY occurs in two distinct forms active and 
 passive. A third form exists, which we may call mixed, since it is 
 brought about by a combination of the procedures necessary for 
 the development of the other two. 
 
 Active immunity may be defined as acquired immunity, due to 
 an attack of the disease in question in its normal form, or in a 
 modified and less severe form of artificial production. The 
 essential feature is that the cells and tissues of the person or 
 animal should be subjected to the action of the bacterium (or its 
 toxin), and by its own efforts, and as a result of an active struggle 
 with it, should become less susceptible to its toxin than before. 
 Active immunity is developed only as a result of an illness of the 
 host, due to the action of the microbe on its cells ; and this illness 
 may be of any degree of severity, ranging from an unmodified 
 attack of the disease which may threaten life down to the most 
 transitory and unimportant reaction due to an injection of a 
 minute dose of a mild vaccine. And one of the great aims of 
 modern preventive medicine is to reduce the severity of the disease 
 necessary to produce acquired immunity to a minimum. The 
 greatest step ever made in this direction was Jenner's substitution 
 of vaccination for inoculation. In each case the effect is the same 
 as regards the resulting immunity (though in different degree), 
 but the disease in the former case is mild and devoid of danger, 
 in the latter severe and dangerous. As a general rule, it may be 
 taken that the severer the disease the stronger and more lasting 
 the acquired immunity. A good example will be quoted when 
 dealing with mixed immunity. This is not necessarily the case, 
 
INTRODUCTORY AND GENERAL 21 
 
 however, for the repeated injections of vaccines which are so mild 
 as not to cause any noticeable general and very little local reaction 
 may induce a high degree of immunity. 
 
 The main methods in which active immunity is acquired are 
 these : 
 
 i. A natural attack of the disease, or an attack which is natural 
 in course, but of artificial induction. The only example of the 
 latter in human medicine is the now disused practice of smallpox 
 inoculation, in which the person to be protected was inoculated 
 with the disease, which ran a perfectly usual course, and was not 
 infrequently fatal. As a rule, however, it was milder than the 
 naturally acquired smallpox, since the infective material was 
 taken from a favourable case, and the operation performed when 
 the patient was in good health and able to get proper attention 
 from the outset. Probably, too, the severity of the disease was 
 somewhat modified by the fact that the virus did not reach the 
 body by the usual route. But the infection was ordinary small- 
 pox, and might start an ordinary epidemic. 
 
 The process is used to a much greater extent in veterinary 
 practice, where an occasional death due to the induced disease is 
 of comparatively little importance if thereby the outbreak can be 
 controlled or the great majority of the flock saved. As a rule, 
 an attempt is made to render the attack as mild as possible, 
 either by (a) limiting the amount of the infective material used, 
 or (b) by introducing it in an abnormal way, or (c) inoculating 
 animals at a time when they are found to be least susceptible, or 
 by a combination of these methods. Thus Texas fever is a 
 disease of cattle due to a protozoon (Piroplasma bigemimim) which 
 is conveyed by the bites of ticks. One of the methods used for 
 the protection of cattle in infected districts is to expose calves 
 whilst still milk-fed to the bites of a few infected ticks ; another 
 is to inject blood from diseased animals (containing the parasite) 
 in small doses direct into the jugular vein. In favourable cases 
 the result is a severe attack of the disease, which, however, is 
 rarely fatal, and is followed after a time by complete immunity. 
 In some cases the disease is but slight, and in them a second or 
 even third dose, in each case larger than the preceding, is required. 
 The mortality from the injections is from 3 to 10 per cent., whilst 
 that of untreated animals in infected areas is about 90 per cent. 
 
 A similar method is in use for combating rinderpest, but here 
 bile from an animal dead of the disease is used as the infecting 
 
22 METHODS OF VACCINATION 
 
 agent, since the blood frequently contains other infective materials 
 which would complicate the issue. 
 
 In pleuro-pneumonia of cattle the severity of the disease is 
 lowered by altering the route of infection. In the natural disease 
 the infection probably enters by the lung, and its course is severe 
 and dangerous. Protection is conferred by inoculating virus from 
 the lung of an animal dead of the disease into the subcutaneous 
 tissue near the tail ; much local swelling results, and general 
 immunity is established. Perhaps, strictly speaking, this method 
 of induction of active immunity should be put in a class of its 
 own ; it is one in which a local is substituted for a general disease, 
 with the obvious result of greatly lessening its severity. 
 
 The material used in the production of artificial immunity of 
 the type we are describing is sometimes called a vaccine. This 
 is undesirable, and it is advisable to use the word virus for material 
 containing the infective agent in its normal virulence, retaining 
 the word vaccine for that in which the bacterium has entirely lost 
 its power of producing the normal disease, whatever the dose and 
 whatever the channel of introduction. The term is a somewhat 
 unfortunate one etymologically, but it is in such general use that 
 it is hopeless to attempt to displace it. 
 
 2. By the use of living cultures of pathogenic bacteria of 
 diminished or altered virulence i.e., of a living vaccine. There 
 are as many modifications of this method as there are ways of 
 mitigating the virulence of a culture, and different methods are 
 applicable to different diseases. 
 
 (a) By the use of vaccines diminished in virulence by passage 
 through animals. The most important example of this is, of 
 course, Jennerian vaccination. It would take us too far to 
 examine the evidence in favour of this view, but it may be taken 
 as fairly proved that ordinary lymph vaccine consists of a culture 
 of the smallpox organism modified by passage through " calves, 
 the modification being of such a nature that it has lost its power 
 of producing a general disease (smallpox), but retained that of 
 causing a local one (vaccinia) otherwise similar in nature. 
 
 We have already referred to the decrease in the virulence of the 
 bacillus of swine erysipelas on passage through rabbits, and the 
 use of these mitigated cultures as a vaccine for pigs. This is a 
 better example of the type of immunity we are considering, since 
 it has to do with a known organism. 
 
 (b) By the use of vaccines in which the virulence is diminished 
 
INTRODUCTORY AND GENERAL 23 
 
 by drying. The only practical example is rabies. There the 
 process of immunization is carried out by means of the use of a 
 series of vaccines of gradually increasing degrees of virulence, the 
 degree dependent on the time for which drying has gone on. It 
 is, of course, necessary to proceed with extreme caution, since the 
 cords that have been dried for but a few days are still infective 
 and virulent, and the amount of natural immunity in man is 
 extremely small, so that an attempt to accelerate the process 
 might be fatal. The method varies somewhat at different labora- 
 tories, but the following may be taken as a type of the procedure 
 used. It is of interest as being the method used in the first case 
 treated that of Joseph Meister. 
 
 Day i. Inoculation with vaccine made by drying the cord for 
 fourteen days. A second injection with cord treated for ten 
 days. 
 
 Day 2. Two injections ; cords dried for eleven and nine days. 
 
 Day 3. One injection ; cord dried for eight days. 
 
 Day 4. One injection ; cord dried for seven days. 
 
 Day 5. One injection ; cord dried for six days. 
 
 Day 6. One injection ; cord dried for five days. 
 
 Day 7. One injection ; cord dried for four days. 
 
 Day 8. One injection ; cord dried for three days. 
 
 Day g. One injection ; cord dried for two days. 
 
 Day 10. One injection ; cord from a rabbit which had died 
 the same day, and which was therefore unaltered in virulence. 
 
 The method in use in France at the present day is almost like 
 this, except that the latter stages are repeated twice, or, in severe 
 cases, three times i.e., on the ninth and fourteenth days in mild 
 cases (and on the nineteenth also in severe ones) injections of 
 nine-day cords are started, and the strength increased rapidly, so 
 that three-day cords are used on the thirteenth, eighteenth, and 
 twenty-first. In Germany the treatment is begun with eight-day 
 cords, the older ones being considered inert. 
 
 (c) By the injection of living cultures modified by heat. The 
 classical example is vaccination against anthrax by means of 
 Pasteur's two vaccines, the method of preparing which is given 
 on p. 1 8. The first vaccine is injected, and is followed by the 
 second in about a fortnight, immunity being established in about 
 another fortnight. 
 
 (d) By the injection of cultures attenuated by prolonged cultiva- 
 tion in vitro. The use of this method in the case of chicken 
 
24 VACCINES OF DEAD BACTERIA 
 
 cholera has been referred to already, and it is the one usually 
 employed in the laboratory, where old cultures are used in pre- 
 ference to more virulent ones in the early stages of immunization. 
 
 (e) By the use of very small doses of living cultures of full 
 virulence. This has been proved possible in anthrax, symptomatic 
 anthrax, and some other diseases. At present the process is more 
 interesting than practically useful, but it has been used clinically 
 in the case of tubercle, treatment being commenced by the injec- 
 tion of a single living bacillus, and promising results have been 
 obtained. 
 
 3; A third class of methods consists in the use of vaccines 
 composed of dead bacteria. The advantages are obvious : the 
 dose is under accurate control ; the disease which it induces is 
 self -limited, so that it is impossible for a general infective process 
 to be produced when used on a person of deficient natural 
 immunity ; and the vaccine is easy to keep in a condition ready 
 for immediate use. Hence this method is mostly used in human 
 medicine, whereas the use of mitigated or unmitigated viruses is 
 mainly confined to veterinary work. The methods used in the 
 preparation of the vaccines varies greatly in the different cases, 
 and here we can only glance at some of the general principles. 
 In preparing the cultures, the most careful precautions have to be 
 taken to insure the purity of the microbe used and absence of all 
 other pathogenic forms, especially perhaps the spores of the 
 tetanus bacillus. The age of the culture has to be determined by 
 the necessities of the case, but as a rule young cultures are 
 preferable. The method by which the bacteria is killed also 
 varies, but heat is generally employed, and as a rule the shorter 
 the exposure and the lower the temperature the better. In other 
 cases the bacteria are emulsified in saline solution and allowed to 
 undergo autolysis at the body temperature, sterility being ensured 
 subsequently by means of heat or chemical antiseptics ; or they 
 may be killed with a minimum of heat, and submitted to autolysis 
 at 37 C. subsequently. 
 
 There are numerous methods of determining the dose to be 
 used, (a) A definite fraction of an agar or other culture of 
 known age may be taken, or, what comes to much the same 
 thing, the growth from so many square centimetres or millimetres 
 of surface of the culture medium. (b) The amount may be 
 judged by the weight, and this is the method used in the case of 
 tubercle. When it is employed with other bacteria it is usually 
 
INTRODUCTORY AND GENERAL 25 
 
 carried out by means of standard loops, each of which will pick 
 up a known amount of growth. (c) In Wright's ingenious 
 method of counting a vaccine a certain amount of the latter is 
 mixed with human blood in definite proportions, and films are 
 prepared and stained. The numbers of red corpuscles and of 
 bacteria in several fields of the microscope are then counted, and 
 (the numbers of red corpuscles in a definite volume of the blood 
 being known) the proportions of the two will permit of the calcu- 
 1 ation of the numbers of the bacteria, (d) Some determine the 
 strength of the vaccine by reference to a permanent standard, 
 usually consisting of a fine suspension of barium sulphate. A 
 strong emulsion of bacteria is prepared and diluted until it 
 matches the standard, (e) The volume of the bacteria in the 
 emulsion may be determined by centrifugalization in a graduated 
 tube, and a certain volume of sediment made up to a certain 
 volume of vaccine. (/) The number of bacteria present in the 
 emulsion may be counted directly by the use of the counting 
 chamber of the hsemocytometer, and this is the method I usually 
 employ. The emulsion is diluted (usually to twenty times its 
 volume) with a dilute solution of methylene blue or other stain, 
 boiled, and a drop placed in the counting chamber and prepared as 
 if it were a blood specimen in which the red corpuscles were to be 
 counted, A ^-inch lens and a high eyepiece are used, and, as a 
 rule, the process presents no difficulty. 
 
 In all cases an addition of a chemical antiseptic is advisable 
 to avoid subsequent contamination. Carbolic acid or lysol (0*25 to 
 0-5 per cent.) are most used; another good plan is to keep a few 
 drops of chloroform at the bottom of the bottle, so that the fluid 
 is always saturated. 
 
 This method is mostly used in plague, cholera, and enteric 
 fever in preventive medicine, and in the treatment of infective 
 processes by Sir Almroth Wright's method in curative medicine. 
 These will be discussed subsequently. 
 
 4. Inoculation with the chemical products or with the toxins 
 of the bacteria, the bodies of the bacteria themselves being 
 removed by filtration or in some other way. This is obviously 
 closely allied to the last method the use of killed cultures. 
 
 It was introduced by Smith and Salmon in the case of hog 
 cholera, and is now chiefly used in the immunization of animals 
 for the production of antitoxic sera. It is considered fully in a 
 subsequent chapter. 
 
26 PASSIVE IMMUNITY 
 
 PASSIVE IMMUNITY, the second form of acquired immunity, is 
 conferred by injecting into a susceptible animal the serum of one 
 which has acquired, an active immunity to the disease in question. 
 It is a kind of second-hand immunity, acquired in virtue of the 
 reception of substances actively formed by another animal which 
 has had to fight against the infecting agent in order to form them. 
 In its production there is no necessary illness, however slight. 
 Such may occur, it is true, but it is not more than would be pro- 
 duced by normal serum, and stands in no necessary relationship 
 to the development of the immunity. And when such illness does 
 occur, it does so after the production of the immunity, and may 
 be very severe when the protection given is but slight, and 
 vice versa. 
 
 For the production of passive immunity it is necessary to 
 inject the serum of an animal which has been artificially immun- 
 ized, that from one which is naturally immune being devoid 
 of action in this respect. To this general rule there are 
 one or two exceptions, which are perhaps more apparent than 
 real. 
 
 Passive immunity is sometimes called antitoxic. The term, 
 however, is not a good one, since there are several varieties of 
 passive immunity, only one of which is due to an antitoxin. 
 
 Passive immunity is specific that is, the serum of an animal 
 which has acquired immunity against one organism will protect 
 a second against that, and against no other. In this, of course, 
 it resembles active immunity, but the two differ in several 
 important particulars. 
 
 i. As regards its production. Active immunity takes some 
 time usually a week or so to develop, dating from the infection 
 or injection of the vaccine, and in many cases at least its 
 appearance is preceded by a negative phase, in which the natural 
 immunity to the organism in question is lowered. But passive 
 immunity is established as soon as the serum has become mixed 
 with the blood of the person or animal injected, and there is no 
 negative phase. 
 
 Hence in severe infections our best hope in the way of specific 
 medication is in the production of passive immunity. It is but 
 recently that the injection of vaccines was thought of in face of 
 an infection already developed, and it is obvious that the method 
 will be useless or dangerous in very severe and rapid infective 
 processes. Passive immunity, on the other hand, can be induced 
 
INTRODUCTORY AND GENERAL 27 
 
 at once and without a negative phase. Unfortunately, it is not 
 always or often possible. 
 
 2. As regards its duration. Active immunity lasts for a long 
 time, the length differing greatly in different diseases and after 
 various methods of induction. In many cases it lasts a year or 
 more. Passive immunity, on the other hand, is always of brief 
 duration, and lasts only about as long as the serum injected is 
 
 FIG. 2. SHOWING THE SEQUENCE OF EVENTS IN THE PRODUCTION OF 
 ACTIVE IMMUNITY. 
 
 An injection of vaccine at b is followed by a decrease in the degree of 
 immunity (negative phase), a rise, and a gradual return to the normal 
 condition. 
 
 actually present in the blood. It depends to a certain extent on 
 the dose of serum given, and also on the species of animal from 
 which it was derived. An animal of a certain species is immu- 
 nized for a longer period by serum from another animal of the 
 same kind than from one of a different species. In general terms 
 the duration of passive immunity is three to six weeks. It is 
 renewable at pleasure, as far as we know indefinitely. 
 
 a 
 
 FIG. 3. SHOWING THE SEQUENCE OF EVENTS IN PASSIVE IMMUNITY. 
 An injection of serum is given at b. 
 
 Hence passive immunity is chiefly of value to ward off* an 
 infection the danger to which is of short duration. Thus in 
 veterinary practice the passive immunity of horses conferred by 
 the injection of tetanus antitoxin is of the greatest possible value 
 before operations, or immediately after the infliction of a wound, 
 horses being so prone to tetanus that in some places any opera- 
 
28 MIXED IMMUNITY 
 
 tion was a matter of great danger before the introduction of this 
 method. 
 
 Passive immunity is also useful as a basis for active immunity 
 This will be described under the heading of Mixed Immunity. 
 
 3. Passive immunity for a given bacterium or its products 
 cannot be made so potent as the active form for the same disease 
 in the same animal species. The reason is obvious : the passive 
 form only occurs in virtue of the presence in the blood of some of 
 the foreign serum, which can never form more than a fraction of 
 the whole fluid. The degree of the immunity may be sufficient for 
 all practical purposes, but can never reach the enormous height 
 met with in hypervaccinated animals. 
 
 4. Active immunity we believe to be developed to some extent 
 in all, or almost all, infections, but the production of passive 
 immunity is impossible in very many cases e.g., tubercle (as far 
 as we know at present), infections with pyocyaneus, glanders, 
 malaria, and many other parasitic organisms. Perhaps in the 
 future we shall be able to procure active sera against all organisms, 
 but at present we have comparatively few of any value. 
 
 MIXED IMMUNITY is a combination of the two forms already 
 described, in which the dangers and delay incidental to the induc- 
 tion of active immunity are avoided by the use of a protective 
 serum. It is really a succession of the two forms, the passive 
 immunity being developed at once as a consequence of the injec- 
 tion of the serum, whilst the active form develops later in conse- 
 quence of the vaccination. The process is sometimes called 
 sero- vaccination. 
 
 It is not of great importance in human pathology, the chief 
 example being the form of typhoid inoculation suggested by 
 Besredka, and not yet used on a large scale. In it the killed 
 typhoid bacilli are submitted to the action of the immune serum, 
 from which they absorb certain protective substances and become 
 modified thereby. It is claimed that this treatment prevents the 
 development of the discomfort that follows the use of ordinary 
 typhoid vaccine, and that the immunity is developed very rapidly. 
 It may be followed by an injection of ordinary vaccine. 
 
 The method is used to a considerable extent by veterinary 
 surgeons, and there are several modifications in the process, the 
 serum being injected either mixed with the virus, or before, or 
 after, or simultaneously in different sides of the body. Thus in 
 the treatment of South African horse-sickness the virus (the blood 
 
INTRODUCTORY AND GENERAL 29 
 
 of diseased animals) may be mixed with the serum from hyper- 
 immunized animals and injected subcutaneously. If the serum 
 and virus are injected separately the animal will in all cases 
 acquire passive immunity ; but unless there is some degree of ill- 
 ness (a " reaction") this will be but temporary, and no active 
 immunity will be superadded. Thus, if the serum be injected and 
 the virus given subcutaneously at the same time, no reaction 
 follows, and the immunity does not last more than a month ; but 
 if the injection is made into a vein a reaction occurs, and active 
 immunity, lasting for about a year, will follow (Stockman). 
 
 The method is also used in the early stages of antitoxin forma- 
 tion, the horse being treated with a mixture of toxin and anti- 
 toxin, the latter being in excess. But here it seems unquestionable 
 that active immunity is acquired, and the mechanism by which 
 this occurs is discussed subsequently. 
 
 FIG. 4. MIXED IMMUNITY. 
 The presence of a negative phase, as shown in the diagram, is not essential. 
 
 LOCAL IMMUNITY. We have hitherto spoken of the body as a 
 whole, assuming that all parts are equally resistant or susceptible. 
 This is not the case, and certain parts are found to have a marked 
 degree of immunity to certain bacteria. Here we have to be sure 
 that we are dealing with regions that are equally exposed to 
 infection. The stomach, for example, is comparatively rarely 
 attacked by infective processes, and this may be due to the fact 
 that the gastric juice is of a sufficient degree of acidity to kill or 
 inhibit most bacteria. Yet here it is probable that this does not 
 account for all the phenomena, and that some degree of true local 
 immunity does exist. Numerous other examples may be quoted. 
 Pneumococcic infections are common in the lungs and pleura, but 
 rarely spread further, and cause disease of the ribs and intercostal 
 muscles ; tubercle is common in the bones and extremely rare in 
 the muscles, whilst Trichina spimlis affects the muscles and never 
 
30 LOCAL IMMUNITY 
 
 attacks the bones, and rarely any other tissues. Some of the best 
 examples may be taken from diseases that spread by continuity 
 from one tissue to another. Thus the gonococcus in either sex 
 spreads along the urethra with ease, but seldom involves the 
 mucous membrane of the bladder ; it practically never attacks the 
 vaginal mucosa (in adults), but spreads from the cervical to the 
 corporeal endometrium, and thence to the Fallopian tubes, but 
 comparatively rarely goes farther and produces a general peri- 
 tonitis. Diphtheria, too, though it may spread in any direction, 
 seldom creeps down the oesophagus. Many other examples might 
 be quoted. 
 
 There are marked differences in regard to local immunity 
 between the child and the adult. The most marked example, 
 perhaps, is in the almost perfect local immunity of the scalp to 
 ringworm in adults, which contrasts so markedly with the absolute 
 susceptibility of children, whereas the susceptibility of the skin of 
 the body to the same parasite is, if anything, greater in the former. 
 In most cases of differing immunity at different ages the child is 
 more susceptible, just as its resistance to general diseases is less, 
 and the few exceptions that may be quoted are perhaps rather 
 apparent than real. 
 
 Local immunity may be natural or acquired. Passive im- 
 munity, of course, cannot be local for long, as any serum which 
 is injected will rapidly diffuse away and be removed by the 
 lymphatics and blood-stream. The cases mentioned above are 
 all examples of natural local immunity. The difference between 
 the reactions of the tissues of children and adults do not neces- 
 sarily point to the acquisition of any active immunity in the sense 
 in which the word has been defined above, but rather to the 
 general rise in resisting power accompanying the general improve- 
 ment in strength and vitality, and in some cases, perhaps, to an 
 actual maturation of the tissues, as in the case of the adult vaginal 
 mucous membrane, which is immune to the gonococcus, whereas 
 the thin and immature infantile membrane is susceptible. The 
 immunity of the adult scalp to ringworm also is not acquired, 
 using the word in the narrow sense, for it occurs apart altogether 
 from an attack of the disease. 
 
 Our knowledge of acquired local immunity is very incomplete ; 
 it is a difficult subject for research, and more attention has been 
 paid to general immunity. A little consideration will demonstrate 
 the fact of its occurrence. For example, when a person develops 
 
 
INTRODUCTORY AND GENERAL 3! 
 
 crops of boils it will often be found that one is undergoing involu- 
 tion whilst another is developing ; hence the cure of the first 
 cannot be due to any general immunity, but must depend on local 
 changes which do not affect the second. A similar line of argu- 
 ment will show the development of acquired immunity to the 
 streptococcus in erysipelas ; the healthy skin is susceptible, since 
 the disease spreads to it, but the process does not extend back- 
 ward into an area already affected, but now cured, or does so but 
 rarely. 
 
 The subject cannot be discussed further with advantage, and 
 will be deferred to a subsequent chapter, when the known factors 
 on which immunity depends have been elucidated. 
 
 There are important non-specific causes for alterations in local 
 immunity, as is the case with general. These practically resolve 
 themselves into the presence or absence of an adequate supply of 
 blood ; the more copious the supply of healthy circulating blood, 
 the greater the resistance to infections, and vice versa. Hence the 
 utility of fomentations and other hot applications in the initial 
 stages of an infective lesion ; hence, too, the application of Bier's 
 method of passive congestion, in which an excess of blood 
 (though partly stagnant) is made to flush the tissues. And there 
 is no doubt that the object of the dilatation of the vessels and 
 acceleration of the flow of blood through them which occurs in 
 the early stages of inflammation is a beneficial process which 
 has this improvement of the local resisting powers as one of its 
 objects, the influx of an increased number of leucocytes and the 
 dilution and removal of the soluble toxins being others. In acute 
 inflammation we may distinguish two stages. In the first, the 
 stage just mentioned, the conservative reaction of the vessels is 
 most obvious, and in the case of a mild infection, or if the 
 immunity is very strong, may suffice to destroy and remove the 
 infective material and its toxin. The stagnation and ultimate 
 cessation of the blood-flow are indications that the irritant is, 
 temporarily at least, getting the upper hand, and, by cutting off 
 the blood-supply, is neutralizing the most powerful defensive 
 factor. The acceleration of the flow may be regarded as physio- 
 logical, the retardation and cessation as pathological. 
 
 The causes of local reduction of immunity by obstruction of the 
 blood-stream are numerous, the most important being traumatism 
 (by injuring the vessels going to the region), endarteritis, throm- 
 bosis, tight bandaging, etc. They need not be discussed at 
 
32 LOCAL IMMUNITY 
 
 length, but it is advisable to point out that severe traumatism, in 
 the form of violent laceration and contusion of a part, is an 
 extremely powerful predisposing agent, and that it acts in two 
 ways, or perhaps more. In the first place, there may be some 
 death of tissues, either in small or large amounts, and in these 
 dead tissues the natural resisting powers are of course in abeyance, 
 so that the bacteria will grow unchecked, as they would in dead 
 culture media ; and, secondly, that the blood does not reach this 
 dead material, and the leucocytes only do so with difficulty. The 
 importance of this is well seen in tetanus. The normal tissues 
 have a considerable degree of resistance to this organism, and 
 infection rarely takes place in a clean incised wound, even in 
 cases in which we can be almost certain that the spores of the 
 tetanus, bacillus have been introduced. 
 
 Another cause of reduced local immunity is the action of 
 irritants on the tissues. Here we must distinguish two cases. 
 If the irritant be but mild, it may be actually beneficial ; it 
 causes the earlier phenomena of inflammation which we have 
 previously referred to as being protective, and may tend to raise 
 the resistance of the part in consequence. Thus, according to 
 many observers (who do not agree precisely on the interpretation), 
 the injection of a small quantity of almost any bland (but never- 
 theless foreign) substance into the peritoneal cavity may protect 
 an animal against a lethal dose of a bacterial culture introduced 
 subsequently; normal saline solution, water, broth, serum, etc., 
 all have this action. But if the irritant be more powerful, so that 
 the tissues are killed and the vessels occluded, or the leucocytes 
 killed, the susceptibility of the region is greatly increased. 
 Chemical antiseptics have this action, especially in certain 
 regions, such as the peritoneum. The same thing may be demon- 
 strated experimentally. Tetanus spores washed free of toxin 
 will not produce tetanus in rabbits, but will do so if an irritant 
 such as lactic or carbolic acid is injected simultaneously. 
 
 A few words may be said here on the phenomena of immunity 
 arid susceptibility in relation to the modifications they cause in 
 the infective processes. Where the immunity is great, or, as we 
 say, absolute, the result of an injection of the infective agent is 
 nil ; there is, of course, some degree of inflammation, but this 
 follows the injection of any fluid, even normal saline solution, and 
 the effect of the bacteria themselves is inappreciable. In this 
 case, therefore, the bacteria are immediately destroyed, and the 
 
INTRODUCTORY AND GENERAL 33 
 
 substances which they produce are without deleterious effect on 
 the cells of the body. In another group of cases, referred to 
 above, the bacteria do not die, but their toxins remain harmless 
 to the host ; this is Ehrlich's immunitas non stevilisans, and it occurs 
 in the case of many of the lower animals which have in their blood 
 various protozoa (trypanosomes, etc.), without thereby suffering 
 the slightest appreciable injury. In man the condition is best 
 seen in its acquired form in the immunity possessed by negroes 
 to the action of malaria parasites, though the plasmodium may be 
 found in the blood. A closely allied phenomenon is in the latency 
 of bacteria. Thus a person may develop an attack of typhoid 
 osteitis years after an attack of typhoid fever, and we can only 
 assume that the bacteria have lain latent in his tissues for this 
 time ; in all probability they have been kept from infecting him as 
 a result of a sufficient degree of immunity, and when this breaks 
 down or wears off a renewed outburst occurs. The gonococcus 
 may be latent in a similar way for periods equally long. Another 
 similar phenomenon is the carriage of infection by persons who 
 remain themselves healthy. Diphtheria is a common example, 
 and it is no rarity to find a person in whose throat diphtheria 
 bacilli are present, but who remains unattacked. Here the 
 immunity suffices to prevent the bacillus from invading the body, 
 but not to destroy it. 
 
 At the opposite end of the scale occur those cases in which 
 immunity is practically absent. Here the result of the introduc- 
 tion of the bacteria is a rapid infection, both local and general, 
 with profound symptoms of intoxication ; the bacteria spread 
 through the tissues just as they would through a good culture 
 medium, and, in addition, invade the blood and multiply therein. 
 This is rarely seen in man, though some examples of septicaemic 
 plague and streptococcal septicaemia from post-mortem wounds 
 approach it closely. It can be produced experimentally in 
 animals, when large doses of virulent cultures are injected. 
 Death follows in a few hours, and the blood is found to be swarm- 
 ing with bacteria. 
 
 Between these two extremes come those cases in which the 
 introduction of the bacterium is followed by the production of a 
 local lesion. This always indicates some degree of local immunity, 
 and may be regarded as an attempt to localize the organism and 
 prevent its further spread. And the nature and severity of the 
 local lesion stand in close relation to the severity of the infection 
 
 3 
 
34 THE LOCAL LESION 
 
 and the degree of the immunity. For example, in severe and 
 rapidly fatal infections from post-mortem wounds i.e., where the 
 infection is virulent and the immunity but slight there is very 
 little local reaction and very little glandular enlargement, the 
 process being septicaemic from the first. Where the infective and 
 protective forces are equally matched the local lesions are more 
 developed; inflammation, and usually suppuration, occur at the 
 site of the wound, and the glands enlarge and may suppurate ; 
 and when the infection is so feeble as to be quite unable to cope 
 with the immunity, the local lesion is the sole result of the infec- 
 tion. Eyre gives a similar example in the results of injecting 
 similar doses of pneumococci into rabbits of different ages. The 
 young animal is most susceptible, and in it death occurs within 
 forty-eight hours from septicaemia, and there is but little local 
 reaction. In half-grown animals the local lesion is more developed, 
 and is gelatinous or fibrinous, containing many leucocytes, and 
 the animal lives several days. In old rabbits quite definite pus is 
 formed, and the animal lives longer, and may recover completely. 
 Hence suppuration may be regarded as a proof that the defensive 
 and infecting forces are fairly balanced, and that either may be 
 victorious in the conflict. 
 
 The other local lesions need not be discussed at length, but the 
 case of tubercle and the allied diseases requires a brief notice. 
 Here the lesion indicates the presence of a very considerable 
 degree of immunity to the toxin, for the structure of a tubercle is 
 exactly similar to that of the cellular reaction to many feebly 
 irritating foreign bodies e.g., unabsorbable ligatures, substances 
 from which it is clear no potent toxin can be given off; but it 
 also indicates that there is a defect in the mechanism by which 
 the bacilli should be removed, since the process is (for a time at 
 least) a progressive one. Here the walling-in of the infected area 
 which occurs as the result of the reaction of the tissues may be 
 taken to be a defensive process, but, as we shall have occasion to 
 see, it is one of doubtful utility. 
 
 EARLY THEORIES OF IMMUNITY. Before turning to the dis- 
 cussion of the nature of immunity in the light of our present 
 knowledge, it will be convenient to insert a short account of some 
 of the early theories of the subject, which are in the main of 
 historic interest only. They have served their purpose as a point 
 of departure for subsequent research. 
 
 Of such nature was Pasteur's theory of exhaustion, the earliest 
 
 
INTRODUCTORY AND GENERAL 35 
 
 attempt at a scientific explanation of the facts of recovery from, 
 and subsequent immunity to, the infectious diseases. Pasteur was 
 a chemist, and was only led to the study of bacteriology by the 
 pursuit of chemical investigations into examining reactions which 
 he proved to be due to micro-organisms. His theory was a 
 chemical one. A certain amount of food is necessary for each 
 bacterium, and when the total amount contained in a given solu- 
 tion is used up the growth of the bacteria must cease. For 
 example, if we take a dilute solution of sugar (containing the 
 necessary salts, etc.), and inoculate it with yeast, the cells will 
 begin to divide and multiply with great rapidity. After a time 
 the growth ceases, and it will not be resumed if we inoculate the 
 fluid with an additional amount of yeast. We may compare the 
 test-tube to the patient, the yeast to the pathogenic organism, and 
 the process of fermentation to the disease, and we may say that 
 the fluid has recovered from the disease and is now immune to it. 
 This immunity depends upon the absence of sugar, which was 
 used up by the yeast cells, and if more sugar be added the process 
 of fermentation may be restarted by a fresh inoculation, or by the 
 yeast still remaining. 
 
 The theory can easily be disproved, from the fact that bacteria 
 may grow well enough in the dead tissues and fluids of immune 
 animals ; and, secondly, because immunity, as we have seen, may 
 be produced (in some cases) by the injection of the chemical pro- 
 ducts of the bacteria, substances which can hardly use up food 
 materials. The theory has, however, been recently revived in a 
 modified form by Ehrlich, who considers that there is sufficient 
 evidence for the occurrence of this form of immunity in certain 
 cases. He calls it atreptic immunity. 
 
 The retention hypothesis of Chauveau is the exact opposite of 
 Pasteur's. Several observers showed that the growth of micro- 
 organisms in fluid media might cease spontaneously whilst 
 abundant food material remained unutilized. This was found to 
 be due to the presence of certain products of metabolism, which, 
 like carbon dioxide in the case of animals, act as poisons to the 
 organism which produces them. For instance, the fermentation 
 of sugar by yeast is found to cease when about 14 per cent, of 
 alcohol is present, and if a strong solution be taken the process 
 will stop at this point, but can be started again if the alcohol be 
 removed by distillation. Here the fermentation is stopped by 
 alcohol, a product of metabolism of the yeast cell, which acts as a 
 
 32 
 
36 THEORY OF RETENTION 
 
 poison on the organism producing it. The theory of immunity 
 based on these facts is obvious. Bacteria growing in the body 
 will yield substances inimical to the continued growth of the 
 organism, so that they will die out and recovery ensue, and the 
 body will remain immune as long as these substances are retained 
 therein. This theory accounts well for the production of immunity 
 by injections of the toxins and other soluble products of bacteria. 
 It is negatived by the fact that bacteria may grow in the blood and 
 tissues of immune animals, and is improbable if we consider that 
 immunity may last for many years, and that it is extremely 
 improbable that substances (necessarily soluble) should be retained 
 in the body for so long a time. 
 
 We shall now proceed to a study of the more modern views, 
 and in doing so it will be convenient to deal with the subjects of 
 immunity to toxins and immunity to bacteria in separate sections. 
 Of course, in most cases they run parallel to one another: an 
 animal contracts a disease because its fluids and tissues cannot 
 kill the pathogenic bacteria offhand, and because its cells are sus- 
 ceptible to the action of the toxin, and vice versa. This is not 
 necessarily the case, however, and the two phenomena may be 
 entirely independent. 
 
 The subject of immunity of toxins is on the whole the more 
 important of the two, the simpler (though complex enough), and 
 the best understood. It will be best to deal with it first. 
 
 
CHAPTER II 
 ON THE NATURE OF TOXINS 
 
 THE fact that the pathogenic action of any organism is dependent 
 entirely, or almost entirely, on that of the ^toxins which it pro- 
 duces renders it necessary to make a brief study of these 
 substances before considering the method in which the infected 
 animal reacts to the organism, and defends itself against infection. 
 In doing so we must distinguish clearly between the specific 
 toxins which are produced by any organism and the non-specific 
 and less important poisons which it may also elaborate. The 
 difference is a fundamental one. Numerous bacteria produce 
 by-products of metabolism, excreta, etc., which are comparatively 
 simple chemical substances of definite composition ; for example, 
 acids, alkalis, alcohol, ptomains, nitrites, etc. These may be 
 poisonous, and may, in some cases at least, play a part of some 
 importance in the production of the symptoms of the disease. 
 The cholera vibrio, for instance, produces nitrites in considerable 
 amount, and since the symptoms of cholera have some resem- 
 blance to those of nitrite poisoning, it is conceivable that those 
 substances may be, to some extent at least, the active causes of 
 the disease, and these nitrites might be regarded as the toxins of 
 the cholera vibrio. This, however, is not the case, and the true 
 toxins are quite different in nature, as is shown by many facts, 
 especially by the proof that cholera vibrios which have no longer 
 the power of producing nitrites may still cause infection in 
 susceptible animals. 
 
 The specific bacterial toxins differ from these poisonous sub- 
 stances in many important particulars. They are, as a rule, 
 formed only in very small amounts, and are extremely powerful. 
 For example, the toxin of tetanus may readily be obtained in so 
 poisonous a solution that T oV TF c - c - w *ll kill a guinea-pig in a day 
 or two, and of this solution only a very small fraction even of 
 the dried residue consists of toxin. They are not simple chemical 
 
 37 
 
3 TOXINS THEIR FRAGILITY 
 
 substances, and their exact nature is as yet unknown. This may 
 be due in part to the minute amounts which are formed, and in 
 part to the difficulties which prevent their being obtained in a 
 pure state ; but there are other reasons, to which we shall revert 
 later, for this complexity. Further, they are, with a few ex- 
 ceptions, very fragile substances, and are readily destroyed by 
 the action of many agents, and especially by heat. Nearly all 
 the bacterial toxins are rendered inert by boiling, and many of 
 them by a short exposure to a temperature of 60 or 70 C. 
 They are usually destroyed by gastric digestion, so that they are 
 without action when administered by the mouth. 
 
 A considerable amount of attention has been paid to this 
 question, since it would be desirable, if possible, to replace hypo- 
 dermic injections of vaccines, etc., by oral or rectal administration. 
 In general terms the statement made above holds good : toxins 
 administered by the mouth are not absorbed as such, and do not 
 produce the characteristic symptoms of the disease. In some 
 cases, however, there is reason to believe that a small amount 
 of absorption, probably of the toxin in an altered form, does 
 occur, and a certain degree of immunity may be produced by 
 the oral administration of killed cultures of typhoid bacilli, and 
 possibly of tubercle bacilli. But this method has only one advan- 
 tage its painlessness over the hypodermic method, whereas its 
 uncertainty renders it extremely undesirable. There can be no 
 doubt that the advantage of giving an exactly measured dose, with 
 the certainty that every particle will be absorbed and act in the 
 way desired, will, under ordinary circumstances, render the hypo- 
 dermic method infinitely preferable. To administer infinitesimal 
 doses of killed tubercle bacilli or of TR to an infant who may be 
 swallowing large doses of living and dead bacilli in milk, sputum, 
 etc., does not appear rational, and the clinical evidence in its favour 
 is entirely unconclusive. In the case of ricin, about a hundredth 
 part of the toxin given by the mouth is absorbed as such i.e. t the 
 minimal lethal dose on oral administration is about 100 times 
 as large as the lethal dose of the same preparation given sub- 
 cutaneously (Stillmarck). Ricin is, however, far more resistant to 
 the action of digestive enzymes than are the exotoxins. 
 
 The most important feature of the bacterial toxins is their 
 relation to immunity. It is possible in all cases to render a 
 susceptible animal immune to their action by the injection of the 
 toxins in suitable doses at suitable intervals, though in some 
 
ON THE NATURE OF TOXINS 39 
 
 cases the task is a difficult one. This is not the case with the 
 non-specific toxins. It is true that in a few isolated instances we 
 are able to increase slightly the resistance of an animal to the 
 simple chemical poisons (e.g., to alkaloids such as morphine), but 
 these apparent exceptions hardly interfere with the utility of the 
 general rule. Further, and more important, an animal immunized 
 to the action of a toxin is also protected against the pathogenic action 
 of the bacterium which produces it, and vice versa. Thus an animal 
 which has been rendered immune to the toxin of tetanus by re- 
 peated injections of that substance is also immune to infection 
 with the living cultures of the bacillus, and an animal which has 
 successfully survived an infection with the tetanus bacillus is 
 thereby rendered in some degree immune to the action of tetanus 
 toxin. This method of immunization with the bacterial toxins 
 (the so-called " chemical vaccination ") is of the utmost impor- 
 tance in practice. It was introduced by Smith and Salmon, who 
 showed that it was possible to immunize pigeons against living 
 cultures of the hog-cholera bacillus by means of the sterilized 
 products of that organism. 
 
 When this method is applicable it supplies us with a test as to 
 the specificity of a toxic substance which we have isolated from a 
 culture of a bacterium, or from the organs of an animal which 
 has been killed by an infection. The substance must be poisonous 
 for animals which are susceptible to the infection in question, and 
 it must be harmless to animals which have been immunized to 
 the organism ; on the other hand, it must immunize animals both 
 to its own action and to that of the bacterium when injected in 
 a living state. These conditions are never fulfilled by the non- 
 specific toxins. 
 
 There are a few apparent exceptions to this rule, but they fail 
 to stand investigation, being based on the fact that it is easier to 
 render an animal refractory to a living organism than to its toxin. 
 Thus an animal which has been injected with the filtered products 
 of certain organisms may be rendered immune to infection with 
 those organisms, but remain as susceptible as before to their 
 toxins. But this is due to the fact that the animal has been 
 immunized but partially ; if the process be carried further the 
 animal will be rendered refractory to both. 
 
 Again, an animal which has been immunized to the toxin of 
 one bacterium remains as susceptible as before to the action of 
 another toxin or bacterium. A horse which has been immunized 
 
40 THE EXOTOXINS 
 
 to diphtheria toxin (e.g., in the production of diphtheria antitoxin) 
 will be just as susceptible to tetanus toxin as a normal animal. 
 In a very few cases the law does not hold. The only well- 
 authenticated example of this sort is the antagonism which 
 animals display to anthrax after injection with the products of 
 B. pyocyaneus. 
 
 These preliminary considerations will serve to show the more 
 important criteria by which the nature of a bacterial product may 
 be determined, and its nature as a true toxin established. 
 
 These toxins were soon found to fall into two main groups the 
 extracellular or soluble toxins, or, as we shall call them, the exo- 
 toxins, and the intracellular insoluble toxins, or endotoxins. We 
 shall consider these substances in turn. 
 
 THE EXOTOXINS. 
 
 The exotoxim are substances which are given off in a free state 
 when the bacteria are grown in a suitable culture medium outside 
 the body, and can usually be separated by simple filtration (through 
 a Pasteur or Berkefeld filter) from the organisms which produce 
 them. We may consider them provisionally as the specific secre- 
 tions or excretions of the bacteria. They are not formed by all 
 pathogenic bacteria that is, in the present state of bacteriological 
 science no suitable culture media have been found in which certain 
 organisms will produce a soluble toxin. The three most impor- 
 tant organisms which do so are the B. tetani, B. diphtheria, 
 and the B. botulismus. These toxins, the first two especially, 
 are substances of the greatest interest, since they have been 
 submitted to a most profound examination, and our knowledge 
 of the structure of bacterial toxins, of their action on the body, 
 and of the production of immunity thereto, is based almost entirely 
 on the results thus obtained. In addition to these, there are sub- 
 stances which are much less toxic if, indeed, toxic at all and 
 which fail to fulfil our definitions of a specific toxin, since an 
 animal which has been immunized thereto is not necessarily 
 immune to the organism, but which have many points in common 
 with the true toxins, and will be considered in this connection. 
 These are the bacterial cytolysins and hsemolysins, T substances 
 
 1 Haemolysis, or the liberation of haemoglobin from red blood-corpuscles, 
 may be brought about by a variety of agents, which fall under three main 
 headings : (i) Simple chemical substances, such as distilled water, ether, 
 acids, etc., which act osmotically, or by a direct solution of the strorna of 
 
ON THE NATURE OF TOXINS 41 
 
 which have the power of dissolving living cells or red blood- 
 corpuscles respectively from susceptible animals. 
 
 In dealing with these substances we will consider firstly their 
 action, secondly their structure, and thirdly what has been estab- 
 lished concerning their chemical relationships with other sub- 
 stances. The last is comparatively unimportant. 
 
 i. Action of Toxins. The results of the injection of a toxin 
 into a living and susceptible animal depend, in most instances, 
 on the dose injected. If, for instance, we inject a large amount of 
 the filtered broth in which the tetanus bacillus has been growing 
 for a month or so, and which in consequence contains tetanus 
 toxin, the animal (a guinea-pig, for example) will develop the 
 rigidities, spasmodic contractions of the muscles, etc., charac- 
 teristic of tetanus ; and these make their appearance after an 
 interval of some hours, during which period the animal shows 
 no symptoms whatever of the disease. Great stress was laid 
 at one time on the occurrence of this " latent period," since it was 
 thought to be peculiar to the bacterial toxins (and to the similar 
 substances of animal and vegetable origin), and to distinguish 
 them sharply from other poisons, alkaloids, etc. This is hardly 
 correct. It is true that in most cases of intoxication by bacterial 
 toxins there is a latent period, but in a few it is practically absent 
 The most interesting example is the " Nasik " vibrio, an organism 
 allied to that of cholera. This produces an exotoxin (though not 
 a very powerful one in the sense that it kills in small doses), 
 which proves fatal on intravenous injection into a rabbit after a 
 period of ten to thirty minutes, and symptoms are produced before 
 this. On the other hand, some of the alkaloids, and notably 
 colchicine, display a well-marked latent period. The phenomenon, 
 therefore, is not absolutely peculiar to, nor characteristic of, the 
 toxins ; but since it is so commonly displayed by them, it calls for 
 some investigation. Moreover, we must assume that part at 
 least of the incubation period of an infective disease is taken up 
 by the latent period of the bacterial toxin, a circumstance which 
 invests it with especial interest. Thus a horse which Madsen 
 
 the corpuscles or of parts thereof; (2) the simple organic haemolysins, which 
 include the bacterial haemolysins dealt with above, the haemolysins of vegetable 
 origin (such as ricin, etc.), and some of the haemolysins of animal origin ; 
 and (3) the compound haemolysins, all of animal origin, which will be dealt 
 with subsequently. These groups differ profoundly in their action, and must 
 be kept quite distinct. 
 
42 ACTIONS OF TOXINS 
 
 was treating for the production of diphtheria antitoxin developed 
 tetanus, and tetanus toxin was found in a sample of blood collected 
 five days before the development of symptoms. 
 
 On diminishing the amount of the toxin which we inject, we 
 find that the latent period becomes gradually longer, and the 
 duration of the disease (i.e., the time between the first develop- 
 ment of symptoms of intoxication and the fatal issue) also lengthens. 
 By diminishing the dose gradually we can find an amount which 
 will just kill the animal in question in a given number of days, 
 and, provided the test animals used are approximately the same 
 in age and weight, we shall find that this amount, the " minimal 
 lethal dose," is fairly constant for animals of the same species. 
 Thus, in the standardization of diphtheria antitoxin the first step 
 is the estimation of the minimal lethal dose of the toxin, and for 
 this purpose it is customary to use guinea-pigs weighing from 
 250 to 280 grammes, and to fix a time-limit of four days. It is 
 found that the minimum lethal dose is the same, within close 
 limits, for all test animals, and that if a series similar in size and 
 weight be inoculated with the same dose, the majority will die 
 within a few hours of one another. This fact enables diphtheria 
 antitoxin to be titrated with some approach to chemical accuracy, 
 the test guinea-pig being used as the indicator. 
 
 On reducing the dose still further, we find that the incubation 
 period is still further prolonged, that the symptoms are less severe, 
 and that death may not take place, or only do so at a later period 
 than that which has been fixed for the minimal lethal dose. Thus, 
 in antitoxin-testing a dose of toxin which does not kill in five days 
 is regarded as a sublethal dose, although death may take place at 
 a later date perhaps much later. 
 
 On giving still smaller doses the symptoms take still longer to 
 develop, are still slighter, and are followed by recovery, and the 
 animal may then present a certain degree of immunity to the toxin 
 and to the organism producing it. On the other hand, under 
 certain circumstances it may be more than usually sensitive to 
 the action of the toxin in question. 
 
 These phenomena present some points of comparison with 
 those which are presented in the action of the soluble enzymes, 
 such as pepsin. In each case an excessively minute amount of 
 the active substance will produce the given effect, and in each 
 the effect is more rapid if a larger amount be used. In either 
 case there is a latent period of longer or shorter duration before 
 
ON THE NATURE OF TOXINS 43 
 
 the peculiar chemical action is manifested. There are several 
 other analogies between the soluble enzymes and the exotoxins. 
 
 (a) The soluble enzymes are, without exception, all produced 
 by living animal or vegetable cells, and are either secreted or 
 excreted by them, or remain locked in their protoplasm. The 
 bacterial toxins, in the same way, are all formed and eliminated 
 by living bacteria ; or, in the case of the endotoxins, retained 
 in the cell. In other words, both extracellular enzymes and 
 exotoxins are products of metabolism given off during the life of 
 a living organism. Further, both substances represent a method 
 in which the organism attempts to modify its environment and 
 render it more suitable : the animal secretes pepsin into its stomach 
 in order to modify the ingested proteids and render them suitable 
 for food, and the tetanus bacillus produces toxin in a living animal 
 because it is in itself but little adapted to grow in living tissues, 
 but can do so easily when these tissues have been injured by the 
 action of toxin. The spores of tetanus which have been washed 
 free from all traces of toxin have no power of producing tetanus 
 when injected into an animal, and are rapidly taken up by the 
 leucocytes, or otherwise dealt with by the tissues ; but if a minute 
 amount of toxin be injected at the same time the bacteria can 
 resist the leucocytes and tissues, which are injured thereby, and 
 continue to grow and produce fresh toxin, giving rise to fatal 
 tetanus. 
 
 (b) It is capable of proof that enzymes commence their action 
 on the substances which they attack by forming a combination 
 therewith. Thus the first effect of the addition of pepsin to fibrin 
 is the formation of a compound between the two substances, as 
 shown by the fact that, if the fibrin be thoroughly washed at 
 a temperature near the freezing-point until all traces of free 
 enzyme are washed away, it will still undergo digestion when 
 raised to the body temperature. Further, the enzyme is less 
 easily destroyed by heat when it has combined with the fibrin. 
 
 In a similar way it is capable of proof that the toxins unite 
 chemically with the cells of susceptible animals. The proof may 
 be deduced from the fact that if toxin be injected intravenously 
 into a susceptible animal it rapidly disappears from the blood, 
 although it does not escape, or only to a very small extent, in the 
 secretions. When the injection is made into insusceptible animals 
 it may disappear by a process to be discussed subsequently, or 
 may persist for long periods. Thus in one case Metchnikoff 
 
44 COMBINATION OF TOXINS AND TISSUES 
 
 was able to demonstrate the presence of tetanus toxin in the 
 tortoise, which is insusceptible to the action of that substance, 
 at a period of four months after the injection. That the disap- 
 pearance which occurs in susceptible animals is actually due to 
 a combination of the toxin with the tissues of the body, and not 
 to its destruction or elimination, is shown by the fact that the 
 tissus of an animal which has been injected with tetanus toxin, 
 but which no longer contains that substance in the blood, may 
 produce tetanus when injected into a susceptible animal. In the 
 case of fowls it seems that this power of combining with tetanus 
 toxin is most marked in the leucocytes. Again, it is possible 
 to reproduce the absorption of tetanus toxin by fresh tissues in 
 vitro. This has been especially studied by Ignowtowsky, who 
 showed that emulsions of liver, kidney, spleen, etc., have the 
 power to absorb tetanus toxin, but that the subsequent injection 
 of these cells will produce the symptoms of the disease. 
 
 It ought to follow logically that the toxin will combine especially 
 with those cells and tissues which are acted upon by it in the 
 living body, and in all probability this is the case. The proof, 
 however, is somewhat difficult. Wassermann apparently proved 
 the point by his demonstration of the fact that tetanus toxin is 
 absorbed and neutralized by an emulsion of the central nervous 
 system, and not by that of any other organ, although, as has 
 been mentioned above, it is absorbed by other tissues. Now, 
 tetanus toxin acts entirely, or almost entirely, on the central 
 nervous system, and this well-known and oft-quoted experiment 
 appears to constitute a proof of the point at issue. The exact 
 interpretation of Wassermann's experiment appears, however, to 
 be doubtful, and it is hardly safe to rely on it as a proof of the 
 point. 
 
 With the bacterial haemolysins, which, although of feeble 
 toxicity, are in every other respect identical with the exotoxins, 
 we are on surer ground. A filtered broth culture of the tetanus 
 bacillus contains the specific toxin (tetanospasmin), and in addi- 
 tion a second substance, which has the power of dissolving red 
 blood-corpuscles when kept at a temperature near that of the 
 body. At a low temperature they do not act in this way ; but if 
 red corpuscles be added in suitable amount to a solution of tetano- 
 lysin at a temperature of o C. and centrifugalized, the supernatant 
 fluid has no longer the power of producing haemolysis. On the 
 other hand, the red corpuscles, even after washing with normal 
 
ON THE NATURE OF TOXINS 45 
 
 saline solution to remove all traces of free haemolysin, are dissolved 
 when raised to the body temperature. In other words, the 
 specific haemolysin of tetanus can form a combination with the 
 structures on which they act. Numerous similar examples will 
 be met with. 
 
 (c) In some of the specific exotoxins, notably that of tetanus, we 
 meet with a similar dependence on a suitable temperature for the 
 development of their toxic action, a property in which again 
 they resemble the soluble enzymes. The most striking example 
 is obtained by a study of the action of tetanus toxin on the frog, 
 which, in common with all cold-blooded animals, is but slightly 
 susceptible to its action. If, however, the frogs be kept in an 
 elevated temperature 30 C. or higher they develop the typical 
 symptoms of the disease after five days or thereabouts. Now 
 Morgenroth has shown that the toxin unites with the central 
 nervous system at a low temperature (8 C.), but without the de- 
 velopment of symptoms. For the production of these a high tem- 
 perature is necessary, exactly as in the case of the combination 
 of tetanolysin with red blood-corpuscles and the solution of the 
 latter. 
 
 (d) These and similar researches lead us to distinguish between 
 two faculties of a toxin that of combining and that of injuring ; 
 and the fact that in some instances these processes can take place 
 at different temperatures leads us to the belief that they are quite 
 different properties of the toxin. In other words, the mere union 
 of a toxin with a cell is not sufficient to cause injury to the latter. 
 This is susceptible of proof. In Ehrlich's elaborate studies on the 
 standardization of diphtheria antitoxin he first obtained a speci- 
 men of diphtheria antitoxin, and determined its minimum lethal 
 dose for test guinea-pigs. For the sake of simplicity we will 
 suppose that for a given sample of toxin this was T ^ c.c. i.e. 9 
 that amount of the filtered broth culture of the diphtheria bacillus 
 would just kill a guinea-pig weighing 250 grammes in four days. 
 Further, let us suppose that we have a standard sample of anti- 
 toxin of which i c.c. just neutralizes i c.c. of toxin (100 lethal 
 doses), so that the mixture of the two causes no symptoms when 
 injected into a test animal. Diphtheria antitoxin is a relatively 
 stable substance, and can be preserved in a dry state, at a low 
 temperature, for long periods if light and air are excluded. It is 
 thus possible to re-test the sample of toxin with a precisely similar 
 solution of antitoxin after some months. When this is done, it is 
 
46 TOXINS AND TOXOIDS 
 
 found that it will have fallen off in potency ; for example, it may 
 take gL c.c. to kill a guinea-pig. It might be supposed that this 
 was due to a complete destruction of half the toxin, but this is not 
 the case. If it were so, we should find that to neutralize i c.c. 
 ( = 50 lethal doses) we should require \ c.c. of antitoxin, since the 
 latter has not altered in potency. As a matter of fact, we find 
 that we still require i c.c. of antitoxin ; in other words, the 
 diminution of the toxic power of the solution has not been accom- 
 panied by a diminution in its combining capacity for antitoxin. 
 The explanation given by Ehrlich, and fully proved by analogy 
 with numerous other similar phenomena, is that part of the toxin 
 has altered into a substance which retains its power of uniting 
 with antitoxin (and, as we shall show later, with the tissue cells), 
 but which has been deprived of its toxicity. Toxin which has 
 undergone this change is called toxoid. Haemolysin also appears 
 to undergo a similar change into haemolysoid, and the rapid loss of 
 
 . #< 
 
 I J 
 
 FIG. 5. A MOLECULE OF TOXIN WITH ITS HAPTOPHORE (a) AND 
 TOXOPHORE (6) GROUPS. 
 
 On the right a similar molecule, which has lost its toxophore group, and 
 become converted into toxoid. 
 
 activity which tetanolysin undergoes is very probably due to a 
 change into that substance. 
 
 The alteration of the toxin to toxoid can be best explained 
 by supposing that the power of entering into combination and the 
 power of intoxication reside in two different parts which we may 
 regard as groups of atoms of the molecule of toxin, and by 
 further supposing that the combining group is a relatively stable 
 one, and that the toxic group is easily destroyed. In the very 
 convenient nomenclature introduced by Ehrlich, and now uni- 
 versally adopted, the group of atoms which has the power of 
 entering into chemical combination with the living protoplas 
 or with antitoxin is called the haptophore grotip, whilst the portion 
 on which the toxic action depends is called the toxophore group. 
 The change of toxin into toxoid, or of haemolysin into haemolysoid, 
 consists in a destruction of the toxophore group, with retention of 
 the more stable haptophore group (Fig. 5.) From what has been 
 said as to the dependence of the phenomena of intoxication on a 
 
ON THE NATURE OF TOXINS 47 
 
 temperature approaching that of the body, it follows that the hapto- 
 phore group can functionate at a low temperature (o to 10 C.), 
 while the toxophore group can only do so at a fairly high one. 
 Looked at in this way, the process of intoxication with an endo- 
 toxin, or of haemolysis with a bacterial haemolysin, may be divided 
 into two stages : in the first place, the haptophore group of the 
 toxin or haemolysin combines with the protoplasm or with the 
 stroma of the red corpuscle, and in the second the toxophore 
 group exerts its action, and the cell is poisoned or the red corpuscle 
 dissolved. The phenomena of tetanus in frogs is thus readily 
 explicable. 
 
 We shall see several other examples of substances in which it 
 is possible to distinguish between a combining and an active 
 group, and the same terminology will be adopted throughout 
 (agglutinoids, complementoids, etc.). 
 
 The change of toxin into toxoid takes place in all exotoxins, 
 but at very different rapidities. Tetanolysin is transformed com- 
 pletely into haemolysoid in a day or so, whilst tetanospasmin, the 
 true toxin of the disease, is much more stable. The process is 
 accelerated by heat, light, and the access of oxygen, and by 
 certain chemical substances which are not sufficiently powerful 
 to destroy the toxin outright. Of these the most important are 
 a solution of iodine in iodide of potassium, and bisulphide of 
 carbon. 
 
 The exotoxins are destroyed outright by heating to the boiling- 
 point (to this rule there are a few exceptions, none of which has 
 been fully examined), by strong acids and alkalis, and by the 
 action of the digestive enzymes. They are, as a rule, precipitated 
 by the substances which precipitate proteids, and destroyed by 
 the substances that destroy those bodies. They have, further, 
 the power of attaching themselves to precipitates, of whatever 
 nature, which are thrown down in fluids containing them ; so that 
 formerly they were thought to be albumins, albumoses, nucleo- 
 albumins, etc., since they were carried down mechanically when 
 these substances were precipitated from a bacterial culture in 
 which they were present along with the exotoxin. In these points 
 again they closely resemble the enzymes. 
 
 They are substances the molecules of which must be small in 
 comparison with those of the coagulable proteids, since they 
 readily pass through filters (of unglazed porcelain permeated with 
 gelatin) which retain the latter. This fact was put to an ingenious 
 
48 TOXINS ANALOGIES WITH ENZYMES 
 
 use by Martin and Cherry in their demonstration that diphtheria 
 toxin and antitoxin combine chemically. 
 
 Enzymes are also substances of small molecule, and pass 
 through similar niters. When injected into suitable animals 
 enzymes give rise to the production of anti-enzymes, which are 
 exactly equivalent to antitoxins. Thus we see that in many 
 points the process of intoxication with the bacterial exotoxins 
 presents close analogies with the destruction of proteids, etc., by 
 enzymes; and to these we might add the suggestion that it is 
 very probable that these exotoxins act, partly at least, by a 
 process of hydrolysis. This suggestion is based partially on the 
 fact that the process of haemolysis is almost certainly one of 
 hydrolysis, and partially on the appearance of poisoned cells, 
 which look as if they had absorbed water and became partly 
 dissolved. 
 
 There is, however, one feature in which the exotoxins and 
 their allies, the bacterial haemolysins, are absolutely different from 
 the enzymes. In the case of the enzymes a molecule attaches 
 itself to the substance to be attacked, water is absorbed, and the 
 whole complex molecule breaks down ; and in this process the 
 molecule of enzyme is set free, and is again ready to attack 
 another molecule. Thus a very small amount of the active 
 substance can decompose a large amount of fermentable substance. 
 The toxins do not behave in this way, and, as far as we know, 
 a molecule of toxin which has united with one molecule of proto- 
 plasm is never set free to attack another. 1 The proof of this is 
 not very direct, and rests mainly on the fact that the amount of 
 toxin necessary to kill two animals of the same species varies roughly 
 with their weight. Thus the minimal lethal dose of diphtheria 
 toxin for a guinea-pig of 250 grammes will not kill one of 400. 
 If the molecule of toxin could attack one molecule of cell 
 substance after another in the same way as an enzyme, we should 
 expect it to do so, though after a longer interval. It must be 
 confessed, however, that this proof is not very striking ; excep- 
 tions frequently occur, since, as a rule, older animals are less 
 susceptible than younger ones in proportion to the body-weight. 
 But it is certainly true with regard to the bacterial haemolysins, 
 since we can test them on the same sample of blood, and when 
 
 1 It may possibly undergo dissociation, and be set free to attack another 
 molecule, but this is a different process : the molecule first attacked is not 
 injured. 
 
ON THE NATURE OF TOXINS 4Q 
 
 this is done we find they obey the law of multiple proportions 
 with great accuracy. Thus the exotoxins differ from the enzymes 
 mainly in the fact that each molecule of the former acts once, and 
 once only. We shall subsequently meet another group of sub- 
 stances, of very similar nature but of animal origin, which have 
 an enzyme-like action, but are destroyed in the process. They 
 are the complements (alexins, etc.), which resemble the exotoxins 
 in many respects, and might well be called the animal toxins. 
 
 On investigating more closely the action of the exotoxins, we 
 find that certain of them exert their pathogenic action mainly on 
 certain cells of the body. The most marked example of this is 
 in tetanus, which practically only affects the cells of the central 
 nervous system, causing in them definite histological changes, 
 and having a pharmacological action almost exactly like that of 
 strychnine. In the case of diphtheria also the action is most 
 marked on these cells ; this is best shown by the occurrence of 
 diphtheritic paralysis (associated with histological changes in the 
 ganglion cells similar to those of tetanus, and subsequent degene- 
 ration of the nerves), which occurs after the action of minute 
 doses of the toxin. 1 We may fairly assume that when but an 
 excessively small amount of toxin is present, it will unite with 
 the cells with which it has most affinity in this case with those 
 of the central nervous ganglia. But diphtheria toxin is not 
 limited in its action, as tetanus toxin is, and can act upon the 
 tissue cells almost without exception. Thus we find that the 
 injection of a large dose of toxin subcutaneously is followed by 
 the production of an acute inflammatory swelling, showing that 
 it can poison the connective tissues, and after death there may be 
 focal necrosis of the liver, degenerative changes in the renal 
 epithelium, fatty degeneration of the heart, etc., showing that 
 the toxin may act on all these organs and tissues. We may 
 regard it as a good example of a general protoplasmic toxin 
 having, as is so frequently the case, a special action on certain 
 cells. The toxins of most diseases come under this heading, the 
 specialized action of the tetanus toxin being unique. 
 
 In some cases we can study the action of the exotoxins and 
 allied substances on isolated cells in vitro, and these are of especial 
 interest from the ease with which they can be investigated, and 
 are of some importance in disease. They are the leucolysins, or 
 leucotoxins, and the haemolysins. 
 
 1 If we accept Arrhenius's view of the interaction of toxin and antitoxin. 
 
 4 
 
50 THE BACTERIAL LEUCOLYSINS 
 
 The leucolysins are substances which are formed by bacteria, 
 and which have the power of killing and dissolving, or partially 
 dissolving, the leucocytes of susceptible animals. Owing to the 
 comparative difficulty of obtaining emulsions of living leucocytes, 
 they have not been submitted to the same thorough examination 
 as have been the bacterial haemolysins ; but the important rela- 
 tions between the leucocytes and immunity lead us to*believe that 
 they are of very considerable pathological interest. The first 
 to be described was that formed by the Streptococcus pyogenes, the 
 action of which on the living leucocytes was shown by an ingenious 
 experiment to occur in vitro, and to be neutralized by means of 
 antileucolysin, this being one of the earliest proofs that toxin and 
 antitoxin form a chemical combination, and that the preventive 
 and curative effects of the latter are not due to some profound 
 influence on the tissues of the living body, by which they are 
 rendered immune before the toxin can attack them. The method, 
 invented by Neisser and Wechsberg, is based on the fact that 
 living leucocytes have the power of deoxidizing and bleaching a 
 solution of methylene blue. When a solution of the products of 
 growth of streptococci is added to an emulsion of living leuco- 
 cytes, together with a little of the dye, and a layer of liquid 
 paraffin added to prevent the further access of air and subsequent 
 oxidation of the methylene blue, the colour no longer disappears, 
 showing that the leucocytes have been killed. If, however, a 
 suitable amount of antileucolysin (obtained by injecting the 
 filtered products of the streptococci into an animal) be added to 
 the mixture the colour disappears, showing that the leucocytes 
 have been protected from the action of the leucolysin, which has 
 now been neutralized by the serum. 
 
 The action of the leucolysins can also be studied microscopi- 
 cally in vitro, when the cells are seen to become more transparent, 
 and their nuclei to become more indistinct, and ultimately to 
 disappear. The dissolving leucocytes look very much like those 
 found in pus. 
 
 Leucolysins are formed by the Streptococcus pyogenes, the staphy- 
 lococcus, and B. pyocyaneus, and probably by other organisms. 
 
 The bacterial hsemolysins are an interesting group of substances 
 which are closely allied to the exotoxins in their reactions, but 
 are little toxic, if at all. The most toxic appears to be that of 
 Streptococcus pyogenes, to which some observers, though not all, 
 attribute feeble poisonous powers when injected into animals. 
 
ON THE NATURE OF TOXINS 51 
 
 At the same time, it is quite certain that these substances play 
 some part in the production of the symptoms of various diseases. 
 The anaemia which develops so rapidly in acute sepsis is well 
 known, and is one of the most constant symptoms of that affec- 
 tion ; it is to be ascribed, in part at least, to the destruction of 
 the red corpuscles by the haemolysins elaborated by the strepto- 
 cocci, staphylococci, or colon bacillus, if these happen to be the 
 infective organisms. The blood of an animal which has been 
 injected with virulent streptococci is found to contain haemolysin, 
 and that this is actually the haemolysin produced by the strepto- 
 coccus is shown by the fact that the action of this serum is 
 restrained by the addition of serum from an animal treated by 
 injections of streptococcic haemolysins. Thus it is proved that this 
 organism elaborates its haemolysin in vivo as well as in vitro ; and 
 several observers have found that it is those species of strepto- 
 coccus which are specially virulent to animals and man that 
 form haemolysins, the harmless ones doing so to a small extent, 
 if at all. The same is true for staphylolysins. Further, when 
 a culture of streptococci which is but slightly virulent and forms 
 but little haemolysin is rendered more virulent by " passage " 
 through rabbits, its power of forming streptocolysin is increased. 
 
 These facts render it certain that some at least of the bac- 
 terial haemolysins act, to some extent, as exotoxins, though the 
 organisms producing them certainly form other and more im- 
 portant specific poisons. We may consider them as accessory 
 toxins of comparatively little pathological importance. 
 
 The similarity in nature of the bacterial haemolysins and the 
 specific exotoxins is shown by the fact that (in the case of strep- 
 tocolysin, and probably in others) they can become converted into 
 htzmolysoids, analogous to toxoids. This is shown as follows : 
 Streptocolysin becomes inert in a week. If a small quantity of 
 blood-corpuscles be added to an excess of this inert solution, and 
 then thoroughly washed and added to a fresh and active solution 
 of streptocolysins, they will not be dissolved ; the corpuscles had 
 evidently become saturated with inert haemolysoid, and are now 
 unable to take up any haemolysin, their combining powers being 
 satisfied (see Fig. 6). 
 
 The chief bacterial haemolysins are those formed by the tetanus 
 bacillus, the staphylococcus, the Streptococcus pyogenes, the B. pyo- 
 cyaneus, B. coli, and the typhoid bacillus. Their more important 
 features will be recapitulated briefly. 
 
 42 
 
TETANOLYSIN AND STAPHYLOLYSIN 
 
 Tetanolysin is formed along with the specific toxin, tetano- 
 spasmin, when the B. tetani is grown in broth, the two substances 
 being formed in variable amounts under different circumstances. 
 It is very unstable, disappearing entirely in a day or two at the 
 room temperature, and being destroyed by heating to 50 C. for 
 twenty minutes. It cannot be obtained free from tetanospasmin, 
 but a solution of tetanus toxin can be deprived of its lysin, and 
 only the specific toxin left, by adding some red corpuscles to the 
 solution, kept at a low temperature, and centrifugalizing them 
 
 FIG. 6. A "SATURATION EXPERIMENT" SHOWING THAT H^MOLYSOID HAS 
 THE POWER OF COMBINING WITH RED BLOOD-CORPUSCLES, AND SHIELD- 
 ING THEM FROM THE ACTION OF H^MOLYSIN. (SCHEMATIC.) 
 
 In the first tube the corpuscles are shown in presence of an excess of old 
 or heated haemolysin ; in the second they are washed clear from this 
 excess, and are apparently unaltered ; in the third active haemolysin is 
 added, but the corpuscles are not dissolved, as they would be in a control- 
 tube with normal corpuscles. 
 
 off; the supernatural fluid will contain tetanospasmin, whilst the 
 tetanolysin will have combined with the corpuscles. 
 
 Staphylolysin appears in alkaline cultures on the fourth day, 
 and reaches its maximum between the tenth and twelfth. It is 
 an unstable substance, but more stable than tetanolysin, persist- 
 ing for a fortnight at the room temperature, and requiring a 
 temperature of 56 C. for twenty minutes for its complete destruc- 
 tion and in this case the destruction appears to be really com- 
 plete, for the injection of the heated solution is said not to lead 
 to the production of an antistaphylolysin. Many normal sera, 
 especially those of man and the horse, contain antistaphylolysin ; 
 
ON THE NATURE OF TOXINS 53 
 
 perhaps this is the reason why slight staphylococcic infections in 
 man are not associated with marked haemolysis. 
 
 Streptocolysin is formed in forty -eight hours when a virulent 
 streptococcus is incubated in broth containing blood-serum or 
 ascitic fluid, and it is a remarkable fact that the nature of the 
 serum used modifies the lysin produced. Thus if ox serum be 
 employed the lysin will act on the corpuscles of the guinea-pig, 
 rabbit, and man, but not those of the ox or sheep ; whilst all 
 these will be dissolved by that grown in broth to which human 
 serum has been added. 
 
 Streptocolysin is less thermolabile than tetanolysin and staphy- 
 lolysin, requiring an exposure of ten hours to 55 C. or of two 
 hours to 70 C. for complete destruction. 
 
 The other bacterial haemolysins i.e., those produced by the 
 B. pyocyanens, B. typhosus, and B. coli are quite different from 
 the foregoing in being thermostable. Thus pyocyanolysin re- 
 sists a temperature of 120 C. for thirty minutes. Typholysin 
 appears to be less resistant, but is definitely thermostable. 
 Colilysin is as stable as pyocyanolysin ; it is not destroyed by 
 a temperature of 120 C. for half an hour, and does not undergo 
 spontaneous weakening for months. It is obvious that these 
 substances are different in character from the other haemolysins 
 and exotoxins, and the fact that (in the case of pyocyanolysin and 
 colilysin, the most heat-resistant of the group) the haemolytic 
 property of the culture only appears when it becomes strongly 
 alkaline and is roughly parallel in degree to the amount of alka- 
 linity, has led some to think that the substances are not the true 
 haemolysins at all, but merely simple alkaline chemical products 
 of growth ; and this is corroborated by the fact that much of the 
 haemolytic power is taken away on neutralization with a weak 
 acid. It appears that this is not the case, since in a culture of 
 B. coli at a temperature of 23 C., the alkalinity reaches its maxi- 
 mum on the fifth day, whilst the haemolytic property does not 
 appear until later. The subject requires further investigation, and 
 at present it is advisable to disregard these substances which differ 
 so much from their allies. 
 
 The chemical nature of the exotoxins has been the subject of 
 much controversy, and is still very imperfectly understood. It 
 will not be discussed at great length, since from the point of 
 view of immunity it is not of very great importance. 
 
 The close analogy between the bacterial exotoxins and certain 
 
54 CHEMICAL NATURE OF TOXINS 
 
 vegetable toxins, such as ricin and abrin (which were thought to 
 be definitely proteid in nature), led, very early in the history of 
 the subject, to the view that these toxins are proteid in nature, 
 and this view was strengthened by the fact that when the diph- 
 theria or tetanus bacillus is grown in an albuminous fluid, proteid 
 substances which are toxic and give the specific reactions of the 
 toxins in question can be precipitated therefrom. Thus Hankin 
 and Sidney Martin found toxic albumoses in bacterial cultures, 
 and apparently succeeded in proving that abrin is an albumose. 
 Brieger isolated a toxalbumin from diphtheria cultures, and 
 Sidney Martin showed that from cultures of the same organism 
 in alkali albumin it is possible to prepare an albumose which 
 he thought to be the specific toxin. Many similar researches 
 were published, and the exotoxins were regarded as being albu- 
 minoid in nature, and the term toxoprotein was applied to them. 
 Several writers Duclaux in particular argued that this was 
 not the case, and thought that these proteid substances merely 
 carried the true toxins with them mechanically on precipitation, 
 just as the precipitates of inert substances such as cholesterin 
 will carry enzymes down with them. This theory was sup- 
 ported by Brieger and Cohn, who purified tetanus toxin from all 
 ordinary proteids, and especially by the researches of Buchner 
 and Uschinsky, who cultivated tetanus and diphtheria bacilli in 
 solutions devoid of all albuminous material, the necessary nitro- 
 genous nutriment being provided by asparagin. Under these 
 circumstances the toxic solution contains neither albumoses, 
 peptones, nor known proteids of any description. The toxins 
 thus formed are present in infinitesimally small amount, and have 
 never been obtained in a pure form, nor submitted to ultimate 
 analysis. It is known, however, that they contain nitrogen, that 
 they are readily destroyed by heat, and that they are dialysable. 
 These considerations lead us to the supposition that they are 
 closely allied to the proteids, and especially to the albumoses or 
 peptones, but form a group differing from any of them, and 
 approximating more closely to the enzymes. That this is the 
 case appears certain from the facts brought out by researches on 
 the antibodies ; all the substances of known chemical composition 
 which lead to the production of antibodies on injection into suit- 
 able animals are either proteids or else substances of indefinite 
 composition similar to the toxins, and apparently all proteids will 
 lead to the production of antibodies on injection into suitable 
 
ON THE NATURE OF TOXINS 55 
 
 animals. These facts lead us to the belief that the exotoxins are, 
 at any rate, allied to the proteids, and form with the enzymes a 
 group of the substances of peculiar composition. 
 
 We have referred above to ricin as a substance once thought 
 to be of definite proteid nature, and a few facts may be given 
 concerning this substance, which is closely allied in every way 
 to the bacterial toxins, and which may be taken as a type of the 
 vegetable toxins or phytotoxins. It occurs in the seeds of various 
 species of Ricinus, and was formerly regarded as being a proteid, 
 since, like the bacterial toxins, it is carried down mechanically 
 with proteid precipitates. Thus Stillmarck regarded it as a globulin, 
 since he prepared it from the seeds by a process which was adapted 
 to the separation of those substances (solution in 10 per cent. NaCl, 
 precipitation with sodium or magnesium sulphate, and dialysis). 
 But Jacoby thought he had succeeded in separating it entirely 
 from its proteid accompaniment, making use of the fact that when 
 a mixture of ricin and the other substances present in the seeds 
 are acted on by trypsin, the active principle is acted on but slightly, 
 if at all ; the ricin itself, in a pure state, is readily digested by 
 trypsin, like the other toxins. Jacoby digested an extract of 
 castor-oil seeds for five weeks, and then added enough ammonium 
 sulphate to render the fluid 60 per cent, saturated, and ricin was 
 thrown down in an almost pure state ; it was purified by repreci- 
 pitation, and then found not to give any of the proteid reactions, 
 though it retained the characteristic toxic properties of the sub- 
 stance. Quite recently, however, Osborne, Mendel, and Harris 
 obtained ricin in a very pure form, and found it to be either 
 proteid in nature or at least inseparably associated with coagulable 
 albumin ; its toxicity was removed by tryptic digestion or heat 
 coagulation. Its great potency ( TTr V^ m g r - being a lethal dose 
 per i kilo of rabbit) suggests that the substance which they 
 prepared was really pure. 
 
 Ricin resembles the bacterial toxins in the following points : 
 It has a period of incubation ; it gives rise to an antitoxin when 
 suitably administered ; it is extraordinarily potent, the lethal dose 
 per kilo of weight (in rabbits) being a minute fraction of a 
 gramme ; it is destroyed by boiling ; and it is much less potent on 
 ingestion than on injection. Its main toxic properties are fever, 
 loss of weight, albuminuria, haematuria, and haemorrhage from 
 the intestine ; death occurs in about twenty-four hours with acute 
 nervous symptoms. It has a most interesting and characteristic 
 
56 THE ENDOTOXINS 
 
 action on the blood, clumping the corpuscles in a peculiar way, 
 even at a dilution of i : 600,000, and also haemolyzing them. 
 
 Further research leads us to believe that the toxin molecule 
 may be, and under ordinary circumstances is, actually of more 
 complex constitution, being combined with a molecule of true 
 proteid. We have already pointed out the fact that streptocolysin 
 differs in its reactions according to the origin of the serum on 
 which it is grown. The best example, however, is derived from 
 diphtheria toxin when grown in broth containing blood-serum or 
 plasma, and subsequently heated. This solution is but feebly 
 toxic, probably from the toxins having undergone a change into 
 toxoids, yet it possesses the power of immunizing an animal 
 against diphtheria, and of stimulating the production of anti- 
 toxin to an unusual degree, but only on condition that it is 
 injected into an animal of the same species as that from which 
 the serum in which the bacillus grew was obtained. Thus horse- 
 serum toxin will stimulate the production of antitoxin in horses, 
 but not in goats or rabbits, and so forth. We are justified in sup- 
 posing that the essential toxin molecule formed by the diphtheria 
 bacillus exists in this fluid in a state of combination with a specific 
 proteid of horse serum, and that the resulting compound molecule 
 differs from that form when the bacillus is grown in goat serum, 
 in which the essential toxic molecule is united with a different 
 proteid. We may suppose that this essential toxin molecule is 
 produced in Buchner and Uschinsky's asparagin solution, but that 
 it is not produced under ordinary conditions, being in a state of 
 combination with proteid materials of more complex structure. 
 These facts render further research into the chemical nature of 
 the exotoxins of comparatively little importance. 
 
 THE ENDOTOXINS. 
 
 In the case of diphtheria and tetanus and a few other organisms 
 the mode of formation of toxins is a perfectly simple one, and 
 one exactly analogous to the formation of soluble enzymes. In 
 most other cases, however, the facts are less easy to understand, 
 and seem to point to the formation of a toxin which remains 
 under normal circumstances locked up in the substance of the 
 bacteria, just as invertase and diastase are contained within the 
 yeast cell, and not excreted by it into the surrounding fluid. A 
 satisfactory theory as to the nature of these toxins is not forth- 
 coming, and the experimental results obtained by various 
 
ON THE NATURE OF TOXINS 57 
 
 observers is very contradictory and difficult to understand, the 
 difficulty being increased by the fact that in the earlier researches 
 the distinction between antitoxic and bacterial immunity was not 
 understood. As a result of this we have to be careful in in- 
 terpreting these early results, so as to make sure that when the 
 author speaks of a serum as containing antitoxin he does not 
 really mean that it contains a protective substance which may 
 not be an antitoxin at all. In many cases the data are not 
 sufficient for us to discover its actual nature. 
 
 The organisms on which the chief amount of experimental 
 work has been done are those of cholera, typhoid, tubercle, 
 anthrax, and the pneumococcus, and it is these which we shall 
 discuss in chief, excluding, however, the consideration of the 
 toxins of the tubercle bacillus for separate consideration in a 
 subsequent chapter. 
 
 The general facts brought out by experiments with organisms 
 such as those of typhoid and cholera are these : The germ-free 
 filtrate of a young and actively growing culture is very slightly 
 toxic, if at all. The nitrate of an older culture is usually feebly 
 toxic, but to a degree which can hardly be compared with that of 
 diphtheria or tetanus ; it may take several cubic centimetres to 
 kill a rabbit or guinea-pig. And even this feeble toxicity is 
 largely discounted by the fact that the nitrate may contain acids, 
 nitrites, etc., which are poisonous, but in no way related to true 
 toxins. Yet in some cases exotoxins do exist in the filtrate, 
 since it is possible to obtain an antitoxin for them. The re- 
 actions of these antitoxins, however, are peculiar, in that the law 
 of multiples does not seem to apply beyond a certain figure. This 
 is well seen in the case of B. pyocyaneus, which forms a sort of 
 connecting-link between cholera and diphtheria, in that it forms 
 a definite though feeble exotoxin, whilst the immunity to it is 
 bactericidal. Wassermann showed that it is possible to produce 
 a true antitoxin against this toxin, and to determine the amount 
 which will just neutralize one lethal dose. He found, however, 
 that a multiple of this amount of antitoxin beyond ten would not 
 protect an animal against a corresponding dose of toxin, With 
 larger doses of toxin even a great excess of antitoxin was power- 
 less to prevent a lethal issue. Similar results have been obtained 
 in the case of cholera. These and other results have led some 
 authorities to consider that these exotoxins are not the specific 
 toxins which the pathogenic action of the bacillus defends, but 
 
58 MACFADYEN'S RESEARCHES 
 
 secondary products of but little importance. Thus, in the case of 
 B. pyocyaneus it is possible to immunize an animal by cautious 
 injections of living organisms, yet its serum has no antitoxic 
 powers against the so-called toxin. 
 
 These facts have turned attention to the bodies of the bacteria 
 themselves, with the result that they have been found to be 
 definitely toxic, although in many cases the toxicity is not great. 
 The theory has therefore been put forward by Pfeiffer espe- 
 cially that under normal circumstances these organisms do not 
 secrete a soluble toxin, but that their protoplasm itself is toxic, 
 and that it is only set free on the death and solution of the cell, 
 thus accounting for the slight toxicity of old cultures, in which 
 such a solution of the cells must take place. The symptom of 
 the disease caused by these organisms is attributed to the solu- 
 tion of the bacteria by the fluids of the body. 
 
 The study of these endotoxins has not left the matter clear. 
 They are present in the bodies of the bacteria, whether the latter 
 have been killed by heat, by antiseptics, or by drying. They are 
 apparently but slightly soluble in water, but can be obtained in 
 solution by autolysis of the bacteria in normal saline solution in 
 the incubator, by grinding the dead bacilli, or by the use of very 
 high pressure (the method introduced by Buchner for the extrac- 
 tion of endo-enzymes from yeast). But it did not appear possible 
 to produce an antitoxin against this poisonous material ; in 
 addition, animals which have been immunized against the living 
 organism might be as susceptible as before to the dead bacteria, 
 or to extracts of them. 
 
 The researches of Macfadyen and Rowland have apparently 
 disproved this, and tend to support the opinion that the endo- 
 toxin is a true toxin, for which an antitoxin can be obtained. 
 They obtained young cultures of various organisms, froze them 
 at the temperature of liquid air, and then ground them (whilst 
 solid) into an impalpable powder. This was made into a paste 
 with normal saline solution and centrifugalized, to remove any 
 solid particles. The juice thus obtained was sterile. It was 
 more powerful than endotoxins prepared in other ways, and it 
 acted very quickly, having a very short period of incubation, if 
 any. Thus, in the case of the typhoid toxin i c.c. killed in three 
 hours and J^ c.c. in less than two days, on intraperitoneal in- 
 jection. It was less active on subcutaneous injection not more 
 so, in fact, than other toxins of the typhoid bacillus requiring 
 
ON THE NATURE OF TOXINS 59 
 
 to y 1 ^ c.c. to kill in seven days. Macfadyen and Rowland 
 found that they could immunize animals against their toxin, and 
 that its serum was antitoxic. These researches are difficult to 
 harmonize with those of other observers. We must admit, how- 
 ever, that it is possible to prepare an antitoxin to the endotoxins. 
 The failure of other observers to do so may be owing to the fact 
 that their toxins were not prepared in so suitable a manner for 
 this purpose, and may have undergone some unknown secondary 
 alterations. 
 
 But these researches do not clear up the whole of the mystery, 
 for some observations of Metchnikoff and others show that the 
 V. cholera: can produce a soluble toxin whilst in the animal, and 
 apparently without being killed in the process. These observers 
 prepared collodion bags, which they filled with cultures of this 
 organism, hermetically sealed, and inserted into the peritoneal 
 cavities of guinea-pigs. The animals died in a few days with 
 the symptoms of cholera intoxication, although no bacteria had 
 escaped from the sacs ; the organisms in that situation were still 
 alive. Control experiments with dead organisms showed that 
 little toxin was present ; the animals remained alive, though they 
 might show some symptoms of toxic action. It appears, there- 
 fore, that the living bacteria do elaborate an exotoxin whilst 
 within the animal body, and that this exotoxin has the power of 
 diffusing through a collodion membrane. Welch has suggested 
 an explanation which cannot be discussed fully here, but which 
 may be mentioned briefly. He points out that when bacteria 
 are injected in living animals the tissues of the latter react and 
 produce substances bacteriolysins, etc. which are injurious to 
 the bacteria, and which determine in part the resistance of the 
 host, and suggests that the bacteria may also react in a similar 
 way to the cells with which they are brought in contact. Just as 
 the animal host only produces its toxins the bactericidal sub- 
 stances when the bacteria are brought into contact with it, so 
 the bacteria may only produce their protective substances the 
 unidentified true toxins when brought into contact with aggressive 
 animal cells. If this is the case it is obvious that we cannot expect 
 to produce these toxins in vitro, except perhaps by cultivation of 
 the bacteria in question in fresh serum from an immunized animal. 
 
CHAPTER III 
 
 THE PHENOMENA OF ANTITOXIN 
 FORMATION 
 
 As a general rule, to which there are important exceptions, it is 
 necessary to make use of susceptible animals for the production 
 of antitoxin. When toxin is injected into animals in which it pro- 
 duces no injurious effects, it either disappears rapidly from the 
 blood or remains for a long time in that fluid or in the tissues 
 without leading to the formation of antitoxin. The most remark- 
 able exception to this rule is the way the cayman reacts to tetanus 
 toxin. The animal is immune, and if kept in the cold (20 C.) the 
 toxin soon disappears from the blood, no antitoxin being formed. If, 
 however, it is kept at an elevated temperature (32 to 37 C.), the 
 toxin disappears as before, but now antitoxin makes its appearance 
 (Metchnikoff). Such cases are exceptional, and when we wish 
 to procure antitoxin, we make use of an animal in which the 
 toxin in question produces symptoms of intoxication. The pro- 
 cess is usually much easier in large animals, such as horses or 
 goats, than in small ones, such as rabbits or guinea-pigs, the 
 immunization of which presents considerable difficulties. We 
 shall take as illustrations of the general phenomena of the pro- 
 cess the methods adopted for procuring diphtheria antitoxin and 
 tetanus antitoxin from horses, since these have become so familiar 
 from their extensive application. 
 
 On injecting a small dose of a potent diphtheria toxin sub- 
 cutaneously into a horse say, \ c.c. under the skin of the 
 neck we find there is a latent interval of a few hours or a 
 day before the development of symptoms ; then there is a lcel 
 reaction, consisting in the formation of a hard brawny mass f 
 inflammatory oedema round the site of the inoculation, and a 
 general reaction, consisting in fever, anorexia, and symptoms of 
 general malaise. These symptoms last a day or two, according 
 to the dose of toxin injected, its potency, and the degree of sus- 
 
 60 
 
THE PHENOMENA OF ANTITOXIN FORMATION 6l 
 
 ceptibility of the animal ; and when they have passed off a small 
 amount of antitoxin will be found in the blood, and the animal 
 will, as a rule, be found to be less susceptible to the action of the 
 toxin than before, so that the injection of the same dose will 
 produce less reaction, both local and general. 
 
 This, however, is not always the case, and careful research 
 leads us to the belief that the appearance of immunity is preceded 
 by a period of hypey sensitiveness, in which the animal betrays a 
 greatly increased susceptibility to the action of the toxin, and this 
 in spite of the fact that it may contain quite large quantities of 
 antitoxin in its blood. Thus it happens not infrequently that 
 after a horse has passed successfully through the early stages 
 of immunization to diphtheria toxin, and has developed far more 
 antitoxin than is necessary to neutralize the doses of toxin with 
 which it is being treated, it yet will die after the injection of an 
 amount which it would appear must be immediately rendered inert 
 as soon as it came into contact with the plasma. Such cases have 
 been reported from the Pasteur Institute, Behring and Kitashima, 
 and others, and by Brieger for tetanus. In the latter an immunized 
 horse died after an injection of tetanus toxin with the typical 
 symptoms of tetanus intoxication, and after death its blood con- 
 tained much free antitoxin. The phenomenon has probably been 
 witnessed by most observers who have been engaged in the manu- 
 facture of antitoxin, though it has become much less frequent since 
 the introduction of modern methods for the early treatment of 
 animals. It is an exceedingly puzzling one, and we shall leave 
 its further interpretation until later ; here it is sufficient to say 
 that Behring's theory of the occurrence of a stage in which the 
 tissues are hypersensitive to the toxin is well established. 
 
 The difficulty of immunizing the small animals of the laboratory 
 to these toxins appears to depend in large measure on the marked 
 development of hypersensitiveness. Thus Behring and Kitashima 
 found that they could kill a guinea-pig with ^J^ of the " minimal 
 lethal dose" of tetanus toxin, if this amount were divided into 
 several doses and given at suitable intervals, and similar facts 
 have been recorded by others. 
 
 The most striking proof of the occurrence of hypersensitiveness 
 in the process of immunization has been investigated by Behring, 
 who pointed out that normal horses show no local effects from the 
 injection of small quantities of tetanus toxin ; their connective 
 tissues are insusceptible to its action. As the animal becomes 
 
62 CHOICE OF TOXINS 
 
 immunized to the action of the toxin this is not the case; the 
 tissues at the site of inoculation react to the poison with the pro- 
 duction of a mass of inflammatory redema similar to that seen in 
 a horse injected with diphtheria toxin. It is obvious that these 
 connective tissues have become more susceptible to the action of 
 the tetanus toxin, and this in spite of the antitoxin with which 
 they are bathed. 
 
 In order to avoid the difficulties arising from the occurrence of 
 hypersensitiveness in the early stages of immunization, the use 
 of unaltered toxin has now been practically abandoned, the follow- 
 ing methods, either alone or in combination, being employed 
 instead : 
 
 1. The use of mixtures of toxin and antitoxin, the latter being 
 present in amount sufficient to neutralize all the toxin, or in 
 excess. This is repeated several times, the amount of antitoxin 
 given being gradually reduced, until at last a small amount of 
 unaltered toxin is given. 
 
 It must not be thought that the immunity which is acquired in 
 this case is simply passive, and due to the free antitoxin which is 
 injected. The process is probably fundamentally different. We 
 shall revert to it subsequently. 
 
 2. The injection of toxoids. This method is of especial advan- 
 tage in the case of tetanus, to which toxin animals are extremely 
 sensitive, and the dangers of the early stages of the process of 
 immunization are very great. The toxin formed in the cultures 
 may be transformed into toxoids by the action of trichloride of 
 iodine, a solution of iodine in iodide of potassium, or by heat, 
 the filtered cultures being exposed to a temperature of 60 C. for 
 a time sufficient to destroy their toxicity. Toxin that has been 
 heated to a temperature much higher than this is completely 
 destroyed, and is useless for the process. 
 
 3. The use of serum toxin, which probably contains toxoids in 
 an unusual condition of activity. This method was introduced 
 by Cartwright Wood, and is now in general use in this country 
 for a part at least of the process of immunization, since it leads to 
 a more rapid production of antitoxin of high potency than can be 
 obtained by other methods in the same time. Ordinary alkaline 
 broth is inoculated with diphtheria bacilli, and incubated for a week 
 at 37 C. Then 15 to 30 per cent, of its volume of serum from an 
 animal of the same species as is to be used in the process of 
 immunization is added, and the incubation continued for a month 
 
THE PHENOMENA OF ANTITOXIN FORMATION 63 
 
 or six weeks. It is then heated to 65 C. for half an hour and 
 filtered. It gives rise to marked febrile reaction and but little 
 local reaction. The initial dose is 200 to 300 c.c. 
 
 In giving these large doses the most convenient method is to 
 use a large wash-bottle, the side of which is graduated in 
 cubic centimetres. To the outflow arm there is attached 2 or 
 3 yards of pressure tubing, in the farther end of which a strong 
 exploring needle is inserted, and firmly wired in place. The 
 pressure is obtained by means of a bicycle pump attached to the 
 inflow tube of the wash-bottle by means of pressure tubing. 
 There should be a lateral branch communicating with a mano- 
 meter, by which the pressure can be regulated. Very high 
 pressure is sometimes necessary, especially in the later stages of 
 the process, when the subcutaneous tissues of the horse's neck 
 become sclerosed and dense from the repeated injections. The 
 apparatus is most easily sterilized by passing strong carbolic 
 lotion through it. 
 
 On testing the blood-serum from time to time, it is found that 
 the amount of antitoxin gradually rises, each injection being 
 followed by an increase in the antitoxic value of the serum. Thus 
 the process is a cumulative one, the antitoxic level being raised 
 step by step until a certain height is reached. This height differs in 
 different animals. Thus Atkinson, in summarizing his experience 
 of 100 horses, found that half of this number gave less than 
 300 units of antitoxin per cubic centimetre, a quarter between 
 300 and 500, whilst three gave more than 800. There appears 
 to be no method of investigation by which the value of a horse 
 as a source of antitoxin can be predicted early in the course 
 of treatment, and the great variability amongst different animals 
 is probably the reason that different observers have come to 
 such divergent opinions as to the best doses to give and the 
 most suitable intervals between each. Here are three chief 
 methods : 
 
 (a) By the use of large doses of toxin, 250 to 500 c.c. every day, 
 or almost every day, leaving an interval of a week or ten days 
 before the bleeding, so as to allow the last injection to produce its 
 maximum effect. 
 
 (b) The use of large injections (similar to the former) at longer 
 intervals five to ten days. 
 
 (c) The use of relatively small doses of weak toxins repeated 
 every day. 
 
64 THE NEGATIVE PHASE 
 
 All these methods have their advocates, and good results can 
 apparently be obtained by all. 
 
 On ceasing to inject toxin, it usually happens that the antitoxic 
 value of the serum commences to decline, and, in the absence of 
 further injections, would probably continue to do so until it had 
 entirely disappeared from the blood. In a few cases, however, 
 a period of antitoxic equilibrium is maintained for some time, the 
 amount of antitoxin lost by the excretions or destroyed in the 
 system being compensated for by a fresh production of the same 
 amount. When this is the case the phenomena resulting from 
 the injection of a single dose of toxin can be traced with ease, and 
 is of great importance, as will appear subsequently. The first 
 effect of the injection is the production of a negative phase, in which 
 the amount of antitoxin in the blood is suddenly and greatly 
 diminished. This production of a negative phase is apparently 
 a general phenomenon, and is found to occur in the development 
 of nearly all antibodies in which it has been investigated. If the 
 dose of the primary substance (toxin, etc.) is very small, the 
 negative phase may be short in duration and very slight in extent, 
 and may be overlooked, or may possibly be omitted altogether. 
 Its explanation is very uncertain and cannot be discussed here, 
 but it must be pointed out that it is not due to the neutralization 
 of the antitoxin in the blood by the toxin injected ; the proof of 
 this is that it is large out of all proportion to the latter. Thus in 
 one reported case the fall in antitoxic value of the serum which 
 occurred in the negative phase would have required an injection 
 of toxin 12,000 times as large as was actually given if it were due 
 to simple neutralization. The length of the negative phase varies 
 in different animals, and can only be learnt by experiment. It 
 appears to be roughly proportional (in the same animal) to the 
 amount of primary substance injected : the larger the doses of 
 toxin, the greater the fall and the longer its duration. It is, 
 of course, synchronous with the toxic symptoms, if any, of the 
 substance injected, since both are due to the action of this sub- 
 stance on the blood and tissues ; but the two do not appear to be 
 mutually dependent : a well-marked negative phase may appear 
 without any other symptoms of disease. 
 
 The negative phase is succeeded by a rise, the positive phase, 
 in which the antitoxic value of the blood reaches and usually 
 surpasses its previous level. It commonly reaches its maximum 
 in about a week, and then commences to decline ; hence it is 
 
THE PHENOMENA OF ANTITOXIN FORMATION 65 
 
 advisable that the animal should be bled for antitoxin after a rest 
 of about a week from its last injection. 
 
 The bleedings are carried out at the laboratories of the Royal 
 Colleges of Physicians and Surgeons in the following manner : 
 The receptacles for the blood are 2-pound glass jam-jars, which 
 are sterilized by heat and covered with parchment paper which 
 has been soaked for some hours in i : 20 carbolic. Two layers 
 of this are used, and the lower one has two radial slits cut in it, 
 leaving a triangular wedge, which can be raised and access to the 
 bottle thus obtained. Twelve or fourteen of these are required 
 for each horse, and each is filled about two-thirds full. 
 
 The side of the horse's neck is shaved and washed with a 
 solution of lysol, or a lysol dressing is put on an hour or two 
 before the operation. The horse is placed in the stocks, and if 
 violent the head is restrained by a twitch. It is then necessary 
 to apply pressure at the lower part of the neck, in order to distend 
 the jugular vein ; this may be done by the thumb of an assistant, 
 or, better, by means of a firm leather plug, which is pressed into 
 the groove in front of the sterno-mastoid muscle by means of an 
 arrangement of straps devised by Dr. Cartwright Wood. In this 
 way the vein is temporarily occluded, and stands out clearly 
 above the region where the pressure is applied. The operator 
 (having sterilized his hands as for a surgical operation) then 
 makes an incision about 2 inches long and above or just in- 
 ternal to the vessel ; this should open the deep fascia, but 
 need not actually expose the vein. He then takes a trocar and 
 cannula having a diameter of about T \ inch, and pushes it firmly 
 downwards into the vein ; success in this is shown by the blood 
 oozing up by the side of the trocar. An assistant now stands 
 ready with a short metal tube which fits inside the cannula 
 and communicates with 2 or 3 yards of indiarubber tubing, with 
 a foot or so of glass tubing at its farther end. The whole has 
 been sterilized by being soaked in lysol or carbolic lotion. A 
 second assistant now reflects half of the outer parchment covers 
 of one of the jam-pots, reflects the triangular strip which has'been 
 already cut in the inner cover, and inserts the glass tube in the 
 opening. The operator then removes the trocar, and the first 
 assistant rapidly fits the metal tube attached to the rubber tubing 
 into the cannula; when this is done quickly hardly any blood 
 escapes. The blood now passes through the rubber tubing into 
 the jam-pot, which rapidly fills. When about two-thirds full the 
 
 5 
 
66 PREPARATION OF ANTITOXIN ON A LARGE SCALE 
 
 assistant pinches the indiarubber tube and places the outflow tube 
 in a second pot. The outer cover is replaced on the first pot, 
 which is removed to a warm place to clot. The process is repeated 
 until twelve or fourteen pots have been filled. 
 
 Horse's blood coagulates slowly, and a well-marked buffy coat is 
 formed. In twenty-four hours this will have contracted, and 
 much of the serum will be squeezed out. In order to draw this 
 off use is made of a wash -bottle, the short tube of which is con- 
 nected with a water-pump, such as is used for filters, by which 
 a partial vacuum can be maintained. The long tube is connected 
 to a piece of indiarubber tubing terminating in a length of glass 
 tubing. A jam-pot is opened by half reflecting the outer cover 
 and lifting the triangular strip cut in the inner one, and the glass 
 tube is inserted. Air is now sucked out of the wash-bottle by 
 turning the tap which puts it into communication with the suction- 
 pump, and the serum siphons over. When all the serum has been 
 abstracted a second jar is treated in the same way, the parchment 
 cover of the first being replaced, and the process is continued with 
 all the jars. In twenty-four hours more serum will have appeared, 
 and the process is repeated, and a small amount may often be 
 obtained on the third day. In this way the total yield of anti- 
 toxin is usually nearly 50 per cent, of the total volume of blood 
 (4i to 5 litres). 
 
 The antitoxin thus obtained is usually sterile, the most careful 
 precautions being taken to prevent contamination. Carbolic acid 
 (0-3 per cent.), or trikresol (0-3 per cent.), or a mixture of the two, 
 must now be added to preserve it. It is then filtered through 
 a Berkefeld filter (not a Chamberland filter, through which it 
 passes with great difficulty, if at all), a low pressure only being 
 used, and finally tested for sterility by means of cultures, and for 
 the presence of toxins by the injection of large (10 c.c. or more) 
 amounts into normal guinea-pigs. 
 
 A specimen is taken at the time of the bleeding, and this is 
 tested for antitoxic value in the manner to be described subse- 
 quently. The results of this testing will give the amount necessary 
 to obtain the required dose, and this amount is placed in sterile 
 tubes or bottles ready for use. In most cases mixtures are made, 
 antitoxin of low potency being mixed with more powerful sera in 
 order to obtain the requisite dose in a given volume of serum. 
 An ingenious machine is used by which the tubes or bottles are 
 filled automatically with the antitoxin in the required amounts. 
 
THE PHENOMENA OF ANTITOXIN FORMATION 67 
 
 In the earlier stages of immunization, as we have seen, each 
 injection is followed (after a negative phase) by a rise in the anti- 
 toxic value of the serum above its previous level. In the stage 
 which now follows this does not occur, or not definitely ; there is 
 a negative phase, but it is found impossible to force the antitoxic 
 value above a certain level, which varies in different horses. 
 This second stage, or period of maintained maximum, varies in 
 different horses, and may last a few months or a year. While it 
 lasts there are, of course, oscillations ; it falls, for instance, if the 
 animal contracts any disease or suffers in general health, but its 
 general average is about the same. 
 
 Sooner or later this state of affairs changes, and the antitoxic 
 value of the serum begins to fall, and cannot be raised or even 
 
 FIG. 7. 
 
 a, b, Normal resisting power ; b, c, period of hypersensitiveness ; c, d, period 
 of rise in immunity ; and d, e, maintained high level thereof. /, g, Normal 
 amount of antitoxin ; g, h, period in which it increases ; and h, i, gradual 
 fall and ultimate (theoretical) disappearance. 
 
 maintained at its former level in spite of very large doses of toxin. 
 It trends steadily downward, although the animal may continue 
 to give useful serum for a long time. Thus in one of Atkinson's 
 best horses (out of a series of 100) the serum contained 1,000 to 
 1,100 antitoxic units per cubic centimetre for ten months, and 
 then gradually sank, but remained above 300 units per cubic 
 centimetre for two years. 
 
 After a prolonged rest the power of manufacturing antitoxin 
 may return, but then only lasts a short time, and cannot be re- 
 newed again. 
 
 The third stage, therefore, consists in a gradual disappearance 
 of antitoxin from the blood, without any loss of the immunity to the 
 toxin. It would seem, indeed, as if the immunity reaches its 
 highest level at this point, in spite of the almost complete absence 
 
 52 
 
68 IMMUNITY NOT DEPENDENT ON ANTITOXIN 
 
 of antitoxin. Thus the two phenomena do not run paripassu with 
 one another. This, is illustrated in the foregoing diagram, which 
 represents in a purely schematic way the period of immunization 
 and utility of an antitoxin horse, the height of the continuous line 
 from the base representing the degree of immunity, that of the 
 dotted line the amount of antitoxin in the blood. 
 
CHAPTER IV 
 
 INTERREACTIONS OF TOXIN AND ANTITOXIN 
 
 STARTING from the facts that a suitable dose of antitoxin will 
 prevent the development of symptoms if toxin is injected shortly 
 before, at the same time, or shortly after, or that if antitoxin and 
 toxin be mixed in vitro and injected subsequently, no symptoms 
 develop, we have to inquire the mechanism by which this is brought 
 about. Two theories suggest themselves at once. The antitoxin 
 might act on the cells of the living body in such a way as to render 
 them insusceptible to the action of the poison, or, in other words, 
 render them immune, or the toxin and antitoxin might unite 
 chemically to form an inert and harmless compound. When the 
 fundamental facts of antitoxic action were first discovered, the 
 majority of pathologists probably inclined to the former alternative, 
 the latter seeming too simple and teleological. A certain amount 
 of experimental evidence was also forthcoming in favour of this 
 view, but as this has a merely historical value it will not be 
 considered. It is now fully proved that toxin and antitoxin form 
 chemical compounds, and that the prophylactic and curative value 
 of the latter is to be explained simply on the grounds that this 
 compound is inert, or devoid of toxic action on the animal cells. 
 The evidence in favour of the occurrence of this chemical com- 
 bination requires brief discussion. 
 
 The first group of experiments pointing in this direction are 
 those in which the toxin and antitoxin are mixed in vitro, and 
 the result tested by means of red blood-corpuscles as indicators, 
 the intervention of the cells of the living body being thus excluded. 
 (Many of these experiments can be repeated on corpuscles which 
 have been heated to a temperature sufficient to destroy the life of 
 isolated body cells, and the possible objection that the corpuscles 
 are " surviving " thus removed.) 
 
 The first of these researches was that of Ehrlich, who showed 
 
 69 
 
70 FILTRATION EXPERIMENTS 
 
 that the agglutinative action of ricin on red blood-corpuscles 
 could be inhibited in vitro by means of the serum of an immunized 
 animal. Kanthack showed that the action of snake -venom in 
 inhibiting the coagulation of blood was similarly prevented in 
 vitro by its appropriate serum, whilst Kossel and others did the 
 same for the haemoglobin of eel's blood, and Ehrlich for 
 tetanolysin. The previous cases were not of true bacterial toxins, 
 and might possibly be open to objection on that account. The 
 experiment of Neisser and Wechsberg on the effect of leucocidin 
 on leucocytes in vitro, and its inhibition by means of an antiserum, 
 is another case in point. It is true that in this case the leucocytes 
 are living, but we can hardly imagine that they have become 
 immunized by the action of the serum, or that the phenomenon 
 can be explained on any hypothesis other than that the toxin and 
 its antiserum have combined. 
 
 The second and most important series of researches are those 
 of Martin and Cherry, who show that several toxins (e.g., that 
 of diphtheria and snake-venom) pass through a porcelain filter 
 which is impregnated with gelatin, whereas their appropriate 
 antitoxins, being composed of larger molecules, do not. (This had 
 previously been proved by Brodie.) They found, further, that 
 when a mixture of toxin and antitoxin was placed on such a filter 
 the first portion of the filtrate was toxic, but that the amount 
 diminished, and all toxicity disappeared a few minutes after the 
 mixture had been made. The inference is clear : the toxin had 
 united with the antitoxin to form a molecule as large as, or even 
 larger than, that of the latter, and therefore, like it, unable to pass 
 through the pores of the filter. These researches have been 
 confirmed by Brodie, and form, on the whole, the most striking 
 direct proof of the union of the two substances yet brought 
 forward. 
 
 Calmette found that snake-venom is more heat-resistant than 
 its antitoxin, withstanding a temperature of 80 or 90 C., whereas 
 the latter is rendered inert at 68 C. He was then able to show 
 that a neutral mixture of the two could be rendered toxic again 
 by exposure to a temperature of 70 C. ; and this fact was used first 
 as an argument against the chemical theory of combination, and 
 secondly as a proof that the toxin is not destroyed when it unites 
 with antitoxin. As a matter of fact, neither inference is 
 necessarily correct, and the experiment was shown by the further 
 researches of Martin and Cherry to constitute a proof of the 
 
INTERREACTIONS OF TOXIN AND ANTITOXIN 71 
 
 chemical theory : for they found that if the mixture were allowed 
 to stand for some time at the temperature of the body before being 
 heated, its toxicity was not restored by a temperature of 70 C. 
 This seems to show that the toxin did not exist as such in the 
 mixture, otherwise it would not have been destroyed by the heat ; 
 it must, therefore, have become combined with the antitoxin, or 
 at any rate modified by it in some way. On the other hand, the 
 experiment does not prove that the toxin is completely destroyed 
 beyond all power of further activity ; it simply shows that, when 
 in a condition of combination with its antitoxin, it is less thermo- 
 stable than when free. Similar facts were adduced by Wasser- 
 mann with regard to the combination between pyocyaneus toxin 
 and its antitoxin, and are capable of a similar explanation. 
 Marenghi has also brought forward somewhat similar results 
 with diphtheria toxin. 
 
 Lastly, Ehrlich has shown that the conditions which favour the 
 occurrence of chemical combinations favour the union of toxin 
 and antitoxin e.g., it is accelerated by heat, and takes place more 
 quickly in concentrated than in dilute solutions. 
 
 This brings us to the question as to whether the combination 
 takes place in accordance with the law of multiple proportions 
 a question of great difficulty, but one which has lead in its 
 elucidation to the discovery of facts of much interest. As far as 
 concerns the action of the haemolysins and other toxins that can 
 be readily tested in vitro, there is no doubt that this question, in 
 its simplest form, must be answered in the affirmative. If it 
 requires x c.c. of a given solution of toxin to dissolve exactly 
 i c.c. of a 5 per cent, emulsion of red blood-corpuscles, then it 
 will require 2#, 3*, 4*, etc., c.c. to haemolyze 2, 3, 4, etc., c.c. of 
 the same emulsion. We assume in each case that the haemolysin 
 is added at once, and not in small consecutive amounts. To 
 study the effect of the partial neutralization of toxin by antitoxin 
 we will briefly outline Ehrlich's famous work on the standardiza- 
 tion of diphtheria toxin, and the conclusions he arrived at in 
 consequence of the results thus obtained. 
 
 We have seen that it is possible to determine with a close 
 approach to accuracy the minimal lethal dose of diphtheria toxin 
 for standard guinea-pigs 4.e., those weighing about 250 grammes. 
 This amount is called the toxic unit (TU), and a toxin of which 
 T ^ c.c. is just sufficient to kill a test guinea-pig in three or four 
 days is considered to be normal toxin of unit strength, and is 
 
72 STANDARDIZATION OF DIPHTHERIA TOXIN 
 
 written DTN. 1 A toxin of half this strength, of which -^ c.c. 
 is the lethal dose, is written DTN . 5 . Toxins of other potencies 
 are numbered accordingly. 
 
 Ehrlich now proceeded to define a unit of antitoxin as the 
 amount that would just neutralize 100 lethal doses of toxin : this 
 is called IU ( = immunizing unit). This amount may be contained 
 in any quantity of the serum ; thus, in that used for clinical work 
 i c.c. contains anything between 300 and 1,000 units, or even 
 more. For the purpose of testing toxins it is convenient to use 
 an antitoxic serum which is much more dilute than this, and an 
 antitoxin of unit strength is defined as one which contains i unit 
 of antitoxin in i c.c. i.e., i c.c. of the antitoxin will just neutra- 
 lize i c.c. (100 lethal doses) of standard toxin. The reaction 
 between these amounts is written thus : 
 
 i c.c. toxin (=100 lethal doses) + i c.c. antitoxin = L , 
 
 where L (L = limes) indicates that the mixture is a truly neutral 
 one, and that it does not kill a susceptible animal within the time- 
 limit, or produce any pathogenic action whatever. 
 
 Now, if, as Ehrlich believes, the affinity of toxin for antitoxin 
 is a powerful one, similar to that of a strong acid for a strong 
 base, it should follow that if to the 100 lethal doses of toxin we 
 add only T 9 ^ of i c.c. of standard antitoxin, then T Jy of the original 
 amount i.e., i lethal dose should remain unneutralized, and the 
 animal should die in the same time as a similar animal which had 
 received i lethal dose and no antitoxin. 
 
 As a matter of fact, this is not what occurs. We find that when 
 we inject the mixture the animal does not die in a short time 
 with the ordinary symptoms of diphtheritic intoxication, but 
 develops local oedema, and possibly paralysis, which may bring 
 about death at a remote period. The same thing happens if we 
 add still less antitoxin to the 100 lethal doses of toxin. To take 
 a particular case, it is not until the mixture contains less than 
 T 7 ^y of i c.c. of antitoxin that the animal dies acutely in the way 
 it does after an injection of i lethal dose of toxin. It seems, 
 therefore, that the whole of the toxicity of the toxin is removed 
 when only three-fourths of the amount of antitoxin necessary to 
 neutralize it has been added, or that a given amount of toxin can 
 
 1 DTN = diphtheria toxin normal. It is also written DTN 1 M 250 =DTN one 
 unit for a guinea-pig (Meerschweinchen) weighing 250 grammes. 
 
INTERREACTIONS OF TOXIN AND ANTITOXIN 
 
 73 
 
 combine with one -fourth more antitoxin than is necessary to 
 neutralize it. 
 
 To account for this Ehrlich supposed that there are really two 
 substances present in the broth in which diphtheria bacilli have 
 been grown. There is the true toxin, which brings about local 
 inflammatory oedema, often going on to necrosis and causing local 
 alopecia, and causing acute death, and toxon, which produces only 
 soft and transient cedema locally and subsequent paralysis. Both 
 these substances combine with antitoxin, but the toxin has the 
 greater affinity for that substance, and when the total neutralizing 
 dose of antitoxin is added in successive small amounts, the whole 
 of the toxin is neutralized first, leaving the toxon free, and this 
 takes place when three-fourths of the whole amount of antitoxin 
 has been added. Ehrlich represents this result in the form of a 
 spectrum, thus : 
 
 25 50 75 100 
 
 FIG. 8. SIMPLE SPECTRUM OF TOXIN. 
 
 The rectangle represents the L dose of toxin i.e., in this simple 
 case i c.c. of the solution. The portion with the greatest affinity 
 for antitoxin is placed at the left hand of the "spectrum"; in 
 this case it is represented by the toxin. On the right are the 
 substances with the least affinity for antitoxin in this case the 
 toxon. 
 
 Further investigation shows that the process is not usually so 
 simple as this. In certain samples of toxin we find that the 
 addition of small quantities of antitoxin causes no alteration in the 
 toxicity of the L dose. Thus, in a case of frequent occurrence 
 it happens that we may add J c.c. of normal antitoxin before 
 any loss of toxicity occurs ; i c.c. of the normal toxin will kill 
 100 guinea-pigs, and i c.c. of the same toxin + J- c.c. of normal 
 antitoxin will still kill 100 guinea-pigs. To explain this, Ehrlich 
 supposed that the solution contains a third substance, prototoxoid, 
 which is entirely devoid of lethal activity, but which has a power 
 of combining with antitoxin even greater than that which toxin 
 possesses. Thus, on the addition of small amounts (up to J c.c.) of 
 the antitoxin, this inert substance will seize on the antibody, unite 
 
74 
 
 STANDARDIZATION OF DIPHTHERIA TOXIN 
 
 with it, and so render it incapable of neutralizing the true toxin. 
 The spectrum of this solution will be represented thus : 
 
 Pntto- 
 toxoid 
 
 Toxone 
 
 FIG. 9. SPECTRUM OF TOXIN. 
 
 In this, as in the other diagrams, the lethal portion of the mixture 
 is shaded, the non-lethal portion left blank. 
 
 Ehrlich found on actual experiment that the constitution of the 
 solution was even more complex than this, and had to assume the 
 existence of yet other bodies. Thus, if the spectrum above were 
 a true representation of the constitution of i c.c. of the solution, it 
 follows that the first quarter and the last quarter of the antitoxin 
 added were without effect, so that the middle \ c.c. completely 
 neutralized the whole of the TOO lethal doses. Now let us 
 imagine this i c.c. of standard antitoxin divided into 200 equal 
 parts, and added part by part to the i c.c. of standard toxin, or 
 100 lethal doses. Then 
 
 The first 50 parts added will combine with prototoxoid, and will 
 
 not affect the toxicity of the mixture ; 
 The next 100 parts added will neutralize 100 lethal amounts 
 
 of true toxin ; and 
 The last 50 parts will combine with toxon. 
 
 Now if the spectrum were as simple as is shown above, and if 
 the toxin were quite uniform in its combining capacity and its 
 toxicity, it would follow that the first -^^ part added after the 
 addition of ^^ part would just neutralize one lethal dose and leave 
 99 lethal doses over. Again, the addition of the amount necessary 
 to neutralize all the prototoxoid ( = -f^ c.c.) + -$ c.c., which would 
 neutralize all the prototoxoid and all the toxin except yj^ part 
 (=i lethal dose), and all the toxon, should leave i lethal dose 
 of toxin free, and the animal should die in the limit of time for 
 i lethal dose. We might represent this as follows : 
 
 149 unnc'jfralised 
 'Toxin ( 
 
INTERREACTIONS OF TOXIN AND ANTITOXIN 75 
 
 in which the oblique shading represents the toxic portions, as 
 before, and the horizontal shading represents the amount neutral- 
 ized by the addition of ^-^ c.c. of antitoxin ; the portion with 
 oblique but no horizontal shading represents the toxic portion 
 which remains unneutralized : it constitutes T J^ of the total 
 shaded portion, and is therefore i lethal dose. 
 
 Such a finding may occur, but is unusual. In most cases we 
 find that the amount of toxin left free on partial neutralization is 
 subject to laws which are far more complex. In a case given by 
 Madsen and described in the same way we find : 
 
 The addition of | parts of antitoxin left free no lethal substance 
 a term which we shall use for the present, instead of " toxin," 
 to denote the portion of the spectrum with the oblique shading. 
 In Ehrlich's language all had been neutralized except the toxon. 
 
 The addition of $ left 5 units of lethal substance free ; it 
 follows that iS~ 2 9 ^j- = 2 fi oi7 had been necessary to neutralize 
 these 5 units. 
 
 The addition of -f^ left 55 lethal units free ; hence, if after 
 the addition of -f^ (as above, leaving 5 lethal doses free) we add 
 an additional -/^, the difference (/<&) will neutralize 50 lethal 
 doses (55-5). 
 
 Hence the additon of will just neutralize the remaining 
 lethal doses i.e., 45. 
 
 To account for facts like these, Ehrlich suggests that the 
 solution contains four or five substances. The first i.e., that 
 which has the greatest power of combining with antitoxin, is called 
 prototoxin ; it is lethal, and it consists of two parts an a part, 
 which is readily changed into inert prototoxoid, and a ft part, 
 which is more stable, but which may, after a time, change into 
 prototoxoid also. These two modifications have exactly the same 
 affinity for antitoxin, so that if they were present in equal 
 amounts, and if all the a modification were changed into proto- 
 toxoid, each addition of antitoxin would go to neutralize active 
 prototoxin and inert prototoxoid in equal amount ; hence half of 
 it would apparently be wasted. 
 
 Secondly, there is deuterotoxin, which also exists in an a and a /? 
 modification, of which the a part is readily transformed into 
 deuterotoxoid, whilst the (3 modification is very stable and is the 
 last lethal substance to disappear. The a and /3 modifications 
 have equal affinity for antitoxin, but this is less than that of the 
 prototoxin. 
 
7 6 
 
 SPECTRA OF TOXINS 
 
 Thirdly, there is tvitotoxin, again in an a and a /3 modification, 
 with less affinity for antitoxin than deuterotoxin, and so are placed 
 on its right in the spectrum. 
 
 It is found, further, that the proportion of a modification to 
 P modification in the above forms of toxin is a simple one, 
 so that the ratio of toxoid to toxin present in any one part of the 
 spectrum is always simple (-J, ^, ^, etc.). 
 
 Fourthly, there is toxon (toxone) or epitoxoid, the characters of 
 which we have seen. 
 
 Lastly, some researches seem to prove that there is yet 
 another body, epitoxonoid, which has still less affinity for antitoxin 
 than has toxon, and which is entirely devoid of lethal or toxic 
 power. It will be left out of the further consideration of these 
 bodies. 
 
 The spectrum of a toxin on this theory is recorded thus : 
 
 Profofaxo/clA 
 
 Trifofoxoicf A 
 
 Toxon 
 
 Profotoxin B Deuterotoxin B Tritotoxin B 
 
 FIG. ii. 
 
 The spectrum of the example given by Madsen and quoted 
 above would be : 
 
 FIG. 12. 
 Another spectrum, given by Ehrlich, is appended : 
 
 DeuferotoxinB Tritoxoxm B 
 
 FIG. 13. 
 
 We must now turn to the experimental results which have led 
 to this idea of the change of the toxin into toxoid ; it has been 
 
INTERREACTIONS OF TOXIN AND ANTITOXIN 77 
 
 referred to several times already, but not fully discussed in order 
 not to interrupt the main line of the argument. 
 
 L has been denned as the amount of toxic solution which is 
 exactly neutralized by i IU of antitoxin, and L + is the amount 
 which, when added to i IU of antitoxin has i lethal dose left un- 
 neutralized. Now if the toxic solution contained a simple sub- 
 stance, we should expect the two quantities to have the following 
 relation in the simple standard toxin of which i c.c. contains 100 
 lethal doses. 
 
 i c.c. toxin(= 100 lethal doses) + i c.c. antitoxin (= i IU) = L . 
 roi c.c. toxin(= 101 lethal doses) + i c.c. antitoxin ( = i IU) = L + . 
 .-. L + -L = o-oi c.c. = i lethal dose. 
 
 This, however, is not the case. If we take a neutral mixture of 
 toxin and antitoxin e.g., of 100 units of the former and i of the 
 latter add to it i lethal dose of toxin, and inject it into an animal, 
 it will not cause death ; there may be transient local cedema and 
 late paralysis, symptoms which are indicative of the presence of 
 free toxon. We must in general add very much more than 
 i lethal dose to the neutral mixture in order to bring about a fatal 
 result. For example, in our standard toxin it might happen that 
 the L + dose was about 1-35 c.c. In other words 
 
 i -oo c.c. toxin solution + i unit of antitoxin = L . 
 I *35 c.c. toxin solution + i unit of antitoxin = L + . 
 L + -L = o-35 c.c. 
 
 This result can readily be explained on Ehrlich's assumption 
 of the existence of substances of differing combining powers for 
 antitoxin. For the sake of simplicity, we will take his earlier 
 nomenclature, and consider the substance as made up of proto- 
 toxoid (with a greater affinity for antitoxin than true toxin has), 
 toxin, and epitoxoid, with little affinity, and corresponding to 
 toxon. The spectrum of the toxin under discussion is : 
 
 150 200 
 
 FIG. 14. 
 
 In this diagram we represent the L dose i.e., i c.c. divided 
 into its component parts. The oblique shading represents, as 
 
7 8 
 
 DIFFERENCE BETWEEN L. 
 
 before,.Jthe acutely lethal portion, and the whole is shaded hori- 
 zontally to show that it is completely neutralized by the i unit of 
 antitoxin. 
 
 Now let us take 1*25 of the same solution and add to it i unit 
 of antitoxin. In this extra 0-25 c.c. of toxin (a quarter of 
 the original amount) there are 12-5 parts of prototoxoid, 25 of 
 toxin, and 12-5 of epitoxoid. There will now be 62-5 parts of 
 prototoxoid, 125 of toxin, and 62-5 parts of epitoxoid. The 200 parts 
 into which we imagine the unit of antitoxin is divided will now 
 neutralize the whole of the prototoxoid (62-5 parts), the whole of 
 the toxin (125 parts), and 12-5 parts of toxon. There will be 
 50 parts of epitoxoid left free, but no toxin. Hence, 1*25 c.c. of 
 the toxic solution is less than the L + dose. The result may 
 be represented thus : 
 
 n-6 parts of epifoxotd 
 
 62-5 parrs 
 
 125 parrs. 
 FIG. 15. 
 
 200 62-5 parrs 
 
 Let us now imagine a third mixture of 1-33 c.c. of the toxic 
 solution and i unit of antitoxin. The 0-33 c.c. of toxin will 
 contain 16-6 c.c. of prototoxoid, 33-3 c.c. of toxin, and 16*6 c.c. of 
 toxon, and the total 1*33 c.c. will thus contain 66-6 c.c. of proto- 
 toxoid, i33'3 c.c. of toxin, and 66*6 c.c. of epitoxoid. The proto- 
 toxoid + toxin ( = 200 parts) will just absorb the whole of the unit 
 of antitoxin, leaving nothing but toxon free. Thus : 
 
 66 6 parrs 
 
 133-3 parrs 
 
 FIG. 1 6. 
 
 ZOO 66- 6 parrs 
 
 Then, if i extra lethal dose of toxin be added to the above 
 mixture, it will find all the antitoxin utilized by substances with 
 a combining affinity as great as, or greater than, its own, and 
 will be left free. Hence, the L + dose is just greater than 
 1 33 c.c. 
 
 All this follows from what has previously been said concerning 
 
INTERREACTIONS OF TOXIN AND ANTITOXIN 
 
 79 
 
 partial neutralization. If, however, we now keep this atrtitoxin 
 for some time, especially if it is exposed to warmth, light, air, 
 or certain chemical substances, we find a great change. The 
 L dose is unaltered : i c.c. is still exactly neutralized by i unit 
 of antitoxin, but we find that this amount is now much less lethal, 
 
 Prototoxoid 
 
 Epitoxoid 
 
 50 parrs 
 
 loo parrs 
 FIG. 17. 
 
 SO parrs 
 
 and the minimal lethal dose may have risen from o'oi c.c. to 
 O'O2 c.c., or higher. 
 
 If the cause for this increase in the lethal doses is investigated 
 by the partial neutralization method described above, it will be 
 found that the results obtained are such as will be readily ex- 
 plicable on the assumption that some of the molecules of toxin 
 
 Profotoxoid. 
 
 Toxoid (SO parts) 
 
 Epiroxoid 
 
 50 parts 
 
 100 parts 
 FIG. 18. 
 
 50 parrs 
 
 have ceased to be poisonous, but have retained their combining 
 power unaltered; whilst the non - poisonous portions of the 
 spectrum are unaltered. Thus, to take the simple case described 
 above, and shown in Fig. 17, in which protoxoid, toxin, and 
 epitoxoid are present in the proportion of 50, 100, and 50. If we 
 keep this, we may find the lethal dose doubled i.e., ^ c.c. instead 
 of T<JTT c - c - But on working out the action of antitoxin, we may 
 find that the first -25 c.c. added removes none of the toxicity, and 
 the last -25 is equally without apparent effect. Thus the middle 
 5 c.c. is used up in neutralizing 50 lethal doses, and of this 
 fraction further investigation shows that each one-fiftieth part 
 neutralizes one lethal dose; the antitoxin, therefore, appears to 
 have fallen off in potency ; but we know that this is not the case. 
 The explanation is that half the molecules have been changed into 
 the non-toxic form described above. The spectrum of this altered 
 form is shown in Fig. 18. 
 
 Thus, in a particular case Ehrlich found the minimal lethal 
 
80 TOXIN AND TOXOID 
 
 dose of a toxin to be 0*003, an d the L dose 0-305 (= 100 lethal 
 doses). Nine months later the L dose was still 0305, whilst 
 the minimal lethal dose was 0-009 c - c - (=33*3 lethal doses only). 
 Thus, the toxin had fallen to one-third of its former toxicity, but 
 had retained its power of combining with antitoxin unaltered ; and 
 it is found that the two numbers usually bear some simple ratio 
 to one another e.g., i - ^ or i J, as in the previous examples. 
 
 We are now in a position to understand all Ehrlich's results, 
 and especially the proof that a molecule of toxin consists of two 
 parts a haptophore group, which has the power of combining 
 with antitoxin or with the protoplasm of the body cells, but which 
 has in itself no lethal action ; and a toxophore group, which has 
 the power, when linked to a body cell by means of the haptophore 
 group, of causing toxic symptoms, probably by a process akin to 
 enzyme action. This toxophore group is unstable, and when it is 
 decomposed the toxin molecule is converted into toxoid ; it then 
 retains its power of uniting with antitoxin and with the tissue 
 cells, but is devoid of toxicity. Further, there are many varieties 
 of toxin, which differ from one another (i) in their avidity for 
 antitoxin, and presumably for the tissue cells ; (2) in the readiness 
 with which they are decomposed into inert toxoid; and (3) in 
 their toxic action, in that true toxins cause rapid death, local 
 inflammation, necrosis, etc., but no paralysis, whilst toxons 
 produce only transient soft oedema and subsequent paralysis. 
 Lastly, Ehrlich supposes that toxin and antitoxin have a great 
 affinity for one another, so that their combination resembles that 
 of a strong acid with a strong base, and the compound toxin- 
 antitoxin molecule when once formed does not dissociate, but 
 remains as a stable substance. It is on this point that his views 
 differ from the more recent ones of Arrhenius and Madsen, who 
 regard the union as being akin to that of a weak acid and a weak 
 base. We hope to be pardoned if, before describing their experi- 
 ments and conclusions, we give a brief outline of the difference 
 between the two reactions. 
 
 According to modern chemical theories, most substances in 
 watery solution decompose into ions, atoms or groups of atoms 
 carrying an electrical charge. Thus, a solution of hydrochloric 
 acid contains ions of H carrying a positive charge and of Cl 
 carrying a negative one. Different substances undergo ionization 
 to a different degree. Thus, HC1 and NaOH are ionized to a 
 great extent, boracic acid and ammonia hydroxide but slightly. 
 
INTERREACTIONS OF TOXIN AND ANTITOXIN 8l 
 
 Now in general it is only free ions which enter into chemical 
 combination. For example, on adding HC1 to NaOH the posi- 
 tively charged H ion combines with the negatively charged OH 
 ion to form water, and the positively charged Na ion combines 
 with the negatively charged Cl ion to form NaCl. The two 
 substances HC1 and NaOH are strongly dissociated, and hence 
 the combination between the two is almost complete. This is an 
 example of the combination of a strong acid and a strong base ; 
 where it was expressed in older phraseology, the substances have 
 a strong affinity for one another. It is to this type that Ehrlich 
 conceives the union of toxin and antitoxin belongs. 
 
 When two substances dissociate but slightly e.g., acetic acid 
 and alcohol or boracic acid and ammonia the reaction takes 
 place in obedience to different laws (the law of "mass action" 
 of Guldberg and Waage). When we ^dd alcohol and acetic 
 acid we get ethyl acetate (ester) and water ; but if we take 
 equivalent combining quantities of the two primary substances, 
 the reaction is not complete, as it is in the case of HC1 and 
 NaOH. On the contrary, the solution will still contain free 
 alcohol, free acetic acid, ester, and water. This is due to the 
 fact that the process is reversible. Acetic acid and alcohol 
 combine on the one hand to form ester and water, and ester 
 and water combine on the other, and dissociate into free acid and 
 free alcohol. 
 
 In the first case, in the combination of a strong acid and a 
 strong base, the reaction is a simple one. If we take 100 com- 
 bining equivalents of NaOH, and add to it 10 combining equiva- 
 lents of NaOH, 10 parts of the alkali will be neutralized, and so 
 on, the whole of the alkali being neutralized when 100 combining 
 equivalents have been added. In the second case the reaction is 
 more complicated, and is expressed by the law that the products 
 of the concentrations of the substances on one side of the equa- 
 tion, divided by the product of those on the other, is a constant 
 (which varies in different reactions, and can be determined by 
 experiment). Thus in the case given : 
 
 Concentration of acid x concentration of alcohol 
 
 = constant. 
 Concentration of ester x concentration 01 water 
 
 It follows that when we add a relatively small amount of acid 
 to a given volume of alcohol, it is practically all used up to form 
 ester, much free alcohol remaining ; but each succeeding addition 
 
 6 
 
82 REVERSIBLE REACTIONS 
 
 of acid is divided into two parts, of which one combines with 
 the alcohol and the other remains free. As we continue to add 
 successive small amounts, this latter uncombined portion gets 
 greater and greater, so that the addition of successive small 
 volumes use up less and less of the alcohol ; and it is only when 
 there is a great excess of acid that all the alcohol is used up. 
 Theoretically, some always remains. 
 
 The difference may be represented graphically thus : 
 
 FIG. 19. 
 
 The line a b represents the neutralization of a given amount of 
 NaOH by its equivalent of acid. It is a straight line, since each 
 successive addition of equal amounts of acid neutralizes the same 
 amount of alkali. 
 
 The line c d represents the reaction of alcohol and acetic acid, 
 or, to take the example used by Madsen, the neutralization of 
 ammonia by boracic acid. It is a hyperbolic curve. It is almost 
 a straight line to begin with, since each small addition of boracic 
 acid is almost all used up by the ammonia, there being enough 
 NH 4 ions to seize on all the boracic ions which are added. 
 Farther along, however, it changes according to the rules already 
 described, and ultimately approaches infinitely near to the base 
 line, but never reaches it. There are always free boracic acid 
 and free ammonia in the solution, though the amount of the latter 
 is infinitely small when the former is in great excess. 
 
 The first suggestion that the reaction between toxin and anti- 
 toxin might be a reversible one was due to Myers in an investi- 
 gation on the haemolytic action of snake-venom, a substance 
 
INTERREACTIONS OF TOXIN AND ANTITOXIN 3 
 
 which gives partial neutralization phenomena quite similar to 
 those we have described as occurring with diphtheria toxin, and 
 also declines in toxic strength, but not in combining capacity, 
 forming toxoids. It was, however, the researches of Arrhenius 
 and Madsen which led to the establishment of the theory on a 
 sound basis. Madsen had previously investigated the constitution 
 of the haemolysin of tetanus (tetanolysin), working on Ehrlich's 
 lines, and had found it to consist of proto-, deutero-, and trito- 
 toxin, and a large amount of toxone. The substance was much 
 more convenient to work with than diphtheria toxin, since a constant 
 emulsion of red blood-corpuscles could be used as test objects 
 instead of guinea-pigs, and thus the experiment could be multi- 
 plied at will ; and when Madsen added the antiserum in small 
 amounts he found that the apparent irregularities due to the 
 presence of these various forms of toxin disappeared, and the 
 curve of neutralization is represented by a curve similar to that 
 given above, as illustrating the reactions between boracic acid 
 and ammonia. Thus : 
 
 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 
 
 FIG. 20. CURVE OF NEUTRALIZATION OF TETANOLYSIN (MADSEN). 
 
 62 
 
8 4 
 
 ARRHENIUS' AND MADSEN's VIEWS 
 
 The heavy line represents the curve of neutralization on 
 Ehrlich's principles, and apparently shows the presence of a 
 large number of varieties of toxin with different combining 
 capacities. When, however, the neutralization was carried out by 
 the addition of very small quantities of the antitoxin at a time, 
 the curve became a hyperbola of the character seen in the dia- 
 gram. Further, having found the value of k by determining by 
 experiment the amount of toxin left free after the addition of 
 a certain amount of antitoxin to a certain amount of the toxic 
 solution in two different instances, and hence, by use of the 
 formula : 
 
 Free toxin free antitoxin _ (toxin x antitoxin) 
 vol. vol. vol. 
 
 the amount of free toxin after any addition of antitoxin could be 
 calculated theoretically. It could then be determined by experi- 
 ment, and the results compared. In one case given by Arrhenius 
 and Madsen this was done, with the following result : 
 
 Amount of 
 Antitoxin added. 
 
 Amount of Toxin 
 (observed). 
 
 Amount of Toxin 
 (calculated). 
 
 0*05 
 
 3-6 7 
 
 3-67 
 
 o-i 
 
 3-13 
 
 2'95 
 
 0*15 
 0-2 
 
 2-32 
 1-62 
 
 2-29 
 172 
 
 0-3 
 0-4 
 
 07 
 I'D 
 
 0-97 
 0-63 
 
 o*45 
 0-27 
 0-18 
 
 1-03 
 0-62 
 0-46 
 0-28 
 0-18 
 
 1*3 
 
 1-6 
 
 0*12 
 
 0-13 
 
 O'll 
 
 2-0 
 
 0'08 
 
 0-09 
 
 The correspondence is certainly very close. 
 
 The case of diphtheria toxin was next investigated, and in the 
 figure which follows the " stair-step " curve, showing Ehrlich's 
 conception of its constitution, shows the presence of proto-, deutro-, 
 and trito-toxins, and of toxon. The curved line is that calcu- 
 lated after the constant of dissociation had been determined by 
 experiment. 
 
INTERREACTIONS OF TOXIN AND ANTITOXIN 
 
 32 
 30 
 28 
 26 
 24 
 22 
 20 
 18 
 16 
 14 
 12 
 10 
 8 
 6 
 4 
 2 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 \ 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 \ 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 v 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 \ 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 \ 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 (\ 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 \ 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 "^ 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 ^ 
 
 ^ 
 
 ) 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 \ 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 \ 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 \ 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 \ 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 \ 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 f 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 \ 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 ^ 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 v 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 2 4 6 8 10 12 14 16 -18' 
 
 FIG. 21. CURVE OF NEUTRALIZATION OF DIPHTHERIA TOXIN (MADSEN). 
 
 This table shows the difference between the observed and calcu- 
 lated results : 
 
 Antitoxin. 
 
 Free Toxin 
 (observed). 
 
 Free Toxin 
 (calculated). 
 
 O'l 
 
 75'I 
 
 75* 1 
 
 0-I 5 
 
 62'6 
 
 627 
 
 0-2 
 
 47-6 
 
 50*6 
 
 0-25 
 
 45-8 
 
 38-6 
 
 0-3 
 
 25*9 
 
 27-3 
 
 o'35 
 
 i7'3 
 
 17*5 
 
 0-4 
 
 9-6 
 
 9'6 
 
 o'45 
 
 5'3 
 
 6-0 
 
 o'5 
 
 3' 1 
 
 4'i 
 
 0-6 
 
 1-6 
 
 2-6 
 
 The conclusion to which Arrhenius and Madsen came was that 
 toxin and antitoxin react like a weak acid and a weak base ; that 
 the reaction is a reversible one ; and hence that the combination 
 of toxin and antitoxin is an unstable one, dissociating into free 
 toxin and free antitoxin. 
 
86 ARRHENIUS* AND MADSEN'S VIEWS 
 
 On this theory many facts which were formerly very difficult 
 to explain become quite simple. Take, for instance, the exact point 
 of neutralization of toxin by antitoxin as seen in the determina- 
 tion of the L dose : this has always been a matter of great 
 difficulty a difficulty formerly explained by assuming the last 
 substances to be neutralized are the toxons, which have a very 
 feeble and indefinite pathogenic action. On the physical chemistry 
 theory the difficulty disappears, because there is no point of exact 
 neutralization. In spite of the presence of an excess of antitoxin, 
 some dissociation of the toxin-antitoxin molecule will always 
 occur, and the mixture will always contain free toxin, though in 
 very small amount. Further, an exactly neutralized mixture con- 
 taining a few lethal doses of toxin is, of course, without action, 
 whereas a large bulk of the same mixture may be toxic ; this 
 also is readily explicable. Then there are some old experiments 
 of Buchner's, which showed that a mixture of tetanus toxin and 
 antitoxin which was neutral to mice would produce tetanus in 
 guinea-pigs, and several others (Roux's and Roux and Vaillard's) 
 of similar nature. The explanation of these is also easy. 
 
 The most interesting explanation of a previously known phe- 
 nomenon which Madsen offers in the light of his new theory is 
 that of the immunization of animals by means of a neutral mixture 
 of toxin and antitoxin. If we follow Ehrlich, and believe that the 
 compound is a stable one, this is very difficult to explain. Madsen's 
 solution is that the mixture contains free toxin, to which the 
 immunizing property is due. 
 
 Again, we can also explain the death of animals from specific 
 intoxication when it occurs in spite of the presence of free anti- 
 toxin in the blood in exactly the same way. It is true that the 
 free antitoxin would tend to inhibit dissociation ; but, on the other 
 hand, the hypersensitiveness of the tissues which occurs at the 
 early stages of the process of immunization and it must be 
 remembered that it is only in these stages that death from in- 
 toxication takes place would render the cells more susceptible 
 to minute amounts of toxin. The explanation may not be a 
 perfect one, but it appears to be the best forthcoming. 
 
 The importance of the whole question from the point of view 
 of immunity rests on this question of the dissociability of the 
 combination, for it is obvious that if this is the case, our views of 
 the action of antitoxin in the animal body will be very different 
 from those we shall hold if we regard the toxin-antitoxin molecule 
 
INTERREACTIONS OF TOXIN AND ANTITOXIN 87 
 
 as an inert one of no further interest ; and this theory of dissocia- 
 tion is open to the objection urged by Nernst, and probably felt 
 by most bacteriologists on the first enunciation of the views of 
 Arrhenius and Madsen. Thus, if the combination of toxin and 
 antitoxin undergoes dissociation in the living animal and the toxin 
 is set free, it will immediately combine with the susceptible cells. 
 The equilibrium will now be disturbed and more toxin-antitoxin 
 molecules will be dissociated, more toxin set free, and more cells 
 poisoned ; and this process will go on until all the toxin has been 
 passed on to the cells and the antitoxin left free. Now this 
 dissociation takes place quickly, so that on this theory it would 
 seem that the antitoxin would only interpose a very temporary 
 barrier between the cells and the toxin, the lethal action of which 
 would be delayed, but in no way inhibited. We will return to 
 this question of dissociation shortly, and meanwhile state briefly 
 Ehrlich's objections to the physical-chemical theory. In the first 
 place, he points out that if we make a mixture of two alkaloids, 
 of which one is haemolytic and the other not, and neutralize them 
 by the addition of a strong acid, the result may be represented by 
 a hyperbolic curve ; this is put forward as a parallel experiment 
 to the neutralization by antitoxin of a substance containing active 
 toxin and inert toxoid. Secondly, some of the curves given by 
 Madsen and Arrhenius do not correspond very closely with the 
 observed results, and this is especially the case at their commence- 
 ment and termination. At the commencement of the curve it is 
 found in many cases that the addition of small amounts of anti- 
 toxin does not influence the toxicity ; this is readily explained on 
 the supposition of the existence of prototoxoid, but hardly on any 
 other hypothesis. The most interesting point, however, is the 
 behaviour of the curves at the termination i.e., in what Erhlich 
 would call the region of the toxons ; here Arrhenius and Madsen 
 usually found figures which were lower than the calculated results. 
 Now Ehrlich holds that traces of toxin do not lead to paralysis, 
 and if the effect of a nearly neutral mixture of toxin and antitoxin 
 were due to dissociation we should expect no paralysis to occur, 
 the toxic action being due to toxin, and not, as Ehrlich thinks, to 
 toxon. Madsen and Arrhenius suggest that the action of this 
 trace of antitoxin may be modified by the presence of antitoxin in 
 excess. Further, Madsen and Dreyer claim to have found a 
 diphtheria poison, of which small quantities would cause paralysis 
 without the addition of antitoxin, so that the question of toxon 
 
88 EVIDENCE IN FAVOUR OF DISSOCIATION 
 
 would not come in. And they also showed that certain mixtures 
 of toxin and antitoxin might act fatally on rabbits in a few days 
 and cause only paralysis in guinea-pigs, but that if a little more 
 antitoxin were added it would act as a toxon on rabbits and be 
 inert to guinea-pigs. To explain this, Ehrlich had to add yet 
 another body to his list of components of the diphtheria poison, 
 and to the proto-, deutero-, trito-toxin, and toxon he added 
 toxonoid, which is inert for guinea-pigs and produces paralysis 
 in the rabbit. The subject will not be followed farther, but 
 enough has been said to show its extreme difficulty. 
 
 To revert to the question of dissociation, Madsen and Walbum 
 have brought forward some definite evidence of its existence, of 
 which the more important are the following. They neutralized 
 ricin with antiricin, and to the neutral mixture added some red 
 blood-corpuscles. After allowing the mixture to stand for some 
 time, the latter were centrifugalized off, when it was found that 
 they had become charged with ricin and the fluid containe(i/free 
 antiricin. / 
 
 Secondly, they made use of the fact that diphtheria toxin is a 
 substance of comparatively small molecule, and can diffuse 
 through gelatin, whereas its antitoxin does so very slowly. They 
 prepared a neutral mixture of the two, and placed it on the 
 surface of a column of gelatin, and after some forty days' contact 
 examined slices at different depths, and found free toxin at a 
 certain distance from the surface. This they explained by assum- 
 ing that it had dissociated from the antitoxin, and diffused down- 
 wards into the gelatin. It was pointed out, however, that if the 
 mixture had been allowed to stand for some time the phenomenon 
 did not occur ; it would seem, therefore, that the combination 
 takes place slowly, but, once formed, does not dissociate. 
 
 There is, however, other and independent evidence in favour of 
 the theory that dissociation of a primary substance and its anti- 
 body does occur. Thus Muir and Morgenroth showed inde- 
 pendently that if red corpuscles are treated with as much 
 amboceptor as they will take up, and then mixed with normal 
 corpuscles, some of the amboceptor will pass to the latter, and 
 on the addition of a suitable complement-containing serum the 
 whole may be dissolved. (In this experiment the red corpuscles 
 correspond to the toxin, whilst the amboceptor is the antibody 
 and corresponds to the antitoxin.) 
 
 Bordet's explanation of the phenomena is quite different. Both 
 
INTERREACTIONS OF TOXIN AND ANTITOXIN OQ 
 
 Ehrlich on the one hand and Arrhenius and Madsen on the other 
 agree that the combination of toxin and antitoxin is a chemical 
 union, and takes place in obedience to the laws of multiple 
 proportions : a single molecule of the one substance always com- 
 bines with the same number of the other on complete neutraliza- 
 tion. Bordet denies this, and compares the phenomenon with the 
 absorption of a stain by a colourable substance. His theory is 
 that a molecule of toxin requires many molecules of antitoxin for 
 its complete neutralization, and that it can be partially neutralized 
 or attenuated by a smaller number. Thus he holds that when a 
 small amount of antitoxin is added to a large amount of toxin, 
 there is not a mixture of free toxin and of molecules of toxin- 
 antitoxin (as would occur if either of the theories already discussed 
 was true), but a uniform solution of toxin of diminished potency. 
 Bordet shows that many of the experimental results of other 
 observers are explicable on his theory, and gives some new facts 
 in support thereof. For example, the amount of an emulsion of 
 red blood-corpuscles which can be haemolyzed by the addition of 
 a given quantity of haemolytic serum can be readily determined. 
 We will suppose that i c.c. of the serum just dissolves all the 
 corpuscles in 5 c.c. of the emulsion, and no more. We might 
 suppose that all the red corpuscles have all their combining 
 valencies exactly neutralized, so that we may regard them as an 
 exactly neutralized toxin-antitoxin mixture. This, however, is 
 not the case, for if we add the haemolytic serum in small amounts, 
 say 0-05 c.c. at a time, we shall find that only a small proportion, 
 perhaps 2 c.c., of the same emulsion can be completely haemolyzed. 
 Thus each corpuscle takes up more haemolysin in the second case 
 than in the first. 
 
 The objection may be raised that a red corpuscle is very 
 different from a molecule of toxin. It can undoubtedly combine 
 with a vast number of molecules of its antibody (haemolysin), but 
 it is quite easy to understand how complete haemolysis may take 
 place if only a certain number of its combining valencies are 
 occupied by antibody. But the molecule of toxin is, as we have 
 already seen reason to believe, much smaller than the molecule 
 of antitoxin ; and although this fact does not in itself render it 
 impossible that the toxin is of higher valency than the latter, 
 it certainly renders it unlikely that it is so much higher in this 
 respect that there can be an indefinite number of stages between 
 free toxin and a fully neutralized one. Bordet has, however, 
 
90 ADSORPTION 
 
 brought forward evidence in favour of his view to which this 
 objection can hardly apply. It will not be discussed here, as it 
 involves certain questions concerning serum haemolysins which 
 have not yet been discussed. 
 
 Bordet's theory supplies an explanation, though hardly an 
 adequate one, of the negative phase. We have already seen that 
 when an animal is producing antitoxin an injection of toxin will 
 cause a fall in the antitoxic value of the serum far greater than 
 can be accounted for by the neutralization effected by the toxin 
 injected : it may be 2,000 times as great, or more. If we assume 
 that the combination does not take place in obedience to the laws 
 of multiple proportions, we can imagine that it may go on very 
 differently in the body, and that in that situation a small amount 
 of toxin may neutralize a large amount of antitoxin. But it is 
 very difficult to believe that this is the true explanation, for it 
 would involve an increase of the neutralizing power of the toxin 
 to 2,000 times that which it has outside the body an increase 
 which certainly seems improbable. Further, we have been asked 
 to imagine a molecule of antitoxin spreading itself out and 
 weakening many molecules of toxin, and we are now asked to 
 imagine a molecule of toxin dividing itself amongst 2,000 molecules 
 of toxin, and completely neutralizing them. 
 
 The last conception which we have to consider is that of Biltz, 
 which approaches very closely to Bordet's. Starting with the 
 idea that toxins and antitoxins are both colloids, he attempts to 
 show that their union is analogous to adsorption rather than to 
 an ordinary chemical union. 
 
 Adsorption is a process akin to solution, and does not necessarily 
 involve a chemical union between the two substances which take 
 part in it. It may take place between colloids and colloids, or 
 between colloids and crystalloids, and in other ways. The two 
 substances taking part in the process are found to be electrically 
 positive and negative (as shown by their moving to the cathode 
 or anode in an electric field) ; thus a colloidal solution of silicic 
 acid (which is negative) is precipitated slowly by K 2 SO 4 , which 
 has a feeble positive charge ; more rapidly by CuSO 4 , which has a 
 stronger one ; and immediately by A1 2 (SO 4 ) 3 , which has a stronger 
 one still. 
 
 Biltz and his collaborators started with the idea that toxins and 
 antitoxins are both colloids. They attempted to find the electrical 
 nature of tetanus toxin by electrolysis, but found that it was 
 
INTERREACTIONS OF TOXIN AND ANTITOXIN QI 
 
 destroyed ; the destruction occurred, however, sooner at the 
 cathode than at the anode, suggesting that it was a negative 
 colloid. They found it to be precipitated by positive colloids, 
 such as colloidal hydrated oxide of iron or chromium, though this 
 action occurred only in vitro, not in vivo. Tetanus antitoxin, how- 
 ever, did not lose its activity when exposed to mineral colloids. 
 
 Further, there is an approach to the phenomenon of specificity 
 in the adsorption of one colloid by another, since certain colloids 
 are only precipitated by certain other colloids ; the specificity, 
 however, is by no means so exact as in the case of the toxins, etc., 
 and their antibodies. 
 
 A further analogy arises in the explanation of the difference 
 between the L and L + dose, which is paralleled by the relation- 
 ship between colloidal ferric hydroxide and arsenious acid. When 
 these two substances are mixed adsorption and precipitation take 
 place ; hence the use of the former substance as an antidote for 
 the latter. Now it is found that if a solution of arsenious acid is 
 just rendered tox*e by the addition of ferric hydroxide, very much 
 more than one lethal dose of arsenic must be added to make the 
 mixture toxic again. 
 
 The further investigation into the evidence which they have 
 adduced would lead us too far into the study of the agglutinins to 
 be undertaken here. It will be dealt with subsequently (see 
 Chapter XII.). 
 
 It seems, on the whole, that no theory is absolutely sufficient to 
 explain all the phenomena, and that as soon after each new one is 
 adduced the supporters of the older ones bring forward evidence 
 which renders it untenable. The probability is, at the time of 
 writing, that Ehrlich's views are generally held, and are open to 
 the fewest objections. They are complicated, it is true, and have 
 had to undergo constant modifications as new facts have arisen ; 
 but the facts themselves are complicated. Yet it must be con- 
 fessed that there are some grave objections to its acceptance in its 
 present form, and it may become yet more involved before it can 
 be fully accepted as a complete explanation. Thus the analogy 
 with other bodies, and the phenomena of the death from intoxica- 
 tion of animals with antitoxin in their blood, seem to point strongly 
 to the theory that the toxin-antitoxin molecule dissociates strongly 
 both in vivo and in vitro. Yet this is not compatible with Ehrlich's 
 views. 
 
CHAPTER V 
 
 THE ORIGIN OF ANTITOXIN THE SIDE-CHAIN 
 
 THEORY 
 
 THE theory that antitoxins are derived from their appropriate 
 toxins deserves a short consideration, since it is so inherently 
 probable. When we consider the large numbers of toxins which 
 give rise to their antitoxins when injected into animals, and see 
 that each antitoxin has a direct action on its own toxin, but none 
 or practically none on others, the most likely explanation of the 
 phenomenon is that the animal tissues have the power of splitting 
 the toxin into two parts, or of otherwise modifying it, and that 
 this modified toxin can combine with unaltered toxin and form 
 antitoxin. Some experimental evidence is forthcoming to support 
 this view. Thus it is found that diphtheria toxin which has been 
 submitted to the action of an electric current loses its toxicity, but 
 retains that of producing immunity on injection. Some thought 
 that this was due to the direct transformation of the toxin into 
 " artificial antitoxin," and it was hoped that a similar change 
 might be brought about in the human body in disease by the use 
 of electricity. The explanation of .the phenomenon is very 
 simple. The electrolysis of the water in which the toxin is 
 dissolved gives rise to oxidizing substances which transform the 
 toxins into toxoids, the immunizing virtues of which we have 
 already seen. There is no method by which antitoxin can be 
 prepared other than by the injection of toxins into suitable 
 animals. 
 
 That antitoxin is not a direct transformation product of toxin 
 appears probable from the following considerations, none of which 
 perhaps is conclusive, but which together make up a body 'of 
 evidence of some importance : 
 
 i. The injection of a certain amount of toxin will give rise, 
 under suitable circumstances, to the formation of much more 
 
 92 
 
THE ORIGIN OF ANTITOXIN THE SIDE-CHAIN THEORY 93 
 
 antitoxin than it can neutralize. Thus Knorr showed that a 
 single unit of toxin might call forth 100,000 units of antitoxin. 
 This can scarcely be accounted for by supposing the transforma- 
 tion of the one substance into the other. 
 
 2. It frequently happens that antitoxin occurs, sometimes in 
 considerable amounts, in the blood of animals that have not 
 received injections of toxin, and have not suffered, as far as 
 known, from the disease in question in other words, of " normal " 
 animals. Thus horses frequently have traces of diphtheria anti- 
 toxin in the blood, as much as 4 units per c.c. having been 
 observed. Metchnikoff suggests that this may be due to the 
 action of " pseudo-diphtheria " bacilli, which are so common. 
 But no one has been able as yet to produce diphtheria toxin or 
 symptoms of diphtheria by means of these bacilli, and it is not 
 usual to regard them as having any connection, other than a 
 superficial morphological resemblance, with the true diphtheria 
 bacillus. 
 
 If we may bring other antibodies into the argument the case 
 against Metchnikoff's supposition is enormously strengthened. 
 The blood of many animals contains agglutinins and hsemolysins 
 often in large amounts, often under conditions in which we may 
 almost exclude the possibility of a preceding infection. Thus the 
 blood of a normal horse will almost invariably clump typhoid 
 bacilli, B. pyocyaneus, the cholera vibrio, and many other 
 organisms. 
 
 3. An animal which has received an injection of antitoxin 
 rapidly eliminates it from the blood, whereas the antitoxin which 
 is formed in the body remains a much longer time. This argu- 
 ment does not carry very much weight, since, as Metchnikoff 
 points out, the toxin might remain for long periods occluded 
 in the cells, and only undergo a gradual transformation into 
 antitoxin. 
 
 4. Roux and Vaillard showed that the whole of an animal's 
 blood might be removed by repeated venesections, and that the 
 newly regenerated blood might contain almost as much antitoxin 
 as was present before the haemorrhage, thus suggesting a new 
 formation. But the objection urged under the last heading is of 
 weight here also, and if we consider it probable that toxin may 
 remain latent in the tissues for long periods without causing 
 symptoms, we shall exclude this piece of evidence from the 
 argument, as well as the observations of Salomonsen and Madsen, 
 
94 CONSTITUTION OF PROTOPLASM 
 
 that the production of antitoxin may be increased by an injection 
 of pilocarpin. 
 
 If we reject this explanation of the phenomenon, and regard 
 the production of antitoxin as being analogous to the process of 
 internal secretion, we must regard it as being due to the action of 
 the living cells when they are stimulated by the toxin. There is 
 at the present time but one theory which suggests how this may 
 be brought about : this is Ehrlich's celebrated side-chain theory, 
 which has played so important a part in the modern study of 
 problems concerning infection and immunity, and which, though 
 highly hypothetical, deserves a close and careful study. 
 
 Ehrlich points out that a cell has two functions. The first 
 is the physiological function, which it enacts in the body secretory 
 in the case of a gland cell, conductive in the case of a nerve fibre, 
 etc. ; and the second is the function of nutrition, necessary to 
 supply the waste which is constantly taking place. We must 
 suppose a similar constitution for each of the complex molecules 
 of living protoplasm which build up the cell ; in each molecule 
 there is a portion which discharges the specific function of the 
 cell, and this portion has to be nourished. The functional portion 
 is the more important, and is called by Ehrlich the active centre 
 (Leistungskern). In our diagrams we shall represent it as forming 
 the central portion of the giant molecule, but it is hardly necessary 
 to caution the reader that this diagrammatic representation has 
 no necessary morphological basis. It is just as permissible to 
 regard this portion as diffused through the nutritive part of the 
 molecule as long as the difference in constitution and action of the 
 two are borne in mind. 
 
 The second portion, that concerned in nutrition, is more 
 important in relation to immunity. We may regard it as having 
 two functions it " seizes " suitable molecules of food substances 
 from the blood or lymph in which it is bathed, and it alters them 
 in such a way as to build them up into living material which can 
 take its place in the molecule of protoplasm, so as to replace that 
 which has been used in the life of the cell. 
 
 The function of " seizing " molecules of food from the sur- 
 rounding tissues implies a selective faculty, for we cannot imagine 
 that all the molecules which circulate in the blood and lymph are 
 suitable for all cells at all times. This apparently intelligent 
 selection of suitable material is, of course, at bottom a chemical 
 process : the food molecule becomes attached to some portion of 
 
THE ORIGIN OF ANTITOXIN THE SIDE-CHAIN THEORY 95 
 
 the cell for which it has a chemical affinity. Now Ehrlich 
 supposes that this affinity for food molecules is situated in certain 
 portions of the molecule of protoplasm in certain groups of atoms 
 which he calls side-chains, receptors, or haptines. [The name " side- 
 chain " was, perhaps, badly chosen. It denotes a possible analogy 
 with complex organic bodies e.g., of the aromatic group which 
 are composed of a central portion (such as the benzene ring) and 
 side-chains, on which many of their reactions depend. The com- 
 parison is not a very close one, and all that is necessary is to 
 regard the molecule of protoplasm as possessing numerous groups 
 of atoms, each of which has an affinity for one of the bodies 
 circulating in the body fluids, and necessary for the life of the 
 molecule in question.] 
 
 On this theory the nutrition of the molecule takes place as 
 follows : A molecule of suitable food substance in the fluid sur- 
 rounding the cell is brought into contact with one of the receptors 
 for which it has a chemical affinity, and the two unite. This 
 is the first step in the process. The food is "anchored " to the 
 cell by means of a receptor, for which it has a specific combining 
 affinity, or which, to use Ehrlich's analogy, fits it like a key fits a 
 lock. The second stage involves a process which we may compare 
 to digestion, by which the food molecule is altered in some pro- 
 found manner, and absorbed, in whole or in part, into the molecule 
 of protoplasm. 
 
 Let us apply this theory to the process by which a cell is 
 poisoned. We will imagine for a moment that the molecule of 
 toxin contains a group of atoms which will unite specifically with 
 the side-chain of one of the body cells. We have already shown 
 that this molecule of toxin contains a haptophore group of mole- 
 cules, in virtue of which it can combine with antitoxin, and a 
 toxophore group, on which its toxic action depends. We can now 
 go farther, and say that the first stage of the intoxication of a cell 
 by means of a true toxin consists in the union of the haptophore 
 group of atoms in the toxin to a receptor of a molecule of proto- 
 plasm, this receptor being one which " fits it like a key fits a 
 lock." Each molecule of protoplasm has innumerable receptors, 
 of which only a certain number are suitable for this toxin. This 
 is the first step. The toxin molecule is now " anchored " to the 
 living cell, and in the second stage the toxophore radicle of the 
 toxin comes into play. We may regard this toxophore group as 
 exerting an enzyme-like action on the protoplasm through the 
 
96 INTOXICATION AND NUTRITION 
 
 haptophore group and the receptor, by which the two are united. 
 Thus: 
 
 CELL 
 
 FIG. 22. CELL MOLECULE WITH RECEPTORS (E, E). 
 
 A, A, Molecules of toxin (B = toxophore, C = haptophore), D = a molecule 
 
 of toxoid. 
 
 The result of this is that the protoplasm is poisoned. If only a 
 few of its receptors are united to toxin molecules the result may 
 be but slight, whereas if more are occupied the functioning 
 centre of the molecule will be affected, and marked symptoms will 
 arise, and if still more molecules of toxin are linked up the mole- 
 cule of protoplasm may be killed. 
 
 The essential point in this process is that it is exactly analogous 
 to natural nutrition, except that in the latter case the receptor 
 unites with a molecule which is of use to the cell, whereas in the 
 former it unites with one which simply resembles the food molecule 
 in having a haptophore group with similar chemical affinities. 
 Thus, intoxication with bacterial toxins is essentially a process of 
 nutrition, but is perverted in its later stages by the nature of the 
 toxophore group of the toxin. 
 
 Now consider the case in which a molecule of protoplasm is 
 attacked by a number of molecules of toxin which is not large 
 enough to kill. A considerable number of receptors will be taken 
 up by toxin, and we must consider these receptors as being thereby 
 rendered useless. The protoplasm, however, has need of these 
 receptors, and the need may probably be more pressing since it is 
 poisoned, and metabolic changes may take place more rapidly, and 
 there is thus greater need for renewed nutriment. Fresh receptors 
 must therefore be formed, and Ehrlich compares their regeneration 
 to the budding forth of fresh tentacles in the hydra, to replace 
 
THE ORIGIN OF ANTITOXIN THE SIDE-CHAIN THEORY 97 
 
 those which have been lost. Now suppose a fresh dose of toxin 
 reaches the cell, and that these new receptors are in their turn 
 taken up by toxin, but yet not in numbers sufficient to kill the 
 cell. The same processes will occur : the receptors will be rendered 
 useless, and a fresh crop of the same nature will be produced. 
 
 Now in accordance with the well-known laws of habit, in virtue 
 of which a part of the body can gradually be trained to perform a 
 function which is difficult at first, but which becomes more easy 
 by usage, the molecule of protoplasm gradually acquires the 
 faculty of producing these receptors more and more easily, in 
 obedience to nicely graduated doses of toxin. If these are 
 repeated in proper amount and at suitable intervals the cell may 
 be trained, so to speak, to produce these receptors, and it may 
 
 FIG. 23. 
 
 ultimately come to do so in excess and far beyond its physiologi- 
 cal requirements. This also is analogous with known biological 
 facts, such as the production of callus in a fractured bone in 
 excess of the amount necessary for repair. We shall revert to 
 this point later. Now we have to imagine that these receptors 
 may be formed in excess so great that they cannot all remain 
 attached to the cell ; some of them are pushed off, so to speak, by 
 the younger ones, which are growing up to take their place. If 
 this occurs, these receptors will pass off freely into the blood. As 
 we have seen, they are so constituted that they will unite speci- 
 fically with the particular toxin that was injected. These cast-off 
 receptors constitute antitoxin, and the formation of that substance is 
 simply due to the pathological separation of the groups of atoms, 
 in virtue of which the toxin molecule is linked up to the living 
 
 7 
 
98 
 
 WEIGERT'S HYPOTHESIS 
 
 protoplasm. The whole process is explained as one of perverted 
 nutrition, and (once admitting Ehrlich's theory of the constitution 
 of the molecule of protoplasm and of the way it is nourished) 
 without introducing any but well-recognized biological pheno- 
 mena. The theory is fascinating in its simplicity, and although 
 there are a few difficulties in the way of its full acceptance as a 
 complete explanation of the facts, it certainly accounts for them 
 far better, on the whole, than any other theory will do if, indeed, 
 an alternative one has been put forth. And it has the great merit 
 in a theory that its application has led to the discovery of new 
 facts. * 
 
 A few remarks are necessary in connection with the question 
 
 FIG. 24. 
 
 of the mechanism in which the over-production of side-chains is 
 produced. Weigert's hypothesis with regard to the nature of 
 hyperplasia as the result of injury or irritation is now well known, 
 and a brief outline will suffice. He imagines that the maintenance 
 of the normal structure and physiological function of a tissue 
 depends upon a condition of equilibrium brought about by a 
 series of mutual restraints exerted by each cell on its neighbours. 
 In the same way, the structure of a single cell, or perhaps of a 
 single compound molecule of protoplasm, depends on a similar 
 equilibrium existing between minute living units. If one of these 
 components is killed or injured, the restraint which it exerts on 
 the surrounding units is removed, and unrestrained growth can 
 
THE ORIGIN OF ANTITOXIN THE SIDE-CHAIN THEORY QQ 
 
 take place ; and Weigert points out that this always goes on to 
 excess, more new material being formed than is necessary to 
 replace the amount lost. The application of this theory to the 
 mode of new formation of receptors is sufficiently obvious. 
 
 We will now consider certain phenomena in the light of the 
 side-chain theory. 
 
 The occurrence of antibodies in the blood of normal animals is 
 susceptible of a ready explanation. The receptors are, in general, 
 united to the cell, but it is easily conceivable that a few may be 
 desquamated accidentally and escape into the blood. It must be 
 remembered that the actual amount (weight or bulk) of these 
 antibodies in the normal blood is probably always infinitesimal, 
 although the effects of this small amount may be striking. Thus 
 the agglutination of typhoid bacilli at a dilution of i to 160 
 (which often occurs with the blood of normal horses) indicates 
 the presence of a very small amount of specific antibody, as is 
 apparent from the fact that the blood may be made to clump at 
 i to 1,000,000 without being altered chemically in any way that 
 can be detected. If an exceedingly minute proportion of all the 
 receptors are shed into the blood, it will probably account for all 
 the antibodies found in that situation under normal conditions. 
 
 The fact that toxoids produce antitoxin 1 is also explicable. We 
 must regard the receptor which is occupied with toxoid as being 
 thereby rendered useless for the protoplasm, although the latter is 
 not poisoned as a result of the union. 
 
 Next arises a most important question, and one which is ap- 
 parently satisfactorily answered on the side-chain theory the 
 fact that certain substances (bacterial toxins in particular) give 
 rise to the production of antibodies, whilst others, such as the 
 alkaloids, glucosides, mineral poisons, poisons of simple con- 
 stitution, such as alcohol, etc., do not. (This may be taken as 
 fairly proved, certain researches which go to prove the existence 
 of antibodies to morphine, alcohol, etc., being inconclusive.) 
 
 The explanation is founded on differences in the way the 
 various substances form combinations with the protoplasm. This 
 latter has, as an integral part of its constitution, certain receptors 
 which have a specific combined affinity for proteids, and we must 
 imagine the union of proteids, whatever be their nature, as taking 
 place by a direct union with these receptors. Other substances, 
 
 1 According to Ehrlich. The fact is not universally admitted, though all 
 agree that they will produce immunity. 
 
 72 
 
IOO NATURE OF UNION OF TOXINS AND TISSUES 
 
 however, need not, and in all probability do not, unite with the 
 protoplasm in this way. Ehrlich, basing his theories on the fact that 
 certain alkaloids and other substances can be extracted from their 
 combinations with organic matter by simple means, has suggested 
 that the union of non-proteid poisons with protoplasm is less firm 
 than in the other case. This seems doubtful, for nothing could 
 be much firmer than the combination of nitrate of silver or similar 
 bodies with the tissues. And we know as a matter of fact that 
 the toxin-protoplasm compound is not necessarily a stable one. 
 Emulsions of tissues will abstract tetanus toxin, and retain it on 
 washing, but give it up again in a free state when allowed to stand 
 for some time in contact with normal saline solution. However 
 this may be and the point is not of importance we may readily 
 admit with Ehrlich that it is highly probable that proteids unite 
 with protoplasm in a manner fundamentally different from alka- 
 loids, etc. The former process follows on physiological lines, 
 whilst the latter is a pathological process entirely, and one which 
 has no counterpart in normal nutrition. The former may be 
 compared to the insertion of the key in the lock, the latter to the 
 violent smashing of the lock. If this is the case, it is easy to 
 see that "chemical" poisons cannot be expected to give rise to 
 the production of antibodies. It is true that if a cell is already 
 secreting antibodies, we might expect that a substance which 
 stimulates the general metabolism of the cell and increases the 
 rapidity of the vital processes might temporarily increase this 
 production, and we have seen that this is the case with pilo- 
 carpin, which stimulates the production of diphtheria antitoxin in 
 an immunized horse. This is an entirely different process, and 
 one that is easily explicable on the side-chain theory. 
 
 It is necessary, therefore, to determine whether all the sub- 
 stances which give rise to the production of antibodies on injec- 
 tion are proteids. We must admit at the outset that in many cases 
 this cannot be proved ; the chemical constitution of toxins, enzymes, 
 and many other antigens is as yet quite unknown. But in the 
 cases in which the chemical composition of the primary substance 
 is ascertained we find it to be invariably of proteid nature ; thus, 
 solutions of any of the coagulable proteids will give rise to the 
 production of precipitins; the proteid substances present in the 
 body of bacteria give rise to the production of agglutinins, which 
 are themselves of a proteid nature, and will, on injection into 
 suitable animals, give rise to anti-agglutinins ; the proteid con- 
 
THE ORIGIN OF ANTITOXIN THE SIDE-CHAIN THEORY IOI 
 
 stituents of the red blood-corpuscles, the substances forming their 
 stroma, call forth the production of haemolysins ; and a great many 
 other cases might be quoted. It may be taken as definitely 
 established that wherever the nature of the antigen is known and 
 falls into one of the known groups, that antigen is a proteid. 
 
 We may almost go a step farther, and say that all proteids, of 
 whatever nature, when injected into suitable animals, will give 
 rise to antibodies ; in fact, some writers actually enunciate this 
 as a law. There are, however, a few exceptions, which are 
 gradually diminishing in face of further researches, and it is 
 highly probable that time will show that the law is valid to the 
 fullest extent. 
 
 Reverting now to the question of the nature of the bodies of 
 unknown constitution which form antibodies i.e., the toxins, 
 enzymes, etc. we may ask whether these are not in reality 
 proteids, in spite of their failure to give the proteid reactions as 
 usually accepted by chemists. The question is largely one of 
 nomenclature. Accepting the definitions usually given in physio- 
 logical textbooks, and based on a study of ordinary proteids, such 
 as egg-albumin, etc., we may admit that they are not; but are 
 these definitions valid ? Might we not define them with greater 
 accuracy as substances from which animal organisms can obtain 
 food material containing combined (" organic ") nitrogen ? In 
 this case we should substitute for coarse chemical experiments 
 in vitro the more delicate reactions of the living animal body; 
 any nitrogenous substance which is recognized by living proto- 
 plasm as being a suitable pabulum for it would be defined as a 
 proteid. If this is the case, we are thrown back at once on 
 Ehrlich's theory, and can define the proteids as substances which 
 unite in a specific manner, haptophore to receptor, with the living 
 molecule of protoplasm. If we do this, we may admit that the 
 toxins and enzymes, whatever their chemical reactions, and 
 whether they are or are not food substances for the cells, combine 
 with protoplasm in the manner which is characteristic of proteids, 
 and are to be regarded as proteids when looked at from this point 
 of view. And we must not forget that many of the substances 
 which we regard as toxins are, as a matter of fact, of value in 
 nutrition to the animal which forms them, and only act as poisons 
 in strange species. Thus, the ichthyotoxin of eel serum must 
 nourish the cells of the eel, but is a powerful toxin to almost 
 all other species. Whether any animal can utilize tetanus toxin, 
 
IO2 CONDITIONS OF ANTIBODY PRODUCTION 
 
 diphtheria toxin, etc., as cell pabulum is doubtful, but there are 
 some experiments which go to show that this is the case. Thus, 
 the rat is relatively immune to diphtheria toxin, and shows no 
 symptoms after the injection of an amount which is lethal for 
 many rabbits, and this immunity is not due to the presence of 
 antitoxin. This being so, it seems clear that the diphtheria toxin 
 disappears from the blood of the rat in virtue of forming a com- 
 bination with the cells of the latter without injuring them, and we 
 may fairly assume that it is of value in nutrition, although, of 
 course, this assumption is not open to direct proof. In a similar 
 way tetanus toxin disappears rapidly from the blood of scorpions, 
 no antibody being formed. 
 
 Thus it appears to be a logical sequel of the acceptance of 
 Ehrlich's theory that we may define proteids as substances which, 
 when injected into suitable animals, give rise to the production of 
 antibodies. We have now to glance for a moment at the ques- 
 tion of the suitability of different animals for the production of 
 antibodies. 
 
 Researches with precipitins show that the proteid substances 
 which are present in the serum of any species of animal are 
 different from those which occur in any other. We must regard 
 the molecule of proteid of any type (say of a globulin) as being of 
 great complexity, and capable of many slight modifications, which 
 are inappreciable to gross chemical tests, but which are perfectly 
 obvious to the living animal cells. Human globulin differs from 
 horse globulin, and this, again, from sheep globulin. Further, 
 some facts go to show that differences exist between the proteids 
 of animals of the same species, and that the proteids in the serum 
 of one man or horse are not exactly the same as those in another 
 man or horse. These differences come in as a result of the last 
 step in the process of digestion ; the food materials are broken 
 down into simpler bodies in the alimentary canal, and built up 
 again in their passage through the epithelial cells which line that 
 structure, and in this process receive certain distinctive features 
 peculiar to the species or to the animal in question. As a result, 
 the body fluids of any animal contain proteids which have been 
 deliberately adapted for the nutrition of the cells of that animal, 
 but which may be quite useless or even toxic for those of another 
 species. The prime requisite of their suitability for nutritive 
 purposes is that the proteid molecules should possess haptophores 
 which " fit " the receptors of the cell molecules ; the second, 
 
THE ORIGIN OF ANTITOXIN THE SIDE-CHAIN THEORY 103 
 
 equally important, is that the proteid molecules shall be " diges- 
 tible " by the cell. If the first requisite fails, the proteid molecule 
 fails to unite with the cells ; it may remain for long periods in the 
 blood (as is the case with tetanus toxin in the blood of Oryctes, 
 where it remains months after injection), or may probably be 
 eliminated with the excretions, or be otherwise dealt with. In 
 this case no antibody can be formed. 
 
 When the injected proteid molecule finds suitable receptors and 
 unites with them, but when the resulting compound is not suit- 
 able for the nutrition of the cell, and cannot be " digested " by it, 
 the case is different. The receptors which are occupied by the 
 molecules of proteid are useless for the cell, and the conditions 
 which we have discussed in dealing with the action of toxins are 
 reproduced, although there may be no toxic action ; the receptors 
 are useless and have to be regenerated, and under suitable circum- 
 stances may be produced in excess and form antibodies. The 
 criteria of the suitability of any animal for the production of an 
 antibody to a given proteid are therefore three in number : 
 
 1. This proteid must not exist naturally in the blood of the 
 animal. 
 
 2. It must possess haptophores which " fit " the receptors of 
 the animal cells. 
 
 3. The compound thus formed must be " indigestible " by the 
 cell and useless for its nutrition. 
 
 These are the theoretical criteria, which are derived from a 
 study of the side-chain theory, and, on the whole, experimental 
 research agrees with them, and thus corroborates the theory to an 
 extraordinary extent. For example, we need scarcely refer to 
 criterion i : we know that precipitins do not occur normally in 
 the blood of any animal to the proteids which circulate in that 
 fluid ; it is only the injection of a foreign proteid which causes 
 their production. Further, the more closely allied are two animal 
 species, the less the production of precipitin when serum from the 
 one is injected into the other. Thus, in order to prepare a potent 
 antihuman serum, the blood of a man must be injected into a 
 rabbit, fowl, etc., not into a monkey. If it is true, as appears to 
 be the case, that the serum of an animal causes the production of 
 precipitins when injected into another of the same species, this 
 does not affect the argument, for these precipitins only occur in 
 minute amount, and simply show that the fluids and tissues of 
 different animals of the same species are not in reality identical. 
 
IO4 CONDITIONS OF ANTIBODY PRODUCTION 
 
 There is nothing surprising in that when we consider how different 
 persons vary in constitution and susceptibility. Again, it is not 
 uncommon to find auto-agglutinins in human blood i.e., sub- 
 stances which agglutinate human red corpuscles. When this is 
 the case, it is found that these agglutinins have no action on the 
 red corpuscles of the persons from whom the serum is taken, but 
 only on those of other individuals ; the stroma of the corpuscles 
 cannot act as an antigen to the cells with which it comes normally 
 into contact. (The way in which these auto-agglutinins are 
 called into existence, and their meaning, if any, or use in the 
 economy, are still unexplained.) 
 
 The existence and mode of formation of the secondary anti- 
 bodies i.e., the antibodies to antibodies point in the same 
 direction. Thus, if we inject typhoid bacilli or their proteid 
 constituents into rabbits or other animals, the specific antibody 
 the agglutinin is produced, and accumulates in the blood in con- 
 siderable amount, but it does not produce anti -agglutinin. If, 
 however, we inject this serum into an animal of another species, 
 and preferably one far removed from the first zoologically, the 
 production of anti -agglutinin occurs. The agglutinin produced in 
 any one species of animal must have, in addition to its peculiar 
 clumping properties, the general constitution of a proteid charac- 
 teristic of that animal, and hence be devoid of the power of 
 forming antibodies whilst in the blood where it was produced. 
 Similar phenomena occur in the production of other haemolysins 
 (amboceptor), which will be referred to subsequently. 
 
 With regard to the second criterion the possession of hapto- 
 phores which fit the receptors of the protoplasm of the animal 
 into which it is injected it is only necessary to say that, as far 
 as can be traced, the substances which produce antibodies do, as a 
 matter of fact, disappear from the blood of the animal in a short 
 time a few hours or days. As has been pointed out, the converse 
 of this is not necessarily true ; the toxin may disappear from the 
 blood, and yet no antitoxin be formed, as in the case of tetanus 
 toxin in scorpions. Here we have assumed that the scorpion 
 contains naturally a proteid with a haptophore closely allied to 
 that of tetanus toxin, that the toxophore of the latter is without 
 action on the cell with which it is linked, and that the latter can 
 use the former as pabulum in the same way as it uses the 
 molecules of normal occurrence in its blood. 
 
 The third criterion is that the proteid molecule shall not be 
 
THE ORIGIN OF ANTITOXIN- THE SIDE-CHAIN THEORY 105 
 
 capable of assimilation by the protoplasm to which it is anchored ; 
 it is not necessary that there should be a toxophore group which 
 injures the cell. In this case the production of the antibody will 
 not be accompanied by any symptoms of disease. The proto- 
 plasmic molecule will have some of its receptors occupied and 
 rendered useless by the foreign proteid, and will have to regenerate 
 others of the same nature, but it will not be injured in any way in 
 the process. It is in this way that we explain the formation of 
 antitoxins as a sequence to the injection of toxoids which have 
 retained their haptophore groups, but lost their toxophores, as 
 well as the formation of agglutinins (to non-pathogenic bacteria), 
 precipitins, etc. 
 
 We pass on to another question that of specificity. We have 
 seen that the antitoxins are, in general, adapted only to neutralize 
 the toxins which lead to their production. To this rule there are 
 a few exceptions, real or apparent. Thus, antirobin serum has 
 also an action against ricin, tetanus antitoxin has apparently 
 some action on snake-venom, anti-snake venom serum neutralizes 
 scorpion venom, etc. The explanation is doubtless that the 
 venoms in question have haptophore groups which closely re- 
 semble one another in their chemical affinities, whilst differing 
 from those of other toxins. Thus, Ehrlich has suggested that 
 robin is the toxon of ricin, in which case the combining portions 
 of the molecules of the two would be identical. 
 
 In any case, the side-chain theory seems to fit quite well with 
 ascertained facts. It explains the specificity ; it is the receptors 
 which unite with the injected toxin which are produced in excess, 
 and which therefore form antitoxin. But in the complex changes 
 in metabolism which must take place in the poisoned cell it is 
 quite easy to imagine that, under certain circumstances, other 
 receptors may either be formed, in slight excess as a result of the 
 general stimulation of the cell, or may be cast off in slight excess 
 as a result of necrotic or autolytic processes taking place therein. 
 This may be the explanation of some of the apparent exceptions, 
 especially of those in which the serum has but slight antitoxic 
 power on the toxin which was not injected, whereas it is much 
 more potent on that which was. 
 
 The next question, and a much more difficult and important 
 one, deals with the site of production of the antibodies. Ehrlich's 
 original idea was that the antitoxins are produced from those cells 
 on which the toxins act i.e., on the susceptible cells. For instance, 
 
I06 TETANUS TOXIN AND BRAIN-TISSUE 
 
 in the case of tetanus, he supposed that antitoxin is formed by the 
 cells in the central nervous system, and explained the great difficulty 
 of immunizing animals to this toxin by pointing out the enormous 
 susceptibility of the cells to the action of this poison ; it is only 
 in the cells of the central nervous system that antitoxin can be 
 formed, and these cells are extremely easily killed by the toxin, 
 and are necessary for life. The chief evidence in favour of this 
 theory is derived from the experiments of Wassermann and of 
 Romer, which we shall now consider seriatim. 
 
 Wassermann's experiment may be regarded as a corollary to 
 that of Ransom, who found that, after injection of tetanus toxin 
 in pigeons (which are, in the ordinary sense of the word, in- 
 susceptible to that substance), he could extract it from all 
 substances except the brain. Wassermann and Takaki found 
 that the mixture of tetanus toxin with an emulsion of the central 
 nervous system was no longer toxic when injected into susceptible 
 animals ; in other words, that the emulsion of central nervous 
 system behaved like antitoxin. Emulsions of other organs are 
 devoid of this power. They will combine with tetanus toxin, but 
 will yield it again when injected into susceptible animals or 
 macerated in salt solution. It appears, therefore, that the central 
 rjervous system is the seat, and the only seat, of an antitoxin-like 
 substance, and it is thus rendered at least probable that when 
 antitoxin appears in the blood it is due to its release from the 
 cells which contain it normally in other words, from the cells of 
 the central nervous system, the cells whichX attacks. The case 
 of tetanus is the only one in which a toxin appears to have a 
 definite selective influence on one group of cells, and is therefore 
 unique in providing a means whereby Ehrlich's supposition as 
 to the origin of antitoxin may be tested. It is no wonder that 
 Wassermann's experiment has been submitted to an extra- 
 ordinarily careful investigation, the results of which we must 
 outline briefly. Some of these experiments seem to point to the 
 truth of Ehrlich's assumption. Thus, Blumenthal showed that 
 after injection of tetanus toxin into living animals the spinal cord 
 lost its property of fixing the toxin in vitro, the receptors of the 
 cells being already occupied with that substance. Further, boiled 
 brain substance loses its power of neutralizing toxin, just as anti- 
 toxin does on being heated. He also showed that the central 
 nervous system of fowls, which are but slightly susceptible to 
 tetanus, has but little power of binding that toxin, so that emul- 
 
THE ORIGIN OF ANTITOXIN THE SIDE-CHAIN THEORY 107 
 
 sions of the brain must remain in contact with the toxin for some 
 time before the latter is neutralized. 
 
 It was urged by many writers that the brain cannot be the site 
 of production of antitoxin, since the injection of tetanus toxin 
 direct into the brain of immunized animals causes the develop- 
 ment of tetanic symptoms. This argument is obviously fallacious. 
 A cell that is secreting antitoxin still possesses side-chains suitable 
 for union with the toxin ; it possesses them, indeed, in increased 
 amount, and is therefore, if anything, more susceptible to the 
 action of the toxin. The antitoxin which is circulating in the 
 blood only prevents the toxin from reaching the cell, acting much 
 in the same way as a lightning conductor. 
 
 Further research, however, seems to prove definitely that the 
 neutralizing properties of brain substance are not due to the 
 presence of a substance having any real likeness to antitoxin. 
 Thus, Metchnikoff showed that when an emulsion of frog's brain 
 is mixed with tetanus toxin no neutralization takes place, although 
 the frog is, under certain circumstances, susceptible to the action 
 of the toxin ; these researches were corroborated by Courmont 
 and Doyon. Arguing from this and other facts, Metchnikoff 
 attributed the fixation fo tetanus -as*itoxin by the central nervous 
 system to the presence in the latter of fatty substances ; these are 
 absent from the brain of the frog. In support of this view, several 
 observers found that substances such as lecithin, cholesterin, 
 tyrosin, etc., have also the power of neutralizing various toxins. 
 
 It was objected to these researches of Metchnikoff and Cour- 
 mont and Doyon that these experimenters did not leave the 
 tetanus toxin in contact with the brain substance for a sufficiently 
 long time. But this seems unnecessary, since the emulsion of the 
 central nervous system of higher animals will neutralize tetanus 
 toxin if the mixture be injected immediately, or even if the 
 two substances are injected separately, though in this case the 
 necessary amount of brain substance is larger. And Danysz 
 showed that if a neutral mixture of brain and toxin be macerated 
 for a long time in salt solution the toxin is again set free, in which 
 again the phenomenon is unlike that presented by antitoxin in its 
 action on toxin. 
 
 The researches of Morax and Marie also point in the same 
 direction. They showed that the fixative power of the brain is 
 almost entirely destroyed by drying, whereas, as we know, that 
 process has practically no effect on antitoxin. The researches of 
 
io8 ROMER'S EXPERIMENTS 
 
 Dmitrevsky are also of great interest, and almost constitute a 
 crucial experiment. 
 
 If Ehrlich's theory is true, and if antitoxin is produced by the 
 budding-off of the receptors in great numbers, it ought to follow 
 that the brain of an immunized animal, which contains these 
 receptors in abnormally great amount, ought to have a greater power 
 of neutralizing toxin than that of normal animals. But Dmitrev- 
 sky's experiments show that this is not the case, and constitute a 
 strong proof against the theory in its original form. It seems fair 
 to conclude that Wassermann's phenomenon is due to some 
 accidental property of the central nervous system, or possibly to 
 the presence of fats, and must not be quoted as evidence of the 
 origin of antitoxin from the cells on which it chiefly acts. 
 
 Romer's experiment was made with abrin, a substance which 
 has the power of causing violent conjunctivitis. He made use 
 of rabbits, instilling small but increasing amounts into the right 
 conjunctiva. After three weeks the animal was killed, and each 
 conjunctiva dissected off, ground up with one lethal dose of abrin, 
 and injected into animals. The right conjunctiva, that into which 
 the abrin had been instilled, was found to act as an antitoxin and 
 to neutralize the abrin, whereas the left was devoid of this power. 
 Of course, if the process were carried on for a long time the abrin 
 would be absorbed into the system, and there would be a general 
 production of antitoxin, but at first the process appears local. 
 
 These researches are interesting, but do not seem to have any very 
 direct bearing on the question at issue. They show what does 
 not require proof that tissues which are not reached by the toxin 
 do not produce antitoxin ; but the question as to whether the 
 latter substance is produced by the cells which are peculiarly 
 susceptible to the toxin is not elucidated. There are in the 
 conjunctiva numerous structures connective-tissue cells, blood- 
 vessels, endothelium, epithelium, leucocytes, etc. and these 
 experiments afford no means of gauging which of these are 
 affected by the toxin or which produce antitoxin. The behaviour 
 of the leucocytes is of especial interest ; they are present in the 
 inflamed conjunctiva in increased amounts, and the question may 
 be asked whether the apparent antitoxic action of the right con- 
 junctiva may not be due to these cells, which are present in but 
 small numbers in the left. This view is rendered more probable 
 from the further researches of Romer, who showed that in 
 immunized animals antiabrin is present in greater amounts in the 
 
THE ORIGIN OF ANTITOXIN THE SIDE-CHAIN THEORY 
 
 organs rich in leucocytes, such as the spleen, lymph glands, and 
 bone-marrow. This view harmonizes well with that of Metch- 
 nikoff, which we shall consider subsequently. 
 
 The chief arguments against the origin of antibodies from the 
 cells which are especially acted on are those of Metchnikoff on 
 the production of antispermotoxin, and from the study of other 
 cytotoxins. The first series of experiments are of fundamental 
 importance, and will be discussed here, although the substances in 
 question are not simple bodies like toxins, but have a much more 
 complex structure. The main facts are as follows : The injection 
 of spermatozoa into living animals is followed by the production 
 and appearance in the serum of certain substances which have 
 the power of immobilizing and clumping the spermatozoa of the 
 species from which the spermatozoa used for the injection was 
 taken ; we may call the substances spermotoxin. If the serum of 
 animals which has been prepared in this way is now injected into 
 animals of another species, the result will be the formation of an 
 antispermotoxin analogous with antitoxin, which inhibits the 
 action of the spermotoxic serum in vitro on the spermatozoa. 
 
 Now on Ehrlich's theory we may assume that this anti- 
 spermotoxin is derived from the cells from which the spermotoxin 
 is derived i.e., from the spermatozoa. But Metchnikoff proved 
 that this is not the case, since the substance is developed as the 
 result of the injection of spermotoxic serum into castrated males 
 or into young animals of either sex. Antispermotoxins, therefore, 
 are not necessarily derived from sperm cells. This is certainly a 
 very striking result, and one that tells against the side-chain 
 theory, but it is not conclusive for this reason the cytotoxins are 
 not usually sharply specific. Thus a cytotoxic serum made by the 
 injection of kidney cells has a profound action on the kidney, but 
 it usually has some haemolytic action also, and may affect other 
 cells as well. There do not seem to be any experiments bearing 
 on the point, but it is at least probable that spermotoxins have 
 some action on cells other than spermatozoa, and if this is the 
 case Metchnikoff 's experiments, considered as a proof against the 
 side-chain theory, fall to the ground. 
 
 The mode of formation of the antihaemolysins also calls for short 
 notice in this connection. The haemolysins are, in some cases at 
 least, more sharply specific than some of the cytotoxins, acting 
 apparently almost exclusively on the red blood-corpuscles. It 
 should follow on Ehrlich's theory that the antihaemolysins are 
 
IIO SITE OF ANTITOXIN FORMATION 
 
 derived mainly from the red blood-corpuscles. This view, 
 however, seems very difficult to believe when we consider that 
 these substances are devoid of protoplasm and nuclei, and do not 
 present any of those appearances indicative of active metabolism 
 or secretion which we should expect in the case of a cell perform- 
 ing so profound a physiological function as the production of an 
 antitoxin. 
 
 Nor is there any evidence, except in the one doubtful case of 
 the action of brain substance on tetanus aa^koxin, of the presence 
 of substances like antibodies in the normal tissues and organs, in 
 spite of the fact that they must contain receptors which would 
 neutralize the toxins to which they are tested. Thus Calmette 
 did not find that emulsions of brain possessed any neutralizing 
 action on snake venom, although that substance certainly acts on 
 the central nervous system, and there are many similar researches 
 on the actions of other emulsions of organs on other toxins, all 
 with negative results. We may quote, for example, those of Blum, 
 who submitted various organs of the horse liver, spleen, etc., all 
 of which are certainly acted on by diphtheria toxin, since they 
 show definite morphological lesions in acute cases of fatal intoxi- 
 cation to prolonged autolysis, and found nothing in the nature of 
 an antitoxin for diphtheria or snake venom in the resulting 
 material. In one case that of autolyzed lymphatic glands of the 
 calf he found a substance which reacted like tetanus antitoxin. 
 
 On the whole, therefore, it would seem that there is no good 
 evidence for the theory that the cells which are acutely and 
 powerfully poisoned by means of toxin are those from which the 
 antitoxin is derived. It is certain, however, that this substance 
 must be formed from cells which are reached and affected in some 
 way by the toxin. The cells which especially require discussion 
 are those of the connective tissues and the leucocytes. 
 
 Attention is drawn especially to the connective tissues from the 
 fact that the most powerful production of antitoxin is usually 
 elicited by a subcutaneous injection of toxin ; intraperitoneal 
 injection is in general less potent, and intravenous injection less 
 still, and may not be followed by any production of antitoxin 
 at all. The same law holds, and even more strongly, in the 
 production of the other antibodies, although some exceptions 
 occur. 
 
 Now in some cases it is true that these toxins have a local 
 pathogenic action on the tissues into which they are injected, but 
 
THE ORIGIN OF ANTITOXIN THE SIDE-CHAIN THEORY III 
 
 in others this is extremely slight, or not more than that due to 
 the injection of any so-called " inert " fluid into that situation. 
 Consider in this connection the reactions of certain animals to 
 tetanus toxins. When this substance is injected into guinea-pigs, 
 the effect is practically the same whether the injection is sub- 
 cutaneous or intracerebral. In rabbits, on the other hand, 
 a very much smaller dose suffices to kill if injected into the brain 
 than is necessary if the injection is subcutaneous. Now rabbits 
 are less susceptible to tetanus toxin than are guinea-pigs, requiring 
 relatively larger doses to bring about a fatal issue. On the other 
 hand, it is easier to prepare tetanus antitoxin from rabbits than 
 from guinea-pigs by the injection of tetanus toxin, though difficult 
 in both cases. Compare these results with those obtained from 
 fowls. These animals are resistant against tetanus toxin (except 
 in absolutely enormous doses) when injected subcutaneously, but 
 are easily affected when the injection is made into the brain 
 or subarachnoid space. The probable explanation turns on the 
 varying affinity of the toxin for different parts of the body. We 
 may assume in all cases that the tetanus toxin acts as a poison 
 only on the brain and spinal cord, and that in the guinea-pigs it 
 has practically no affinity for the subcutaneous tissues, and 
 that when injected into this situation it passes ultimately to the 
 brain almost without loss. In rabbits some of the toxin is united 
 to the tissues, and only a part reaches the central nervous system, 
 whilst in fowls the affinity of the tissues for the toxin is so great 
 that the whole of the latter is absorbed and the brain escapes 
 injury. Now it is practically impossible to obtain antitoxin by 
 injecting toxin into the guinea-pig, difficult to obtain it from the 
 rabbit, but easy to obtain it from the fowl, so that it would appear 
 that it is the tissues which are not especially vulnerable to the 
 toxin which yield it. If this is true, it is no argument against the 
 validity of the side-chain theory. It simply indicates that the 
 cells which are profoundly affected with toxin are thereby 
 rendered unable to produce antitoxin in virtue of the toxic action 
 of the poison, the other conditions being suitable. We know this 
 to be the case from the fact that we can produce tetanus 
 antitoxin from guinea-pigs as a result of the injection of toxoid, 
 which we must assume to unite entirely with the cells of the 
 central nervous system, just as the toxin does, though without 
 injuring them. We may assume, therefore, that antitoxin is 
 produced from cells which unite with toxin or toxoid, but which 
 
112 SIGNIFICANCE OF LEUCOCYTOSIS 
 
 are not too profoundly injured thereby to perform their functions 
 of self -nutrition in a normal manner. 
 
 The question whether we can narrow down the issue to one 
 particular set of cells which always occur in the connective and 
 other tissues to wit, the leucocytes is a more difficult one. We 
 have already adverted to the researches of Romer, who found that 
 antiabrin was present in immunized animals in greatest amount 
 in the organs that are rich in leucocytes, and Metchnikoff has 
 brought forward a whole series of researches which point strongly 
 in the same direction. The discussion of some of these will be 
 deferred to a subsequent chapter, since they hardly seem to 
 indicate that the leucocytes form antitoxin, though they do go 
 to prove that they discharge another and equally important 
 function in the production of immunity to toxins. Here it will 
 only be necessary to point out some well-known and important 
 facts which appear to have a bearing on the question of the 
 origin of antitoxin. We allude especially to the chemotactic 
 attraction for leucocytes which is show r n by almost all toxins 
 when not present in too great an amount. In virtue of this 
 attraction the most marked and constant feature of intoxication 
 is the presence of an excess of leucocytes, and this is, in general, 
 most marked when there is a balanced contest between the toxin 
 and the host i.e., when the latter is neither destroyed at once 
 nor shows but little effect of the injection. It is practically a 
 general rule that wherever the host is victorious in the struggle 
 with the toxin there is an excess of leucocytes ; and even where 
 the fatal issue is not averted there is some leucocytosis, unless 
 the intoxication is a very grave one and very rapidly fatal. This 
 is well known in human pathology in the infective processes, 
 where a diminution of the number of leucocytes is a bad sign, 
 whereas hyperleucocytosis is a good sign in so far as it indicates 
 that the infection, though intense, is being combated by the 
 patient by the best means at his disposal. Many researches have 
 indicated that the same law holds in these intoxications. Thus 
 Chatenay, in an exhaustive study on the reactions of the more 
 important toxins, finds that a dose that is rapidly fatal brings 
 about a fall in the leucocytes, whereas smaller doses cause hyper- 
 leucocytosis ; thus, in the guinea-pig about 100 lethal doses are 
 required to prevent the occurrence of the latter. Now we 
 can scarcely imagine that so widespread and constant a phe- 
 nomenon is without some meaning or without some use to the 
 
THE ORIGIN OF ANTITOXIN THE SIDE-CHAIN THEORY 113 
 
 organism, and since it always occurs and is most marked under 
 conditions in which antitoxin is formed, it is highly probable that 
 the leucocytes may be, entirely or in part, the origin of the 
 antitoxin. But we must not forget that in many cases the 
 leucocytes play other parts in the struggle against the infections ; 
 for example, it is plausible to argue that their presence in excess in 
 infected areas is an attempt to combat the bacteria themselves, and 
 not the toxins which they produce. In this case their access to 
 areas into which a toxin had been injected might be useless, and they 
 might simply be attracted there under the mistaken supposition that 
 there would be bacteria to combat as well as toxins. But this 
 view is rendered improbable in face of the numerous facts that 
 have been brought out by the French school with regard to the 
 absorption of toxins and other poisons by the leucocytes. Thus 
 Metchnikoff showed (and it is a fact of the utmost importance) 
 that if an aseptic exudate be produced in a fowl that has been 
 injected with tetanus toxin, that .substance can be demonstrated 
 in large amount in the leucocytes of the exudate. Again, Vaillard 
 and Vincent have shown that if tetanus bacilli or spores washed 
 free from toxin are injected into a guinea-pig, they become 
 surrounded by leucocytes, and that under such circumstances no 
 tetanic symptoms occur. 
 
 With regard to the action of other poisons the facts are still 
 * more striking. Thus rabbits are far more susceptible to the 
 v intracerebral injection of atropin than to the same substance 
 injected into the circulation; and when the injection has been 
 made in the second manner, atropin can be demonstrated from 
 the leucocytes, whereas it is not present, or only in very small 
 amounts, in the plasma and red corpuscles. Similar facts have 
 been found for arsenic and other poisons. Still more striking are 
 the researches of Besredka with regard to arsenic. He studied 
 first the action of the trisulphide of arsenic, an almost insoluble 
 substance, yet very poisonous. When this was injected in small 
 amounts into the peritoneal cavity of a guinea-pig, there was a 
 marked increase in the mononuclear leucocytes in that situation, 
 and in a short time these cells had ingested the whole of the 
 arsenic, which could be recognized as fine granules in their 
 protoplasm. These gradually became smaller and ultimately 
 disappeared, being probably converted into a non-toxic compound 
 perhaps by some process analogous with antitoxin formation. 
 In a second series of experiments he tested the action of the 
 
 8 
 
114 ACTION OF LEUCOCYTES ON TOXINS 
 
 same substance when shielded from the leucocytes by being 
 enclosed in permeable bags before being placed in the peritoneum ; 
 in this case the animal died, the arsenic doubtless becoming 
 dissolved, and thus escaping the action of the leucocytes. He 
 found also that substances which diminished the number of the 
 leucocytes in the peritoneal fluid aided the action of the arsenic, 
 whilst those which caused a leucocytosis diminished it. He found 
 similar facts with relation to the action of a soluble salt of arsenic, 
 and showed that in animals which had been immunized to that 
 substance the arsenic is especially taken up by the leucocytes. 
 There can be no doubt, therefore, that the leucocytes play a role 
 of the utmost importance in the defence of the body against the 
 ordinary poisons. 
 
 All things being considered, we may deduce that antitoxin is 
 formed from any cell with which the toxin can combine, provided 
 that that cell is not too profoundly injured in the process, and 
 may attribute to the leucocytes an important, though not an 
 exclusive, role in this process. It is hardly necessary to say that 
 this does not disprove the side-chain theory ; we must regard the 
 leucocytes as being cells which are specially told off for the 
 defence of the organism against infections and intoxications. 
 They are, in consequence, specially immune to the action of 
 toxins, being able to resist amounts which are injurious to the 
 more highly organized tissues ; that this is the case is proved 
 by their invasion and persistence in a living state in areas in 
 which the tissues are profoundly injured by a toxin. It is not 
 difficult to believe that these resistant cells, which have a special 
 predilection for intoxicated areas in which antitoxin is especially 
 required, should be the source of that substance. We have, then, 
 only to regard them as possessing suitable haptophore groups and 
 as being relatively insusceptible to the toxophore groups both of 
 which are inherently probable to see that they fulfil in a special 
 way the criteria which the side-chain theory demands for cells 
 which are to produce antitoxin when exposed to the action of 
 toxins. 
 
CHAPTER VI 
 IMMUNITY TO TOXINS 
 
 THE mechanisms by which the injurious effects of toxin in the 
 blood and tissues are combated are exceedingly complex, and 
 may be divided roughly into two series. In the first and less 
 interesting, though perhaps not less important, the mechanism 
 may be called (for want of a better term) non-specific. It acts 
 equally on all or many toxins, and recovery from the effects of the 
 poison is not necessarily or usually followed by any immunity 
 thereto. For example, the dilatation of the vessels and accelera- 
 tion of the blood-stream which takes place in an inflamed focus 
 may be regarded as one of the natural provisions for avoiding the 
 too intense action of a toxin in one small area. The poison pro- 
 duced is rapidly swept into, and diluted by, the whole blood- 
 stream, and if not produced in large amounts may not reach any 
 tissue in amount sufficient to cause much damage. Perhaps, also, 
 the proteolytic action of the enzymes frequently produced (mainly 
 from the polynuclear leucocytes) in the inflammatory focus are of 
 some importance. Toxins are very fragile bodies, and it is ex- 
 tremely probable that the potent enzymes which occur in all fluids 
 rich in polynuclear leucocytes may bring about their destruction in 
 considerable amounts. 
 
 The more purely chemical processes are hardly known in their 
 application to true toxins, but they have been largely studied in 
 regard to the methods by which the organism combats deleterious 
 substances produced in the course of metabolism, or absorbed 
 from the alimentary canal. The latter are for the most part 
 bacterial products, though not true toxins. It is, however, quite 
 possible that these latter substances are destroyed by the same 
 processes, which Herter classifies as (i) oxidation, (2) hydration 
 and dehydration, and (3) various syntheses. 
 
 As an example of the process of oxidation, we may consider the 
 formation of indol, a toxic substance, which is formed by B. coli 
 
 "5 82 
 
Il6 NEUTRALIZATION OF POISONS 
 
 and other organisms in the alimentary canal, and which probably 
 plays some part in the production of the symptoms of auto-intoxi- 
 cation due to abnormal digestive processes occurring in the small 
 intestine. It undergoes oxidation to indoxyl, a much less toxic 
 substance, which then combines with sulphate of potash to form 
 indoxyl-potassium-sulphate, or indican. There is reason to believe 
 that this process takes place to a very large extent in the liver, 
 one of the chief functions of which is the destruction or neutraliza- 
 tion of poisonous substances conveyed to it by the portal circula- 
 tion. Thus the toxic dose of nicotin and of some other alkaloids 
 is much less when the injection is made into the general circulation 
 than if the substance be injected into the portal vein. This, it 
 must be noticed, is not due merely to the elimination of the poison 
 in the bile, though that may happen. The liver cells have the 
 power of actually destroying certain of the alkaloids. Thus 
 hyoscyamine mixed with surviving liver pulp is rapidly destroyed. 
 In the same way, indol, phenol, and skatol, all toxic substances of 
 bacterial origin, cannot be recovered by distillation after contact 
 with liver cells. It appears, therefore, that much of the immunity 
 to non-specific poisons is dependent on the functional activity of 
 this organ, which is, so to speak, interpolated in the blood-current 
 in order that it may rid the circulation of certain poisons, and 
 that these are eliminated in the bile or submitted to various 
 chemical modifications, in the course of which they become inert, 
 or at least less toxic. 
 
 Examples of the neutralization of poisons in the body are 
 numerous, the most important being, perhaps, the formation of 
 urea, a relatively non-toxic substance, by the synthesis of ammonia 
 and carbonic acid, followed by dehydration, this process taking 
 place mainly or entirely in the liver. Other syntheses, however, 
 take place in other organs for example, the union of glycocoll 
 and benzoic acid is performed in the kidney. And we shall see 
 subsequently that the leucocytes and leucocytic organs have a 
 very special and important duty to perform in dealing with poisons 
 of all sorts, both organic and inorganic, though the exact chemical 
 processes that they bring about are unknown. It will be more 
 convenient to defer the consideration of these cells for the 
 present. 
 
 The exact application of these "non-specific" processes of 
 detoxication to the bacterial toxins has not yet been worked out, 
 but there can be little doubt of its great importance. Compare, 
 
IMMUNITY TO TOXINS 117 
 
 for example, the severity of the symptoms due to an abscess due 
 to B. coli and the lack of symptoms due to the absorption of the 
 toxins of this organism which occurs from the alimentary canal, 
 and very probably from bacilli which actually make their way 
 through its walls into the lacteals. There can be but little doubt 
 that the symptoms of intoxication in the former case are due to 
 the direct access of the bacterial poisons into the blood-stream 
 without having first to traverse the liver. Again, in a case of 
 diphtheria of moderate severity virulent bacilli are often present 
 in abundance so great, that by comparison with the toxin-forming 
 powers of the organisms in vitro we might expect a rapidly fatal 
 issue from the toxin absorbed, and yet the symptoms of general 
 intoxication may not be severe. In all probability only a small 
 fraction of the toxin formed actually reaches the distant tissues. 
 Some may be dealt with by the zone of leucocytes which under- 
 lies the layer of diphtheria bacilli, but it is also possible that some 
 of the toxin is destroyed in the blood-stream, perhaps by a process 
 of oxidation. These non-specific methods of dealing with toxins 
 are matters more for the physiologist than for the pathologist, 
 depending as they do simply on the perfect discharge of the 
 normal functions of the body. They are of great importance 
 greater, perhaps, than pathologists usually realize, the interest 
 attaching to them being so much less than that which is connected 
 with the study of the antitoxins and similar bodies. They are to 
 be regarded as the first line of defence against poisons of all sorts. 
 When a small dose of a specific bacterial toxin gains access it is 
 probable that the natural physiological methods in daily use for 
 dealing with the natural poisons (formed in metabolism or absorbed 
 from the alimentary canal) are in most cases applicable to it also, 
 and no specific process has to be brought into action. In confir- 
 mation of this view is the fact that diphtheria antitoxin is not 
 necessarily found in the blood after natural recovery from diph- 
 theria, suggesting that in these cases the processes at work are 
 non-specific. 
 
 But when the toxin is present in greater amount this process 
 may fail, and it may do so in consequence of the action of the 
 poison on the tissues and organs normally concerned in the defence 
 of the body. The neutralization of toxins, etc., in the liver and 
 other regions can be carried out best when these organs are in a 
 state of high functional activity, and when this is impaired some 
 of the deleterious substances will escape their action. Thus we 
 
Il8 FORMS OF IMMUNITY TO TOXINS 
 
 get a vicious circle. The poison injures the liver or other organ 
 and impairs its defensive powers. More poison is allowed to 
 circulate in the blood, and the liver is injured still more. When 
 this occurs we are thrown back on the specific methods of dealing 
 with the toxins, which take longer to come into action, and which 
 may be regarded as the second line of defence. In one case, 
 however, we must regard them as of chief importance. Thus in 
 tetanus the toxin is produced locally, and ascends the nerve - 
 trunks without entering the blood-stream or being carried to the 
 liver or leucocytic organs. Here, then, in all probability, specific 
 methods have to be resorted to, and the chance of recovery depends 
 on their early development. 
 
 The question of specific immunity to toxins, and of recovery 
 from intoxication with true toxins, may be considered under three 
 heads : 
 
 1. Acquired immunity, due to disease or to vaccination with 
 toxins or toxoids (chemical vaccination). 
 
 2. Antitoxic or passive immunity, due to the injection of anti- 
 toxic serum, or possibly to its natural occurrence in the blood. 
 
 3. Natural immunity, not due to vaccination, and not accom- 
 panied by antitoxin in the blood. 
 
 It will be convenient to consider them in this order. 
 
 Natural recovery from toxic diseases such as diphtheria and 
 tetanus is not necessarily accompanied by the appearance of anti- 
 toxin in the blood at least, not in demonstrable amounts. Further, 
 it appears from the researches of Abel that antitoxin, when it is 
 formed at all, does not make its appearance until about the eighth 
 day of the disease, at which period the severity of the toxic 
 symptoms may have already begun to decline. What may be 
 called the simple antitoxic theory cannot be maintained. Under 
 natural conditions the process of recovery is not due simply to the 
 production of antitoxin, though it is possible that this comes into 
 action in prolonged cases. 
 
 Further, acquired immunity to toxins is not due solely to the 
 presence of antitoxin in the blood. If it were so it should develop 
 proportionately to the development of antitoxin, and the two should 
 persist for the same length of time, and disappear together. This, 
 however, is not the case. We have already seen that animals in 
 the early stage of immunization to diphtheria and tetanus frequently 
 present a decreased amount of resistance, or hypersensitiveness, 
 to these substances, and may die with symptoms of acute intoxica- 
 
IMMUNITY TO TOXINS IIQ 
 
 tion, in spite of the presence in the blood of amounts of antitoxin 
 sufficient to neutralize the toxin many times over. Similar appear- 
 ances may be seen in animals in which death from acute intoxica- 
 tion does not occur. Thus, in two horses which were treated at 
 the same time for the production of diphtheria antitoxin, one 
 showed but little sign of the action of the toxin, and improved in 
 general health during the treatment it developed only 5 units of 
 antitoxin per cubic centimetre of serum ; the other suffered severely 
 in general health, and had ultimately to be killed it developed 
 70 units per cubic centimetre. Similar phenomena are often met 
 with, and, as a general rule (to which there are numerous excep- 
 tions), the presence of a large amount of antitoxin in the blood 
 indicates susceptibility rather than immunity. The facts of the 
 later stages of antitoxin-formation may also be borne in mind. 
 Sooner or later in the history of any antitoxin horse there comes a 
 time when the amount of antitoxin begins to diminish, and would, 
 in all probability, disappear entirely if the injections were con- 
 tinued ; yet these horses are extremely resistant to the action of 
 toxin more so, in fact, than animals with much antitoxin in the 
 blood. The degree of immunity, therefore, is not measured by 
 the amount of antitoxin in the blood, and we might argue that the 
 two are not related in any way. This, however, is absurd, in 
 view of the ascertained action of antitoxin in neutralizing the 
 effects of toxin in vitro, or of protecting against a dose surely fatal. 
 We may consider the role of antitoxin under two heads : (i) in 
 immunity, and (2) in recovery from disease. 
 
 Considering first the question why an animal dies in spite of the 
 presence of an excess of antitoxin in the blood, we must regard 
 the simplest and most probable explanation as one on which the 
 toxin-antitoxin molecule is looked upon as dissociable. This is 
 always the case on Arrhenius and Madsen's theory of the inter- 
 action of the two substances, whilst on the colloid theory the dis- 
 sociation only takes place for a short time after the compound has 
 formed, the union between the two substances gradually becoming 
 firmer and firmer. On this supposition the failure of antitoxin is 
 readily explicable : the two substances unite, and the inert mole- 
 cule is formed ; this dissociates, and the toxin-molecules, which 
 happen to be set free in the neighbourhood of susceptible cells, 
 unites with them. The removal of some of the molecules of toxin 
 allows more dissociation to take place, and ultimately the whole of 
 this substance is passed on to the tissues. We must assume that 
 
I2O ROLE OF THE LEUCOCYTES 
 
 the compound formed between the toxin and the tissues does not 
 dissociate, as, indeed, appears probable. 
 
 But if this is the case, the effect of antitoxin in the blood would 
 be merely to delay the action of the toxin ; assuming this, how 
 are we to explain the preventive effect of this substance in passive 
 immunity and its curative effect in disease ? For it is only in com- 
 paratively rare cases in the early stages of antitoxin formation 
 that the phenomena under discussion occur, and in all other con- 
 ditions the presence of a sufficient amount of antitoxin in the 
 blood constitutes a perfect safeguard against the action of its 
 corresponding toxin. It is probable that the leucocyte is the all- 
 important factor necessary for the destruction of these specific 
 toxins, whether previously neutralized by antitoxin or not. We 
 must look upon the toxin-antitoxin molecule as one which can be 
 easily ingested and destroyed by the leucocytes, and the neutraliza- 
 tion of toxin by antitoxin as the first step in a double process, the 
 second being the destruction of the compound by the leucocytes. 
 If antitoxin is absent, the leucocytes may still deal successfully 
 with the toxin, if the latter be not too virulent, nor present in too 
 large an amount ; but if the leucocytes make default, the presence 
 of antitoxin may delay the lethal issue, though it is powerless to 
 avert it. We may, perhaps, compare antitoxin with opsonin, 
 which unites with bacteria and renders them suitable for ingestion 
 by the white corpuscles. Metchnikoff and his school have paid 
 great attention to the role of the leucocytes in intoxication, and 
 have brought forward very important and suggestive evidence, 
 pointing to the white corpuscles, and especially the large lympho- 
 cytes (macrophages) as the main source of antitoxin. This 
 question is dealt with elsewhere, and at present we have to discuss 
 only the role of the leucocytes in dealing with the toxins, either 
 alone or when neutralized by antitoxin. 
 
 The evidence proving the importance of the leucocytes in deal- 
 ing with unaltered toxins is abundant, and some of the more striking 
 facts brought forward by the French school have been briefly 
 summarized in the last chapter. The evidence for the removal 
 of toxin in combination with antitoxin by leucocytic action is less 
 direct, since it is obviously impossible to recognize this compound 
 by chemical or microscopical processes whilst in the leucocyte. 
 But it has been shown that where toxins are introduced in com- 
 bination with solid substances, such as carmine or brain tissue, 
 they become surrounded by large numbers of leucocytes, and the 
 
IMMUNITY TO TOXINS 121 
 
 particles are taken up by these cells. Further, the injection of 
 toxin into an animal which already contains antitoxin in the blood 
 brings about a more or less marked leucocytosis. It is true that 
 the local leucocytosis brought about by the injection of neutral 
 mixtures of the two may be but slight, but we must remember 
 that substances in a state of solution are quickly absorbed and 
 carried to the lymph glands or bloodvessels, in either of which 
 situations leucocytes occur in plenty. Further, there are always 
 some leucocytes in the tissues, and the number may be sufficient 
 to deal with the amount of toxin-antitoxin injected, which, con- 
 sidered as mere weight, is always very small. 
 
 The effect of antitoxin on the toxin of B. pyocyaneus may also be 
 cited as a phenomenon capable of explanation on this supposition. 
 A few lethal doses of toxin are fully neutralized by antitoxin, the 
 law of multiple proportions holding up to a certain point. When, 
 however, more than ten lethal doses are injected the law does not 
 hold, and no amount of antitoxin will avert the fatal issue. Here 
 we must assume that the antitoxin has but a slight affinity for the 
 toxin, so that dissociation takes place rapidly, and the ultimate 
 cause of the destruction of the toxin to be leucocytic activity. 
 The single lethal dose is just more than the leucocytes can deal 
 with ; but when antitoxin is injected simultaneously the leucocytes 
 have time to come into action, and the toxin, gradually set free by 
 dissociation, is dealt with in detail instead of in a single dose. 
 There are, however, limits to this process, and when more than 
 ten lethal doses are present fully combined with toxin we must 
 imagine that dissociation goes on so rapidly that more toxin than 
 one lethal dose is set free before the toxin-antitoxin molecules can 
 be taken up. If this explanation is highly theoretical, it seems 
 the best that is available at present. 
 
 The difference between the effect of toxin in animals with anti- 
 toxin in the blood, but otherwise normal, and its effect in animals 
 in the early stage of antitoxin formation, is explicable if we regard 
 the hypersensitiveness as occurring in the leucocytes themselves 
 as well as in the tissues. When antitoxin is injected into a normal 
 animal, the leucocytes of which are functionally active, the con- 
 ditions for the immediate destruction of the inert molecule are 
 present, and no dissociated toxin gains access to the susceptible 
 tissues. But in hypersensitive animals the leucocytes will be 
 unable to deal with the molecule, or will perhaps be killed by toxin 
 liberated by dissociation taking place within their protoplasm, and 
 
122 ROLE OF THE LEUCOCYTES 
 
 the toxin will ultimately reach the tissues. And it is probable 
 that the beneficial effect of early as compared with late injections 
 of diphtheria toxin may be dependent in part on the fact that in 
 this disease me leucocytes are very apt to suffer degenerative 
 changes which doubtless impair their defensive powers. Ewing 
 believes that the variations in the staining capacity of these cells 
 (loss of chromatin being an early and easily recognized sign of 
 their degeneration) might be utilized as a means of prognosis, and 
 points out that in cases which recover the staining quality of many 
 of the leucocytes undergoes a rapid improvement after the injec- 
 tion of antitoxin, whereas in fatal cases this change could not be 
 detected. As a rule the total number of leucocytes is diminished 
 immediately after the injection of antitoxin, but in some fatal 
 cases the previously existing leucocytosis may remain or even 
 increase. This fall in the total number of leucocytes is probably 
 to be accounted for by the neutralization of the toxin, so that 
 fresh white corpuscles are no longer attracted from the bone- 
 marrow by positive chemotaxis, whilst the old and injured cells 
 remain in the spleen or other internal organs, no longer circulating 
 in the blood. In cases which die the reduction in the number of 
 leucocytes may be followed by a subsequent rise. 
 
 Here also we must refer to numerous researches, emanating 
 more especially from the French school, which go to show that 
 substances which are certainly devoid of true specific antagonism 
 have nevertheless the power of preventing or diminishing the 
 action of toxins in virtue of causing an increased inflow of leuco- 
 cytes to the region into which the injections are made. This 
 question has been more especially investigated in regard to the 
 effect of this artificially produced local leucocytosis in causing 
 local bacterial immunity, but Calmette and Delecarde have shown 
 that the peritoneal injections of ordinary broth cause a certain 
 degree of immunity to abrin, whereas normal saline solution has 
 no such power. The former substance is a powerful agent in 
 attracting leucocytes, whilst the latter is much less potent in this 
 respect, though by no means devoid of activity. Substances which 
 have the power of increasing the action of the leucocytes in this 
 way are sometimes termed "stimulins," but the word should be 
 avoided. The action of leucocytes in any region may be in- 
 creased by many methods, the chief of which are : (i) attracting 
 larger numbers by means of substances having a positive chemo- 
 tactic action ; (2) stimulating the activity of the leucocytes ; and 
 
IMMUNITY TO TOXINS 123 
 
 (3) altering the nature of the substances on which they are to act. 
 If the term "stimulin" is to be used at all, it should be rigidly 
 restricted to one that acts in the second way, and there is as yet 
 no strict proof that such a body occurs. Substances acting in the 
 third way are called opsonins, and, if the theories of antitoxic 
 action here outlined are correct, antitoxins have this action also. 
 
 Turning now to the process of recovery from bacterial intoxi- 
 cations in natural disease, we find but little evidence that the 
 formation of antitoxin plays any active part in the process. The 
 conditions are not as a rule suitable for the production of this 
 substance, since the constant flow of the toxin into the circulation 
 would lead rather to a summation of negative phases and the pro- 
 duction of hypersensitiveness. Further, the process of recovery 
 may have commenced before antitoxin appears in the blood a 
 phenomenon which, when it occurs, we may regard rather as a 
 means of preventing reinfection than as a mechanism for the cure 
 of the original attack of the disease. (It may be pointed out that 
 in the case of diphtheria the studies of Ruth Tunnicliffe lead us to 
 believe that the process of recovery runs parallel to, and is due 
 to a rise in, the opsonic index, and that the main factor in the cure 
 of the disease is the removal of the bacilli by a process of phago- 
 cytosis, and consequent cessation of the absorption of toxin.) In 
 prolonged diseases it is possible that the production, and especially, 
 perhaps, the local production of antitoxin, may play some part in the 
 process. In other cases, perhaps, the toxin is dealt with by some of 
 the simpler chemical processes considered already and submitted to 
 various changes of oxidation, etc., and thus rendered inert ; but of 
 this we have but little knowledge, the main studies of the methods 
 in which the body deals with toxins having been devoted to the 
 phenomena occurring in highly immunized animals and brought 
 about by their sera. 
 
 Some experiments designed by Wassermann as evidence in 
 support of the side-chain theory may be alluded to in this con- 
 nection. He found that the injection of tetanus toxoids into 
 highly susceptible animals (guinea-pigs) has the power of render- 
 ing them partially immune to tetanus toxin for a short period. 
 Thus, when the toxoid was injected, followed in an hour's time 
 by unaltered toxin, the lethal dose of the latter was greater than 
 in a normal animal. On the other hand, after a day or two the 
 animal became more susceptible, being killed by less than the 
 minimal lethal dose for normal guinea-pigs. This he explained 
 
124 RECOVERY FROM INTOXICATIONS 
 
 by supposing that the receptors of the sensitive cells were occupied 
 by the inert toxoid, so that part of the cells would be unable to 
 combine with the true toxin. The subsequent susceptibility he 
 explains by the over-production of new receptors. Now when 
 we consider that in acute disease of natural occurrence the pro- 
 duction of toxin (in cases that recover) is only temporary, and is 
 stopped sooner or later by the destruction of the bacteria, it seems 
 probable that some such process may occur and serve to shorten 
 the period during which the cells are exposed to the action of the 
 toxin. The true toxins are so fragile that it is highly probable 
 that a certain proportion of them are converted into toxoids during 
 or after the process of absorption into the blood-stream, and by 
 occupying their receptors diminish the susceptibility of these cells 
 to the true toxin, which, circulating in the blood, may be dealt 
 with by the leucocytes, liver, or other organs. Afterwards anti- 
 toxin would appear in the blood, but it would have no relation to 
 the early slight immunity of the tissues. 
 
 We may therefore recognize the following stages in the process 
 of recovery from a bacterial intoxication : 
 
 1. In the first the processes are mainly non-specific, the chief 
 being probably leucocytic action ; the dilution of the toxin in the 
 general blood-stream, and its elimination, either in a natural con- 
 dition or after it has undergone some chemical alteration ; the 
 partial destruction and conversion of toxin into toxoids, and sub- 
 sequent " blocking " of side-chains in highly important tissues. 
 In mild cases these may be sufficient for the restoration of health, 
 and no immunity, or the merest trace, may follow. 
 
 2. Where the process lasts longer antitoxin begins to be pro- 
 duced, and in all probability the time which must elapse before 
 this occurs varies greatly in different conditions, depending on : 
 (a) the constitution of the patient, (b) the dose of the toxin, and 
 (c) on the nature of the toxin. Thus, with regard to the last 
 point, it may be remarked that it appears to be extremely 
 difficult to prepare antitoxins to the endotoxins, and it is highly 
 probable that in recovery from such diseases as cholera and 
 typhoid fever the production of antitoxin is slight or absent. 
 Such diseases owe their symptoms mainly to the production of 
 endotoxin, and are combated mainly by the removal of the 
 bacteria which produce them. In the cases in which antitoxin is 
 produced, recovery at this stage depends on its presence, together 
 with the functional efficiency and sufficient numbers of the leuco- 
 
IMMUNITY TO TOXINS 125 
 
 cytes, and the continuance of the factors by which the toxin is 
 combated in the first stage of the infection. 
 
 Passive antitoxic immunity consists in the artificial production 
 of this stage. 
 
 3. In the third stage, which is only reached in hyperimmunized 
 animals submitted to treatment for long periods, the conditions 
 are quite different, and do not appear to depend in any way on the 
 action of antitoxin, but approach more nearly to the state of 
 natural immunity to be treated subsequently. In this condition the 
 antitoxin in the blood falls greatly, and would probably disappear 
 entirely if the treatment were carried further, yet the degree of 
 immunity is very great. There are two or three theories which 
 have been invoked to explain this form. 
 
 In the first place, if we accept the side-chain theory, we may 
 ascribe it to the absence of receptors suitable for the toxin which 
 is being injected, and may suppose that the repeated and pro- 
 longed stimulation of the production of this particular receptor 
 has led firstly to a hypertrophy, and secondly to an atrophy of 
 these organs. This is readily conceivable, and might be compared 
 with the sequence of hypertrophy and failure of the heart so 
 frequently met with in Bright's disease, etc. In this case the 
 toxin would be unable to " anchor " itself to the cells, which would 
 thus escape its action, and the result would be one form of tissue 
 immunity. There is but little experimental evidence bearing 
 directly on this theory either for or against. The state is one 
 rarely seen even under experimental conditions, though of great 
 theoretical interest. It is found, however, that during the immuni- 
 zation of the rabbit with eel serum (a potent haemolytic agent) an 
 antitoxin or antihaemolysin is produced, whereas the corpuscles 
 themselves, if carefully washed from all traces of serum, are as 
 susceptible as before. If, however, the injections are repeated, 
 the antitoxin disappears, and, as Tchistovitch showed, about this 
 time the red corpuscles themselves become immune, not being 
 haemolyzed by eel serum, even although all traces of their own 
 serum is washed out. This we may fairly suppose to be due to a 
 loss of the receptors with which the molecules of eel serum 
 combine, and this may be taken as an experimental demonstration 
 of the occurrence of this form of immunity, which was predicted 
 by Ehrlich on theoretical grounds. Metchnikoff, it is true, 
 objects to this hypothesis, pointing out that if the receptors are so 
 necessary for the nutrition of the molecule it is impossible that 
 
126 TISSUE IMMUNITY TO TOXINS 
 
 these latter should continue to live after the receptors had been 
 lost. But this seems based on a misconception of Ehrlich's 
 theories of cell-nutrition. We must imagine each molecule to be 
 provided with a very large number of varieties of side-chain, each 
 adapted to the seizure of a different form of food-molecule, so 
 that the complete loss of one variety does not imply that the cell 
 will suffer in nutrition. The only result is that more work will 
 be thrown on the receptors that remain. There is nothing im- 
 probable in this. It is in the highest degree likely that so 
 important a function as nutrition should not be dependent on the 
 functional integrity of every part of the molecule. The occurrence 
 of tissue immunity from loss of receptors is at least possible. 
 
 An alternative theory asserts that the cells of the body retain 
 their power of combining with toxin, but lose their susceptibility 
 to the action of the toxophore group of the latter, at the same time 
 losing their power of producing antitoxin. We may explain the 
 latter phenomenon by saying that the receptors in question 
 become sessile and incapable of being shed. There is experi- 
 mental evidence that some receptors are naturally of this nature, 
 and it is quite possible that ordinary deciduous receptors might 
 change in this way. As we shall see, tissue immunity in which 
 the toxin becomes anchored to the cell, but without injuring it or 
 stimulating it to produce antitoxin, is met with in some forms of 
 natural immunity, and it is quite possible that it may occur in the 
 process of hyperimmunization by vaccines, though direct proof is 
 lacking. Another process of somewhat similar nature may occur, 
 which has also its analogue in natural immunity. Thus when 
 animals are immunized to tetanus they still retain their suscepti- 
 bility to that toxin if the injection be made directly into the 
 brain. It is obvious, therefore, that the cells of the central 
 nervous system are still susceptible. Why, then, do the animals 
 not develop tetanus if the injections be made subcutaneously ? 
 We exclude, of course, the case in which antitoxin is present in 
 the blood and the leucocytes are functionally active. It will be 
 shown subsequently that some forms of natural immunity depend 
 upon the fact that some cells in the body unite with the toxin, and 
 have, indeed, a great affinity for it, but are not injured thereby, 
 whereas others have an affinity for it, but are killed by its action. 
 Obviously, if the toxin be injected into tissues of the former class 
 it will be absorbed, none will reach the susceptible second class of 
 cells, and no toxic symptoms will result. Now let us suppose 
 
IMMUNITY TO TOXINS 127 
 
 that in immunizing a susceptible animal (rabbit or guinea-pig) 
 to tetanus the connective tissues acquire receptors suitable for 
 the toxin in question. They will absorb this toxin, be shed, and 
 the animal will develop antitoxin, whilst the tissues previously 
 susceptible the brain and cord will remain as susceptible as 
 before. This is probably what occurs, though the critical proof 
 the demonstration of an increase in the toxin-absorbing power of 
 the tissues in acquired immunity, and thus of an increased number 
 of receptors does not appear to be forthcoming. Dmitrevsky 
 investigated the amount of tetanus toxin neutralized by brain 
 tissue before and after immunization ; but since, as we have seen, 
 this process is probably different in nature from the neutralization 
 of toxin by antitoxin, his researches have not much weight in this 
 connection. 
 
 The third explanation is that of Metchnikoff, that the leucocytes 
 themselves have become immune to the toxin, and have thus 
 acquired the power of dealing with that substance in large 
 quantities, so that the tissues are sheltered from its action. This 
 theory simply shifts the immunity back to the leucocytes, and 
 thus does not really solve the problem, which, it must be 
 admitted, is unsolved whatever theory we may adopt, and perhaps 
 insoluble in the present state of our knowledge of the physical 
 chemistry of living matter. We may, however, combine it with 
 perfect propriety with the side-chain theory, and argue that the 
 leucocytes may have behaved in the same way as we have 
 supposed the tissue cells to have done in the last paragraph that 
 is, to have retained, and perhaps increased, their receptors, whilst 
 losing their susceptibility to the toxophore group. Here, again, 
 we are confronted with the absence of any definite evidence either 
 way. There seems to be no experimental proof to show whether 
 leucocytes from a hypervaccinated animal have any increased 
 resistance to the poisonous action of toxins or any increased 
 power of combining with them. This is greatly to be deplored, 
 and might easily be remedied now that Sir Almroth Wright has 
 taught us a simple and convenient method of working with living 
 leucocytes. As far as our experimental knowledge goes, there is no 
 great difference in leucocytes from immunized and normal animals, 
 and the researches on opsonins have led to a tendency to disregard 
 the possible variations in the immunity of the leucocytes, since 
 the researches of Bulloch and others showed that leucocytes from 
 very varied sources would take up the same number of bacteria 
 
128 IMMUNIZATION OF LEUCOCYTES 
 
 under identical conditions. Subsequently, as we have seen above, 
 some slight indications of a difference have been found. But on 
 Metchnikoff's theories there ought to be a very marked difference, 
 and we should expect that, the leucocyte being par excellence the 
 cell devoted to the protection of the animal body against infections, 
 it would be the first to acquire increased resistance in acquired 
 immunity, and we should expect, e.g., leucocytes from a convales- 
 cent case of pneumonia or from an animal vaccinated against the 
 pneumococcus to take up far more pneumococci than leucocytes 
 from a normal person under similar conditions, whereas the 
 difference, if one exists, is extremely slight. A careful considera- 
 tion of the conditions of opsonic experiments leads to the con- 
 clusion that the results obtained are not to be regarded in any 
 sense as an index of the immunity of the leucocytes. Ledingham 
 has shown that the number of bacteria taken up by the leucocytes 
 depends on the extent to which the former have been altered by 
 the action of serum, and not on the temperature at which the 
 phagocytosis takes place. Thus, if the bacteria are sensitized by 
 serum, mixed with leucocytes, and part kept at 18 C. and part at 
 
 -*- c*^^. ^^esoX<_tt. 
 
 37 C., the number of leucocytes is the same in the two cases. 
 Now at the lower temperature no active movements of the 
 leucocytes take place, so that we cannot regard the ingestion of 
 the bacteria as being entirely, as was formerly assumed, an active 
 process brought about by a seizure or surrounding of the 
 organism by pseudopodia, as can be seen so readily in the 
 amoeba. Such a process does occur to some extent, and can be 
 seen to occur under suitable conditions ; but it is also true that 
 bacteria can be seen to make their way into a cell which has 
 been watched continuously under a high power of the microscope, 
 and in which no movement of any sort has been witnessed. We 
 are led, therefore, to believe that the phagocytosis which occurs 
 in opsonin experiments in vitro is a process allied to agglutination 
 rather than to an actual physical seizing of an organism. It is 
 one which can take place without an actual conscious if we may 
 use the term movement of the leucocyte towards its prey. 
 An immunized leucocyte would be immune to bacteria which it 
 had ingested, to the endotoxins liberated in the process of 
 bacteriolysis, whether taking place within the leucocyte or outside 
 it, and to endotoxins ; and it would exert its physiological functions 
 of movement (pseudopodia- production and chemotaxis) equally 
 well whether these toxins were present or absent. Or the effect 
 
IMMUNITY TO TOXINS I2Q 
 
 of a given toxin might be reversed in the immunized leucocytes, 
 so that in place of being repelled, as under normal conditions, it 
 might be attracted. As far as phagocytosis depends on the 
 chemotactic attraction of leucocytes and the active suggestion of 
 organisms, we might expect it to be greatly increased if these 
 cells became immune. But there is no reason to see how a 
 similar increase need occur if it depends on a process akin to 
 agglutination, and resulting, perhaps, from a change in the surface 
 tension between the leucocyte and the organism. And that this is 
 the way in which the vast majority of the bacteria are taken up 
 in ordinary opsonin preparations appears probable. Active move- 
 ments of leucocytes are rarely seen in emulsions prepared for the 
 opsonic technique and examined in normal saline solution on the 
 warm stage. In other words, the opsonic index, though useful as 
 an index of certain activities of the serum, would appear not to 
 throw much, if any, light on the immunity of the leucocytes. 
 That must be sought by other means, notably by experiments in 
 vivo, and these lead us to the belief that the leucocytes may 
 become immune and may play a part of importance in acquired 
 immunity. 
 
 But the facts which have been previously recorded concerning 
 the local reaction in animals which are being immunized to tetanus 
 toxin tend to support MetchnikofFs hypothesis, though the evi- 
 dence is somewhat indirect. We have already pointed out that 
 when normal horses are injected with small doses of this toxin 
 there is at first no local reaction, or but slight, and that as the 
 animal becomes more immune this local reaction becomes more 
 manifest a phenomenon which we have attributed to the 
 hypersensitiveness of the tissues. Now the local reaction is 
 inflammatory in nature, and the tumefaction which occurs is due 
 partly to oedema and partly to an access of leucocytes, so that it 
 would appear that in the production of immunity these cells 
 acquired the power of invading a tissue which is permeated with 
 toxin.. If this is the case, we may assume that they have become 
 immunized to this toxin, and that they have changed in regard to 
 their chemotactic properties. This collection of leucocytes in 
 large numbers at the region of inoculation of toxins, where they 
 may form a sterile abscess of considerableextent, is a frequent 
 phenomenon in the production of diphtheria toxin ; and here, again, 
 it seems not improbable that the leucocytes have become, like the 
 tissue cells, more immune to the toxin. There are no inherent 
 
 9 
 
130 BIOLOGICAL SIGNIFICANCE OF ANTITOXIN 
 
 difficulties in the supposition, and it certainly favours the 
 explanation of the phenomenon. 
 
 It will be remarked that the tendency of modern thought has 
 been rather to minimize the importance of antitoxin in the process 
 of natural recovery and in the subsequent immunity, and to regard 
 its appearance rather as an epiphenomenon, though doubtless one 
 that may under certain circumstances be of advantage to the 
 patient. Let us consider briefly the probable significance of anti- 
 toxin-formation in the animal economy. Is it a purposive pheno- 
 menon, developed and selected by natural selection with a view to 
 defend the species against natural dangers ? This was the natural 
 assumption when antitoxin was thought to play a role of the 
 utmost importance in natural recovery and acquired immunity. 
 There are great difficulties in the way of accepting such a hypo- 
 thesis. In the first place, immunity to bacterial infections is more 
 prevalent in the lower rather than in the higher animal types. 
 Doubtless very great susceptibility to a widespread infective 
 agent would operate unfavourably to the prospect of survival of 
 any animal species, but actual experience seems to show that 
 immunity to ordinary infections is a far less potent factor in 
 evolution than those studied by Darwin and his school. It would 
 appear in the case of tetanus at least that susceptibility is acquired 
 as the animal rises in the zoological scale as a secondary, though 
 perhaps necessary, result of the possession of a nervous system of 
 a certain degree of complexity. Secondly, the immunity due to a 
 successful struggle against a disease is an acquired factor, and as 
 such not transmitted, according, at least, to the majority of modern 
 authorities. It might be objected that the capability of producing 
 antitoxin might be a spontaneous feature, and one capable of being 
 transmitted by heredity. This is probably true, but in this case it 
 could only become a powerful agent in evolution if the disease 
 were prevalent and an attack usual in the life-history of many of 
 the individuals. This might occur in the case of tetanus, though 
 here, as we have seen, the tendency in evolution is for the production 
 of susceptibility rather than immunity ; but it is quite impossible 
 to see how the power of forming an antitoxin to eel serum after 
 the injection of that substance (to take one example of many 
 which might be quoted) can be of advantage to any animal unless 
 its habitat happens to be a pathological laboratory. Lastly, if the 
 acquirement of immunity be of great importance in natural 
 selection, we should expect those species to survive which de- 
 
IMMUNITY TO TOXINS 131 
 
 veloped natural, rather than the power to develop acquired, 
 immunity, since the former would be always available, whilst the 
 latter only come into action after the animal has successfully sur- 
 mounted the hazard of an attack of the disease. 
 
 It appears more likely that the power to produce antibodies 
 under certain circumstances is to be regarded as one of the 
 essential properties of some forms of living protoplasm, and that 
 its occasional value in the cure or prevention of disease is a mere 
 accident. Thus the injection of eel serum leads to the production 
 of antitoxin in all mammals, as far as is known, and this could 
 only be of advantage to the animal if (i) eel serum happened to 
 gain access to the tissues ; (2) the animal recovered ; and (3) eel 
 serum again reached the tissues within a certain period. This is 
 extremely unlikely to occur. Nor is natural immunity from 
 poisons necessarily dependent on antitoxin ; in fact, the common 
 vegetable and other poisons do not lead to a production of anti- 
 bodies, though if they did the possession of these substances 
 would doubtless be of great value to animals in a wild state. If 
 there is any advantage attaching to the power of forming anti- 
 bodies, it is probably in regard to the solution of organized bodies, 
 such as bacteria, or their sensitization previously to phagocytosis. 
 It is quite conceivable that the advantage accruing from the 
 possession of the power of forming antibodies has led to the 
 selection of animals whose protoplasm has the property of develop- 
 ing an antibody to any foreign proteid, the useful side of this 
 property being the formation of bacteriolysins and opsonins, 
 the useless corollary being the production of antitoxins and 
 precipitins. 
 
 The theory of passive antitoxic immunity does not present any 
 difficulties of importance. It may be pointed out that it comes on 
 as soon as the antitoxin reaches the blood-stream i.e., at once if 
 the injection be intravenous, and after a delay of some duration if 
 it be into the subcutaneous tissues or peritoneum. 
 
 Much attention has been paid to the question of the feasibility 
 of administering antitoxin by the mouth and rectum. In general 
 there is no doubt that this is useless, and under ordinary conditions 
 it is not absorbed as such, being probably digested, and thus 
 deprived of its peculiar characters. Under certain circumstances 
 this appears not to be the case ; thus it is held that absorption 
 from the stomach may take place in young animals. Hence, 
 perhaps, some of the undoubted benefit of the use of fresh raw 
 
 92 
 
132 PASSIVE IMMUNITY 
 
 milk (in which the antibodies have not been destroyed by heat) in 
 the feeding of infants. It seems also that absorption of antitoxins 
 can take place in the intestine, and that the process may occur 
 when gastric digestion is impaired or is prevented by artificial 
 means ; thus McClintock and King obtained successful results (in 
 animals) in 93 per cent, of cases after the administration of a 
 mixture of diphtheria antitoxin, opium, and of a saturated solution 
 of salol in chloroform. It is possible that something of the same 
 sort may occur in disease where the digestive powers are notably 
 enfeebled, but this does not justify its exhibition in this way in 
 cases of disease where every hour is of the utmost value. Recent 
 researches have also shown that in some cases animals may be 
 immunized per rectum ; possibly success is only attained when the 
 antitoxin goes sufficiently high up. 
 
 As regards the duration of the immunity conferred by a single 
 dose of antitoxin, our knowledge is not very exact in the case of 
 human beings. According to von Behring, Goodman, and others, an 
 animal injected with diphtheria antitoxin retains its immunity for 
 rather more than three weeks, and the duration is not markedly 
 affected by the size of the dose given for example, 20 units of 
 antitoxin per kilogramme immunized a rabbit twenty-three days, 
 500 units twenty-six days. According to Goodman, the degree of 
 immunity falls rapidly for the first two or three days, and then more 
 slowly. According to him also, the immunity is greater than the 
 amount of antitoxin in the blood would lead one to suppose, and it 
 continues when antitoxin is no longer demonstrable in that situation. 
 The duration of the immunity appears to depend upon the source of 
 the antitoxin injected, being longer if the serum is homologous 
 i.e., derived from an animal of the same species : thus von Behring 
 showed that horses immunized by horse-serum antitoxin retain 
 their passive immunity almost as long as if they had acquired 
 active immunity from toxin injections, whereas the effect in 
 rabbits, etc., as we have seen, is more transient. 
 
 But little need be added to what has been already mentioned 
 incidentally with regard to the curative power of antitoxin. It 
 acts, in the main, by neutralizing all toxin present in a free state 
 in the blood or subsequently formed by the bacteria. In general, 
 as has been pointed out, it does not repair the damage that the 
 toxin has already done, nor remove the latter from its combination 
 with the susceptible cells. It is perhaps hardly safe to assume that 
 there is no trace of such an action, and it is by no means improb- 
 
IMMUNITY TO TOXINS 133 
 
 able that a very large excess of antitoxin present in the blood may 
 have some slight power of attracting toxin from the tissues when 
 the union is but recent. This, however, is quite unproved, but it 
 is certain that cells which have been acted on by the toxin are not 
 benefited by the subsequent action of the antidote. 
 
 It is hardly necessary to point out that antitoxin is devoid of 
 bactericidal action, and that the removal of the infective agent is 
 brought about by other mechanisms in most cases, perhaps, by 
 phagocytosis. Thus we shall see, in the case of diphtheria, that 
 there is a rapid rise in the opsonic index aj^out the time of the 
 improvement in the local lesions ; and we can hardly doubt, though 
 direct evidence is lacking, that the preservation of the leucocytes 
 from the deleterious action of the toxin when the latter is neutral- 
 ized by means of antitoxin also plays its part in facilitating 
 phagocytosis. 
 
 We now turn to the subject of natural immunity, an exceedingly 
 difficult one, and one that is very far from being understood. What 
 follows is for the most part extremely hypothetical. 
 
 It must be borne in mind that, though we speak of animals as 
 susceptible or as immune to a given toxin, no hard-and-fast line 
 can be drawn between the two conditions, and in some cases an un- 
 broken series can be constructed between the most susceptible and 
 the most resistant species. This is well seen in the case of tetanus. 
 According to Knorr, the horse is the most susceptible animal 
 to the toxin of this disease ; the guinea-pig requires twice as much 
 toxin per kilogramme of body- weight to constitute a lethal dose, 
 the goat 4 times as much, the mouse 13 times, the rabbit 2,000 
 times, and the hen 200,000 times. Von Behring confirms these 
 figures in general, but finds the mouse rather more susceptible 
 than the horse. With the exception of the fowl these animals are 
 all " susceptible." Of the " insusceptible " animals the hen is sus- 
 ceptible to large doses, but is also affected by much smaller ones 
 if the injection be intra-cerebral. Other animals are still more 
 resistant, such as the tortoise, lizard, cayman, larva of Oryctes, 
 etc. The effect of other toxins on different species of animals 
 has not yet been fully investigated. 
 
 It is necessary to realize that, before we can say that an animal 
 is insusceptible or immune to the action of a given toxin, it is 
 necessary for the animal to be kept, after injection, at a tempera- 
 ture at which this toxin can act. This subject has already been 
 mentioned in connection with the functions of the haptophore and 
 
134 NATURAL IMMUNITY TO TOXINS 
 
 toxophore groups. We have already seen reason to believe that 
 the former can functionate (i.e., the molecule of toxin can unite 
 with the cell) at a low temperature, whereas the enzyme-like 
 action of the toxophore radical can only act at the temperature of 
 the human body, or one approximating thereto this is in the case 
 of tetanus. It is apparent, therefore, at the outset that there are 
 two possible explanations for immunity to toxins : firstly, the 
 toxin may find no cell with which it can unite or, to use Ehrlich's 
 terminology, no cell receptor having a combining affinity for the 
 haptophore molecule of the toxin molecule ; and, secondly, the 
 union takes place, but the cell is not injured as a consequence 
 that is, the toxophore group is without action on the cell proto- 
 plasm. 
 
 1. Immunity due to an absence of suitable receptors. 
 
 This has been proved to exist in several cases. Most of these 
 are due to the researches of Metchnikoff, who, however, does not 
 express the fact in these words, but contents himself by saying 
 that the toxin does not combine with the tissues. Thus, when the 
 larva of Oryctes is injected with tetanus toxin, no symptoms 
 develop, and if the blood of the animal be collected several months 
 afterwards, it will be found to contain free toxin, as shown by the 
 fact that it will tetanize susceptible animals. These experiments 
 were carried out at a temperature of 30 to 36 C. Similar facts 
 were observed in lizards ; -^ c.c. of blood taken two months after 
 the injection of tetanus toxin produced fatal tetanus in a mouse ; 
 the blood of a turtle (Emys orUcularis) contained toxin no less 
 than four months after injection. In these cases the explanation of 
 the immunity is clear, however we choose to express it : the toxin 
 has no chemical affinity for the living protoplasm of any part of 
 the animal, and is, therefore, powerless to injure it. 
 
 2. Immunity due to the insusceptibility of the cells to the 
 action of the toxophore group. 
 
 Perhaps the best example of this is in the case, so often referred 
 to, of the action of tetanus toxin on the cold frog, but other 
 striking examples have been discovered by Metchnikoff. Thus, 
 when the alligator is kept at ordinary temperatures (about 20 C.) 
 and injected with tetanus toxin, that substance rapidly disappears 
 from the blood, yet without the production of any tetanic symptoms, 
 and without the appearance of antitoxin in the blood. If, how- 
 ever, the animal is kept at a temperature of 32 to 37 C., it pro- 
 duces antitoxin with great rapidity, though still without the 
 
IMMUNITY To TOXINS 135 
 
 production of tetanus. Taking these two experiments together, 
 we may regard them as constituting a definite proof of the existence 
 of this form of immunity. The theory that the toxin disappears 
 because it is anchored to the cells appears to be clearly demon- 
 strated from the fact that antitoxin is produced, an occurrence 
 which we could scarcely explain on any other hypothesis. 
 
 But it is hardly necessary to look for experimental proof of the 
 occurrence of this form of immunity, since the fact that the blood 
 of many species of animals is toxic for other species appears to 
 constitute a ready-made demonstration of the fact. The most 
 striking example is given by eel serum, a substance which is toxic 
 for nearly all animals, except, of course, the eel itself. The serum 
 of the horse is perhaps the least toxic of all sera, but even this 
 has some poisonous properties. Whatever theory we may hold 
 with regard to the mechanism by which the cells of an animal 
 are nourished, it is hardly possible to avoid the conclusion that the 
 proteid substances of the plasma must combine with the living 
 protoplasm. And it is the proteids which make eel serum toxic 
 to other animals. It appears, therefore, that the immunity of the 
 eel to its own serum depends upon the fact that its protoplasm is 
 not injured by the toxophore group, although the molecules of the 
 toxin and protoplasm become united. (The toxicity of eel serum 
 is dependent on a more complex structure than that of ordinary 
 bacterial toxins, being, in fact, a cytolysin or cytotoxin, but this 
 does not affect the argument.) 
 
 A consideration of the use of these poisonous sera to the 
 animals which produce them may perhaps enable us to analyze 
 the process a step further, and to attribute their immunity (e.g., 
 of eels to eel serum) to the power which their cells possess of 
 using these " toxic " substances as sources of nourishment ; and 
 if this be so, we might perhaps apply this suggestion to explain 
 the form of immunity to toxins under discussion. Thus, in the 
 case of the alligator injected with tetanus toxin and kept in the 
 cold, it is possible that the poison which combines with the cells 
 is " digested " by them and used as nourishment. If this is so, we 
 must assume that the molecule of toxin has, as far as its hapto- 
 phore group is concerned, a close chemical affinity with the 
 proteids normally present in the animal's blood and used by it in 
 cell nutrition ; and, further, that the toxophore group is not only 
 innocuous to the cell, but is also no bar to the assimilation of the 
 whole molecule by it. We have seen that in the case of the frog 
 
136 NATURAL IMMUNITY TO TOXINS 
 
 the toxophore group only acts at a raised temperature. In the 
 alligator we must assume that the elevation of the temperature 
 has a similar but less marked effect ; it brings the toxophore group 
 into a state of activity in which it is inassimilable by the molecule 
 of cell protoplasm to which it is anchored, although the latter is 
 not injured in any way. The result is, of course, the production 
 of antitoxin, which approximates to the formation of the pre- 
 cipitins which occurs when non-poisonous foreign proteids are 
 injected. 
 
 Thus it would appear that at the last analysis this form of 
 immunity is in reality an expression of cell nutrition. A cell 
 which can nourish itself on a given toxin is naturally immune to 
 its action. It is hardly necessary, however, to say that there is 
 no direct proof that any cell can extract nourishment from a 
 bacterial toxin. 
 
 There is a third, and in some respects more interesting and 
 important, form of immunity to toxins which is readily conceivable 
 on theoretical grounds, and the occurrence of which is capable of 
 experimental proof. We may define it as immunity due to the 
 fact that some of the cells are insusceptible to the action of the 
 toxin, but have a great combining affinity therewith, and thus 
 shield from its action the susceptible cells, in which the combining 
 affinity is less. We have already referred to the most striking 
 and best known example, that of the immunity of the fowl to 
 tetanus toxin injected subcutaneously as compared with its sus- 
 ceptibility when the injection is made direct into the brain. Some 
 authorities have attempted to explain this on the assumption that 
 there^is a barrier en route which prevents the access of the toxin 
 to the central nervous system. But if by this we are to imagine a 
 physical barrier in the shape of a layer of endothelium or other 
 tissue between the blood and the cells of the brain, the explanation 
 is inadequate, since it would leave unexplained the nature of the 
 immunity of this living barrier, nor would it explain the rapid 
 disappearance of the toxin from the blood. The true explanation 
 is certainly that the toxin combines rapidly with the cells of the 
 body, or perhaps, as we shall see later, the leucocytes, and is 
 thereby prevented from gaining access to the central nervous 
 system. These relatively unimportant cells we must imagine to 
 have the same nutritive relations to the molecules of toxin as have 
 the cells of the heated alligator ; the two can unite, but the proto- 
 plasm is neither injured nor nourished. It seems probable that this 
 
IMMUNITY TO TOXINS 137 
 
 affords a sufficient explanation of the differences in susceptibility 
 of the " susceptible " animals which figure in Knorr's list, and that 
 the cells of the central nervous system in the warm-blooded verte- 
 brates differ but little in their relation to tetanus toxin. Thus, in 
 the case of the horse and the guinea-pig little toxin is bound to the 
 body cells, and practically the whole amount makes its way to the 
 brain wherever the injection is made. In the rabbit the body cells 
 can absorb more, so that if only a small dose is given none may 
 reach the more important and susceptible organs. The fowl marks 
 the other end of the scale ; the tissue cells must have an enormous 
 avidity for toxin, and, unless absolutely gigantic doses are given, 
 absorb the whole of it. 
 
 The objection may be raised that, the receptors of the body cells 
 being of the same nature as antitoxin, the resulting combination 
 of body-cell receptor and toxin should undergo dissociation, the 
 poison being gradually passed on to the central nervous system, 
 in the cells of which dissociation does not occur, since poisoning 
 takes place ; this refers to the case, e.g., of the fowl. It is true 
 that such a process appears to occur in vitro. If tetanus toxin be 
 mixed with emulsions of many of the tissues, the two enter into a 
 loose combination, so that if the fragments of toxin-charged tissues, 
 after thorough washing in normal saline solution, are allowed to 
 soak in that fluid, toxin is gradually liberated. It is only in the 
 case of the central nervous system that stable non-dissociable 
 compounds are formed. Several interpretations of the apparent 
 anomaly might be suggested. It is, for instance, very probable 
 that here, as in so many other phenomena in immunity, the 
 real defensive cell is the leucocyte. Thus, Metchnikoff found that 
 if he injected tetanus toxin into the fowl, and then, after an appro- 
 priate interval, excited an aseptic exudate composed largely of 
 leucocytes, the fluid thus obtained would excite tetanus in a sus- 
 ceptible animal. Perhaps the chain of phenomena is as follows : 
 The toxin first unites with the connective and other tissue cells, 
 and so the amount that the leucocytes have to deal with at first is 
 greatly diminished ; dissociation takes place, and there is a steady 
 stream of toxin from the tissues to the blood. This is dealt with 
 by the leucocytes, which thus have time to increase in numbers 
 (and even in the insusceptible fowl the result of the injection is to 
 cause a marked leucocytosis), and perhaps to become immune. It 
 is useless to deny that these phenomena are difficult to harmonize 
 with the side-chain theory as first expounded, or that Metchnikoff 
 
138 NATURAL IMMUNITY TO TOXINS 
 
 has brought forward very strong (though not conclusive) evidence 
 in support of his theory of the leucocytic origin of antitoxin ; and, 
 as we shall see, as far as our knowledge goes, we are led to believe 
 that all antibodies are derived from the spleen, bone-marrow, and 
 other organs rich in leucocytes. 
 
 The study of the affinity of bacterial toxins and of other poisons 
 to various tissues and organs of the body is destined to play a part 
 of great importance in our ideas of pathology and pharmacology. 
 It has been especially investigated by Ehrlich, and in connection 
 with immunity and susceptibility to toxins he recognizes four 
 conditions which may occur : 
 
 1. In which no specific receptors occur in any part of the 
 animal. This is the first form of natural immunity which we 
 have discussed ; the animal is absolutely immune, and cannot 
 produce antitoxin. 
 
 2. Receptors are present, but only in tissues on which the 
 poison does not act, or in tissues of little importance. Here the 
 animal is immune, but antibodies may be produced. This is the 
 case of the fowl vis-a-vis the tetanus toxin. 
 
 3. The receptors are distributed over various parts of the 
 organism, and are present in the organs which are sensitive to the 
 action of the poison. Here there is a relative immunity, and the 
 degree of intoxication depends largely on the region into which 
 the toxin is introduced. These animals may be immunized, 
 though the process is one of some difficulty, owing to the sensitive- 
 ness to the toxin of vital organs, and, of course, antibodies may 
 be produced. 
 
 4. The receptors are present only in vital organs which are 
 sensitive to the poison. Here the animal is very susceptible, and 
 immunization very difficult, involving the use of extremely minute 
 doses or of toxoids. The action of tetanus toxin on the guinea- 
 pig and horse provides examples. 
 
CHAPTER VII 
 BACTERIOLYSIS AND ALLIED PHENOMENA 
 
 UP to the present we have dealt entirely with the mechanism by 
 which the injurious effects of the infective bacteria are nullified. 
 It is obvious that this is only one, though perhaps the most im- 
 portant, aspect of the question. We have now to study how the 
 bacteria themselves are removed. 
 
 The bactericidal properties of the blood attracted attention very 
 early in the history of bacteriology, and long before the beginnings 
 of the science Hunter showed that blood had the power of resisting 
 decomposition longer than other animal fluids. It was, however, 
 the controversy which took place between Metchnikoff and the 
 humoralist school which first focussed attention on this question, 
 and led to the discovery of the alexins by Buchner, Nuttall, and 
 others. The controversy is best discussed subsequently, and it is 
 sufficient now to point out that it was found that the circulating 
 blood had the power of killing certain bacteria, and that this 
 property was even more marked in the serum. Thus, according 
 to Lubarsch, 16,000 virulent bacilli will kill a rabbit if injected 
 intravenously i.e., the blood has not the power of killing this 
 number ; yet i c.c. of fresh serum will destroy this number or 
 more. It is obvious that the bactericidal substance or substances 
 occur in the blood, and to a greater extent in the serum. 
 
 The properties of these bactericidal substances or alexins were 
 investigated, and it was found that they were fragile bodies, readily 
 destroyed by a moderate temperature (55 C.), and that they 
 disappeared spontaneously if the serum were kept for a few days. 
 They were destroyed by acids and alkalis, and what was most 
 important of all was that they were selective in their action i.e., 
 those from a certain animal might be potent antiseptics as regards 
 certain bacteria and inert towards others, whereas the serum of 
 another species might have quite different actions. They appeared 
 to be formed by the breaking down of leucocytes ; hence their 
 
 139 
 
140 
 
 appearance in the blood after clotting (when fibrin ferment is also 
 liberated from these cells), and their absence from fluids containing 
 none, such as the aqueous humour. 
 
 It was obvious that this was a foundation for a theory of 
 immunity, but it soon became apparent that it did not explain the 
 whole of the phenomena. An animal might be immune to a 
 certain organism, yet its serum might not be bactericidal for it, or 
 it might be susceptible and yet possess suitable alexins. Thus, 
 rabbits are very susceptible to anthrax, yet have serum which 
 kills the bacilli in large numbers, whilst the dog is much less 
 susceptible, though its serum is very slightly bactericidal. Such 
 facts prevented the alexic theory of immunity from making head- 
 way until the discovery of Pfeiffer's phenomenon. 
 
 Pfeiffer found that when cholera vibrios were injected into the 
 peritoneal cavity of a highly immunized guinea-pig they were not 
 only killed, but dissolved, and this with great rapidity. The 
 vibrios are first rendered immotile, and then lose their proper 
 shape, becoming converted into spherical masses which stain 
 badly. In a little while they become smaller, and disappear alto- 
 gether, no trace being left. 
 
 Pfeiffer showed that the phenomenon could also be produced 
 by injecting into the peritoneum of a normal guinea-pig a mixture 
 of serum from an immunized animal and the culture of vibrios. 
 This happened when an old specimen of serum in which the 
 alexins had disappeared spontaneously was used, or a fresh 
 specimen that had been heated to 60 C. Lastly, he found that 
 if an old immune serum were injected into the peritoneal cavity 
 and allowed to remain for a while, it regained its bactericidal 
 powers, and could then dissolve the vibrios in vitro at the body 
 temperature. The phenomenon was specific i.e., if serum from 
 an animal immunized against cholera were used as described 
 above, the vibrios of cholera were dissolved, but no others. If 
 typhoid serum were used, typhoid bacilli showed degenerative 
 changes, though they were not completely destroyed, but there 
 was no effect on cholera vibrios. 
 
 The explanation which suggested itself to Pfeiffer was this : 
 There is in the serum of highly immunized animals a substance 
 which can exist either in an active or inert state. In the blood- 
 serum or peritoneal fluid whilst in the body it occurs as an active 
 substance, but it passes into the inert condition when kept for a 
 few days, or very rapidly when heated to 60 C., and this inactive 
 
BACTERIOLYSIS AND ALLIED PHENOMENA 141 
 
 substance can be rendered active again by contact with the living 
 endothelial cells of the peritoneum. 
 
 The true explanation was given by Bordet, who showed that 
 two substances are necessary for the phenomenon. The one is the 
 inert thermostable substance which occurs in the serum of highly 
 immunized animals, but not in normal animals, and which does 
 not disappear on keeping. The second is a thermolabile sub- 
 stance which occurs in fresh serum, whether from a normal or 
 from an immunized animal. This he showed by demonstrating 
 that, whereas neither heated nor stale immune serum alone, nor 
 fresh serum alone, had the power of leading to the production of 
 Pfeiffer's reaction in vitro, this was caused when the two were 
 used in combination. When a drop of fresh serum from a normal 
 animal, some heated immune serum, and the cholera vibrios are 
 mixed together and incubated, the whole train of phenomena was 
 repeated. 
 
 The thermolabile substance taking part in the process was 
 soon identified as the alexin of the earlier investigators, and the 
 new substance was called by Bordet substance sensibilatrice. His 
 theory was that alexin alone does not unite with the bacteria, 
 unless these have been previously sensitized by the action of the 
 substance in the immune serum. It was shown that the substance 
 sensibilatrice has the power of uniting with the bacteria as follows : 
 A culture exposed to the action of heated immune serum and 
 washed by repeated centrifugalizations with normal saline solution 
 is apparently unaltered, but the bacteria are dissolved by the 
 action of normal serum, which has no action on unsensitized 
 bacteria. Bordet supposed, therefore, that the immune serum 
 alters the constitution of the bacteria in some way, so that the 
 alexin can combine with them subsequently. He compared the 
 process to the opening of a lock, which can only be effected by 
 means of two keys, of which one (the sensibilatrice) must be 
 turned before the other (alexin) can be introduced. 
 
 Bordet found that the essential facts could be reproduced 
 exactly if red blood-corpuscles were substituted for bacteria. It 
 was previously known that the serum of some animals possesses 
 the power of liberating the haemoglobin (haemolysis) from the red 
 corpuscles of other species, just as the serum of some animals 
 can destroy certain bacteria. Bordet showed that the guinea-pig 
 serum has normally no action on the red corpuscles of the rabbit, 
 but that it becomes haemolytic to the latter after a few injections 
 
142 HAEMOLYSIS BORDET'S DISCOVERIES 
 
 of rabbit's blood. Just as an animal which was formerly destitute 
 of that power becomes able to dissolve the cholera vibrio after 
 a few injections of that organism, so a guinea-pig, the serum of 
 which had formerly no action on the rabbit's corpuscles, acquires 
 the power of dissolving them as the result of an injection or two 
 of rabbit's blood ; hence, following the analogy with the cholera 
 vibrio, the guinea-pig is said to be " immunized " to the rabbit's 
 corpuscles, though there is here no question of the avoidance of 
 any deleterious action. Further and hence the importance of 
 these discoveries in the theory of immunity Bordet showed that 
 the laws which govern bacteriolysis by means of immune sera 
 were apparently identical with those governing Pfeiffer's pheno- 
 mena. The fresh immune serum is haemolytic ; it loses its power on 
 heating to 55 C., and it regains it on the addition of fresh normal 
 serum. Further, the action is, to some extent at least, a specific 
 one. An animal (A) injected with corpuscles of another species 
 (B) will dissolve the corpuscles of that species, and may also have 
 some action on those of animals closely allied zoologically. It is 
 evident that bacteriolysis and haemolysis are closely akin, and as 
 researches on the latter phenomena are infinitely more easy than 
 on the former, much work intended to elucidate the mechanism 
 of immunity has been carried out on red corpuscles. This is to 
 some extent regrettable, since the processes, though similar, are 
 not identical, and it is unsafe to argue from one to the other 
 without experimental verification. 
 
 It was this analogy between bacteriolysis and haemolysis that 
 led Ehrlich to an investigation of the latter phenomenon, and 
 his researches led to a flood of new light being thrown upon the 
 subject. Ehrlich introduced fresh names for the substances which 
 Bordet had shown to be necessary for the phenomenon, and it 
 will now be convenient to give a list of the various terms which 
 the theories or caprices of various writers have applied to 
 each. 
 
 The thermostable substance has been called : 
 
 Substance sensibilatrice, or simply sensibilatrice. 
 Immune body. Philocytase. 
 
 Amboceptor. Immunisin. 
 
 Fixator. Desmon. 
 
 Intermediary body. Copula. 
 
 Interbody. Preparator. 
 
BACTERIOLYSIS AND ALLIED PHENOMENA 143 
 
 Whilst the thermolabile substance is spoken of as : 
 
 Alexin. Complement. 
 
 Addiment. Cytase. 
 
 We shall speak of the substances as amboceptor or immune 
 body and as alexin or complement respectively. Objection may be 
 
 FIG. 24. THE CONSTITUENTS OF FRESH IMMUNE SERUM, ON EHRLICH'S 
 
 THEORY. 
 
 a = complement, b = amboceptor, d being its complementophile, and c its cyto- 
 phile groups. Below, a red blood-corpuscle, showing its receptors. 
 
 This and the following figures are modified from Ehrlich, and are intended to 
 illustrate his theories of haemolysis. The black figures represent normal 
 substances (corpuscles, complement), the white ones antibodies. It need 
 hardly be said that they are absolutely diagrammatic. 
 
 FIG. 25. THE IMMUNE SERUM SHOWN IN FIG. 24, AFTER HEATING. 
 The complement is destroyed, and amboceptor only remains. 
 
 FIG. 26. NORMAL SERUM CONTAINING COMPLEMENT, BUT NO AMBOCEPTOR. 
 
 taken to Ehrlich's terms "amboceptor" and "complement," since, 
 as we shall show, they imply a theory, but they are too firmly 
 rooted to be displaced. 
 
 Ehrlich applied his side-chain theory to the study of the 
 phenomena, and argued that amboceptor must be an antibody 
 to the receptors of the red blood-corpuscle. If this is the case, 
 it ought to unite with the corpuscles, and Ehrlich showed in the 
 
144 
 
 HAEMOLYSIS EHRLICH'S RESEARCHES 
 
 following way that such actually occurred : He worked with the 
 serum of a goat which had been injected with and was haemolytic for 
 the red blood-corpuscles of a sheep. He heated this immune serum 
 to 56 C. to destroy complement, and added some sheep's cor- 
 puscles ; the mixture was then centrifugalized, the supernatant fluid 
 then pipetted off, and replaced by normal saline solution. The red 
 corpuscles were to all appearance unaltered, but it was now found 
 
 FIG. 27. HEATED IMMUNE SERUM ADDED TO RED BLOOD - CORPUSCLES, 
 
 WHICH ARE APPARENTLY UNALTERED, BUT ARE IN REALITY " SENSITIZED." 
 
 FIG. 28. EFFECT OF ADDITION OF FRESH NORMAL SERUM TO SENSITIZED 
 
 CORPUSCLES. 
 
 Complement is now linked up to the corpuscles, and haemolysis takes place 
 when they are incubated at 37 C. 
 
 that if a small amount of normal goat serum were added and the 
 mixture incubated, haemolysis occurred. It was evident, therefore, 
 that the corpuscles underwent some change in virtue of their stay 
 in the heated immune serum, though no alteration was obvious. 
 
 In a further experiment he showed that the change consisted in 
 the abstraction of the amboceptor from the fluid. This appeared 
 from the fact that if the supernatant fluid from the last experi- 
 ment were pipetted off, fresh normal goat's serum added (to supply 
 complement), and the mixture tested with sheep's corpuscles, no 
 
BACTERIOLYSIS AND ALLIED PHENOMENA 145 
 
 haemolysis occurred. Evidently it had been removed by the first 
 addition of corpuscles. 
 
 Red corpuscles, therefore, have a combining affinity for ambo- 
 ceptor, and in further experiments Ehrlich showed that this 
 combination takes place at ordinary temperatures or at low ones, 
 down to o C. Expressed in the language of the side-chain theory, 
 amboceptor has a haptophore group with a combining affinity 
 for the receptors of the corpuscle, bacterium, etc., with which it 
 
 '*'** 
 
 * t ~ 
 
 FIG. 29. NORMAL GOAT SERUM (COMPLEMENT), PLUS SHEEP'S CORPUSCLES. 
 
 No combination. This is shown as follows : The mixture is centrifugalized, 
 and to the corpuscles heated immune serum (amboceptor) was added 
 (Fig. 30), whilst to the supernatant fluid corpuscles and heated serum 
 were added (Fig. 31). 
 
 FIG. 30. THE CORPUSCLES FROM THE PREVIOUS EXPERIMENT INCUBATED 
 
 WITH HEATED IMMUNE SERUM. 
 
 No solution, showing that no complement had been abstracted in combination 
 
 with them. 
 
 unites. We shall see reasons for believing that it may have a 
 second haptophore group, and shall distinguish this first by the 
 name of cytophile haptophore. 
 
 Ehrlich now investigated the behaviour of the second substance 
 the complement with the red corpuscles by a precisely similar 
 method, and found that the two had no power of entering into 
 combination. Thus, normal goat's blood (containing complement) 
 was added to sheep's corpuscles, and the mixture centrifugalized. 
 To the corpuscles heated immune serum (amboceptor) was added, 
 but there was no haemolysis. Again, to the supernatant fluid 
 sheep's corpuscles and heated serum were added, and haemolysis 
 
 10 
 
146 
 
 HAEMOLYSIS EHRLICH'S RESEARCHES 
 
 i 
 
 occurred ; complement had evidently not been withdrawn from the 
 fluid. Complement, therefore, will not unite with red corpuscles 
 direct. It has no haptophore group with an affinity for the 
 receptors of the latter. 
 
 This led Ehrlich to the theory that the complement united with 
 the red corpuscle only indirectly by means of the amboceptor. 
 
 FIG. 31. THE SUPERNATANT FLUID FROM FIG. 29, TESTED WITH HEATED 
 IMMUNE SERUM AND SHEEP'S CORPUSCLES. 
 
 Solution takes place, showing that the complement had not been removed. 
 
 FIG. 32. FRESH IMMUNE SERUM (OR A MIXTURE OF HEATED IMMUNE 
 SERUM AND FRESH NORMAL SERUM) ADDED TO CORPUSCLES AT o C. 
 
 Amboceptor unites therewith, complement does not. 
 
 He pictured the latter as having two haptophore groups : a cyto- 
 phile, which we have already mentioned, and a complementophile, 
 with which the complement could unite after the former had 
 seized on a receptor of the red corpuscle. The process of haemo- 
 lysis was supposed to take place as follows : In fresh immune 
 serum or in a mixture of heated immune serum and fresh normal 
 serum (i.e., of amboceptor and complement) the two substances 
 occur independently of one another. When the mixture is kept 
 in the cold, and red corpuscles are added, the cytophile groups of 
 the amboceptor molecules attach themselves to the receptors of 
 
BACTERIOLYSIS AND ALLIED PHENOMENA 147 
 
 the red corpuscles, but the molecules of the complement still 
 remain free, and do not attach themselves either directly or in- 
 directly to the corpuscles. Nevertheless, there is a change in the 
 combining affinities of the complementophile haptophore. 1 This 
 is shown by the fact that if the mixture be raised to the body- 
 heat, the molecules of complement attach themselves to these 
 haptophores, exert a digestive or haemolytic action through the 
 amboceptor on the red corpuscles, and haemolysis occurs. Hence, 
 on Ehrlich's theory, the complement does not attack the cell or 
 corpuscle direct (as Bordet holds, and as is implied in the term 
 substance sensibilatrice for amboceptor), but it unites itself to 
 one part of the molecule of amboceptor after another part of the 
 latter has combined with the corpuscle. We will defer the 
 discussion of these theories for the present. 
 
 For the sake of simplicity, we have assumed that complement 
 will not unite with amboceptor until the latter has combined with 
 its antigen. Ehrlich, however, assumes (on no very clear evidence) 
 that the two substances may enter into a loose and easily dis- 
 sociated chemical combination. This is hastened by heat and 
 retarded by cold. The union between corpuscle and amboceptor 
 is a firm one, which takes place at low temperatures, and which 
 does not tend to dissociate, and after the combination amboceptor 
 has a stronger affinity for complement, though even then a firm 
 union only takes place at 30 C. or over. 
 
 Ehrlich explains the method of formation of amboceptor on the 
 side-chain theory in this wise : He points out that for the 
 absorption of substances of small molecule by the cell protoplasm 
 the simple receptors, which, when cast off, constitute antitoxin, 
 may suffice ; but he says that when a giant proteid molecule (e.g., 
 of albumin) is anchored, it requires the action of a digestive 
 ferment before it can be brought into a condition to be of use 
 in the nourishment of the living cell. Hence it must be first 
 broken down by means of a proteolytic enzyme, and it is of this 
 nature that he imagines the complement to be. He holds, there- 
 fore, that the receptors or side-chains which are adapted to seize 
 molecules of coagulable proteid possess two haptophore groups, 
 the one to seize the nutrient material, and the other to seize a 
 molecule of digestive enzyme from the surrounding blood or 
 lymph. When the formation of such receptors is stimulated to 
 
 1 Ehrlich does not make a definite statement as to this increase of affinity, 
 but it seems a necessary deduction from the facts as he interprets them. 
 
 10 2 
 
148 HAEMOLYSIS EHRLICH'S RESEARCHES 
 
 excess, and they break loose, they retain both their haptophore 
 groups and constitute amboceptor. The stimulation and over- 
 production is, of course, produced by the molecules of the substance 
 injected (the specific antigens), be they red blood-corpuscles, 
 bacteria, or, as we shall see, a whole host of other cells. 
 
 As a rough illustration of the nature of these seizing arms 
 Ehrlich compares them with the tentacles of Drosera, which 
 seize the nutrient material and then secrete a digestive fluid. 
 The analogy is not exact, since the receptors do not form the 
 digestive enzyme, but simply select it from without. 
 
 Ehrlich compares the action of the haemolysins with that of 
 the toxins, and points out that in each case there is a haptophore 
 group adapted to seize on the red corpuscle to be dissolved or the 
 cell to be poisoned, and an actively functional group with functions 
 resembling those of an enzyme. He says that the haemolysins 
 are practically toxins in two parts, and that a combination of these 
 parts is necessary before any action can be effected. 
 
 These discoveries and theories of Ehrlich led to a series of 
 further researches, all of which were directly suggested by them, 
 and the results of which appear to corroborate them to the full. 
 Whatever the ultimate fate of the side-chain theory, there can be 
 no doubt that it has lead to an enormous increase of our knowledge 
 on a most intricate subject. These researches have in many 
 cases but little direct bearing at present on the subject of 
 immunity, but they are far too important to be passed over 
 without mention. We shall, therefore, summarize them as briefly 
 as is possible when dealing with so complicated a subject. We 
 will deal first with the explanation of the haemolytic action which 
 the serum of some normal animals has on the corpuscles of other 
 species ; for instance, normal goat serum will dissolve the red 
 corpuscles of the guinea-pig. This was shown conclusively to 
 depend upon the same mechanism of amboceptor and complement 
 as in the artificial immune sera. The proof was as follows : 
 Goat serum was mixed with guinea-pig's corpuscles, and the 
 mixture kept at o C., and then centrifugal ized. The theory would 
 lead us to believe that the amboceptor had combined with the 
 corpuscles, but that the complement had remained free. If this 
 were so, the supernatant fluid would be found to have lost 
 amboceptor. To test this a further addition of corpuscles was 
 made, and the solvent power of the serum for these was found 
 to be lowered. Evidently, then, sera which are naturally haemo- 
 
BACTERIOLYSIS AND ALLIED PHENOMENA 149 
 
 lytic to certain red corpuscles are so in virtue of containing 
 amboceptors with cytophile haptophore groups which fit the 
 receptors of these cells and complements which can dissolve 
 them. He was also able to show, by an ingenious experiment, 
 that when a normal serum can haemolyze the corpuscles from 
 different species of animals, it does so by the action of different 
 amboceptors, and in some cases of different complements also. 
 (,We must imagine, therefore, that normal serum contains numerous 
 antibodies of the amboceptor type, and adapted to dissolve 
 numerous foreign substances when they gain access to the blood ; 
 and it seems reasonable to believe that these normal antibodies 
 and those which are formed as a result of the presence of the 
 foreign substances play a part of the greatest importance in 
 immunity. \ 
 
 The question then arose, Do animals possess or form haemolysins 
 adapted to the solution of their own corpuscles in other words, 
 an autohtsmolysin ? For instance, if a person sustains a large 
 internal haemorrhage, and the blood is absorbed, is he thereby 
 stimulated to the production of a substance which dissolves his 
 own red corpuscles ? Ehrlich pointed out that such a phenomenon 
 is unknown, and set out to investigate the reason of its non- 
 occurrence. He selected goats as his experimental animals, and 
 injected into them large amounts of mixed blood-corpuscles from 
 other goats. In a week's time the serum was found to be power- 
 fully haemolytic, but only towards the corpuscles of other goats, 
 never towards its own; thus the serum of the first animal 
 experimented on was found to dissolve the corpuscles of goats i, 
 2, 4, 5, 6, and 9 easily ; those of 3 and 8 less powerfully ; and 
 those of 7 and of itself not at all. A second animal was treated 
 just like the first, and it also developed a haemolysin, but this 
 did not act on its own corpuscles, nor on those of other goats 
 in the same way as that of the first ; evidently a different series 
 of amboceptors had been formed. Ehrlich speaks of a haemolysin 
 formed by the injection of the corpuscles of a different species 
 as heterolysin, that which acts on the blood of the same species 
 as an isolysin ; one which would act on the blood-corpuscles of 
 the same animal would be called an autolysin, but this is never 
 formed. 
 
 In attempting to discover the reason for this non-formation 
 Ehrlich first proved that the amboceptor of the isolysin was 
 anchored by the corpuscles which it dissolved, but not by those 
 
150 HAEMOLYSIS ISOLYSIN 
 
 of the goat from which it was derived. An obvious explanation 
 would be that .the latter did not possess any receptors which it 
 would fit. Another suggestion might be that it had such receptors, 
 but that they were already fully occupied by amboceptors occur- 
 ring in the serum ; but in this case they would attract complement, 
 and solution would ensue. By a method 1 which will not be 
 described here, since it would involve anticipation of facts not 
 yet described, Ehrlich was able to prove the former view the 
 correct one. The immunity, therefore, of the corpuscles of 
 an animal to its own isolysin is due to a complete absence 
 of suitable receptors in its corpuscles, and, we may add, in the 
 entire organism. 
 
 He deduced, therefore, that each blood-corpuscle possesses 
 numerous side-chains with haptophore groups, each of which, 
 when injected into a living animal, is able to combine with a 
 suitable receptor. If we take a particular variety of haptophore, 
 which we will call a, we can see that there are two possibilities 
 after the injection ; it may find no receptors a, in which case 
 there will be no antibody formed, or it may find such receptors. 
 In the latter case there are also two possibilities : there may be 
 receptors a only, or there may be receptors a and haptophore side- 
 chains a. 
 
 If there are only receptors a and no side-chains a, the injection 
 of corpuscles with side-chains a, will lead to an overproduction of 
 receptors a, and amboceptor will be produced. This will act as a 
 hsemolysin to the corpuscles injected, but not to those of the 
 animal itself, since they do not contain side-chains to which it can 
 attach itself ; it will be an isolysin, not an autolysin. 
 
 In the second possible case Ehrlich points out that the condi- 
 tions for the production of an autolysin do occur, and such a 
 substance might be produced and might do serious harm to the 
 animal. But it would be produced at first only in small amounts, 
 and the result might be that it would combine with receptor a, 
 which might then be stimulated and cast off and would form anti- 
 autolysin. 
 
 1 He first showed that the injection of an amboceptor from one animal into 
 another of a different species would cause the production of an anti-ambo- 
 ceptor, and then showed that the injection of a serum containing isolysin 
 into a goat whose corpuscles it dissolved produced anti-isolysin. These 
 corpuscles contained receptors, since they fixed the isolysin. In the goat 
 from which the isolysin came there was, of course, no production of anti- 
 isolysin. Hence, he argued, it had no suitable receptors in its entire body. 
 
BACTERIOLYSIS AND ALLIED PHENOMENA 151 
 
 There are, therefore, three possibilities : no formation of haerno- 
 lysin, the formation of an isolysin, and the formation of an anti- 
 autolysin. In other words, if autolysins were ever developed they 
 would be followed immediately by the production of their specific 
 antibody. 
 
 But Ehrlich assumes and our previous account of the iso- 
 agglutinins leads us to be ready to accept his assumption 
 unhesitatingly that each corpuscle in every animal has numerous 
 side-chains, , (3, 7, etc. If such a corpuscle is injected into 
 an animal of the same species, a may lead to no result, finding no 
 receptors ; ft may lead to the production of an isolysin, and 7 to 
 that of an anti-autolysin. The existence of the latter substance 
 could not, of course, be proved, since it was never possible to 
 prepare autolysin. 
 
 Hence what Ehrlich calls the " pluralistic conception of the 
 cellular immunity reaction." There are, he says, in each bac- 
 terial cell numerous side-chains, to each of which an antibody 
 is theoretically possible, and an ideal curative serum would 
 contain all these antibodies. But in some cases, perhaps in most, 
 only a few are found, and others cannot be developed ; for 
 instance, in some animals it is impossible to produce anti -enzymes 
 by the injection of enzymes. He explains this in two ways. 
 The specific receptors may be of peculiar constitution, so that they 
 cannot be cast off from the cell in the process of immunization : 
 these he calls sessile receptors. Secondly, it is conceivable that 
 the side-chains in question are normally produced by the animal 
 cells, and that no antibody is produced to them, or, at any rate, that 
 none accumulates. 
 
 But when either or both of the conditions arise, we may be able 
 to get the antibodies from a second animal which we are unable 
 to do from the first. Thus, if we imagine that the typhoid bacillus 
 has twenty different sorts of side-chains, we may be able to get 
 antibodies to some of them from the dog, to others from the rabbit, 
 etc. Hence he suggests as an important principle in the forma- 
 tion of curative sera to use many animal species, mixing the sera 
 of those which produce the antibodies required for use. (This, it 
 must be pointed out, is not what is meant by a polyvalent serum, 
 which is a serum formed by the injection of many strains of the 
 same organism.) 
 
 Leaving this subject for the present, we will pass on to Ehrlich's 
 researches into the nature of complements. Buchner, in his 
 
152 HAEMOLYSIS COMPLEMENT 
 
 studies on the alexins, assumed that the serum of each animal 
 contained one alexin, though the alexins of different animal 
 species were different. This view was also held by Bordet and 
 others, and constitutes the " Unitarian " view of alexin or com- 
 plement. Metchnikoff holds that there are two alexins, or, as he 
 calls them, cytases macrocytase, which acts specially on red 
 blood-corpuscles, cells, etc., and i /jSaeJ by the large mononuclear 
 cells ; and microcytase, which acts on bacteria, and is formed by the 
 polynuclear leucocytes. 
 
 As against these views, Ehrlich advances the theory of the 
 multiplicity of the complements, the proof of which appears entirely 
 satisfactory. According to him there are numerous complements 
 in the serum of every animal, and these differ from one another in 
 their haptophore groups, so that one can reactivate one ambo- 
 
 * 3' # 
 
 FIG. 33. SHOWING SOME OF THE CONSTITUENTS WHICH CAN BE DEMON- 
 STRATED IN THE SERUM OF A GOAT WHICH HAS BEEN INJECTED WITH 
 SHEEP'S CORPUSCLES. 
 
 a, b, and c are the different complements, a' , b' , and c' the amboceptors which 
 unite them to the corpuscles of the sheep (A), rabbit (B), and guinea pig (C). 
 
 ceptor, another another. They differ in some cases in other 
 respects. The first example which he was able to adduce in 
 which there were at least two complements in a sample of serum 
 was that of a goat which had been injected with sheep's corpuscles, 
 and which dissolved those of the sheep, guinea-pig, and rabbit. 
 But when this serum was heated to 56 C. for three-quarters of an 
 hour, it was found to have no action on the corpuscles of the latter 
 animals, whilst that of the former was unchanged. Evidently 
 then, the complement which activated the amboceptor combining 
 with the sheep's corpuscles was thermostable ; that acting on the 
 others, thermolabile. This was the first known example of a 
 thermostable complement. In a later experiment he was able to 
 prove that the complements taking part in the haemolysis of 
 rabbit's corpuscles and that taking part in the solution of guinea- 
 pigs were different : the latter passes through a Pukall's filter, 
 the former does not. 
 
BACTERIOLYSIS AND ALLIED PHENOMENA 153 
 
 These methods of heating, filtration, etc., of course only separate 
 the complements in certain lucky cases, and the more general 
 method involves the removal of some of these bodies by means of 
 sensitized red corpuscles. Thus fresh goat serum will reactivate 
 heated normal goat serum in its action on rabbits and on guinea- 
 pig's red corpuscles, the amboceptors for which are contained in 
 untreated animals. It will also reactivate the heated serum 
 of goats injected with rabbit's corpuscles, ox corpuscles, or dog's 
 corpuscles. Ehrlich and Sachs found that if they added this fresh 
 serum to rabbit's corpuscles, the complements which took part in 
 the two latter reactions were absorbed, whilst the others were 
 unaltered. Evidently, therefore, two complements at least are 
 present in goat serum. When the serum was allowed to act 
 on guinea-pig's corpuscles, the complements which reactivated 
 the normal amboceptors for rabbit's and guinea-pig's corpuscles 
 were removed, also that which activated the artificial ox ambo- 
 ceptor. By an elaboration of these methods Ehrlich and Sachs 
 were able to demonstrate that these five hsemolytic actions depend 
 upon five different complements, each perfectly distinguishable 
 the one from the other. The subject will not be pursued farther, 
 though there is an abundance of evidence pointing to the same 
 end that the serum of every animal contains a very large number 
 of complements, some of which take part in one reaction, some in 
 another. As a rule, complements which take part in haemolysis 
 have no action on bacteria, or but little, and this is all the truth in 
 MetchnikofT's theory of macro- and microcytase. 1 
 
 Ehrlich lays it down as a law that amboceptors from a certain 
 species of animal are best complemented by complements from 
 this species, and this is true in general ; Muir has adduced some 
 exceptions. 
 
 The main experimental basis for the Unitarian theory was 
 furnished by Bordet and Gengou. They found that if they added 
 "sensitized" bacteria i.e., bacteria which had been placed in 
 heated immune serum in fresh serum, all the complements were 
 removed, and the fluid would no longer dissolve sensitized red 
 corpuscles, so that evidently the haemolytic complement had been 
 removed. Again, Bordet showed that if he added fresh serum to 
 sensitized red corpuscles and allowed solution to take place, all 
 complements, bacteriolytic as well as haemolytic, were removed. 
 These facts are not disputed, and we shall have occasion to refer 
 
 1 See p. 286. 
 
154 HAEMOLYSIS COMPLEMENT 
 
 to them again, as they are of great importance. Their interpreta- 
 tion is, however, still uncertain, and they cannot outweigh the 
 very precise demonstrations of numerous complements by Ehrlich 
 and his school. According to them the receptor, which, when 
 broken off, constitutes amboceptor, may have more than one com- 
 plementophile group, each adapted to seize and utilize different 
 complements. We must suppose that it can discharge its function 
 if it is supplied with one of these complements ; this is called the 
 dominant. It may also seize other complements, so that all its 
 affinities are supplied, and this is what takes place in Bordet's 
 
 FIG. 34. PLURICEPTORS ATTACHED TO A CORPUSCLE, AND SHOWING THE 
 DOMINANT (a) AND NON-DOMINANT (b) COMPLEMENTS. 
 
 FIG. 35. SOME OF THE CONSTITUENTS OF NORMAL GOAT SERUM. 
 
 a Complement which dissolves sensitized sheep-corpuscles; b = that which 
 dissolves rabbit's corpuscles ; c = the normally occurring amboceptor for 
 rabbit's corpuscles. The next two diagrams shows how this is shown to 
 be a biceptor. 
 
 and Gengou's phenomenon ; these complements, which are not 
 essential to the lytic action, are called non-dominant or subordinate 
 complements. 
 
 Ehrlich and Sachs give an example of this. The amboceptor 
 of normal goats for rabbit's cells and that obtained by injecting 
 goats with ox corpuscles are, of course, both sensitized with 
 normal goat serum. Now if we take rabbit's corpuscles and add 
 them to fresh serum (amboceptor-complement), it is found that 
 both the complements which might theoretically be present are 
 removed, and the fluid will no longer reactivate heated immune 
 serum for ox corpuscles. But if the action is allowed to go on for 
 a short time only, and the corpuscles are then centrif ugalized down 
 and removed, it is found that only the non-dominant complement 
 
BACTERIOLYSIS AND ALLIED PHENOMENA 155 
 
 is removed, and the supernatant fluid will still reactivate the action 
 of heated immune serum on rabbit's corpuscles. This is not the 
 invariable rule, and in other cases the non-dominant complements 
 are not seized until the dominant has been anchored. In either 
 case we can see an alternative explanation for the facts observed 
 by Bordet and Gengou. Ehrlich points out that the presence of 
 the power to bring these many ferments into relation with the 
 giant molecule of the protoplasm must be of advantage in cell 
 nutrition, as enabling the greatest possible effect to be produced, 
 and quotes Hoffmeister as showing that the liver cell contains ten 
 different ferments, and Delbruck as attributing five to the yeast 
 cell. 
 
 The discovery that the haemolytic action of snake venom 
 depends on the mechanism of amboceptor and complement made 
 
 FIG. 36. SHOWING EFFECT OF ADDITION OF RABBIT'S CORPUSCLES TO THE 
 SERUM SHOWN IN FIG. 35. 
 
 The amboceptor unites with the corpuscle, and all the complements are with- 
 drawn. The supernatant fluid loses its power of dissolving ox corpuscles 
 previously sensitized. (See also Fig. 37.) 
 
 by Flexner and Noguchi, and the excellent subsequent work of 
 Kyes, made us realize that the process might be even more 
 complex. Flexner and Noguchi "^showed that snake venom 
 contains two substances, one of which is thermostable, not being 
 destroyed at 90 C., and which in itself has no power of bringing 
 about solution, and which is evidently equivalent to amboceptor. 
 It may be reactivated by the other thermolabile substance or by 
 the complements of normal serum. For example, horse corpuscles 
 can be dissolved by heated venom and fresh ox serum, ox blood 
 corpuscles by heated venom and guinea-pig serum, and so on. 
 Kyes found that some corpuscles, washed from all trace of 
 serum, could be dissolved by cobra venom alone ; others required 
 the concomitant presence of fresh serum of the same or other 
 species. Thus the corpuscles of man, the dog, guinea-pig, etc., 
 are dissolved by the venom alone, whilst those of the ox, sheep, 
 and goat are not. He formed the theory (for reasons which will 
 
HAEMOLYSIS ENDO-COMPLEMENT 
 
 not be discussed) that the solution of corpuscles without the action 
 of serum was due to a complementing of the venom amboceptor 
 by means of complements contained in the corpuscle itself, which 
 he called endo- complements. This he proved experimentally. He 
 argued that if he dissolved the red blood-corpuscles which contain 
 endo-complements by means of distilled water, the solution should 
 act as though it contained free complements, and should reactivate 
 venom amboceptor, combined with corpuscles which it was unable 
 to dissolve without the aid of serum. This was found to be the 
 case. A laked solution of human corpuscles mixed with venom 
 would haemolyze the corpuscles of the ox, sheep, and goat ; but a 
 solution of ox corpuscles and venom would not dissolve the cor- 
 puscles of the goat or sheep. He showed that these endo- 
 complements are thermolabile, though more resistant than most 
 
 FIG. 37. SHOWING EFFECT OF SHORT EXPOSURE OF RABBIT'S CORPUSCLES 
 TO THE SERUM SHOWN IN FIG. 35. 
 
 The non -dominant complement is absorbed, and the supernatant fluid no 
 longer dissolves sensitized ox corpuscles, whilst retaining its action on 
 sensitized rabbit corpuscles. (In most cases the dominant complement is 
 removed first.) 
 
 of those in sera. They are destroyed at 62 C. in half an hour. 
 Kyes gave an additional demonstration of his theory by showing 
 that if rabbit's corpuscles are soaked for twenty-four hours in 
 normal saline solution, the endo-complement dissolved out, and 
 could be demonstrated in the fluid, whilst the corpuscles were no 
 longer haemolyzed by snake venom without the addition of serum. 
 He holds, therefore, that the corpuscles contain endo-complements 
 able to dissolve them, but only when brought into organic relation 
 with their protoplasm by means of amboceptor. 
 
 He next went on to demonstrate that a definite chemical 
 substance lecithin can act as a complement to snake venom, 
 and that either it or the serum-complement could act as a 
 dominant so as to bring about solution, or that the two might act 
 together. Lastly, he was able to prepare a combination of cobra 
 venom and lecithin, which he calls cobra-lecithid, which is quite 
 different in its physical and chemical characters from either of its 
 
BACTERIOLYSIS AND ALLIED PHENOMENA 157 
 
 components, and which acts haemolytically on all red corpuscles. 
 It also acts much more quickly than ordinary solutions of venom 
 even when activated with lecithin, and it is very thermostable, 
 resisting boiling for six hours. It is evidently a definite chemical 
 substance and of great theoretical interest, as being apparently 
 an amboceptor-complement compound preparable in a pure state. 
 The analogy between the complements and the exotoxins leads 
 to the inquiry whether the production of an anticomplement occurs 
 when an active serum is injected into an animal of another species. 
 Ehrlich and Bordet both proved this to be the case. He injected 
 fresh horse serum into goats, and obtained a serum which would 
 prevent the normal complementing action of horse serum. 1 He 
 easily showed that this was not due to any action which it exerted 
 on the red corpuscles (rabbit's) themselves or on the amboceptor. 
 
 FIG. 38. SHOWING THE EFFECT OF ADDING TO SENSITIZED RABBIT'S 
 CORPUSCLES (a, b) A MIXTURE OF FRESH HORSE SERUM (CONTAINING 
 COMPLEMENT, c), AND SERUM FROM A GOAT WHICH HAD BEEN INJECTED 
 WITH HORSE SERUM AND CONTAINED ANTICOMPLEMENT, d. 
 
 The complement and anticomplement combine, and the corpuscles are 
 unaffected ; this is shown by the fact that they will dissolve on the 
 addition of fresh complement. 
 
 It was clear, therefore, that it acted as an anticomplement. The 
 question now arose, Was this action exerted on the haptophore or 
 the zymophore group ? The following experiment shows that it 
 acts on the former : Sensitized red corpuscles were treated with a 
 rmxture of normal serum and of serum containing anticomplement: 
 no solution took place. The corpuscles were then centrifugalizecj. 
 ott andTresh serum added : haemolysis occurred. Evidently the 
 anticomplement had not acted on the zymophoric group, for if it 
 had the haptophore group would have combined with the ambo- 
 ceptors of the sensitized corpuscles, and would have shielded 
 them from further action when fresh serum was added. What 
 
 1 There is a possible fallacy here, to be mentioned under our description of 
 Bordet and Gengou's phenomenon. It may be simply a binding of the 
 complement in the precipitate. This is discussed later, and at present 
 Ehrlich's views will be set out as if they were definitely proved. 
 
158 HAEMOLYSIS ANTICOMPLEMENT AND COMPLEMENTED 
 
 occurred was that the haptophore radical of the complement had 
 been " blocked " by the anticomplement. 
 
 In a few cases anticomplements which will act on some only 
 of the complements of a sera can be produced, and from the study 
 of these additional evidence in favour of the multiplicity of com- 
 plements has been adduced. 
 
 As regards the nature of these anticomplements, this is readily 
 explicable on the side-chain theory. Ehrlich supposes that when 
 a foreign serum is injected into an animal it may find no receptors 
 for which it has an affinity ; in this case it forms no anticomple- 
 ment, and such cases are known to occur. Or it may find receptors 
 which it can activate completely, just as the normal complement 
 of the animal can do ; in this case also there will (under ordinary 
 circumstances) be no formation of anticomplement. Lastly, it 
 may find receptors with which it can combine, but the resulting 
 combination may be useless in the nutrition of the cell ; in this 
 
 FIG. 39. SHOWING EFFECT OF ADDITION TO SENSITIZED CORPUSCLES OF A 
 MIXTURE OF COMPLEMENT AND COMPLEMENTOID. 
 
 The former unite with the complementophile haptophore groups of the ambo- 
 ceptor ; the latter, which have decreased in combining affinity, do not. 
 
 case the receptor will be cast off, and will constitute anti- 
 complement. 
 
 Lastly, Ehrlich was able to show that in some cases an 
 auto-anticomplement may be formed i.e., an antibody which can 
 neutralize the complement normally present. The proof for this 
 is elaborate, and will not be given here. 
 
 The change of toxins into toxoids is paralleled by the change of 
 complements into complementoids. We have previously spoken of 
 the complements as being destroyed by heat, but this is not quite 
 correct ; they lose their zymophore groups, but retain their hapto- 
 phore portions, though these appear to lose some of their combining 
 affinity. The proof of the existence of complementoids is simple : 
 when injected into animals they call forth the production of 
 anticomplements, just as toxoids call forth the production of 
 antitoxin. 
 
 For some time it was found impossible to demonstrate the 
 
BACTERIOLYSIS AND ALLIED PHENOMENA 159 
 
 presence of complementoids in test-tube experiments, and this 
 was owing to the fact that the affinity of the haptophore group of 
 the complements suffered loss during the process of heating, so 
 that when a mixture of complement and complementoid was 
 added to sensitized red corpuscles, the former combined just as 
 well as if the latter were not present. " Blocking " of the com- 
 plementophile group of the amboceptor appeared not to occur ; 
 but Ehrlich and Sachs were able subsequently to demonstrate 
 such a phenomenon in this wise : Dog serum contains an ambo- 
 ceptor for guinea-pig's corpuscles, and this can be activated either 
 by dog's or guinea-pig's serum. New when a mixture of heated 
 dog's serum, guinea-pig's corpuscles, and guinea-pig's serum was 
 made, solution took place ; but when the corpuscles and heated 
 serum were added together, allowed to stand, and then centrifuga- 
 lized and removed, no solution occurred on the addition of fresh 
 
 FIG. 40. DEMONSTRATION OF " BLOCKING " OF COMPLEMENTOPHILE GROUPS 
 BY COMPLEMENTOIDS. 
 
 a = complementoid, and & = amboceptor in heated dog serum; r = rabbit's 
 corpuscles (see text). 
 
 guinea-pig's serum. Ehrlich proved 1 that the combination of 
 amboceptor and corpuscles took place as usual, and that it was then 
 followed by a blocking of the free arms of these amboceptors by 
 the complementoids present in the heated dog's blood. In this 
 case, therefore, the complement had not suffered any appreciable 
 change in combining capacity as a result of its conversion into 
 complementoid. 
 
 It follows, therefore, that when we heat fresh serum, or allow it 
 to stand a few days at the room temperature, we do not get rid 
 of the complements altogether. This can be done, as shown by 
 Von Dungern and others, by shaking the serum with cells (liver, 
 kidney, etc.), yeast, fungi, certain bacteria, powdered charcoal, 
 etc. This abstraction of complements by inert bodies is a non- 
 specific process entirely unlike its fixation in haemolysis, etc., 
 
 1 The proof is complete and conclusive, but too long to give here. 
 
i6o 
 
 HAEMOLYSIS ANTI AMBOCEPTOR 
 
 and Ehrlich compares it to a process of physical absorption. The 
 presence of the phenomenon must be borne in mind, since it must 
 make us careful how we interpret the mere disappearance of 
 complement from a fluid after it has acted on articulate sub- 
 stances. The true test of specificity is its action on these 
 substances, not its absorption by them. The yeast cells are in no 
 ways injured through having absorbed complement. 
 
 We have not yet finished our study of the complements, but it 
 will be convenient to continue it later, and to conclude here Ehrlich's 
 studies on the antilysins. Anticomplement has been already 
 discussed, and we will now turn to anti-amboceptor. 
 
 This is a substance of nature different to any that we have 
 previously studied, in that it is an antibody to an antibody. It is 
 prepared by injecting an immune serum into an animal other than 
 that from which it was derived. Thus, if an antityphoid (bacterio- 
 lytic) serum procured by immunizing a horse with typhoid bacilli 
 be injected into a rabbit, the serum of the latter acquires the 
 power of neutralizing the bacteriolytic action of the former. 
 Antibodies to antibodies cannot always be produced; thus, anti- 
 antitoxin is not yet known. Again, Metchnikoff was unable to 
 prepare antispermotoxin by injecting a cytolytic serum for sperma- 
 tozoa into the guinea-pig. In this case there can be no question of 
 the absence of suitable receptors, for the animal's spermatozoa are 
 attacked in vitro by the serum injected. Hence Ehrlich made 
 the assumption of the presence of sessile receptors, which are not 
 cast off when the suitable antibody is injected. 
 
 In studying the anti-amboceptor concerned in haemolysis, 
 Ehrlich first prepared a haemolytic serum by injecting a rabbit 
 with ox blood. When the serum of the latter was found to be 
 powerfully haemolytic, it was injected subcutaneously into a goat : 
 120 c.c. was given during an interval of two months. At the end 
 of this time this goat serum was powerfully antihaemolytic. This 
 was shown as follows : 0-00125 c.c. of (immune) rabbit serum was 
 found to haemolyze completely i c.c. of a 5 per cent, emulsion of 
 ox corpuscles, provided that sufficient normal guinea-pig serum 
 is added to provide complement. Now three times this amount 
 (0-00375), plus 0-5 of the goat serum, was added to i c.c. of the 
 emulsion of corpuscles, allowed to act at 40 C. for an hour, centri- 
 fugalized, and 0-15 c.c. of normal guinea-pig serum (complement) 
 added to the corpuscular sediment. No solution took place, so 
 that 0-5 c.c. of the anti-antiserum had completely neutralized three 
 
BACTERIOLYSIS AND ALLIED PHENOMENA l6l 
 
 dissolving doses of the antiserum. A control test showed that 
 normal goat serum was without action. 
 
 Theoretically, two anti-amboceptors are possible : one which 
 will combine with the cytophile group of the amboceptor, and one 
 which will seize on the complementophile. In his earlier studies 
 Ehrlich thought that the former was that actually produced when 
 immune sera are injected into animals of another species. He 
 later altered his opinion, and now holds that the substance 
 actually formed is an antibody to the complementophile hapto- 
 phore of the amboceptor. He was led to this conclusion by a 
 discovery of Bordet's, which was adduced as evidence against the 
 amboceptor theory, but which was ingeniously adapted to its 
 defence by Ehrlich. The discovery was as follows : Normal 
 rabbit serum when injected into a guinea-pig leads to the production 
 of an anti-antibody, which neutralizes the haemolysin formed by 
 
 FIG. 41. THE COMPLEMENTOPHILE HAPTOPHORE GROUPS OF AN AMBO- 
 CEPTOR "BLOCKED" BY ANTIAMBOCEPTOR (a, a}. 
 
 immunizing with ox blood. This, of course, is readily explicable 
 if normal rabbit serum contained an amboceptor for ox corpuscles, 
 but this is not the case. Ehrlich argued as follows : An anti- 
 amboceptor is obviously formed, but the normal rabbit serum 
 injected contains no amboceptor i.e., no cytophile groups ; hence 
 the ant -amboceptors produced must be anticomplementophile. 
 For this to happen it is necessary that this normal rabbit serum 
 must contain complementophile groups, and this is obviously the 
 case, since other amboceptors, each with a complementophile 
 group, are present. Therefore, on the assumption that these 
 groups are similar, even when they occur in different amboceptors, 
 the case is clear. But are these complementophile groups similar ? 
 Does not this contradict Ehrlich's views on the multiplicity of 
 complements ? To avoid this difficulty, Ehrlich falls back on his 
 polyceptor theory. All or most amboceptors are really poly- 
 ceptors, differing only or mainly in their cytophile groups, and 
 possessing numerous complementophile groups, which are similar 
 or identical in different cases in the same species. But if this 
 
 ii 
 
l62 RECAPITULATION OF EHRLICH'S THEORIES 
 
 is the case, anti-amboceptors should be non-specific ; they are 
 
 directed against the complementophile groups, which are the same 
 
 in all amboceptors from the same species. This subject has not 
 
 yet been fully investigated, but Pfeiffer and Friedberger have 
 
 shown that the antibody to the immune body against the cholera 
 
 vibrio also acts against the antityphoid serum. Ehrlich also 
 
 showed that if anti-amboceptor be added to sensitized cells^these 
 
 are protected against the subsequent addition of complement. 
 
 /This, of course, is attributable to the fact that the anti-amboceptor 
 
 I unites with the free complementophile groups, and " blocks " them 
 
 [^against the subsequent access of the complement molecule. 
 
 Ehrlich leaves undecided the question as to whether an anti- 
 cytophilic anti-amboceptor is ever produced, but holds that it 
 might be formed if by any process we could destroy the cytophile 
 haptophore of the amboceptor molecule. Under ordinary circum- 
 stances the complementophile group has a greater affinity for its 
 cell receptor than has the cytophile for the receptors with which 
 it could combine ; union takes place between the former, and the 
 latter is, so to speak, pulled out of the way of its affinity. 
 
 It is now advisable to recapitulate briefly some of the main 
 points in Ehrlich's theory of the structure of the bacteriolytic and 
 haemolytic sera. Firstly, as regards complements : Of these there is 
 very great number, and each is especially adapted for the solution of 
 one or more varieties of cells, which it can dissolve in the presence 
 of a suitable amboceptor ; it is known as the dominant comple- 
 ment. Other complements, however, may help in the process, 
 and these are termed non-dominant. In general terms the comple- 
 ments, which are especially active in haemolysis, have but little 
 action on bacteria, and vice versa. Secondly, as regards the ambo- 
 ceptor : This is really a polyceptor, and is so constituted that it 
 can combine with the cell to be dissolved, on the one hand, and 
 with a large number of molecules of complement, on the other. 
 
 Of the further complications of the theory which Ehrlich has 
 introduced to explain new phenomena as they arise, of the 
 amboceptoids, of loose and firm union, etc., we do not propose 
 to speak. That the theory needs these complications in order to 
 account for the phenomena is not necessarily in its disfavour, for 
 the phenomena themselves are complex in the extreme. And it 
 must be regarded as a strong argument in its favour that Ehrlich 
 has again and again deduced results from this theory which sub- 
 sequent research has shown to be correct ; and very few hypo- 
 
BACTERIOLYSIS AND ALLIED PHENOMENA 163 
 
 theses can be adduced which have been so rich in leading to the 
 discovery of new facts. 
 
 It will now be necessary for us to glance briefly at some 
 alternative hypotheses, and it must be pointed out that the com- 
 paratively short notice they will receive must not be taken to 
 imply that there is less to be said in their favour. The trend of 
 modern research tends on the whole against Ehrlich's views ; yet 
 in the history of immunity his researches will always be regarded 
 as of the greatest interest and value. 
 
 It may be pointed out that the cast-off receptors of red 
 corpuscles, bacteria, etc., may combine with the complemento- 
 phile groups of an amboceptor, and thus appear to act as a 
 cytophilic anti-amboceptor. 
 
 Bordet holds that the immune body is not an amboceptor at 
 all i.e., that it does not act as a link between the corpuscle or 
 bacterium and the complement, but that it sensitizes the former 
 and renders it susceptible to the action of the latter. On Ehrlich's 
 theory, amboceptor unites with cell and complement with ambo- 
 ceptor ; on Bordet's, both substances unite with the cell direct. 
 On the latter theory many, if not all, of the results observed by 
 Ehrlich and others are explicable, and there appears to be no 
 experimentum crucis by which the truth can be determined. A diffi- 
 culty in the way of accepting Ehrlich's amboceptor theory is this : 
 there is no proof that complement and immune body ever unite 
 unless the latter has already combined with a cell, or with anti- 
 amboceptor, or with free receptors. The only example to the 
 contrary is supplied by Ryes' cobra-lecithid, which, though of 
 great interest, can hardly be quoted as a case in point, since 
 lecithin differs so markedly from the ordinary thermolabile 
 complements of serum. Another case (in the deviation of the 
 complements) in which this process appears to take place is 
 extremely complicated, and the evidence in favour of the direct 
 union of complement and antibody quite unsatisfactory. It 
 follows, then, that we must either assume that the mere union 
 of the cytophile group of the antibody with some other object 
 increases the affinity of the complementophile group for comple- 
 ment (as has been done here), or we must agree with Bordet that 
 cell and complement unite directly, but only after the latter has 
 been prepared by the action of immune body. A very important 
 observation of Muir's is of interest in this connection. Muir 
 showed that after red corpuscles had been saturated with immune 
 
 II 2 
 
164 HAEMOLYSIS BORDET'S VIEWS 
 
 body and then with complement, some of the immune body, but 
 none of the complement, might be dissociated from the combina- 
 tion, and become free in the fluid. It is difficult, though perhaps 
 not quite impossible, to reconcile this experiment with Ehrlich's 
 theory, whilst it is really explicable on that of Bordet ; but Muir 
 (whose recent work on this difficult point should be consulted) 
 has not come to a definite conclusion as to the nature of the 
 combination. 
 
 Bordet also holds peculiar views on the subject of the union 
 of corpuscles and immune body. According to Ehrlich, the com- 
 bination is due to a strong chemical combining affinity between 
 the two substances, and the resulting compound is a stable one, 
 which is not readily dissociable. Cells thus activated possess a 
 strong affinity for complement, and the whole process is a chemi- 
 cal one, dependent on ordinary chemical laws and obeying the 
 laws of multiple proportions. This was, on the whole, confirmed 
 by Muir, who proved that corpuscles combined with multiple 
 doses of immune body took up multiple doses of complement ; 
 but he also showed that the corpuscle-antibody compound did 
 dissociate, so that when corpuscles which had been saturated 
 with immune body were placed in contact with normal corpuscles, 
 some of the amboceptor left the sensitized and attached them- 
 selves to the normal. Bordet adduced evidence to show that the 
 combination is of a remarkable nature, and dependent on obscure 
 chemico-physical reactions. He took a sample of haemolytic 
 serum, and determined the amount of blood which it could 
 haemolyze when the two were mixed together. We may assume, 
 on Ehrlich's theory, that all the suitable receptors in the cor- 
 puscles were saturated with amboceptor. But Bordet showed 
 that if the corpuscles were added a little at a time, a very much 
 smaller dose might take up all the immune body. Hence he 
 compared the process to the staining of filter-paper when im- 
 mersed in a dye. If the paper be added at once, it will be stained 
 a uniform colour, whereas if it be added a little at a time, the first 
 pieces will be stained deeply, the subsequent ones less and less, 
 until the dye is completely absorbed. It would appear to explain 
 the phenomenon equally well if we assumed that the corpuscles 
 could be haemolyzed by fewer amboceptors than they could take 
 up, and there is some experimental evidence of this. Thus Muir 
 showed that after red corpuscles have been dissolved by haemo- 
 lytic sera, the addition of more immune body will lead to the 
 
BACTERIOLYSIS AND ALLIED PHENOMENA 165 
 
 absorption of more complement. It may be added, however, that 
 many of the recent researches on antibodies point to them as 
 being governed by the very complex laws, as yet not fully under- 
 stood, which regulate the actions of colloids on one another 
 and on crystalloids. This is discussed, though very briefly, in a 
 subsequent chapter. 
 
 As regards the evolutionary significance of the facts of cytolysis 
 (using the word to cover the solution of bacterial and all other 
 cells), there are two theories which, though apparently widely 
 different, have, on ultimate analysis, much in common. We have 
 seen how Ehrlich explains the process of cell nutrition, the role 
 which he attributes to complement, and the method in which he 
 conceives the complex antibodies to be formed. On his theories, 
 therefore, the fundamental process is one of cell nutrition, and 
 we may imagine it to have become adapted to serve as a means 
 of immunity during the course of natural selection : the animals 
 which could make use of their mechanism of cell nutrition as a 
 means of defence against invading micro-organisms would survive 
 and perpetuate their species ; the others could not. 
 
 We may add a few more considerations on this process of 
 cell nutrition. We must assume that the food molecules of 
 proteid which circulate in the blood are in an indifferent form, 
 and are equally well adapted for the nourishment of cells of the 
 brain, liver, etc., this method of transmission of nourishment 
 having been found the most convenient and economical. The 
 first requisite for the nutrition of the cell is that one of these 
 molecules shall be brought into close relations with its protoplasm. 
 Ehrlich speaks of this as being due to the selective action of the 
 cell receptors, but we must not be misled by terms or diagrams, 
 however convenient they may be to enable us to form a mental 
 picture of the process. Ehrlich simply means that certain 
 specialized molecules or groups of molecules have a chemical 
 combining affinity for certain food molecules. This union is the 
 first requisite, and in some cases it may be sufficient, and the 
 molecule may be forthwith incorporated with the cell protoplasm. 
 In most cases, however, this is not so, and the cell has to 
 transform an indifferent molecule of proteid material into a form 
 suitable for assimilation into itself. We now know that most, if 
 not all, of the activities of a living cell in metabolism are dis- 
 charged through the agency of enzyme action, and that each of 
 the metabolic actions of a cell appears to depend on a special 
 
l66 CELL NUTRITION 
 
 enzyme. It is natural, therefore, that the cell should make use of 
 soluble ferments in preparing the molecule which it has seized. 
 
 We now (thanks to the researches of Fischer, Hierf elder, and 
 others) know that the action of these enzymes is strictly specific, 
 and dependent on a stereo-chemical relation between the ferment 
 and the body acted on, and that it is necessary in all cases for a 
 union to be brought about between the two substances before the 
 specific action is effected. Thus, if the two could unite in the blood, 
 the molecules would be broken down before they reach the cells, and 
 the food material would reach it in an indifferent form, which, 
 however well adapted to some cells, might be useless to others. 
 The substances, therefore, do not unite in the blood-stream at all, 
 but the cell first seizes a molecule of food-stuff, and then one or 
 more ferments which can change it into the exact form which the 
 cell requires. Thus, the process of cell nutrition, according to 
 Ehrlich, consists in the establishment of a link between the food 
 and a suitable ferment after the former has been selected by the 
 cell. 
 
 We may inquire why the cell does not form its own ferments. 
 In certain cases it may do so ; the existence of the endocom- 
 plements lends support to this view. But it is readily conceivable 
 that it may be more economical in every way for the animal 
 to limit this process of enzyme formation to certain specialized 
 cells or tissues, just as pepsin is formed by the cells of the gastric 
 glands. This is probably the case, and all experience goes to 
 show that the cells to which this duty is delegated are the 
 leucocytes. 
 
 Ehrlich, therefore, conceives of the cytotoxic mechanism of 
 amboceptors and complements as being adapted, in the first place, 
 to cell nutrition, and, secondarily, to the defence of the body by 
 bringing about solution of foreign cells. Metchnikoff 's views may 
 be summarized as follows: He holds that both the substances 
 taking part in cytolysis are ferments, and that both are adapted 
 for intracellular digestion. His views on phagocytosis will be 
 discussed subsequently, and here it will be sufficient to give the 
 merest outline. He has shown that in the lowest Metazoa e.g., 
 in the Coelenterata digestion is an intracellular process entirely ; 
 the hypoblastic cells lining the alimentary canal seize food particles 
 from the lumen and digest them, but form no secretion like those 
 of the higher animals. In the cells which have seized and are 
 digesting food particles in this way he was able to demonstrate 
 
BACTERIOLYSIS AND ALLIED PHENOMENA 167 
 
 ferments allied to pepsin or trypsin, which had evidently been 
 formed to digest the ingested food material. He then showed 
 the importance of this process in the absorption of cells, etc., in 
 the tissues of higher animals, and demonstrated that the two 
 processes are altogether similar. Hence his views on the nature 
 of the cytolysins arise easily and naturally. He holds that com- 
 plement, or, as he calls it, cytase, is the digestive secretion of 
 the leucocytes, and that, under ordinary circumstances, it is 
 retained within the leucocytes ; it is only set free when leucocytes 
 are dissolved (phagolysis), either as the result of an injection of a 
 foreign substance or in the process of clotting (this theory, as we 
 shall show, is held by many other authorities). Thus in Pfeiffer's 
 phenomenon the first result of the injection is phagolysis, and the 
 ferments set free in the process immediately attack the cholera 
 vibrios. Metchnikoff has proved clearly that the injection of 
 almost anything into the peritoneal cavity leads to a diminution in 
 the number of leucocytes, but the proof of the existence of 
 phagolysis under these circumstances is less convincing. Cytase, 
 therefore, is a digestive ferment adapted to deal with large masses 
 of food substance rather than with molecules, as Ehrlich supposes, 
 and is normally intracellular, being formed especially for intra- 
 cellular digestion. 
 
 Metchnikoff sees in amboceptor, or fixator, a substance alto- 
 gether analogous to enterokinase, and acting, like it, as an 
 accessory digestive ferment, which has for its object the linking 
 of the more potent ferment to the food to be digested. He 
 regards it also as being formed in the leucocytes, and for this 
 reason (amongst others, which will be enumerated subsequently) : 
 He states that the amount of fixator or amboceptor produced 
 is proportional to the amount of phagocytosis which occurs during 
 the absorption of the antigen. For example, he states that when 
 defibrinated goose blood is injected into the guinea-pig sub- 
 cutaneously there is but little phagocytosis, the blood being 
 dissolved extracellularly, and but little immune body is produced ; 
 but when the injection is made into the peritoneum there is much 
 phagocytosis and much development of antibody. There is cer- 
 tainly a remarkable difference between the various tissues as 
 origins of antibodies, and, as a rule, the subcutaneous and con- 
 nective tissues are most potent in this respect, the peritoneum 
 next, and the circulating blood worst ; but there is no sufficient 
 evidence to show that this depends on the amount of phagocytosis 
 
168 METCHNIKOFF'S VIEWS 
 
 which occurs in these different situations. The site of the forma- 
 tion of an immune body will be referred to again. 
 
 Thus, whilst Ehrlich sees in the substances taking part in 
 cytolysis evidence of the nutrition of the living molecules of cell 
 protoplasm, Metchnikoff sees in them ferments which were also, 
 in their first appearance in the animal economy, designed for the 
 elaboration of the nourishment of the body or of certain cells 
 therein, the difference being that, according to him, their primitive 
 function was the digestion of large particles as well as of molecules. 
 But in the highly-organized animals with which we have chiefly 
 to deal this function has been changed, for in them phagocytes do 
 not ingest bacteria and red corpuscles for the sake of the nourish- 
 ment they contain, but to rid the body of invaders ; the body as a 
 whole is nourished through the agency of other extracellular 
 digestive ferments, which are secreted into the alimentary canal. 
 It follows, therefore, that cytase is unnecessary in the circulating 
 blood, and, on this supposition, does not usually exist in that situa- 
 tion. Metchnikoff admits that immune body does so exist and has 
 indeed supplied a remarkable proof of the fact, since he showed 
 that the spermatozoa of immunized guinea-pigs are combined 
 with immune body, and only need the addition of fresh serum for 
 cytolysis to occur. Cytase and fixator, on MetchnikorPs explana- 
 tion of the phenomena, must be regarded as substances which in 
 the evolution of the animal kingdom were first developed as 
 digestive juices, but which now are entirely subservient to the 
 defence of the body against invaders. According to Ehrlich, they 
 are both in daily use in nourishment, and their defensive function 
 is of less importance than their value in cell nutrition. In either 
 case, the conception of immunity, as being fundamentally a process 
 of nutrition, is a most striking one. 
 
 We have now to turn to a phenomenon which is of the highest 
 theoretical importance, and which bids fair to be of great practical 
 value in diagnosis. We have already referred to the fact that 
 Bordet, the chief advocate of the Unitarian theory of complement, 
 showed that if red corpuscles be sensitized with amboceptor and 
 added to fresh serum, the latter is deprived of all complementary 
 activity. The same phenomena occur with sensitized bacteria. 
 When, for instance, typhoid bacilli which had been acted on by 
 heated typhoid serum were added to fresh blood, allowed to act, and 
 then centrifugalized, it was found that the supernatant fluid had no 
 longer the power of dissolving red corpuscles sensitized by suitable 
 
BACTERIOLYSIS AND ALLIED PHENOMENA l6g 
 
 amboceptor. These facts are admitted by Ehrlich, but he shows 
 that they do not constitute any real evidence against the pluralist 
 conception, since in some cases it may be shown that, though all 
 complements are absorbed, this may be at different rates, and by 
 stopping the process at a proper time, some may have disappeared, 
 whilst others are left. 
 
 The practical importance of these observations arises from the 
 fact that they give us a method by which we can demonstrate the 
 presence or absence of an antibody to a given antigen, or of an 
 antigen to a given antibody. For example, the sensibilatrice or 
 amboceptor for the tubercle bacillus is very difficult to demonstrate, 
 since the organism is so resistant that it is never obviously dis- 
 solved, even partially, in the most potent serum we can obtain. 
 Nor are bactericidal experiments more promising, owing partly to 
 the resisting power of the organism and partly to the technical 
 difficulties. The only evidence (apart from the presence of 
 agglutinins) which we have in favour of the formation of specific 
 antibodies to tuberculosis is derived from an application of Bordet's 
 phenomenon. The experiments were carried out as follows : A 
 guinea-pig was injected with the bacillus of avian tuberculosis, to 
 which it is but slightly sensitive, and the blood was examined by 
 mixing it with an emulsion of the bacilli. If amboceptors were 
 present they would combine with the bacilli, and draw to them all 
 the complements of the fluid, which would thus lose its power of 
 activating suitably sensitized red corpuscles. This was found to 
 occur, and Bordet and Gengou deduced that the guinea-pig had 
 formed antibodies to the slightly virulent tubercle bacilli. When, 
 on the other hand, the guinea-pigs were injected with virulent 
 human tubercle bacilli, no such antibodies could be demonstrated. 
 
 As an example of the recognition of an antigen by means of 
 Bordet's phenomenon, we shall quote Bruck's demonstration of 
 the presence of tuberculin, or at least of some derivative of the 
 tubercle bacillus, in the blood of patients suffering from general 
 tuberculosis. Here the problem is changed. We have a specimen 
 of serum which we want to test, not for the antibody, but for the 
 antigen. The procedure is as follows : The serum is heated and 
 mixed with a serum known to contain antibodies to the tubercle 
 bacillus (antituberculin of Hochst). To this mixture is added 
 fresh guinea-pig's serum, and lastly sensitized red corpuscles. It 
 is found that no haemolysis takes place. In a control experiment, 
 in which normal human blood took the place of that from the 
 
170 THE BORDET-GENGOU PHENOMENON 
 
 patient, haemolysis occurred ; it followed, therefore, that the 
 patient's blood contained derivatives of the tubercle bacillus, or 
 (as we may fairly say) its toxins. 
 
 The technique of these experiments is somewhat difficult, but 
 the results have been so important, and the method seems so 
 likely to play a part of importance in clinical diagnosis, that it will 
 be discussed in a further chapter. 
 
 Gengou's phenomenon is similar to Bordet's. It requires a 
 little anticipation of facts to be discussed subsequently concerning 
 the precipitins. These are antibodies obtained by the injection 
 of proteid solutions, and having the power of uniting with these 
 proteids to form insoluble precipitates. Gengou showed that in 
 this combination of antigen and antibody the same absorption of 
 complement occurred as in the case of sensitized red corpuscles 
 or bacteria. The process is an extraordinarily delicate one, and 
 it has been shown to be demonstrable with as little as o-oooooi c.c. 
 of the antigen (in this case normal human serum), and may occur 
 when the serum used is so dilute that no visible precipitation 
 occurs. 
 
 It appears, further, that this process of fixation of complement 
 is a general one, occurring whenever an antigen and its antibody 
 unite, whether the antigen occurs in solid or liquid form, and 
 whether the resulting compound forms a precipitate or remains in 
 solution. It has been shown by Nicolle and by Armand-Delille 
 to occur in the neutralization of tetanus and diphtheria toxins by 
 their appropriate antitoxins, and by Pozerski in the interaction of 
 papain and its antiferment. It has been shown by Guedini that 
 when hydatid fluid is mixed with the serum of an animal which 
 has been injected therewith a similar phenomenon occurs, and 
 this fact has been suggested and applied by Weinberg, Parvu, and 
 Lanbry to the diagnosis of hydatid cysts in man. 
 
 The theoretical importance of this phenomenon arises from the 
 light which it throws on the difficult subject of complementoids 
 and anticomplements. We have seen that heated serum has no 
 complementary activity, but that its injection into suitable animals 
 appears to call forth the presence of anticomplements. A little 
 consideration will show that this apparent anticomplementary 
 action can be explained equally well by the absorption of the 
 complements in a specific precipitate. We will take a particular 
 case. 
 
 Goat serum heated to 56 C., and therefore containing no active 
 
BACTERIOLYSIS AND ALLIED PHENOMENA 
 
 complement, is injected into a rabbit. This is supposed to develop 
 an anticomplement in virtue of the complementoids it contains ; 
 we know that it also develops a precipitin which combines 
 with the proteids of' goat serum, and in doing so entangles any 
 complement which may be present. Now the complement of 
 goat serum can dissolve ox corpuscles when sensitized by a 
 suitable amboceptor (e.g., serum of a rabbit which has been 
 injected with ox corpuscles). When, however, the serum of the 
 normal goat is mixed with that of the rabbit which has been 
 
 <=V<7-txT"~ 
 
 injected with rabbit serum and used in this way, no haemolysis 
 occurs. This Ehrlich explains on the supposition that the rabbit 
 serum contains anticomplement, but it is also explicable, as 
 Moreschi and Gay have shown, on the supposition that the com- 
 plements are all absorbed in the precipitate (perhaps an invisible 
 one) formed in the mixture. 
 
 It is obvious that either interpretation may be the correct one, 
 or that both processes may come into play. Moreschi has 
 investigated the process further, and his results tend to show that 
 the experiment is best explained on the absorption of complement 
 theory, and not by the presence of anticomplement. One of his 
 experiments, and that very ingenious, will be described. He 
 injected a rabbit with hen's egg albumin, and obtained a precipitin 
 to that substance ; this he found to act as an anticomplement 
 to fowl serum, but not to that of the rabbit, guinea-pig, or goat, 
 to which, of course, it was not a precipitin. He found, however, 
 that if he added to any of these latter sera a minute trace (^30 ws 
 of its volume) of egg-albumin, it appeared to become so, the 
 explanation being that a precipitate was formed, and that the 
 complements were entangled in it. 
 
 The phenomenon has also been invoked to explain some extremely 
 interesting researches of PfeifTer and Friedberger, who thought 
 they had demonstrated the presence of antibacteriolytic substances 
 in normal serum. They prepared a mixture of cholera vibrios 
 and normal serum, which they allowed to stand for some time, and 
 then removed the bacteria by centrifugalization. This would 
 remove any amboceptor which the serum might contain, and they 
 thought that they could demonstrate that an anti-amboceptor 
 still remained. They added to the serum thus prepared some 
 anticholera serum in suitable amount and a lethal dose of cholera 
 vibrios, and injected the whole into an animal, which invariably 
 died ; the amount of antiserum was sufficient to protect it if no 
 
172 THE BORDET-GENGOU PHENOMENON 
 
 serum which had been exhausted by the action of cholera vibrio 8 
 had been added. The anti-amboceptor which thus appeared to 
 be present in normal serum seemed to be strictly specific ; a 
 serum which had been weakened by the action of cholera vibrios 
 had no action on antityphoid serum, and vice versa. 
 
 Besredka attempted to explain these findings on the assumption 
 that " free receptors " passed from the bacteria into the normal 
 serum, and, when subsequently mixed with antiserum, combined 
 with it, and so prevented its union with the living bacteria. They 
 showed, in reply, that normal saline solution had no such action ; 
 but this is hardly conclusive, since receptors are dissolved from 
 the bacteria much less powerfully in normal saline than in serum. 
 
 According to Sachs, who demonstrated a similar occurrence in 
 haemolysis, the inhibiting substance which actually occurs in the 
 serum is an anticomplement. Gay, however, believes that the 
 whole series of phenomena can be explained by the absorption of 
 the complements in a specific precipitate. Thus, in Sach's 
 experiment normal rabbit serum heated to 55 C. was treated with 
 sheep's corpuscles. It was then removed, and added to a mixture 
 of heated serum immune to sheep's corpuscles (obtained from a 
 rabbit treated with sheep's corpuscles), fresh guinea-pig's serum, 
 and sheep's corpuscles. No haemolysis occurred, as it did in the 
 control experiment, in which no " exhausted " normal serum had 
 been added. His explanation was that the normal serum con- 
 tained amboceptors which combined with sheep's corpuscles, 
 leaving other amboceptors which have a greater affinity for 
 complement than those combined with the corpuscles used as a 
 test. Gay's explanation (which is almost certainly the correct 
 one) is quite similar to Moreschi's explanation of the anti- 
 complements. He believes that the immune serum contains also 
 a precipitin to sheep's serum, and that in the " exhaustion " of the 
 heated normal serum by the sheep's corpuscles some sheep's 
 serum was added to it, the corpuscles in Sach's experiment not 
 having been thoroughly washed. Thus a serum precipitate was 
 formed, and the alexins or complements combined therewith, so 
 that no haemolysis occurred on the subsequent addition of sensitized 
 corpuscles. He showed that if thoroughly washed corpuscles 
 were used for the exhaustion the phenomenon did not occur, and 
 gives other experimental proof. 
 
 Gay has also attempted to explain the process of deviation of 
 the complements in bacteriolysis (the Neisser-Wechsberg pheno- 
 
BACTERIOLYSIS AND ALLIED PHENOMENA 173 
 
 menon) in a similar way. The process is described at length on 
 a subsequent page, but consists essentially in this : when bacteria 
 are acted on by an excess of amboceptor, no bacteriolysis may 
 occur, although sufficient complement is present. Gay's researches 
 on this point have not been fully published at the time of writing, 
 but he apparently thinks that soluble portions receptors may 
 pass off from the bacteria into the fluid in which the latter are 
 suspended, form a precipitate with the serum, and absorb the 
 complement. Much evidence will be required before this can 
 be established ; it might perhaps explain the phenomenon in 
 vitro, where the available amount of complement is limited, but 
 the effect is also demonstrable in the peritoneum, where we should 
 expect as much complement to be forthcoming as is required. 
 
 There is no doubt that the discovery of the absorption of the 
 complements in serum precipitates has rendered uncertain many 
 of the deductions in the process of haemolysis which have emanated 
 from the Ehrlich school, and the whole series of phenomena 
 requires re-investigation in the light of the observations of 
 Moreschi and Gay. 
 
 Experiments with bacteriolytic sera soon showed that, though 
 they protected against specific infections, they only did so in 
 certain doses. It is, of course, readily understandable that too 
 small an amount might be without action, but it was also found 
 that too large a dose might be equally inefficacious. Thus, Loffler 
 and Abel obtained a serum which protected against B. coli, and 
 found that when a lethal dose of this organism was given the 
 animal was only protected when it had received between 0-02 and 
 0*25 c.c. of serum, larger as well as smaller doses being equally 
 without effect. Similar results have been observed with sera 
 against cholera, dysentery, malignant oedema, and other organisms, 
 and seem to be general in dealing with bacteriolytic sera as 
 opposed to antitoxin. 
 
 They can also be obtained in vitro, and since this was first 
 shown by Neisser and Wechsberg, the phenomenon is often called 
 after them. Their method was as follows : They worked with 
 several organisms, amongst others with V. Afetchnikovi, and with 
 the serum of a rabbit which had been immunized against this 
 organism, and had acquired strong bactericidal powers. Equal 
 amounts of a broth culture of the vibrio were placed in a series 
 of test-tubes, and to each a dose of heated immune serum was 
 added. In all cases a uniform amount of normal rabbit serum 
 
174 
 
 THE NEISSER-WECHSBERG PHENOMENON 
 
 (complement) was added, and the mixture made up to constant 
 volume with sterile normal saline solution. It was then incubated 
 for three hours at 37 C., and finally 5 drops from each tube 
 were plated out on agar and incubated. The result is shown on 
 the table. 
 
 Amount of Broth 
 Culture. 
 
 Heated Immune 
 Serum. 
 
 Fresh Normal 
 Serum. 
 
 Colonies Developing. 
 
 
 I C.C. 
 
 
 Infinity. 
 
 
 O'5 C.C. 
 
 
 Infinity. 
 
 
 0-25 c.c. 
 
 
 Many thousands. 
 
 
 O'l C.C. 
 
 
 Several hundreds. 
 
 
 o'O5 c.c. 
 
 
 About 100. 
 
 rTHHJ c - c - in a U 
 
 0-025 c - c - 
 
 0-3 c.c. in all 
 
 About 50. 
 
 cases. 
 
 o*oi c.c. cases. 
 
 None. 
 
 
 0-005 c.c. 
 
 
 None. 
 
 
 0-0025 c.c. 
 
 
 About 100. 
 
 
 O'OOI C.C. 
 
 
 Infinity. 
 
 
 o'ooo5 c.c. 
 
 
 Infinity. 
 
 This shows that when g-^Vzr c.c. of a one-day-old broth culture was 
 treated with 3 c.c. of fresh normal serum (complement) and in- 
 creasing doses of immune serum, there was no appreciable bacteri- 
 cidal action when more than 0-25 c.c. of the latter was used, or 
 with less than o-ooi c.c. When the amount was between o-i and 
 0-025 c.c., or about 0-0025 c.c., there was appreciable action, and 
 when it was between o-oi and 0-005 c.c. it was complete. 
 
 The same authors also demonstrated a very remarkable phe- 
 nomenon of exactly the same nature. A normal serum which has 
 a certain amount of bactericidal action (owing to the presence of 
 complement and of a small quantity of amboceptor) may have its 
 action increased, diminished, or nullified by the addition of a 
 powerful immune serum. This is shown by the following table, 
 which demonstrates the action of normal guinea-pig serum on 
 V. Nordhafen by itself and after the addition of variable doses 
 of heated immune serum. 
 
 This table shows that there is a definite relation between the 
 amounts of amboceptor and of complement which have to be 
 present to produce a maximal effect. In the case of the normal 
 serum, it is evident that there is more complement than can be 
 used by the amount of amboceptor present, and that the bacteri- 
 cidal action of the mixture increases as immune serum is added. 
 0-05 c.c. of normal serum was without obvious bactericidal effect 
 on c.c. of the culture, whereas the same amount plus 0*01 c.c. of 
 
BACTERIOLYSIS AND ALLIED PHENOMENA 
 
 175 
 
 heated immune serum destroyed a very large number of the 
 bacteria. When, however, the immune serum is added in larger 
 proportion than this it does harm instead of good: 0-5 c.c. of 
 
 
 
 Number of Colonies after addition of Variable Amounts of 
 
 Amount of 
 
 Amount of 
 
 Heated Immune Serum. 
 
 
 Normal 
 
 
 Culture. 
 
 Serum. 
 
 No Serum 
 added. 
 
 i c.c. added. 
 
 o'i c.c. added. 
 
 0*01 c.c. added. 
 
 r-b- c.c. 
 
 I C.C. 
 
 None. 
 
 Many 
 
 A few. 
 
 None. 
 
 in all 
 
 
 
 thousands. 
 
 
 
 cases. 
 
 0-5 c.c. 
 
 None. 
 
 Almost in- 
 
 About 100. 
 
 None. 
 
 
 
 
 finity. 
 
 
 
 
 0-25 c.c. 
 
 A few. 
 
 Infinity. 
 
 Several 
 
 A few. 
 
 _ 
 
 
 
 
 hundreds. 
 
 
 
 O'l C.C. 
 
 Several 
 
 Infinity. 
 
 Infinity. 
 
 About loo. 
 
 
 
 thousands. 
 
 
 
 
 
 0-05 c.c. 
 
 Infinity. 
 
 Infinity. 
 
 Infinity. 
 
 Many hun- 
 
 
 
 
 
 dreds. 
 
 
 o - o25 c.c. Infinity. 
 
 Infinity. 
 
 Infinity. 
 
 Infinity. 
 
 fresh serum completely sterilizes the amount of the culture used, 
 whereas after the addition of i c.c. of immune serum its action 
 is barely noticeable. An excess of amboceptor shields the bacteria 
 from the solvent action of the complements. These test-tube 
 experiments explain the results obtained in vivo, and enable us to 
 form some idea as to the mechanism of the process. 
 
 Neisser and Wechsberg offered the following explanation : They 
 assume that the molecules of complement and amboceptor unite 
 in the mixture before the latter has attached itself to the bacteria. 
 Owing to the excess of amboceptor, there will not be sufficient 
 molecules of complement to go round, and it will follow that a 
 variable proportion of molecules of amboceptor will be uncom- 
 plemented. Unless we suppose that the union of the complement 
 has changed the affinity of the cytophile group of the amboceptor 
 for the bacterium, it will follow that not all the amboceptors which 
 attach themselves to the bacteria will be charged with comple- 
 ment, and therefore able to exert a bacteriolytic or solvent action. 
 To take a concrete case, let us suppose that there are twice as 
 many molecules of amboceptor as of complement. Half the 
 molecules of the former will be complemented, and it will follow 
 that, though all the (appropriate) receptors of the bacterium are 
 occupied by amboceptor, only half of these will have their comple- 
 mentophile groups occupied, and this may not be enough to 
 injure it. 
 
176 DEVIATIONS OF COMPLEMENT 
 
 They also imagine the possibility of the combination altering 
 the affinity of the cytophile group of the amboceptor for the cell. 
 It may diminish it, in which case fewer complemented molecules 
 
 FIG. 42. FIRST POSSIBILITY. 
 
 The affinity of the amboceptor for complement is unaltered, as a result of 
 the union of the former with a bacterium. 
 
 of amboceptor would attack the bacterium than ever, and the 
 phenomenon of deviation of the complements would be even more 
 marked. The union might also (conceivably) increase this affinity ; 
 in this case the amboceptors which were complemented would 
 
 FIG. 43. SECOND POSSIBILITY. 
 
 The affinity of the amboceptor for complement is diminished, as a result of the 
 union of the former with a bacterium. 
 
 seize on the bacterial receptors, to the exclusion of those which 
 were not, and the phenomenon of deviation of the complements 
 would not occur. This Neisser and Wechsberg think might occur 
 in the case of haemolysis, for in this process deviation has not 
 
 FIG. 44. THIRD POSSIBILITY. 
 The affinity of the amboceptor for complement is increased. 
 
 been demonstrated, and solution occurs even when there is a 
 great excess of amboceptor. In the case of haemolysis by snake 
 venom, however, Myers and Stephens showed that the process 
 might only take place when medium doses of venom were added, 
 
BACTERIOLYSIS AND ALLIED PHENOMENA 177 
 
 amounts too large or too small being without effect. This was 
 also corroborated by Kyes and Sachs, who showed that in 
 presence of an excess of venom (amboceptor) the addition of a 
 haemolyzing amount of complement might be without effect. 
 Now Kyes had already shown that venom does unite directly with 
 one of the substances (lecithin) which can complement it, and 
 this appeared strong evidence in favour of Neisser and Wechs- 
 berg's explanation. But Noguchi afterwards showed that the 
 protective action of large doses of venom protects the red cor- 
 puscles against the action of haemolytic agents other than comple- 
 ments. Corpuscles so treated are not dissolved by distilled water 
 or by tetanolysin. It appears, therefore, that strong solutions of 
 venom have a curious hardening effect on red corpuscles, render- 
 ing them resistant to haemolytic agents of all sorts. This effect 
 is apparently entirely different from the protection of a bacterium 
 by excessive doses of amboceptor, and should not be used as 
 evidence to support Neisser and Wechsberg's explanation. 
 
 Morgenroth has attempted to explain the absence of the devia- 
 tion of the complements in haemolysis by alleging that in this case 
 the amboceptors and complements do not unite until the former 
 have united with the red corpuscles ; this, of course, is the point 
 at issue. He finds that by adding anti-amboceptor (for the cyto- 
 phile group) to a mixture of amboceptor and complement the 
 phenomenon of deviation can be reproduced. But his experiment 
 can probably be explained by means of Gay's observations, to be 
 explained subsequently, and in any ease his anti-amboceptor would 
 only act as a free receptor of a red corpuscle, and the amboceptor 
 attached to it would be in the same condition as one anchored to 
 a corpuscle, which we know can absorb complement. In any case, 
 his experiment proves little or npthing. 
 
 Some experiments by Meakins afford the best evidence in 
 favour of the alteration in combining affinity undergone by ambo- 
 ceptor after union with complement, and call for short reference, 
 though their interpretation is somewhat uncertain. Meakins 
 experimented with corpuscles which had been very thoroughly 
 washed so as to remove all trace of serum. The importance of 
 this precaution will appear subsequently. He found that when 
 he added a large dose of heated immune serum and a small dose 
 of normal serum (complement) to corpuscles thus washed, and 
 allowed them to act, no haemolysis occurred. The corpuscles, 
 however, were sensitized ; for when they were centrifuged down, 
 
 12 
 
178 NEISSER-WECHSBERG PHENOMENON IN HAEMOLYSIS 
 
 washed, and treated with more normal serum, haemolysis took 
 place. It thus appears that, in the first instance, the corpuscles 
 took up only those amboceptors which had no attached comple- 
 ment. It would appear, therefore, that amboceptor may unite 
 with complement whilst free, and that when it has done so its 
 affinity for the corpuscle is diminished. This is one of the 
 conditions which Neisser and Wechsberg imagined, and which 
 they showed would make the phenomenon of deviation even more 
 marked. And Meakins shows, though not in a very clear way, 
 that it does actually take place with corpuscles that are well 
 washed. The reason why it does not take place under ordinary 
 circumstances is explicable on the basis of Gengou's reaction. 
 
 But this theory cannot be regarded as entirely satisfactory. It 
 assumes, in the first place, a direct union between complement 
 and amboceptor before the latter has united with the cell or 
 bacterium, and of this there is no definite evidence apart from 
 this phenomenon. Ehrlich, it is true, supposes a feeble union 
 between the two which easily dissociates, even at low temperatures, 
 so that a cell which has been placed in contact with a mixture of 
 the two at o will become saturated with amboceptor devoid of 
 complement. But this appears to be a fatal objection to Neisser 
 and Wechsberg's explanation ; for if the hypothetical amboceptor- 
 complement combination dissociates readily at a low temperature, 
 it should do so still more at a high one. Now the receptor- 
 amboceptor-complement will not dissociate, or only to a very 
 slight extent, since lytic action will at once commence. It follows 
 that the amboceptor-complement molecules will gradually all 
 dissociate, and the complements will sooner or later find their 
 way to the anchored amboceptors, and there remain. The process 
 may be delayed, but after a time all the attached amboceptors 
 will become complemented. And if, as seems implied in Ehrlich's 
 writings, the affinity of the complementophile group becomes 
 raised in virtue of the union of the cytophile group, with its 
 appropriate receptor, this process will take place all the more 
 quickly. 
 
 It appears much more likely that the Neisser- Wechsberg 
 phenomenon is an example of a reaction of a kind which has 
 already attracted much attention, and which may possibly modify 
 fundamentally our views on the antibodies, toxins, etc. They will 
 be referred to again when we deal with the agglutinins, and it is 
 sufficient to say here that in other cases, when there is no question 
 
BACTERIOLYSIS AND ALLIED PHENOMENA 179 
 
 of two bodies, and therefore of the validity of Neisser and 
 Wechsberg's explanation, antibodies and other substances have 
 their action diminished or suspended when they are present in 
 excess ; thus with a great excess of agglutinin there may be no 
 agglutination. And Detre and Sellei have shown that phenomena 
 which are apparently somewhat similar occur in the haemolysis 
 induced by mercury perchloride. 
 
 In view of the important role in immunity which must be 
 ascribed to alexin and, as we shall see, it acts not only as a 
 bacteriolytic agent, but also plays a part of great importance in 
 phagocytosis an inquiry into its source becomes of paramount 
 importance. The theory was put forward very early in the 
 history of the subject, before the mode of action of alexin and its 
 dependence on immune body were understood, that it was derived 
 from the leucocytes, either by a process of secretion or disintegra- 
 tion. This was first suggested by Hankin, who prepared a 
 substance having bacteriolytic powers from extracts of the 
 lymphatic glands and spleen ; this, however, was probably not a 
 true alexin. Experimental evidence of a more convincing kind 
 soon followed ; thus Denys, Van de Velde, and others, caused the 
 production of aseptic exudates rich in leucocytes by injecting 
 various irritants into the pleural cavity of animals, and found that 
 the more leucocytes present the greater the bactericidal action of 
 the fluid. Buchner performed similar experiments, using aleuron 
 emulsions to produce the exudate, and found that the richly 
 cellular material had a greater bactericidal effect than blood or 
 serum ; in his experiments he avoided the possibility of phago- 
 cytosis (which probably explained some of the results of other 
 investigators) by freezing the fluids before determining their 
 potency. A large number of other researches were made about 
 this time, and all pointed in the same direction, and in the early 
 nineties it was fairly generally held that the alexins were given off 
 in some way or another by the leucocytes. This view was strongly 
 held by Metchnikoff, whose microcytase must be regarded as 
 identical with the alexin or complement which acts on the 
 bacteria; and this microcytase he holds to be produced by the 
 polynuclear leucocytes under certain circumstances, which will be 
 discussed subsequently. 
 
 With the discovery of the compound nature of the bactericidal 
 substances the question entered on a new phase, and new methods 
 of investigation were seen to be necessary. Since then very 
 
 12 2 
 
l8o ORIGIN OF COMPLEMENT 
 
 many researches have appeared, the point at issue being whether 
 the alexin or complement is formed from the polynuclear leuco- 
 cytes (for there is no evidence pointing to the lymphocytes). 
 Some of the most important of these must be briefly summarized. 
 
 1. Various observers have investigated the relation between 
 the number of leucocytes in a given fluid and the amount of 
 complementary action. Thus Bulloch, working with hsemo- 
 complement, found its amount closely proportionate to the number 
 of polynuclear leucocytes. His figures are very convincing, and 
 the only alternative explanation of his results is that the injection 
 of any substance which stimulates leucocytosis stimulates at the 
 same time the hypothetical complement-producing organ or tissue. 
 Similar observations have been made under natural conditions 
 by Longcope, who found that in disease in man the occurrence 
 of hyperleucocytosis is accompanied by a rise in the amount of 
 complement. On the other hand, Guseff's investigations on 
 similar lines were entirely negative, and he was unable to trace 
 any parallelism between the two ; and Briscoe, dealing with 
 peritoneal fluids, arrived at a similar result. These results may 
 be taken as typical of the whole. A series of experiments by an 
 able worker is apparently conclusive in one direction, and appears 
 to establish the point beyond controversy, but is immediately met 
 with another on similar lines, pointing to a diametrically opposite 
 result. 
 
 Other investigations on similar lines are those of Bordet, who 
 found that the oedema fluid poured out in passive congestion in 
 an immunized animal a fluid poor in cells contains immune 
 body, but no complement, and that the aqueous humour is entirely 
 devoid of both substances. This latter fact has been corroborated 
 by Levaditi, who adds that when, after tapping, the aqueous 
 humour becomes rich in leucocytes, it acquires alexin at the same 
 time. 
 
 2. Many observers have attempted to prepare complement 
 from emulsions of polynuclear leucocytes, from bone-marrow, 
 or from lymphoid organs. The results have been diverse in the 
 extreme, and this diversity appears due in part to the fact that 
 substances other than true alexins or complement may cause, 
 or play some part in the causation of, both haemolysis and 
 bacteriolysis. In particular, we may refer to the important role 
 of lecithin and its allies in haemolysis, and possibly in the destruc- 
 tion of bacteria also. It is quite possible, for instance, that the 
 
BACTERIOLYSIS AND ALLIED PHENOMENA l8l 
 
 haemolytic complement prepared by Levaditi from autolyzed 
 lymphoid tissue was one of these lipoid substances ; it was 
 soluble in alcohol and thermostable. The substance prepared by 
 Morgenroth and Korschun from extracts of various organs was 
 probably similar in nature, since they showed it to be thermostable, 
 and not to give rise to the production of antibodies on injection. 
 It may be at once admitted, as pointed out by Conradi, that 
 autolytic changes occurring in various organs give rise to the 
 production of bactericidal substances ; these may, perhaps, be 
 organic acids or other simple substances, but they do not help us 
 in the inquiry as to whether complement (having the power to 
 dissolve sensitized bacteria or red corpuscles) is formed from 
 the leucocytes. 
 
 The most important researches on this point are those of 
 Schattenfroh and Petrie, and they are mutually contradictory. 
 Schattenfroh washed the leucocytes thoroughly in saline solution, 
 added it to the heated serum, and obtained a bactericidal effect. 
 His results were corroborated by Lastchenko. Petrie's researches 
 were carried out with great care, and he was especially particular 
 to remove every trace of complement adhering to the leucocytes 
 by repeated washing in normal saline solution. (Ascher had 
 previously shown that some trace might remain when the process 
 had only been carried out three times.) The leucocytes were 
 obtained from aleuron exudates, and after washing they were 
 frozen to the temperature of liquid air, and ground to an im- 
 palpable powder, the method used being that of Macfadyen for 
 the preparation of endotoxin. His results were entirely negative, 
 and he failed entirely to reactivate heated bactericidal serum by 
 any of his extracts. Lambotte failed equally, working on lines 
 more like those of Schattenfroh. 
 
 Petrie's experiments seem conclusive on the point which he 
 investigated, and it seems quite certain that polynuclear leucocytes 
 do not contain alexin or complement as such ; they do not, how- 
 ever, negative the possibility that they may contain a precursor of 
 this substance, which may either be secreted or set free during 
 the natural solution of the leucocyte, in either case being converted 
 into an active form during the process. Both these views have 
 been widely held, and we shall discuss them later. 
 
 3. In the process of coagulation of the blood large numbers of 
 the leucocytes, and especially the polynuclears, are disintegrated 
 and partially dissolved, and these disintegrated leucocytes are 
 
l82 ORIGIN OF COMPLEMENT 
 
 generally accepted as the source of the fibrin ferment. If the 
 complements are also set free during the solution of the leucocytes, 
 there should be far more present in the serum than in the circu- 
 lating plasma ; it might happen, indeed, that the plasma might be 
 devoid of any complementary action, though this does not neces- 
 sarily follow. 
 
 The difficulty of investigating this subject is very great, and 
 the evidence is mostly indirect. As far as it goes, it seems to 
 point to the fact that the plasma is alexin-free, or, at any rate, 
 poorer than the serum. The main direct researches into this 
 subject are those of Gengou and Falloise, and here, again, they 
 point in a diametrically opposite direction. Gengou (whose results 
 have been accepted in their entirety by Metchnikoff) worked with 
 plasma obtained by preventing coagulation by collecting the blood 
 in paraffined tubes and centrifugalizing forthwith a method cer- 
 tainly less open to objections than any dependent on the addition 
 of anticoagulants, but difficult in execution. He found that plasma 
 thus prepared was devoid of bactericidal action, though the serum 
 from the same animal possessed it. In some cases the plasma 
 betrayed some action, perhaps because a partial coagulation had 
 taken place, but it was always less potent than the serum. These 
 results have been opposed by numerous investigators, using various 
 methods of inhibiting coagulation. Thus, Falloise used intra- 
 venous injections of peptone, the addition of sodium oxalate or 
 fluoride ; Petterson used oxalate and citrate, and Hahn histon, 
 and in all cases no appreciable difference in alexin content between 
 the plasma and serum was found. But this is hardly a fair test. 
 Peptone is generally believed to be a leucotoxic or leucolytic 
 agent ; and in the case of the citrated or oxalated blood it is 
 obvious that (if leucocytes are the origin of fibrin ferment) some 
 leucolysis has occurred, since the cell-free fluid coagulates on the 
 addition of calcium. Falloise also worked with paraffin tubes on 
 Gengou's method, and found that the plasma contained as much 
 haemolytic complement as the serum. Lastly, both he and 
 Lambotte worked with plasma obtained by centrifugalizing (or 
 by allowing sedimentation to occur in) the blood in a vein isolated 
 between two ligatures and kept at a low temperature, and with 
 the same results. Delezenne, however, criticizes these results by 
 pointing out that plasma obtained in this way coagulates instantly 
 in glass tubes at the ordinary temperature, a phenomenon in- 
 dicating that fibrin ferment is present, and that leucocytes have 
 
BACTERIOLYSIS AND ALLIED PHENOMENA 183 
 
 become disintegrated, or have at least performed an act of secretion 
 which they do not perform in the living blood. The conditions, 
 therefore, are not altogether natural. 
 
 We are forced to rely to a large extent on indirect evidence, and 
 this, as far as it goes, is strongly in favour of Gengou's view. 
 
 4. Ainley Walker's experiments dealt with the amount of com- 
 plement present in the successive amounts of serum squeezed out 
 from a clot, and showed that this gradually rises, the first few drops 
 containing but little, and the quantity gradually increasing until it 
 attains its maximum in about twenty-four hours. Other explana- 
 tions are possible, but it is at least probable that the cause of this 
 increase is the destruction of the leucocytes which is known to 
 occur during the process of coagulation, and that the formation of 
 complement is quite analogous with that of fibrin ferment. The 
 serum first formed is weak in complement, the destruction of the 
 leucocytes having only just commenced ; and it seems a fair 
 deduction that, if we could examine the plasma when no leucolysis 
 has occurred, we should find that it contains much less, or none 
 at all. 
 
 This view is supported by Levaditi in a series of researches on 
 the fate of cholera vibrios injected into the circulatory system of 
 immunized guinea-pigs, care being taken to avoid as far as possible 
 the destruction of leucocytes. Under these circumstances he 
 finds that the Pfeiffer phenomenon does not take place, or does so 
 much more slowly ; the vibrios circulate in the blood for half an 
 hour or more without showing the least trace of granular trans- 
 formation, and are rapidly taken up by the leucocytes. In a 
 similar series of experiments on haemolysis he was able to prove 
 the existence of sensitized red corpuscles in the circulation, 
 although the animal did not present the least trace of haemo- 
 globinuria, and was of opinion that this was due to the absence of 
 free alexin in the plasrrr. The anaemia due to the injection of 
 haemolytic amboceptor he believes to be brought about by the 
 destruction of the red corpuscles within the macrophages of the 
 spleen. 
 
 As a third item of indirect evidence we may quote the experi- 
 ments of Lubarsch and others, to the effect that an animal may be 
 killed by the intravenous injection of a smaller number of bacteria 
 (e.g., of anthrax) than are destroyed by a small quantity (i c.c. or 
 less) of serum. The simplest explanation is that the blood contains 
 an abundance of immune body, and the failure of the defensive 
 
184 ORIGIN OF COMPLEMENT AND IMMUNE BODY 
 
 mechanism is due to lack of complement. It would, however, be 
 unwise to attach too much importance to this proof. 
 
 The difficulties in coming to a conclusion on this subject are 
 great, but on the whole it would appear that the evidence is some- 
 what in favour of the views of Metchnikoff, and that in all proba- 
 bility there is no free alexin, or but little, in the plasma, and that 
 the main source of this substance is the polynuclear leucocyte, 
 but we cannot regard this as definitely proved. This being so, 
 the discussion as to whether the formation of alexin is a vital 
 secretory process or a phenomenon of cell death and solution 
 cannot be regarded as of great importance. Here again the 
 experimental work is most inconclusive, Lastchenko holding that 
 living leucocytes give off alexin when suspended in heated serum, 
 whilst Lazar found that it is only set free when some of the 
 leucocytes are destroyed. Kanthack's experiments may perhaps 
 be quoted at this point. He found that anthrax bacilli immersed 
 in frog's lymph become surrounded by eosinophile cells. After a 
 time these cells discharge their granules, and the bacilli soon 
 begin to show signs of injury, becoming less refractile and losing 
 their sharp-cut outline. After this these leucocytes move away 
 from the bacilli, from which we might argue that they are un- 
 injured, and that the solution of their granules is a vital action, 
 and exactly equivalent to the secretion of pepsin by the gastric 
 cells. This, of course, does not exclude the possibility that alexin 
 might also be liberated during the process of phagolysis, as 
 Metchnikoff maintains. 
 
 As regards the origin of the immune body the evidence is unani- 
 mous, showing that it originates from the lymphoid tissues, and 
 probably from the mononuclear leucocytes. It is found that if 
 animals be injected with cholera vibrios, killed at varying intervals 
 afterwards, and extracts made of the different organs, the first sites 
 in which the antibodies make their appearance are the lymphoid 
 organs, especially the bone-marrow, spleen, and lymphatic glands. 
 Similar facts have been observed for other bacteria and for red 
 blood-corpuscles (Pfeiffer and Marx, Deutsch, Wassermann). And 
 in Bulloch's experiments the amount of haemolytic amboceptor was 
 found to run roughly parallel with the number of mononuclear 
 leucocytes present in the blood. In all probability this increase of 
 circulating mononuclears must be regarded in such cases as an 
 indication of the activity of the lymphoid organs, which are to be 
 regarded as the main source of the protective antibodies. This is 
 a generalization of the highest importance in immunity, and it is 
 
BACTERIOLYSIS AND ALLIED PHENOMENA 185 
 
 fortunate that the evidence in its favour is clear and direct ; and it 
 may be added that the proof is strengthened by the demonstration 
 of the fact that the agglutinins are formed in the same region. It 
 seems clear that the main, if not the only, function of the lympho- 
 cyte is the elaboration of the defensive antibodies. 
 
 There is no doubt that immune body, like the other antibodies, 
 circulates as such in the plasma. The most striking proof is that 
 of Metchnikoff in regard to spermotoxin, but other evidence is 
 available, and the point is not disputed. 
 
 Methods of Researches on Immune Bodies and 
 Complements. 
 
 The methods employed in the investigation of the haemolytic 
 sera are fairly simple in theory, though, as a rule, somewhat 
 tedious in practice, owing to the necessity, in most cases, for 
 quantitative work (so that slight degrees of haemolysis may not be 
 overlooked), and for numerous controls. The animal used in the 
 preparation of the immune serum will naturally depend on the 
 corpuscles to be employed, but in most cases the rabbit is most 
 convenient. The corpuscles used for immunization should be 
 collected in normal saline solution containing about i per cent, 
 sodium citrate, centrifugalized, and rewashed three or four times 
 in normal saline solution, so as to remove every trace of serum, 
 complements, etc. If this is not done the resulting serum may be 
 of great complexity, and results obtained by its action misleading. 
 The emulsion used for the injection may contain about half its 
 volume of corpuscles, and some 10 c.c. (or more in the case of 
 large rabbits) may be injected into the peritoneum. In most 
 cases three such injections at weekly intervals will cause the pro- 
 duction of a powerful haemolytic serum, but if there is any doubt 
 5 c.c. or so of blood may be withdrawn from the marginal vein of 
 the ear, and used to test the progress of the immunization. The 
 ear is well rubbed to make it hyperaemic, shaved, and washed with 
 alcohol. A small puncture is now made with a flat surgical 
 needle or fine knife, taking care that the vessel is merely incised 
 and not completely divided. As a rule the blood will now flow 
 quickly, drop by drop, and a sufficient amount may be collected in 
 a sterile test-tube. If the flow is sluggish the blood may be 
 milked out by passing the finger gently along the vein. 
 
 When the immunization is complete the rabbit is usually killed, 
 and as much blood as possible is collected. The best way to do this 
 
l86 HAEMOLYSIS METHODS OF RESEARCH 
 
 is to administer chloroform, and insert a cannula into the carotid 
 artery ; an easier method is to stick a small piece of capillary 
 glass tubing through the wall of the heart whilst it is still beating, 
 the animal being, of course, under chloroform. In either case the 
 blood is led into a sterile tube, allowed to clot preferably in the 
 incubator, as the retraction of the clot is thereby increased and 
 the clear serum 'withdrawn by means of a sterile pipette, and 
 stored in sterile tubes or small flasks, which may be plugged with 
 cotton- wool or hermetically sealed. Where the serum is to be heated 
 to destroy complement this may be accomplished in a thermostat, 
 or more simply by sealing it in a narrow tube which is tied to the 
 bulb of a thermometer, and placed in a beaker of warm water, and 
 the temperature regulated by hand by the application and removal 
 of a spirit-lamp. By this means any desired temperature can be 
 maintained within very close limits, and the necessity for main- 
 taining several thermostats or of altering the regulator is 
 removed. 
 
 The emulsion of corpuscles to be employed in the actual testing 
 is prepared as above, and must be rewashed at least four times, 
 since it is found that traces of complement may remain after three 
 washings. The emulsion is usually made up so that it contains 
 5 per cent, of corpuscles. This is readily accomplished by per- 
 forming the last centrifugalization in a graduated tube, going on 
 until the volume is constant. The bulk of the corpuscles is then 
 read off, and, the necessary amount of normal saline solution 
 being added, the corpuscles thoroughly mixed in. It is advisable 
 that aseptic precautions should be observed throughout, but as 
 this is troublesome it can often be omitted. It must be remem- 
 bered, however, that many common bacteria produce haemolysis, 
 and that if the mixtures of corpuscles, serum, etc., be incubated 
 for long periods fallacies may arise from this cause. 
 
 The actual experiment in its simplest form is carried out as 
 follows : The necessary amounts of 5 per cent, emulsion of 
 corpuscles, heated serum, and complement are placed in a narrow 
 test-tube, and in most cases normal saline solution is added to 
 bring the whole up to a definite volume. This is now incubated 
 for two hours, being stirred or shaken once or twice in the mean- 
 time. It is now removed and placed in a vertical position in the 
 ice-chest for twelve hours or so and examined. If there is com- 
 plete haemolysis the fluid will be deeply coloured, and there will be 
 no sediment, or only a minute deposit of stromata. With partial 
 haemolysis the fluid will be less deeply-coloured, and there will be 
 
BACTERIOLYSIS AND ALLIED PHENOMENA 
 
 i8 7 
 
 a deposit of undissolved corpuscles, and when there is no haemo- 
 lysis the fluid will be untinged. 
 
 Modifications of the process are numerous, and almost every 
 investigator has his own. Thus it is very convenient to make the 
 mixtures by means of one of Wright's pipettes. The whole is 
 sucked into the pipette, which is sealed and incubated. At the 
 end of the process the mixture may be expelled on to white filter- 
 paper. Any unaltered corpuscles will form a solid dark deposit in 
 the centre of the drop, whilst the fluid which soaks through the 
 paper will be tinged or colourless, according to the amount of 
 haemolysis which has taken place. The amount of haemolysis may 
 also be determined more accurately by comparison of the super- 
 natant fluid with a series of colour-standards previously prepared. 
 The mere presence or absence of the phenomenon may be readily 
 shown by dropping some of the fluid on to filter-paper. 
 
 The amount of immune body present may be determined by 
 some such process as the following : In each of a series of narrow 
 test-tubes (having a mark to indicate 2 c.c.) is placed i c.c. of the 
 emulsion of corpuscles, and then varying amounts of the heated im- 
 mune serum e.g., o-ooi c.c., 0-0025 c.c., 0-005 c - c -> etc -> or more if 
 the serum be a weaker one. As these small amounts are not easy to 
 measure accurately, the serum may be diluted ten or a hundred 
 times with normal saline solution and suitable multiples, these 
 amounts taken in the case of the smaller doses. The actual 
 measurements are done with graduated pipettes, which can be 
 procured from any instrument-maker. The complementing serum 
 is then added : the amount necessary to dissolve i c.c. of fully- 
 sensitized serum should have been previously determined by a 
 few rough tests (we will suppose it to be 0-2 c.c.). Lastly, sufficient 
 normal saline is added to bring the volume of each tube up to 2 c.c., 
 and the whole series treated as above. Thus 
 
 No. 
 
 Emulsion of 
 Corpuscles. 
 
 Heated Immune 
 Serum. 
 
 Fresh 
 Serum. 
 
 Haemolysis. 
 
 I. 
 
 ICC. 
 
 o-ooi c.c. 
 
 O'2 C.C. 
 
 None. 
 
 2. 
 
 
 0-0025 c.c. 
 
 
 M 
 
 3- 
 
 
 0-005 c.c. 
 
 
 5 i 
 
 4- 
 
 
 0-0075 c.c. 
 
 
 Trace. 
 
 5- 
 
 
 O'OI C.C. 
 
 
 Partial. 
 
 6. 
 
 
 0-025 c - c - 
 
 
 Complete. 
 
 7- 
 
 
 0-015 c c - 
 
 
 
 
 8. 
 
 
 0-0175 c.c. 
 
 
 > 
 
 9- 
 
 
 O'O2 C.C. 
 
 
 f , 
 
 10. 
 
 
 0-025 c ' c - 
 
 
 
 
l88 BACTERIOLYSIS METHODS OF RESEARCH 
 
 Here 0-0125 c>c - f ^he immune serum contained sufficient 
 immune body to sensitize fully i c.c. of a 5 per cent, emulsion of 
 corpuscles i.e., a given volume of serum will sensitize 1-25 of its 
 own volume of corpuscles. 
 
 The determination of the amount of complement is made by an 
 inversion of this method. Thus Gay, who has made numerous 
 investigations as to the amount of complement present in human 
 serum, proceeds as follows : The sensitizing serum is derived from 
 a rabbit which has been injected with ox corpuscles. This is 
 heated, and the amount necessary for complete sensitization of a 
 definite amount of ox corpuscles is determined; thus in his 
 experiment 0-7 c.c. saturated 7 c.c. of a 5 per cent, emulsion. A 
 series of tubes, each containing i c.c. of a 5 per cent, emulsion of 
 fully-sensitized corpuscles, is prepared, and varying doses of the 
 serum to be tested are added ; the amount, which is small, is pre- 
 pared by dilution with normal saline to such an extent that the 
 actual bulk added is 0*1 c.c. The subsequent treatment is as 
 above. Gay and Ayer find that on the average about -$ c.c. has 
 to be added to bring about complete haemolysis, the limits being 
 i 1 ^ and g^ c.c. 
 
 Quantitative researches on the bacteriolytic action of the serum 
 are very much more difficult. The actual determination of the 
 amount of bactericidal action is by no means easy, and the results 
 obtained are of very little importance, since the serum may be 
 very deficient in complement, and deviation may occur. The 
 method which has been chiefly employed is that of plating out 
 after the bacteria and serum have been allowed to act together at 
 incubator temperature for a given period. The method is briefly 
 as follows : The emulsion of bacteria must be of constant strength. 
 As a rule, it is sufficient to take a twenty-four-hour broth culture, 
 and to dilute it to the same degree in all experiments ; or the 
 same loop may be employed throughout, or some one or other of 
 the counting methods which have been described may be used. 
 The emulsions should be dilute, so that all the bacteria may 
 be killed. - Klien recommends i : 8,000 of a twenty-four-hour 
 broth culture in the case of B. typhosus. Lastly, normal saline 
 solution is better than broth as a diluting agent, since it diminishes 
 the chance of error owing to the multiplication of bacteria during 
 the somewhat lengthy process of preparing the dilutions. 
 
 The actual process is as follows : Measured small amounts of 
 the serum to be tested are placed in a series of tubes, a uniform 
 
BACTERIOLYSIS AND ALLIED PHENOMENA 189 
 
 amount of the emulsion added, each tube made up to a definite 
 volume, and all incubated for one to four hours. At the end of 
 this period a uniform quantity is withdrawn from each, and 
 plates prepared either by mixing with melted agar, or gelatin 
 where suitable, or by smearing over ready-poured agar plates. 
 The amount must, of course, be the same in each case, and may 
 be easily withdrawn by means of one of Wright's pipettes, which 
 is sterilized after use by being washed out several times with 
 boiling water or oil at 150 C. The plates are then incubated, and 
 the colonies which develop after twenty-four or forty-eight hours 
 are enumerated, and the amount of serum which kills all or the 
 greatest number of bacteria is noted. 
 
 Certain controls are necessary, the main being (a) a tube 
 inoculated as above, but without the addition of serum ; and 
 (b) a tube also containing bacterial emulsion, and also a relatively 
 large amount of heated serum. The main error comes in from 
 the reduction of the number of colonies in consequence of aggluti- 
 nation, but this can be discounted in some measure by comparison 
 with the plate prepared from control (&). 
 
 Other methods are employed, notably that of Wright, for which 
 the original article should be consulted. The value of the pro- 
 cesses is not great, since it does not tell us even the actual 
 bactericidal value of the circulating blood (since we do not know 
 the amount of complement which is available) nor the amount of 
 immune body. In some cases a serum containing a large amount 
 of the latter substance will show little or no bactericidal power in 
 vitro, owing to the deficiency in complement, and may require the 
 addition of a hundred times its volume of normal serum to be 
 fully complemented. 
 
 To determine the relative amount of immune body present, the 
 principle of the method used for the measurement of the haemolytic 
 amboceptor is adopted, a series of mixtures of constant amounts 
 of bacterial emulsion and fresh normal serum is prepared, and 
 varying amounts of the heated immune serum to be tested are 
 added, the whole made up to uniform volume, and treated as 
 above. Here a further control is necessary, since the fresh 
 normal serum may contain some immune body or be otherwise 
 bactericidal. One of Neisser and Wechsberg's examples of this 
 process has been already quoted. 
 
 The determination of the amount of bactericidal complement is 
 simple enough theoretically, and follows the same lines as that 
 
IQO THE CYTOLYSINS 
 
 for the determination of the haemolytic complement. In actual 
 practice these procedures are all so tedious that most of the 
 measurements of complement have been made on the latter 
 variety ; the two are believed to have the same origin, and there 
 is no reason to think that the one does not run parallel to the 
 other. Gay and Ayer employ a more direct method, adding 
 varying amounts of the serum to be tested to a definite volume 
 (0*5 c.c.) of a suspension of cholera vibrios, prepared by emulsify- 
 ing four twenty-four-hour agar cultures in 10 c.c. of normal saline, 
 and subsequently adding a sufficient sensitizing dose of serum 
 from an immunized rabbit. The action is allowed to go on for 
 one and a half hours at 37 C., films prepared, stained, and 
 examined as to the degree of the changes undergone by the 
 vibrios. They found that ^-^ c.c. of normal human serum was 
 sufficient to cause a complete Pfeiffer's reaction in 0-5 c.c. of 
 cholera emulsion tested as above, whilst when y^^ c.c. was used 
 there were distinct changes. 
 
 The Cytolysins. 
 
 Bordet's discovery of acquired haemolytic powers, arising from 
 the injection of foreign red corpuscles, proved the starting-point 
 of a most interesting series of researches, for it was soon shown 
 that the phenomenon was not an isolated one, but that it might be 
 produced when almost any animal cell took the place of the red 
 corpuscles. Thus, Metchnikoff in 1899 prepared a leucotoxic serum 
 by the injection of the cells from the spleen of a rat (mostly 
 lymphocytes) into a guinea-pig. The serum of the latter agglu- 
 tinated and partially dissolved the leucocytes, the lymphocytes 
 being most affected. Besredka studied the subject, and found 
 that, as in the hgemolysins, two substances one thermostable 
 (sensibilatrice or amboceptor) and one thermolabile (alexin or 
 complement) took part in the reaction. He studied the speci- 
 ficity of the substance, and found it was not sharply specialized 
 in its action to leucocytes of the animal used for the source of the 
 antigen ; it would attack those of most animals, but not man. It 
 was toxic, 3 c.c. of serum being a lethal dose. He also prepared 
 an antileucotoxin. 
 
 The next cytolysin to be prepared (by Landsteiner, and inde- 
 pendently by Metchnikoff) was spermotoxin. This was a very 
 suitable subject for study, since its action could be readily 
 
BACTERIOLYSIS AND ALLIED PHENOMENA IQI 
 
 observed, the cells on which it acted being motile ; and it must be 
 pointed out that these cytolysins do not cause complete solution of 
 the cells. A red blood-corpuscle is a remarkable object, and 
 macroscopic evidence of its (partial) solution is easily obtained. 
 It is otherwise with the cytolysins, and here refined histological 
 methods are often necessary for the demonstration of a solvent 
 action. Agglutination of a suitable suspension of the cells is, 
 however, invariably present, and is easily observed. Further 
 evidence is also obtainable by observing the action of the serum 
 on live animals and the disturbances in function which it produces. 
 In the case of the spermotoxin, the spermatozoa are rendered 
 immotile, and are agglutinated, but are not dissolved. 
 
 Several interesting phenomena were brought to light by a study 
 of spermotoxin. Thus, Moxter showed that its action is. not 
 sharply specific, since a spermotoxic serum is also haemolytic. 
 MetchnikofT thought that this non-specificity is only apparent, 
 since haemolytic sera are not spermotoxic ; and he succeeded in 
 removing the haemolytic substance from the serum by the addition 
 of red corpuscles, leaving the spermotoxin intact. 
 
 It may be pointed out here that similar results have been 
 obtained with the other cytolytic sera ; they are not sharply 
 specific, all being haemolytic, and some attacking several cells, as 
 well as those which have been used as their antigens. This 
 subject has been thoroughly investigated by Pearce. Some of 
 his results may be briefly epitomized. Haemolytic sera act, of 
 course, most strongly on the red corpuscles, which they lake, and 
 give rise to haemoglobinuria. They also produce fatty degeneration 
 of the renal epithelium and necrosis of the cells of the liver. 
 With very small doses there may be no haemoglobinuria, bile- 
 pigment being present in the urine, but the lesions of the liver 
 and kidney are also present. A serum prepared by the injection 
 of kidney cells, thoroughly washed, so that no blood was injected 
 with them, was haemolytic in vitro, but did not produce haemo- 
 globinuria. It caused albuminuria, with presence of casts and 
 granular degeneration of the liver cells. A serum similarly pre- 
 pared from the suprarenal glands had no action on them, but 
 produced granular or fatty degeneration of the kidney and liver. 
 An animal injected therewith showed immediate pallor of the 
 mucous membranes and cardiac and respiratory failure. He 
 found that hepatotoxins and pancreatotoxins were without specific 
 action, behaving simply like haemolysins. 
 
TRICHOTOXIN, HEPATOTOXIN, NEPHROTOXIN 
 
 It is obvious that these results are readily explicable if we 
 assume that the red corpuscles and tissue cells have receptors in 
 common, but that a particular sort of receptor is most abundant in 
 a particular species of cell. But, according to Beebe, sera which 
 are much more sharply specific can be prepared if, instead of 
 injecting the cells themselves, we employ the nucleo-proteid pre- 
 pared from them ; the method had also been employed by Bierry 
 and Pettit in the case of the nucleo-proteids of the liver and 
 kidney. 
 
 Another serum which was prepared early in the history of the 
 subject was trichotoxin, the cytotoxin for the ciliated epithelium. 
 This also, as Von Dungern showed, had a haemolytic action, 
 though he considered that there were no red corpuscles in the 
 substance used for the injections. 
 
 Hepatotoxin is produced by the injection of emulsions of liver 
 cells or of nucleo-proteid prepared from the liver. It causes con- 
 gestion of the liver, fatty or granular degeneration of the proto- 
 plasm, and dilatation of the bile canaliculi. If the serum has 
 been prepared by means of nucleo-proteid, no other organ is 
 affected. But the effects of hepatotoxin may also be produced by 
 nephrotoxic and lienotoxic serum, etc. 
 
 A considerable amount of interesting work has been done on 
 nephrotoxin, and the questions which have arisen are far from 
 having been settled. It is produced in the usual way, by injection 
 of animals with a fine emulsion of kidney cells (well washed 
 to remove blood-corpuscles, etc.) from a foreign species. It 
 produces albuminuria (but no glycosuria, according to Bierry), 
 and symptoms having at least some resemblance to uraemia (coma, 
 etc.) are occasionally produced. These symptoms are not specific, 
 and are frequently caused by injections of other cytolysins (SlpHpo- 
 toxin, etc.), or of emulsions of foreign cells. We have already 
 pointed out that Beebe and others have claimed to be able to 
 produce a truly specific nephrotoxin by means of injections of 
 nucleo-proteid from the kidney. 
 
 Of more interest is the question of the possible formation of 
 an autonephrotoxic body, which might conceivably be produced 
 when part of a kidney becomes disorganized whilst in the living 
 body. It has been thought, for instance, that when a toxin acts 
 on the kidneys it produces death and subsequent solution of the 
 renal epithelium, and that these soluble substances, being absorbed 
 into the system, call forth an autonephrotoxin, which reacts on 
 
BACTERIOLYSIS AND ALLIED PHENOMENA 
 
 the kidney, dissolving more cells, which produce more of the anti- 
 body, a vicious circle being thus produced. Hence a pathology 
 for nephritis and uraemia on quite new lines was suggested by 
 Ascoli and Figari and Lindeman, etc. Thus the cardiac hyper- 
 trophy of renal disease is supposed to be due to a spasm of the 
 peripheral vessels and increase of blood-pressure due to the 
 nephrotoxic serum ; the nervous symptoms on the supposition 
 that there is a neurotoxin produced concurrently with the nephro- 
 toxin, and spontaneous recovery by the production of an anti-auto- 
 nephrotoxin, a substance for the existence of which there is a 
 little evidence. 
 
 There is a certain amount of experimental confirmation of this 
 theory. Thus Lindeman treated dogs with potassium bichromate, 
 causing nephritis, and found that the serum of these animals 
 (though free from bichromate) was toxic for other dogs. Again, 
 Le Play and Corpechot found that the injection of renal tissue 
 (of the guinea-pig) into the rabbit produced important organic 
 lesions : great increase in volume, fibrosis of the connective 
 tissues, cystic dilatations of the tubules, and desquamation of the 
 renal epithelium. That these changes may be due to the produc- 
 tion of a nephrolysin appears possible from the fact that when 
 these injections are made in gravid animals similar appearances 
 may be seen in the kidneys of the foatus, suggesting that the 
 nephrolysins traverse the placenta (Charrin and Delaware). 
 Albarran and Bernard also found that renal tissue is lethal on 
 injection, but Pearce denies this, and holds that their animals 
 were killed by bacterial infection. Further, Nefedieff ligatured 
 one ureter (in the rabbit), and found changes similar to those 
 seen in chronic nephritis. His results, might, of course, have 
 been due to the formation of a nephrotoxin in consequence of 
 the disintegration of the renal cells subsequent to ligature of 
 the ureter ; but Albarran pointed out that, according to Nefedieff 
 himself, the second kidney was unaffected at a time when the 
 serum was nephrotoxic, as tested on other animals. Sheldon 
 Amos failed to reproduce Nefedieff's results; according to her, 
 ligature of one ureter causes death after an average period of sixty- 
 nine and a half days in the guinea-pig, and fifty-two days in the 
 rabbit. There may be lesions on the control side, but if so these 
 are slight, and the liver is also affected. But that these results are 
 due to the action of a nephrotoxic serum appears most unlikely, 
 from the fact that when the whole pedicle of the kidney, or the 
 
IQ4 GASTROTOXIN 
 
 artery and vein, are ligatured, no such results follow, though the 
 whole substance of the kidney is absorbed. These and other 
 researches make it very doubtful whether the facts observed in 
 nephritis are explicable on the nephrotoxic theory alone, but 
 further information on the subject is needed. 
 
 The degree of specificity of the nephrotoxic serum is not yet 
 settled. According to Pearce, the lesions which it produces may 
 be caused by other sera. This has been confirmed by other 
 observers, but Woltmann, though in accordance with Pearce on 
 the main question, thinks that nephrotoxin does exhibit some 
 degree of specificity : it produces marked congestion of the 
 medulla and swelling of the cortex, results not seen with other 
 sera. Beebe also finds nephrotoxic sera produced by the injection 
 of nucleo-proteid prepared from the kidney cause renal lesions, 
 whereas other cytotoxic sera produced by the injection of other 
 nucleo-proteids do not. 
 
 Gastvotoxic serum is especially interesting in view of its possible 
 action in the production of gastric ulcer. It has been very 
 thoroughly studied by Bolton, and was prepared by injecting 
 rabbits with emulsions or extracts of guinea-pig's gastric mucous 
 membrane into the rabbit. The serum thus obtained was injected 
 into guinea-pigs, and was found to be lethal, even in small doses 
 (i to 5 c.c.) ; a dose of 10 c.c. usually caused death in twenty-four 
 hours. The lesions were confined to the stomach, and were 
 striking and characteristic. They consisted of patches of necrosis 
 extending down to the muscularis mucosae, and often surrounded 
 by a haemorrhagic infiltration of the surrounding tissues. After 
 a time this necrotic tissue disappeared, leaving an ulcer presenting 
 some resemblance to the ordinary acute gastric ulcer. These 
 appearances (necrosis, etc.) were not seen if the acidity of the 
 gastric juice was neutralized by alkalis. No very definite action 
 could be demonstrated on gastric mucous membrane in vitro, but 
 isolated cells exposed to the action of the serum became hyaline 
 in appearance, resembling shadows. Further, the serum had a 
 powerfully agglutinating action on gastric cells, and produced a 
 precipitate in clear solutions obtained by filtration through a 
 Berkefeld filter. 
 
 Interesting facts were discovered as regards its specificity. It 
 is haemolytic, but this appears to be due to the fact that it 
 contains haemolysin as well as gastrotoxin. This is shown as 
 follows : If the serum is heated it loses its power to produce the 
 
BACTERIOLYSIS AND ALLIED PHENOMENA 
 
 characteristic necrosis of the stomach, so that its immune body 
 cannot be reactivated by guinea-pig alexin; but the latter body 
 can reactivate haemolysin prepared by immunizing rabbits with 
 guinea-pig's corpuscles. If the serum is placed in contact with 
 an emulsion of guinea-pig's mucous membrane, it becomes 
 innocuous, both immune bodies being absorbed; but when 
 saturated with red corpuscles, it loses its haemolytic power, and 
 retains its necrotizing properties. 
 
 Rabbits injected with emulsions of rabbit's mucous membrane 
 develop a gastrotoxin which acts on guinea-pigs, but not on the 
 rabbit itself. Similarly for guinea-pigs treated with emulsions of 
 mucous membrane from the same species : their serum becomes 
 gastrotoxic for the rabbit, not for the guinea-pig. 
 
 To account for these remarkable facts it is suggested that the 
 gastrotoxin has two cytophile groups one which combines with 
 the gastric cells of the animal which produces it, and one which 
 combines with those of the other species. Thus the gastrotoxin 
 of the rabbit has a cytophile group, a, which has an affinity for 
 rabbit's gastric cells, and a second, b, which unites with those of 
 the guinea-pig. During the process of immunization the animal 
 produces an anti-immune body, which combines with the cytophile 
 group a, but not with b. This is readily explicable on the side- 
 chain theory. It follows, therefore, that the gastrotoxin is never 
 efficacious against the species which produces it, being always 
 neutralized as regards these cells by a partial anti-antibody. 
 
 A nti -intestinal serum has been prepared. It is extremely toxic, 
 causing gangrene of the mucous membrane and death. Less 
 powerful sera cause non-fatal diarrhoea. 
 
 Syncytiolysin, or placentolysin, has been obtained by injections of 
 emulsions of placental tissue. According to Liepman the serum 
 thus obtained will give a precipitate with a solution of placental 
 tissue, with blood from the umbilical vein, or even with that of 
 a gravid woman, but not that of a non-gravid woman or a man ; 
 hence he proposed a serum test for pregnancy. But his results, 
 which seemed highly improbable, have been disproved by 
 Weichardt, who showed that the serum thus obtained acts equally 
 well on placental solutions and on all human blood. The question 
 of the action of the placenta when injected (in a fine emulsion) 
 into the tissues is of some importance in connection with a possible 
 pathology for eclampsia and the nephritis of pregnancy. Most 
 authorities (though not all) find that the animal thus treated 
 
 132 
 
196 SYNCYTIOTOXIN, NEUROTOXIN 
 
 develops nephritis and lesions of the liver. Now it is known that 
 in some cases at least fragments of the placenta break loose and 
 circulate for a time in the blood during pregnancy, and it is not 
 difficult to suppose that dissolved products of these cells are 
 constantly being absorbed. Hence it seems possible that some 
 at least of the cases of nephritis during pregnancy and of eclampsia 
 may be produced in this way ; and Weichardt produced symptoms 
 resembling those of eclampsia by macerating placental tissue with 
 syncytiolysin, and injecting the result into normal rabbits. Hence 
 it was hoped that an antitoxin for puerperal eclampsia and 
 nephritis might be produced by immunizing animals with placental 
 tissue, so as to produce a serum which would dissolve the circulat- 
 ing placental cells, and prevent the destruction of the cells of the 
 liver and kidneys. This does not seem to have been put into 
 practice, and there are numerous theoretical objections which 
 might be raised. 
 
 Prostatotoxin has been prepared by Jungano by injecting an 
 emulsion of the prostates of young dogs into rabbits. The serum 
 clumps emulsions of prostatic cells, and when injected in vivo 
 produces fatty and granular degeneration of the epithelial cells 
 of the gland and a leucocytic infiltration of the stroma; it is 
 apparently fairly specific, there being no obvious lesion of other 
 organs. 
 
 Neurotoxin has been prepared by Delezenne, Centanni, Delille, 
 and others, by the treatment of one animal with the brain sub- 
 stance from another, which is often in itself somewhat toxic, so 
 that the process does not always succeed. It causes a remarkable 
 series of phenomena indicative of profound intoxication of the 
 nerve centres. These usually begin with somnolence and torpor, 
 which come on shortly after the injection, and may last some 
 hours, being succeeded by convulsive crises, in which there are 
 tonic and clonic spasms ; there may be one such attack, or a 
 series, with coma between each. The temperature is lowered, and 
 death usually occurs in one to twenty- four hours. The histological 
 changes are marked, and affect the ganglion and cortical cells; 
 they indicate a profound degree of destruction of these structures 
 (neurolysis). The substance is most active when injected into 
 the brain direct ; when introduced into the veins it is innocuous, 
 but forms an anticytolysin. 
 
 Schmidt has prepared a serum which he claims to be more or less 
 specific for the peripheral nerves. A guinea-pig which is injected 
 
BACTERIOLYSIS AND ALLIED PHENOMENA IQ7 
 
 with an emulsion of the sciatic nerves of frogs develops in its 
 serum a substance which leads, when injected into frogs, to the 
 rapid production of symptoms of paralysis, which may become 
 complete, and resemble Landry's paralysis in man. Most of the 
 animals die in from twelve to forty-eight hours, and their nerves 
 show fragmentation of the axis cylinders, multiplication of the 
 nuclei in the sheath of Schwann, etc. The serum is also 
 haemolytic for frog's corpuscles, but neither normal serum nor a 
 simple haemolytic serum produce these paralytic symptoms. 
 
 The suggestion has been made that sympathetic ophthalmia 
 might be due to a specific cytotoxin formed by the disintegration 
 and absorption of the iris and ciliary body in the injured eye 
 (Bram Pusey). There is a certain amount of experimental proof 
 in favour of this interesting theory. Thus Le Play and Corpechot 
 prepared an ophthalmotoxic serum, and found that animals 
 injected therewith were less resistant than normal animals to 
 injections of B.pyocyaneus into the anterior chamber. The subject 
 has been more fully investigated by Golovine, who prepared his 
 serum by injecting into rabbits an emulsion of the ciliary bodies 
 of the dog (twelve to twenty in each animal). The ophthalmo- 
 toxic serum thus obtained was tested by injection into the anterior 
 chamber. It led to the production of a slight pericorneal injection, 
 a fibrinous exudate into the anterior chamber, and some appear- 
 ances of iritis. Microscopically it was found that the ciliary 
 processes presented evidence of inflammation and degeneration, 
 being infiltrated with leucocytes containing granules of pigment. 
 There was also marked evidence of degeneration of the epithelium 
 covering these processes. When the serum was injected into the 
 veins the macroscopic effects were not observed, but similar 
 microscopic changes were noted in the epithelium. 
 
 The pigment taken up by the leucocytes was derived from the 
 ciliary processes, which may become almost absolutely decolourized. 
 Hence Golovine holds that his serum contains not only a specific 
 cyclotoxin, but also a pigmentolysin. 
 
 Other cytolytic sera have been prepared, but are not of much 
 interest. A reference may be made to thyrotoxic serum, which 
 has been used in the treatment of exophthalmic goitre, though 
 without any considerable success. Indeed, the use of cytolytic 
 sera has proved most disappointing in practice. An anti-epithelial 
 serum which was very early suggested as a cure for cancer, but 
 proved inefficacious, and others have been tried. There are very 
 
198 BACTERICIDAL SERA 
 
 many problems connected with cytolytic action that are unsolved, 
 and there can be but little doubt that future research in this 
 direction will yield results of great pathological importance, both 
 in theory and in practice, and whether the therapeutical advance 
 will take the form of a potent serum or of a juster knowledge of 
 the inner processes of the body in disease the future will show. 
 
 THERAPEUTIC APPLICATIONS OF BACTERICIDAL SERA. 
 
 The discovery of the great therapeutic value of diphtheria anti- 
 toxin naturally led to attempts at antitoxin treatment of other 
 diseases, but it was soon found that it was impossible to prepare 
 a potent toxin, and therefore antitoxin, in the great majority of 
 cases. The discovery of Pfeiffer's phenomenon, and the sub- 
 sequent researches on bacteriolysis and haemolysis, with the 
 demonstration of the nature of substances at work, indicated that 
 the problem was to be solved, if at all, on other lines, and anti- 
 sera were made by injecting the bacteria themselves into suitable 
 animals. The process need not be described at length, and of 
 course slight modifications are necessary in different cases. In 
 general the early part of the treatment consists in the injection 
 of small doses of dead or avirulent bacteria, or in some cases 
 (e.g., anthrax) of a more virulent vaccine and of a protective serum 
 from an already immunized animal. The animal (horses, donkeys, 
 or goats, are usually employed) is thus immunized, and now large 
 doses of virulent bacteria are given in order to stimulate the 
 production of antibodies to as great an extent as possible. This 
 part of the treatment is often prolonged, and may last for a year 
 or more. At the end of this time the animal is bled in the 
 manner described above, and the serum used for protective or 
 curative purposes. In some cases it is standardized, the usual 
 method being to determine the amount which will just protect 
 a small animal from a lethal dose of living bacteria, or from some 
 multiple thereof. Thus Sclavo's serum is tested by injecting 
 1*6 c.c. into six rabbits, each of which receives shortly afterwards 
 a known dose of virulent bacilli ; if three of the animals survive, 
 and the rest have their lives greatly prolonged (as compared with 
 controls), the serum is considered to be efficacious. Antistrepto- 
 coccic serum may be standardized in a similar way : according to 
 Hewlett, 0-05 c.c. should suffice to preserve a rabbit from ten 
 lethal doses of living streptococci injected intravenously. In 
 other cases a somewhat more refined method is adopted, and the 
 
BACTERIOLYSIS AND ALLIED PHENOMENA IQQ 
 
 amount of antibody present is estimated. In the case of anti- 
 typhoid serum the simplest method is to measure the degree of 
 agglutination, which may rise as high as i : 1,000,000. This 
 cannot be taken as an absolute criterion of the amount of bactericidal 
 substance present, but in the great majority of cases the two 
 antibodies are developed roughly proportionately, and the agglu- 
 tination may be taken as a fair guide. Of course, the bacteriolytic 
 potency may be worked out by the method already described, 
 taking care that a sufficient amount of complement is added, and 
 that there is no deviation. This is probably the best method, and 
 is sometimes employed; thus Shiga found that O'oooi c.c. of his 
 antidysentery serum when reactivated by 0-3 c.c. of fresh serum 
 would kill all the bacilli in ^^ milligramme of a one-day-old agar 
 culture. 
 
 The results of tests of this nature have been to show that 
 extremely potent sera can be obtained against typhoid bacilli, 
 cholera vibrios, dysentery bacilli, and perhaps streptococci ; sera 
 of less but still of some power against plague bacilli, anthrax 
 bacilli, pneumococci, the gonococcus, and the meningococcus ; 
 whilst the results with staphylococci and tubercle bacilli have 
 been to all intents and purposes negative. 
 
 The method of action of these sera is not quite settled. In 
 some cases there is an abundance of bactericidal immune body, 
 and there is no reason to doubt that, when employed as a prophy- 
 lactic agent, this becomes complemented in the animal body, and 
 causes bacteriolysis of the infecting organism. This is certainly 
 the case with the sera directed against typhoid fever, cholera, and 
 dysentery. In other sera, which are, nevertheless, of definite 
 protective and even curative value, this effect cannot be demon- 
 strated. This is the case with anti-anthrax serum. Here we 
 have to assume either that the substance owes its value to the 
 presence of opsonins or of anti-endotoxin, or possibly (in some 
 cases) that it may contain free toxins, or at least specific antigens, 
 and act as a vaccine, producing active rather than passive 
 immunity, as was suggested by Wright in the case of Calmette's 
 typhoid serum, which is prepared in a manner somewhat different 
 from that just described. There is some reason for thinking that 
 Sclavo's serum acts opsonically, and with regard to the presence 
 of anti-endotoxin, it may be pointed out that the prolonged course of 
 immunization usually employed may lead to the production of 
 this substance in small amounts. 
 
200 CLINICAL FAILURE OF BACTERICIDAL SERA 
 
 These sera, which for the purpose of convenience we shall 
 consider together as if they were all bactericidal, are in general 
 protective, but not curative. Thus the clinical use of antityphoid 
 and anticholera serum has shown them to be quite worthless or 
 even dangerous ; dysentery serum is of distinct value if used 
 early in the attack, and some of the other sera are of some value, 
 and their use is discussed in the final section of this book. In 
 general terms, however, and comparing them with diphtheria 
 antitoxin, we may say that they have proved most disappointing 
 in practice. The reason for this failure requires some discussion. 
 
 Antibacterial sera as ordinarily used are, of course, devoid of 
 complement, which has usually disappeared long before use ; it 
 is rendered inert on keeping, and is especially susceptible to the 
 antiseptics commonly added as a preservative. The first sugges- 
 tion is that the failure of the serum is due to lack of complement : 
 the union of the amboceptor and bacterium is supposed to take 
 place as usual, but the necessary alexin is not forthcoming. This 
 may be due to one of two causes : in the first place, there may be 
 (as is known to occur in certain diseases) a deficiency in the 
 amount of complement in the serum; in the second place, that 
 which is there may be unsuitable in nature. 
 
 As regards deficiency in complement, this has been found to 
 occur in certain diseases, and is very probably a common occur- 
 rence in pathological conditions and states of malnutrition in 
 general ; but when we consider the comparatively small amount 
 necessary to activate a large dose of sensitized bacilli, there is no 
 reason to think that it ever falls below that level. Again, the 
 facts known concerning the immunity of the dog and other 
 animals to anthrax are of such a nature as to render it improbable 
 (in this case, at least) that deficiency of complement can really 
 be of much importance ; for dog's serum contains abundance of 
 amboceptor, yet no suitable complement, and is devoid of bacteri- 
 cidal action. We shall see reasons for believing that amboceptor 
 may possibly act as opsonin, in some cases at least, without the 
 concurrence of complement, and this is probably the explanation 
 of the immunity of the dog to anthrax. 
 
 The other explanation, that of Ehrlich, is that the complements 
 present in human serum may be unsuitable to reactivate serum 
 derived from a horse, ass, or goat, or other animal used as the 
 source of the immune body. To obviate this, he has proposed 
 the use of sera from several species of animals, in the hope of 
 
BACTERIOLYSIS AND ALLIED PHENOMENA 2OI 
 
 finding one that can be reactivated by human complement, and 
 has suggested the use of serum from the higher apes, the com- 
 plements of which closely resemble those of man. These 
 explanations are not very satisfactory. Thus Shiga's antidysentery 
 serum is certainly readily complemented by human blood; and 
 although it has certainly some beneficial action, it is useless in 
 the chronic stages of the disease, and this although the amount 
 injected must be very much greater than is necessary to dissolve 
 all the bacilli present in the body. 
 
 Another possible cause of failure is the deviation of complement. 
 If we admit the action of the bactericidal substances by no 
 means undisputed in the natural process of recovery from 
 disease, we can easily see how it is that this process does not 
 occur under normal conditions. Thus, when infection with the 
 typhoid bacillus occurs, there is at first little or no amboceptor 
 in the blood. The small amount present is quickly seized by 
 the bacteria and removed, and although a few bacilli may be 
 killed, the great majority flourish unchecked. But amboceptor 
 is soon put out in gradually increasing amounts, and at first is 
 used up as soon as it is formed. The two processes, proliferation 
 of bacilli and increase in the amount of amboceptor, now progress 
 side by side, and on their relative rapidity depends the outcome 
 of the disease. At first bacillary proliferation takes place more 
 rapidly than the production of antibody, and the symptoms 
 gradually become more and more severe. After a time the 
 antibodies are released in larger and larger amount, and (in a 
 favourable case) a time arrives when there is exactly enough 
 for all the bacteria present. We must assume that enough 
 complement is available, and in this case it is easy to see how 
 it can never become deviated ; for all the amboceptor is rapidly 
 linked up to the bacilli, and does not accumulate in excess in the 
 blood. It does seem possible, however, that an accumulation of 
 amboceptor might conceivably determine a relapse, bacteria 
 which escaped destruction owing to their having lain in the 
 tissues, gall-bladder, or other inaccessible region, being now free 
 to grow in the blood owing to the removal of complement by 
 deviation. 
 
 But when a dose of bactericidal serum, containing, it may be, 
 many times more immune body than is necessary for the solution 
 of the bacilli present, is suddenly thrown into the circulation, 
 the conditions are quite different. Here there is an excess of 
 
2O2 FAILURE OF BACTERICIDAL SERA 
 
 immune body relatively both to the bacteria and to the com- 
 plement, and deviation of the latter may occur. Hence it is at 
 least conceivable that a dose of bactericidal serum may be 
 injurious in that it actually inhibits the normal bacteriolytic 
 processes that are at work in the blood-stream. We have already 
 quoted processes exactly parallel in describing the experimental 
 proof of the deviation of complement. 
 
 Another suggestion that has been made is to use perfectly fresh 
 immune serum, or to reactivate it by fresh serum from a normal 
 animal. But this seems not to be successful, and apparently 
 alien complements rapidly unite with the tissues of the animals 
 into which they are injected, and so become inert. 
 
 It would seem that no explanation based on deficiency in com- 
 plement will be found satisfactory : the facts concerning the 
 action of the dog's serum on anthrax bacilli appear to offer a 
 crucial experiment settling this point. Nor if the added ambo- 
 ceptor really acts as opsonin would the question of complement 
 come in. The most satisfactory explanation appears to be that 
 the sera do not actually come in contact with the bacteria in the 
 lesions, though they may, and very probably do, tend to sterilize 
 the blood, and so prevent further generalization of the infection. 
 This question of the accessibility of the bacteria in the lesions 
 to the substances circulating in the blood is probably one of prime 
 importance in immunity and recovery, and we shall meet with it 
 again in dealing with the opsonins. It seems to meet the facts 
 of the case very well with regard to the action of the serum in 
 dysentery. In acute cases it is of value ; and here the bacilli are 
 lying in regions which are fairly accessible to the blood. In chronic 
 dysentery it is almost useless, and in this form of the disease the 
 bacilli are shielded by a dense and impermeable layer of inflam- 
 matory tissues. And in cholera the bacilli are mostly lying in the 
 intestinal tract ; probably a few do gain access to the blood and 
 tissues, and are immediately destroyed. In typhoid fever the 
 bacilli are found in the blood early in the disease, and later, 
 roughly at the period at which antibodies make their appearance 
 in large amounts, they disappear. But there are always large 
 numbers in the lymph glands and spleen, regions in which it is 
 almost certain they are shielded from the action of the blood. 
 This explanation appears far more satisfactory than any depending 
 on deficiency in complement. 
 
 If the bacteria in the blood-stream are actually dissolved by the 
 
BACTERIOLYSIS AND ALLIED PHENOMENA 203 
 
 added bactericidal substances, a new danger is involved that of 
 the liberation of a large amount of endotoxin. This substance 
 we believe not to be liberated when bacteria are dissolved within 
 the leucocytes, but to be set free when extracellular solution takes 
 place. The essential fever of the early stages of typhoid fever 
 is very probably due to the endotoxin set free by the solution 
 of the bacilli in the circulating blood, and any sudden addition to 
 this amount occurring before the tissues have become immunized 
 or trained to produce anti-endotoxin may be fraught with danger. 
 There can be little doubt that if sero-therapy has any future 
 triumphs in store, they will be in the direction of the production 
 of anti-endotoxins. 
 
 The various antibacterial sera in common use are considered in 
 the last section. 
 
CHAPTER VIII 
 THE AGGLUTININS 
 
 AN exceedingly interesting and important group of antibodies, 
 which were discovered by Gruber and Durham in 1896 (though 
 their effect had been observed by Charrin and Roger in 1889 
 in the case of B. pyocyaneus), 1 are called the agglutinins, since 
 they have the power of agglutinating their antigens, or causing 
 them to adhere in masses. Their effect is best seen after the 
 addition of the serum of a patient convalescent from typhoid fever 
 (or of an animal which has been injected with typhoid bacilli) 
 to a living culture of the organism. The bacilli, which at first are 
 actively motile and are distributed uniformly throughout the fluid, 
 first lose their motility, and then individuals may be seen to move 
 nearer and nearer to one another, until they come into close contact. 
 It often happens, especially in weakly agglutinating sera, that this 
 approach of two bacilli may be seen to occur before their paralysis 
 has taken place. They then revolve rapidly round a common 
 axis, giving the observer the impression that they are united 
 together by a sort of invisible link, which they struggle to break. 
 This process continues, and fresh individuals are attracted to the 
 groups, until at last all the bacilli, instead of being scattered equally 
 throughout the fluid, are collected into masses, the intervening 
 fluid being free. The process may also be watched with the naked 
 eye, and the emulsion, which is at first uniformly turbid, will be 
 seen to lose its homogeneity, and take on a finely granular 
 appearance. This at first can only be realized by comparison 
 with a control specimen to which no serum has been added, but 
 in a little time it will be obvious that flocculi of bacilli are being 
 formed, and that between these flocculi the fluid is clearing. Soon 
 
 1 The effect had also been observed by Metchnikoff in the case of V. Metchni- 
 kovi in 1891 ; he was inclined to regard it as a general phenomenon, but failed 
 to find it in another case. Similar appearances had also been seen by Issaeff 
 in 1893. 
 
 204 
 
THE AGGLUTININS 205 
 
 (if the emulsion is thick enough) all the organisms will be found to 
 have collected into a single mass or a few masses, the rest of the 
 fluid being quite clear. Finally, these masses will sink to the 
 bottom of the vessel, and it will be noted that if the bacilli in 
 the control specimen also sink (as happens with killed organisms), 
 the masses will be much more voluminous than the deposit of 
 unagglutinated bacteria. A microscopic examination of the de- 
 posit in the two cases will show why this is. In the deposit of 
 dead bacilli the separate rods have sunk down slowly, and have 
 packed themselves closely together, and will be found, to a very 
 large extent, to lie horizontally side by side. In the agglutinated 
 mass the bacilli point indifferently in all directions, and the explana- 
 tion suggests itself that they have been drawn forcibly together by 
 a centripetal force, and have not had time to adapt themselves so 
 as to take up as little room as possible. A result of this is that it 
 is easy to distinguish between a specimen that has agglutinated 
 and one in which the bacilli have simply settled, even although the 
 actual occurrence of the phenomenon has not been witnessed. 
 
 The reaction is given with the serum of immunized animals, and 
 is a general one. It is given with nearly all species of bacteria, 
 though to a very different extent in different cases, with red blood- 
 corpuscles, leucocytes, and with cells of all kinds. The occurrence 
 of motility is not necessary for it, and dead bacilli will clump 
 almost or quite as well as living ones. The reaction is, in general, 
 specific, and a serum which is strongly agglutinating as regards 
 one species of organism may be entirely devoid of action on others. 
 Hence it was proposed by Gruber and Durham as a test for the 
 identification of bacteria, and is of great value. Thus, when a 
 bacteriologist has isolated a culture of an organism resembling 
 B. typhosus from a patient suspected of having typhoid fever, or 
 from a sample of water supposed to be contaminated, the first step 
 in the identification is made by observing whether it is clumped by 
 a serum known to have agglutinating powers over typhoid bacilli 
 and not over others. Other tests are necessary for its complete 
 identification, but these are slower, and for some purposes un- 
 necessary. The clinical diagnosis of cholera by means of cultures 
 from the stools is carried out in the same way, and sera adapted 
 for either purpose can be obtained commercially. 
 
 The reaction, however, is not an absolutely specific one, and it 
 is found that a given immune serum may clump not only the 
 culture used in its production, but also those of closely allied 
 
2C>6 SPECIFICITY OF AGGLUTININS 
 
 species. Thus typhoid serum clumps B. coli, the paratyphoid and 
 paracolon bacilli, the B. psittacosis, and others. This is called a 
 group reaction, and is of profound interest in classification. It is 
 not, as might be thought, a hindrance to the practical application 
 of the process as a method of identification of the nature of a 
 culture, since it is found that the action is exerted much more 
 strongly on the organism used for the immunization than on 
 others. This is determined by ascertaining the dilution necessary 
 to bring about agglutination in a certain time at a given tempera- 
 ture. For example, we may find that certain specimens of anti- 
 typhoid serum will agglutinate typhoid bacilli at a dilution of 
 i : 10,000 in one hour, whilst B. coli is not affected if the dilution 
 is greater than i : 50. In the practical use of this serum we 
 should not be certain that a given culture was one of B. typhosus 
 unless it reacted at i : 1,000 or more. 
 
 The explanation of these group reactions on Ehrlich's theory 
 offers no difficulties. Agglutinin is, as will be shown, a specific 
 antibody to the molecules of protoplasm contained in the bodies of 
 the injected cells. In each cell these will be of many varieties, 
 and to each a specific antibody will be produced. We must 
 imagine a typhoid bacillus as containing a large number of one 
 particular sort of molecule, a smaller one of another, whilst in the 
 colon bacillus these relations will be the reverse. A typhoid serum, 
 therefore, will contain much agglutinin which acts on the typhoid 
 molecules, and a little which acts on a few of those present in 
 B. coli ; it will agglutinate the former strongly, the latter feebly. 
 But the colon serum will contain antibodies to a few only of the 
 molecules present in the typhoid bacilli, and will clump it only in 
 strong dilutions. 1 
 
 Agglutinins are formed, as we have seen, as the result of the 
 
 1 There are a few noteworthy exceptions which have been recorded to these 
 general rules. In a few cases of tuberculosis the power of agglutinating 
 B. typhosus has been seen to rise, and Park has quoted a case in which an 
 animal immunized against staphylococci increased ^its power against the same 
 bacillus from i : 10 to i : 160. In interpreting these results we must always 
 wonder whether they might not be explained by a rise in the sensitiveness of 
 the culture used. But this objection does not apply to the observations of 
 Posselt and Sagasser, who obtained an agglutinin which acted on bacteria 
 other than those used for the injection, and which was not removed from the 
 serum by these bacteria. And some cases have been recorded in which a 
 serum had less action on its own antigen than on others. All these exceptions 
 are rare and not full)' investigated, and do not affect the general law. 
 
THE AGGLUTININS 2O7 
 
 injections of their specific antigens. They are also frequently 
 present apart from any interference. For example, normal human 
 serum clumps the second vaccine of anthrax powerfully, and in 
 most cases has a feeble action on both B. typhosus and B. coli. 
 Horse serum is very rich in agglutinins, clumping typhoid and 
 coli bacilli, the B. pyocyarmts, and the cholera vibrio, often in 
 dilutions as high as i : 100. In most cases agglutinins are present 
 in small amounts in the serum of infants and young children, and 
 become more abundant in later life. This suggests that they may 
 be formed in part, at least by a process of auto-inoculation with 
 bacteria, principally, perhaps, from the intestine. We have 
 already seen, however, that on Ehrlich's theory the presence of 
 antibodies in normal animals is readily explicable without such 
 assumption. 
 
 The injections of bacteria or cells of any sort leads to the pro- 
 duction both of agglutinins and of cytolysins, and in most cases of 
 haemolysis or bacteriolysis agglutination occurs as the first step in 
 the process. The question arises, therefore, whether they are the 
 same substance. It is easy to show that they are not, since sera 
 which contains agglutinin do not necessarily contain immune body, 
 or vice versa. In sera obtained by artificial immunization, of course, 
 the two are almost invariably formed side by side, and it is only 
 by special processes that we can obtain the one without the other. 
 Thus Frouin claims that if dried dog's corpuscles are washed with 
 acetone and injected into a rabbit, they cause the production of 
 agglutinin ; but no haemolysin. The residue from the evapora- 
 tion of the acetone, on the other hand, yields haemolysin, but no 
 agglutinin. But in sera from normal animals it is quite common 
 to find the one without the other. Thus the serum of healthy 
 human beings frequently clumps normal human corpuscles, but 
 haemolysis is extremely rare. The converse process haemolysis 
 without agglutination also occurs; and with regard to antibacterial 
 sera of artificial origin, Frankel and Otto found that when a dog 
 was fed on typhoid cultures it developed agglutinin, but no immune 
 body. Lastly, in many cases the action of agglutinin is destroyed 
 at a lower temperature than that of immune body, although both 
 substances are in a marked degree thermostable. We shall have 
 to discuss the effects of heat on agglutinin more fully subsequently. 
 
 There is, as a matter of fact, a kind of antagonism between 
 agglutination and cytolysis. Cells which are crowded firmly 
 together are naturally shielded, more or less, from the solvent 
 
208 AGGLUTININS IN IMMUNITY 
 
 action of the fluid in which they are suspended ; and equally 
 naturally cells which are dissolved do not show ordinary agglu- 
 tination, though, as we shall see, they show a similar phenomenon. 
 
 The formation of agglutinins follows laws similar to those 
 governing the formation of other antibodies. After each injec- 
 tion there is a negative phase, followed by a rise, which, as a rule, 
 attains its maximum in about a week. In the case of typhoid 
 fever no agglutinin can be demonstrated, as a rule, during the 
 first week ; there is then a steady rise, which usually attains its 
 maximum at the commencement of convalescence. After this 
 the amount tends gradually downward, and disappears after a 
 time, which varies between a few months and several years. 
 On a single occasion the author has seen a marked drop in the 
 amount precede a relapse, during which a second rise occurred. 
 This was obviously a negative phase, and the occurrence of the 
 relapse might have been foretold therefrom. 
 
 Bacteria which have been acted on by agglutinin are not altered 
 thereby in appearance, viability, or virulence, and the process does 
 not appear to play a part of much importance in immunity. Two 
 suggestions have been made in this respect : Gruber thought it 
 caused the outer layer of the bacillus to swell up, so that it could 
 be attacked by alexin, and Walker suggested that the clumping 
 of the bacilli might render them more easily taken up in large 
 numbers by the leucocytes. Possibly, also, the paralysis is the 
 essential feature of the process, as a reaction of immunity, since 
 we should expect non- motile bacteria to be more easily ingested 
 by phagocytes. It is interesting in this connection to notice that 
 the bacteria for which strong agglutinating sera are obtainable 
 are all highly motile (B. typhosus, coli, and pyocyaneus, vibrios). 
 The recent researches on the thermostable opsonins have caused 
 a certain amount of attention to be directed to the agglutinins 
 from this point of view, but nothing is definitely proved. 
 
 That agglutinin, in common with the other antibodies, unites 
 directly with its antigen may be shown in several ways. In one 
 an agglutinating serum cooled to o is added to a culture similarly 
 cooled, and the mixture kept on ice. The bacteria will gradually 
 settle down without agglutinating, and the supernatant fluid may 
 be pipetted off. This may be tested in the ordinary way, and 
 will be found to have lost much of its agglutinating power. The 
 bacteria, if suspended in warm saline solution, will immediately 
 clump. Evidently, therefore, the agglutinin has been removed in 
 
THE AGGLUTININS 2Og 
 
 combination with the bacteria. Further, it is clear that we may 
 distinguish two properties of agglutinin (that of uniting with antigen 
 and that of clumping), and that these are discharged at different 
 temperatures : the agglutinin unites at o, and only exerts its 
 specific action at higher temperatures. We may express this in 
 Ehrlich's terminology by saying that it possesses a haptophore 
 group which functionates at o, and an ergophore group which 
 only acts in the warm. 
 
 Another proof is as follows : It was shown by Bordet that 
 agglutination only takes place when certain salts are present. Of 
 these sodium chloride appears to be the most generally efficient, 
 but Crendiropoulo and Amos have shown the calcium chloride 
 has a special adjuvant action in the agglutination of cholera 
 vibrios. To this subject we shall return. The proof of the union 
 between bacteria and their agglutinins is made as follows : 
 Bacteria are added to clumping serum, and the precipitate collected 
 and washed and shaken in a large quantity of distilled water. 
 No agglutination occurs until salt is added, when it takes place 
 rapidly, according to the thickness of the emulsion. In this case 
 also the two substances must have entered into the combination. 
 
 The substance with which agglutinin combines i.e., that 
 which calls forth its production in the living animal is evidently 
 not a toxin, since an agglutinating serum has, as such, no protec- 
 tive action. We know some of its characters. It is formed, of 
 course, in the bodies of the bacteria, and in young cultures is 
 entirely intracellular. In older cultures, however, it diffuses out, 
 being probably set free by a process of autolysis, and passes into 
 solution. This is especially the case in broth cultures, and this is 
 one of the reasons why, if liquid cultures are used in agglutination 
 tests, they must be young ; in agar cultures there is less diffusion 
 of the agglutinable substance, and the need is not so great. Its 
 presence may be proved in two ways : In the first place, this 
 filtrate, if injected into animals, will bring about the production 
 of agglutinin, as we should expect. In the second place, this 
 fluid, when added to a powerful clumping serum, will cause a 
 precipitate. This is Kraus's reaction, and it is a most interesting 
 phenomenon. It is best seen when the fluid portion of broth 
 culture of B. typhosns or V. cholera (at least a month old and 
 filtered through a Berkefeld filter to remove all solid particles) is 
 added in various proportions to a strong immune serum. Under 
 such circumstances the fluid will gradually become opalescent, or 
 
210 AGGLUTINOID 
 
 even opaque, then granular, and finally flocculent. It presents a 
 most extraordinary resemblance to the clumping of an ordinary 
 culture, but a microscopic examination will show the flocculi 
 consist of amorphous granules instead of bacteria. It has been 
 suggested that it is due to a clumping of cilia which have passed 
 through the filter (Nicolle), but the phenomenon has since been 
 observed in the case of the pneumococcus (Panichi) and other 
 non-flagellated organisms. The agglutinable substance is thermo- 
 stable. It does not appear to be given off in all cases, and some- 
 times all attempts to get Kraus's reaction are unsuccessful. 
 
 This substance is the antigen of agglutinin, and our nomen- 
 clature would be more uniform if we were to call it agglutin and 
 its antibody anti-agglutin, but the terms are too firmly fixed to be 
 altered. We shall call it agglutinable substance, or agglutinogen. 
 
 The fact that heated serum still agglutinates shows that alexin 
 or complement plays no part in the process, but we have already 
 explained how we know that the molecule of agglutinin possesses 
 an ergophore or zymophore group. This group, as is the case 
 with the corresponding groups of the toxins and complements, is 
 less resistant than is the haptophore group, and is destroyed at 
 70 to 75 C. The substance left is called agglutinoid, and is 
 analogous to toxoid and complementoid. Its existence is demon- 
 strated thus : Heated serum (or serum which has been kept for a 
 long time) is added to a culture of bacteria. No agglutination 
 takes place. The bacteria are then centrifugalized off and placed in 
 a strongly agglutinating serum, but are found not to clump. It 
 is evident, therefore, that the bacteria have their receptors 
 occupied by some substance which prevents the union of the 
 agglutinin. The agglutinoid has combined with the agglutinogen, 
 and excludes the unaltered agglutinin. 
 
 In some cases at least agglutinoids, which have a stronger 
 affinity for bacteria than has normal agglutinin, may be present. 
 In this case, if bacteria be added to a mixture of the two sub- 
 stances, no agglutination occurs. The pro-agglutinoids (as they are 
 termed, the expression being taken from the prototoxoids) seize 
 on the agglutinable substance in the bacteria before the 
 agglutinin can do so. If to this mixture more bacteria be added, 
 more pro-agglutinoid will be taken up, until it is all exhausted, 
 and then any fresh bacteria that are added will be clumped. 
 This is one explanation of a phenomenon which is fairly frequently 
 observed (if looked for) in the clinical diagnosis of typhoid fever, 
 
THE AGGLUTININS 211 
 
 and is probably a source of error often overlooked : the serum 
 clumps at a high dilution, and not at a low one. The author has 
 observed it three times in the last four years. Another explana- 
 tion, which is probably more often the true one, is that in the low 
 dilutions partial bacteriolysis takes place, and the partly dissolved 
 bacteria do not clump. The reason for this conclusion is that the 
 clumping may occur in low dilutions in the cold, when bacterio- 
 lysis does not take place. Yet other explanations have been 
 given. 
 
 Certain non-specific substances may bring about clumping 
 which has a close superficial resemblance to that caused by 
 agglutinin. This was first showed by Malvoz in the case of the 
 action of chrysoidin on V. cholera. He also showed that certain 
 stains, such as fuchsin, vesuvin, and safranin, and some anti- 
 septics, such as formalin (in fairly large amounts), corrosive 
 sublimate, and peroxide of hydrogen, have this action. Mineral 
 acids also possess this property, and also certain salts. In the 
 case of cholera vibrios, Ruffer and Crendiropoulo found calcium 
 chloride to have a powerful action, sodium phosphate to have a 
 very slight one. This must not be confused with the effect of 
 salts in favouring the action of agglutinating serum. 
 
 We are now in a position to discuss the mechanism of the 
 process. Numerous theories have been propounded. Thus 
 Gruber thought that the external membrane of the bacterium 
 became " sticky," so that organisms once brought into contact 
 remained adherent. But no visible alteration of the organisms 
 or red corpuscles can be seen. Further, it would not account for 
 the approach of two non-motile cells, which certainly appears to 
 take place in clumping, and would not explain why the cells or 
 bacteria were brought into contact in the first instance. Nicolle 
 propounded a similar theory. He, however, showed that when 
 inert and insoluble particles, such as of talc, were suspended in old 
 filtered cultures of typhoid bacilli (Kraus's fluid), and serum 
 added, they appeared to clump just as typhoid bacilli did, and it 
 is difficult to reconcile this with his theory. Dineur thought that 
 the flagella of the bacilli might have an adhesive material 
 deposited on them ; but many non -flagellated bacteria clump, to 
 say nothing of red corpuscles. Others have thought that Kraus's 
 reaction is the fundamental phenomenon, and that the bacteria, 
 etc., are entangled in it . like the particles of talc in Nicolle's 
 experiment. But no obvious precipitate can be seen in stained 
 
 142 
 
212 MECHANISM OF AGGLUTINATION 
 
 films of clumped bacteria, whereas Kraus's precipitate is easily 
 demonstrated ; besides which, agglutination can be perfectly 
 easily demonstrated in young cultures (the fluid portion of which 
 will not precipitate with specific serum) or with carefully washed 
 bacteria. This explanation, though ingenious, may be disre- 
 garded, r 
 
 Bordet's view is undoubtedly the correct one.( It explains agglu- 
 tination as being due to a change in the molecular relations 
 between the objects and the fluids which bathe it in other words, 
 it is practically an effect of surface tension^ It takes place in 
 many cases other than those in which it is produced by specific 
 sera acting on bacteria, red blood-corpuscles, etc. : thus, these 
 objects can be made to clump by the action of many aniline stains, 
 acids, antiseptics, etc. An emulsion of clay in distilled water will 
 remain turbid for a long time, but will rapidly clear, owing to the 
 formation of aggregates of particles, when salt is added. This 
 phenomenon (which explains the formation of mudbanks at the 
 mouths of rivers, where admixture of fresh and salt water occurs) 
 is of especial interest in view of the necessity for the presence of 
 salts in specific agglutination. Many bacteria, especially the 
 tubercle bacillus, clump spontaneously without the addition of 
 serum. In some cases this can be avoided by using a fluid poor in 
 or free from salt to make the dilution, as in Sir Almroth Wright's 
 method of estimating the agglutinating power of the serum on the 
 tubercle bacillus. A process fundamentally similar can be seen if 
 wooden matches smeared with grease are thrown on to the surface 
 of water, and may also be seen in the gathering together of 
 bubbles on the top of any fluid. 
 
 Two phenomena are involved : the approach of the particles the 
 one to the other, and their adhesion subsequently. The former 
 depends on certain physical laws investigated by Korn and others, 
 and not yet fully elaborated, in virtue of which two elastic 
 particles suspended in an inelastic fluid in which vibrations are 
 taking place tend to approach one another. It is probably fair to 
 assume that these conditions are always present in the case of 
 bacteria suspended in a fluid medium, and that, even in the 
 absence of any agglutinin, the individual organisms will tend to 
 approach one another and to form aggregates. But in the case 
 of most organisms the aggregates thus formed are quite instable, 
 breaking up when the slightest shaking of the fluid takes place. 
 Here the force of surface tension is all-important. It is a force 
 

 THE AGGLUTTNINS 213 
 
 which is generated wherever a fluid comes into contact with any 
 other substance, whether solid, liquid, or gas, and which acts 
 exactly as if the surface of the fluid in question were in a state of 
 tension, like a stretched film of indiarubber. If a relatively 
 small amount of any fluid be suspended in another fluid of the 
 same specific gravity with which it does not mix, it will assume 
 the form of a sphere : this is because the sphere has a smaller 
 surface for a given volume than any other solid body, and the 
 hypothetical film on the surface continually contracts until this 
 figure is assumed. Hence leucocytes, and most other free cells 
 consisting of fluid or semi-fluid protoplasm, tend to assume a 
 spherical form when in a resting condition ; hence also, of course, 
 the spherical form of soap-bubbles, oil-globules, etc. Now consider 
 the case of two spheres acted on by surface tension and just 
 touching one another ; for example, take two drops of oil 
 suspended in a fluid of about the same specific gravity. If we 
 regard the surface of the two spheres as continuous, it is obvious 
 that it is much larger than it would be if the two drops coalesced 
 to form a single sphere. (It is roughly larger in the proportion of 
 4:3.) The film, therefore, will contract until the two globules are 
 drawn into a single drop, with double the volume of each original 
 globule, but with a much smaller superficies than that of the two 
 separately. This process will take place whenever two bodies, 
 neither or both of which are wetted by the fluid, are brought in 
 contact or very close together : when one is wetted and the other 
 not, they tend to repel one another. The force of surface tension 
 only extends for an exceedingly minute distance into the fluid 
 from the surface, and therefore does not draw the substances 
 together if they are a finite distance apart. Its action comes into 
 play when the two bodies touch one another in one point, so that 
 the surfaces between the two bodies and the fluid join to become 
 one at this point. Thus, if two red blood-corpuscles touch one 
 another obliquely at one point, they become drawn together, and 
 slide the one on the other until they oppose as small a surface as 
 possible to the surrounding fluid. This, of course, is when the one 
 lies flat on the other, as in rouleaux formation. Two wooden 
 discs enclosed in a small indiarubber bag would act precisely 
 similarly. 
 
 The exact way in which the agglutinin affects the surface 
 tension between the bacteria and the fluid in which they lie is not 
 quite clear, and raises difficult questions in molecular physics, some 
 
214 MECHANISM OF AGGLUTINATION 
 
 of which are glanced at in our section on Colloidal Chemistry. It 
 is intimately concerned with the subject of solubility. If a body 
 is soluble in a fluid i.e., if the molecules of the latter have a 
 greater affinity for those of the former than these have for one 
 another there will be no sharp line of demarcation between the two : 
 between the solid and the liquid there will be a zone in which mole- 
 cules of both substances are present, and this will shade gradually 
 off into the solid body on the one hand and the fluid on the other. 
 Here, then, there will be practically no surface between the two, 
 and surface tension will be small or absent ; ..and in a general way 
 substances present in a fluid which dissolves them have no 
 tendency to clump. Thus to prepare an emulsion of an oil, a 
 solution of a soap or of an alkali is used, and the emulsions thus 
 formed are comparatively stable ; but if the fluid be made acid, 
 the surface tension is increased, and the globules quickly run 
 together or clump. Now it is clear from the fact that the fluid 
 part of bacterial emulsions will give Kraus's reaction, and will 
 lead to the production of antibodies on injection, that a certain 
 amount of solution does take place. That agglutinin actually 
 renders the bacteria less soluble appears clear from the phenomena 
 of Kraus's reaction, though here the insoluble precipitate is formed 
 on and in the bacteria, rather than in the fluid. And the complete 
 absence of clumping which occurs when bacteriolysis takes place 
 (though there, is a large amount of agglutinin in the serum used) 
 is an indication of what takes place when the bacteria are rendered 
 more soluble, instead of less, by means of an antibody. Insolubility 
 does not account for the whole of the phenomena, but it is a feature 
 of great importance. 
 
 As regards the nature of agglutinin, all we know is that it is 
 precipitated with the globulins, and may be of that nature. It 
 does not dialyze, and is digested by trypsin, etc. 
 
 It appears to be formed in the lymphoid organs, red marrow, 
 and spleen, being found early in those organs after injections of 
 cholera vibrios (Pfeiffer and Marx). Metchnikoff found that the 
 peritoneal exudate might be richer in agglutinins than the blood, 
 and thought they came from the cells (leucocytes and endothelial) 
 in that fluid. The subject has also been investigated by Van 
 Emden, Deutsch, and Ruffer and Crendiropoulo, who all confirm 
 Pfeiffer and Marx as to the early presence of these substances in 
 the lymphoid tissues after inoculation. 
 
 So far the study of the agglutinins has not presented much 
 
THE AGGLUTININS 215 
 
 difficulty, but further research has shown it to be full of com- 
 plexities. We will glance briefly at some more recent researches 
 on the subject, the exact explanation and significance of which 
 are not ascertained beyond dispute. 
 
 Several facts go to show that the agglutinin of B. typhosus is not 
 a simple substance, but that two or more bodies are concerned. 
 (It may be mentioned that this bacillus has been studied more 
 than any other in this connection.) Thus Joos, after a series of 
 ingenious researches, came to the conclusion that the bacillus 
 contains two agglutinogens, and that each has its corresponding 
 agglutinin. The agglutinogen which is present in largest amount 
 (and which he calls a) is thermolabile, being destroyed at 62 C., 
 leaving only agglutinogen /3, which is thermostable. An animal 
 injected with living cultures will contain agglutinins (a and /3) 
 against both the substances. The first will combine with agglu- 
 tinogen a only, whilst the second will combine with both substances. 
 The two substances differ in their thermostability : a is thermo- 
 stable, but /3 loses its power of agglutination at 62 C. A couple 
 of examples of the facts which this complicated theory was intro- 
 duced to explain may be given. The serum of a horse treated 
 with living typhoid bacilli (and therefore containing agglu- 
 tinins a and /3) clumped a living culture at i : 20,000, and a 
 heated one at i : 1,000. When the supernatant fluid of this last 
 dilution was tested with heated typhoid bacilli, no agglutination 
 took place (agglutinin /3 had been removed), whereas it would 
 clump living bacilli readily enough. Agglutinin a was present in 
 larger amount than /3, and had not all been removed at this 
 dilution. Again, when heated serum is added to heated bacilli 
 there is no agglutination, since the thermolabile agglutinin /3 is 
 destroyed. The agglutinin a, it is true, is not destroyed, but its 
 agglutinogen (which is thermolabile) is. But when living bacilli 
 are now added clumping occurs, since the agglutinin a can find 
 unaltered agglutinogen a to affect. 
 
 Smith and Reagh (and their researches have been, in the main, 
 corroborated by others) found that typhoid bacilli and other 
 flagellated bacilli might form two agglutinins the one acting 
 on the agglutinogen of the bodies of the bacilli, the other on that 
 of the flagella. The subject has also been investigated in a some- 
 what similar way by Buxton and Torrey, who find also two 
 agglutinins the one to a substance which remains attached to the 
 body of the bacillus, whilst the other can be separated from it by 
 
2l6 THE " PRO-ZONES " IN AGGLUTINATION 
 
 a temperature of 72, followed by filtration. The action of the two 
 is specific. If the filtrate be injected into animals, the serum 
 which results clumps ordinary typhoid bacilli well, but has little 
 action on those from which the separable substance has been 
 removed. The serum obtained by injection of the bacilli deprived 
 of separable substance is weaker, and has an equal action on the 
 bacilli whether normal or heated and deprived of soluble substance. 
 
 It is evident that the subject is a complicated one, and this 
 is even more clear from the researches of Dreyer and Jex- Blake 
 on the agglutination of B. coli by its specific serum. Investigating 
 first the behaviour of the bacilli when heated, they found, as other 
 observers had done, no alteration at 60 C., but a sudden diminu- 
 tion in the power of undergoing agglutination when heated to 
 70 C. This, of course, i<s usually ascribed to the complete or 
 partial destruction of the agglutinogen, though this explanation is 
 incomplete, since (as Eisenberg and Volk had previously found) 
 the bacilli which do not clump will still combine with agglutinin. 
 
 But Dreyer and Jex-Blake found that the agglutinability is 
 partially or completely restored by prolonged heating to 100 C. 
 After exposure to this temperature for a period of from two to 
 thirteen hours, the susceptibility of the bacilli to the serum might 
 be as great, or almost as great, as at first. This is an extra- 
 ordinary fact, and one for which no adequate explanation is 
 forthcoming. The only parallel is the behaviour of megatheriolysin 
 (a bacterial haemolysin), also investigated by Dreyer. This is 
 destroyed, or at least rendered inert, at 60 C., and reactivated at 
 the boiling-point. 
 
 These authors also adduce evidence to show that the " zones of 
 inhibition," or "pro- zones," described by Eisenberg and Volk as 
 occurring with heated serum, cannot be explained by the assump- 
 tion of the presence of " agglutinoids " with a high affinity for 
 agglutinogen. The evidence against this view is briefly this : If 
 it were true, the more the serum were weakened by the heat 
 (i.e., the greater the production of agglutinoid), the larger should 
 be the zone of inhibition, and vice versa. This they found not 
 to be the case, for a serum which had not been appreciably injured 
 by the heat might have a large zone of inhibition. They also 
 found exactly similar zones in investigating agglutination caused 
 by means of acids, in which case, of course, nothing of the nature 
 of agglutinoids could occur. Thus, in a series of experiments it 
 was found that when orthophosphoric acid was added to a definite 
 
THE AGGLUTININS 217 
 
 volume of emulsion of B. coli, agglutination occurred when between 
 118 centigrammes and 4 centigrammes of the acid was present, 
 and when between i-i milligrammes and 0*001 milligramme, but 
 not with intermediate amounts. 
 
 Similar phenomena (i.e., the presence of zones of inhibition) can 
 be seen in many of the actions of coagulants on colloid emulsions 
 or solutions, such, for instance, as the precipitation of gum mastic 
 from water by ferric chloride or trisulphide of arsenic. This 
 case also, as Dreyer points out, shows a marked analogy with 
 the clumping of coli bacilli by phosphoric acid, since in each case 
 the zone of inhibition becomes smaller when the agglutinating sub- 
 stance (bacilli or particles of gum) become more numerous. This 
 analogy between the agglutination of bacteria and the flocculation 
 of colloids has been investigated by Bechhold, Neisser and 
 Friedemann, and Biltz, and is a subject of the utmost interest, 
 and one which bids fair to revolutionize our views on the inter- 
 relations of the antigens and antibodies. At present our knowledge 
 of the subject is hardly sufficiently advanced to justify an account 
 of the experiments and theories on which these views are based, 
 and the original papers must be consulted for further information. 
 
 To revert again to the question of the specificity of the reaction : 
 The subject is a complex one, and we have already seen that the 
 phenomenon of the "group reaction " leads us to the supposition 
 that bacteria of different species must possess molecules of the 
 same nature. Further study shows that there are differences in 
 this respect between bacteria of the same species, which are in- 
 distinguishable the one from the other by ordinary morphological 
 and chemical tests, but which have had different origins. Thus it 
 is found that if the serum of an animal which is strongly immu- 
 nized to a given culture of V. cholera: be tested against cultures of 
 various stocks, that which was used for the injections will be 
 clumped most powerfully, the others to variable degrees. These 
 apparent anomalies, though inconvenient in practice, tend to make 
 us regard the reaction as more specific rather than as less 
 sharply specific, that is, as regards a certain sort of chemical 
 substance which is formed in greater degree by certain races of a 
 given species than by others. 
 
 This modified specificity, sharp as regards chemical substances, 
 but not as regards bacterial species, is well illustrated in 
 Castellani's absorption reaction. If an agglutinating serum which , 
 e.g., clumps the typhoid bacillus powerfully, and a colon less 
 
218 
 
 CASTELLANl'S ABSORPTION REACTION 
 
 strongly (and which was obtained by immunizing an animal 
 against typhoid) be saturated with typhoid bacilli, centrifugalized, 
 and retested, it will be found that when the serum has lost its 
 power of agglutinating that organism, it will also be bereft of 
 power over the B. coli. On the other hand, if it be saturated 
 with colon bacilli until all its agglutinating power on that 
 organism is removed, its action on B. typkosus will be intact or 
 but slightly reduced. The mechanism by which this is brought 
 about is readily understandable by means of the following diagram 
 (Bolduan). 
 
 Fig. i represents a typhoid bacillus, shown as if it consisted of 
 three varieties of proteid material, A, B, and C, of which A is 
 present in largest amount. The agglutinin formed by its injection 
 will consist of three substances, each specific for its own proteid, 
 and the antibody to A (which we may call the main agglutinin) 
 
 B C 
 
 B 
 
 
 Typhoid Bacillus 
 
 Colon Bacillus 
 E H 
 
 Dysentery Bacillus 
 
 FIG. 45. (After Bolduan.) 
 
 will be most abundant. Fig. 2 represents a colon bacillus, and 
 is shown as consisting of four forms of proteid, of which B is 
 common both to it and to the typhoid bacillus. It follows that 
 the antityphoid serum will clump this bacillus as well as that of 
 typhoid, though not in so high a dilution ; the serum only acts in 
 virtue of its anti-B agglutinin, of which it possesses but a small 
 amount, and this can only act on proteid B, which forms but a 
 small part of the colon bacillus. 
 
 If the serum in question be saturated with typhoid bacilli the 
 whole of the agglutinins are removed in combination with the 
 bacilli, anti-B amongst them, and the serum will become devoid 
 of agglutinating action on both bacilli. 
 
 But if it be saturated with colon bacilli, the only effect will be 
 to withdraw the anti-B agglutinin ; the agglutinins to A and C 
 will remain, and consequently the serum will only lose its clump- 
 ing power on the typhoid bacillus to a slight extent. After 
 
THE AGGLUTININS 2IQ 
 
 saturating the serum with all the allied bacteria on which it can 
 act, we might theoretically, at least remove all the partial 
 agglutinins, leaving only the main agglutinin, which acts on the 
 proteid formed by the typhoid bacillus, and by it only, and so 
 obtain a truly specific clumping serum. 
 
 Castellani's test seems to be generally correct, though excep- 
 tions have been recorded. 
 
 Further, bacilli of the same stock can be made to vary greatly 
 in their sensitiveness to the action of agglutinin by various 
 methods. These have been carefully studied by Bordet, Nicolle, 
 Kirschbruch, and others, and it has been found that the 
 sensitiveness of typhoid bacilli is diminished by washing, by 
 culture at high temperatures (40 C.) or low temperatures, by the 
 addition to the medium of minute traces of antiseptics, and is 
 less in old cultures and in those that have been recently isolated 
 from the living animal. It is found in general that bacilli just 
 isolated from a typhoid patient clump badly, and that they 
 gradually increase in sensitiveness for six months on cultivation in 
 artificial media : a very faintly acid medium is the most suitable. 
 Sometimes a culture alters very rapidly without obvious cause, 
 and this is a possible source of error in the clinical application of 
 Widal's reaction. The author in one case found a culture which 
 was clumped by a certain serum at i : 60 was clumped by the 
 same serum at i : 250 three days later. 
 
 Another method by which bacteria can be made to diminish in 
 their sensitiveness to clumping is by cultivation in specific 
 immune serum. This was first observed by Ainley Walker, and 
 since it is of some theoretical interest in connection with Welch's 
 theory of the nature of the unknown toxins, requires a short 
 description. He found that when typhoid bacilli were grown in 
 immune serum (of course, devoid of complement) diluted with 
 broth, they gradually lost their agglutinability, and became more 
 virulent. Thus both of the effects of" passage " were reproduced 
 in vitro, and Walker further found that bacilli thus treated re- 
 moved the agglutinin from the serum in which they were grown, 
 and believed his results could be best explained on the supposition 
 that the bacilli produced specific anti-agglutinins. This, of course, 
 tends to corroborate Welch's interesting suggestion that some of 
 the organisms for which no exotoxins have been discovered exert 
 their lethal action by producing antibodies against the blood of 
 their host. 
 
22O IMMUNITY OF BACTERIA TO AGGLUTININS 
 
 The subject has been investigated by others, and their results 
 do not altogether corroborate those of Walker. Thus Miiller 
 found the same inagglutinability after culture in diluted typhoid 
 serum, but did not find any evidence .of the formation of an anti- 
 agglutinin ; on the contrary, he found that bacilli thus treated 
 had less power of weakening the action of typhoid serum than 
 had normal bacilli. We might explain his results by saying 
 the bacilli had lost their agglutinable receptors, or agglutinogen. 
 Bail, working with a different method, obtained comparable 
 results, though he explained them quite differently. He, as well 
 as Landsteiner and others, noticed that bacilli grown in the 
 immune serum grew into long branching threads, losing their 
 bacillary character entirely. This has been thought to be a sort 
 of end-to-end agglutination. 
 
 The change is a more or less permanent one. Thus Park and 
 Collins found that cultures (of B. coli and B. dysenteric) which 
 had lost their power to agglutinate might require to be grown for 
 months on ordinary agar before they retained their normal sensi- 
 tiveness. The change is evidently a very profound one, and one 
 that is handed on for many generations. 
 
 The subject has recently been carefully investigated by Marshall 
 and Knox, working with B. dysenteric. They found the same loss 
 of agglutinability when the bacillus was grown on immune sera, 
 but the same change also occurred when normal horse serum was 
 used ; and they further showed that the alteration was a rapid 
 rather than a gradual one, as Walker had found in the case of 
 B. typhosus. With regard to the mechanism of the process, they 
 proved conclusively that the modified bacilli had no power of 
 uniting with the agglutinin, as Miiller had also found. It is quite 
 clear, therefore, that the loss of agglutinability is due to the loss 
 of appropriate receptors, in this case at least. They point out, 
 apropos of Welch's hypothesis of toxins, that a bacterium which 
 attempted to protect itself against its host by the formation of 
 antibodies would have but a poor chance of surviving, whereas 
 by the simple process of losing some of its receptors tbey can 
 nullify some of the protective mechanisms possessed by the latter. 
 
 Their explanation of the process by which these resistant 
 bacilli are produced is interesting and suggestive. They do not 
 think, with Walker, that the bacilli acquire a new character 
 i.e., the power of producing antibodies and transmit it to their 
 descendants, but that there is simply a process of natural selection. 
 
Of 
 
 ^^ 
 
 THE AGGLUTININS 221 
 
 In any culture of bacteria it will be found that some individuals 
 are more easily agglutinated than others. In the early cultures 
 in clumping serum the susceptible forms grow, but they sink to the 
 bottom in a dense network. The less sensitive forms also grow 
 from slight granules or a diffuse turbidity through the fluid. If 
 the subcultures are inoculated from this fluid, the change to the 
 non -clumping form occurs more quickly than if the masses were 
 used for the transfer. There is therefore a process of selection 
 of the non-clumping forms, and in time all the susceptible bacilli 
 become eliminated. It is this process, though in a more marked 
 form, that accounts for the increased virulence of the bacteria, 
 which is brought about by passage. For here all bacteria which 
 are not resistant to the bacteriolysins and to the phagocytic 
 action of the leucocytes will be killed off, and will not propagate 
 their species. We may therefore conclude on theoretical grounds 
 that virulent bacilli are those having (along with a potent toxin) 
 few receptors which can be attacked by amboceptor and opsonin. 
 We have seen that Pfeiffer and Friedberger's experiments do 
 not tend to corroborate this (in the case of amboceptor), but that 
 they cannot be regarded as conclusive. 
 
 The increase in the virulence of the organism by cultivation in 
 immune serum has been generally corroborated, though Roger 
 found the opposite to occur when streptococci were cultivated in 
 antistreptococcic serum. 
 
 The hamagglutinins occur in normal and in immunized 
 animals. Those in the latter call for no especial notice ; they are 
 developed after injection of red corpuscles side by side with 
 immune body, and their presence can be demonstrated if the 
 serum be heated or if the experiment be performed in the cold. 
 The normal agglutinins which one species may possess for the 
 red corpuscles of another species call for no special notice. 
 
 The iso-agglutinins are, however, worthy of a short description. 
 They are common, and have been most studied in human blood, 
 and will be found to occur to a greater or less extent in most 
 specimens of human sera. The phenomena they produce are 
 entirely similar to those produced by a specific serum in a typhoid 
 culture. Under the microscope the corpuscles will be seen to run 
 together into masses which are quite unlike the ordinary rouleaux 
 of shed blood in that the corpuscles are approximated together 
 without any trace of definite arrangement. When a strong serum 
 is used the cohesive force may be so great that the corpuscles 
 
222 THE H^MAGGLUTININS 
 
 become absolutely fused together into a solid mass, in the centre 
 of which the outlines of the original corpuscles cannot be made 
 out. The macroscopic appearances are similar to those of a 
 Widal's reaction, the emulsion of corpuscles becoming granular 
 and flocculent, and clear rapidly, with the deposition of a red mass 
 at the bottom of the vessel. With a powerful serum a column of 
 emulsion 2 inches high may be completely cleared in five minutes, 
 and the clumping, as observed in a drop of the fluid on a slide, 
 may be complete in a few seconds. 
 
 It will be found that a certain serum does not clump all human 
 corpuscles equally well, and facts have been observed quite 
 comparable to those found by Ehrlich in the case of isohsemo- 
 lysin. According to Landsteiner (whose researches have been 
 corroborated by Hektoen), human bloods may be divided into 
 three groups, which Hektoen describes as follows : 
 
 Group i. Here the corpuscles are not agglutinated by sera of 
 the other groups, whilst the sera agglutinate the corpuscles of 
 both groups. 
 
 Group 2. In this group the corpuscles are agglutinated by the 
 sera of the other groups, whilst the sera agglutinate the 
 corpuscles of Group 3, but not of Group i. 
 
 Group 3. The corpuscles are agglutinated by all other sera, 
 and the sera agglutinate the corpuscles of Group 2, but not of 
 Group i. 
 
 A few specimens of blood are found which do not fit exactly 
 into any of these categories. 
 
 It will be noted that in none of these groups is there an 
 agglutination of red corpuscles by its own serum i.e., an example 
 of the presence of an auto-agglutinin. This substance, however, 
 may occur in the blood, but apparently only in disease. The 
 best example (in fact, the only one in which it has been positively 
 demonstrated) is in pernicious anaemia. In this disease it often 
 happens that the washed corpuscles are immediately and strongly 
 clumped by the serum of the same patient, and the same 
 phenomenon may occur when citrated plasma is used instead of 
 serum. Whether similar phenomena occur in the body, and if 
 not, the reason for its absence, is quite unknown : it is exceedingly 
 difficult to imagine that clumps similar to what we see in vitro 
 can be formed in the circulation, for they appear to be much too 
 large to pass through the capillaries. The only plausible explana- 
 tion is that the auto-agglutinin does not exist as such in the 
 
THE AGGLUTININS 223 
 
 circulation, only coming into existence as the blood is shed. If 
 this is the case, it appears clear that its production is not the 
 result of clotting, since -the clumping may occur before coagula- 
 tion has taken place ; indeed, if the blood is carefully watched as 
 it flows from a skin puncture in a marked case of pernicious 
 anaemia, it maybe seen to become " streaky," pale and dark areas 
 being present, and a microscopical examination will show that 
 this is due to clumping of the corpuscles, which thus appears to 
 take place immediately the blood leaves the vessels. 
 
 The researches of Gay would appear to show that the clumping 
 of the red corpuscles by serum is not necessarily due to the 
 presence of an agglutinin at all, but may be caused by variations 
 in the tonicity of the corpuscles and serum. Thus he found that 
 in the bloods which have non-agglutinable corpuscles there is a 
 higher molecular concentration than in the other groups, indicating 
 a greater amount of salts both in the corpuscles and in the sera. 
 There are also differences in tonicity, though less marked, between 
 the members of the other groups. He also finds that if a serum 
 which is hypertonic to a certain sample of corpuscles, and which 
 therefore clumps them, is examined after contact with the 
 corpuscles in question, its tonicity is decreased until it reaches 
 that of the serum which normally accompanies those corpuscles, 
 which, of course, it now no longer agglutinates. This is a simple 
 saturation experiment, the old explanation of which would have 
 been that the agglutinin had all been removed by contact with the 
 corpuscles, but which Gay explains by absorption of salts by the 
 corpuscles. Lastly, a simple hypertonic solution of NaCl and 
 CaCl 2 , and according to Peskind a large number of acids and acid 
 salts, gave rise to appearances suggestive of clumping, though not 
 identical therewith. His researches are highly suggestive, and it 
 may be that we shall have to modify our ideas of the normal 
 iso-agglutinins ; but the subject requires further investigation. 
 
 Rouleaux formation ] presents some resemblances and also some 
 differences. Red blood-corpuscles, when washed free from serum 
 and suspended in normal saline solution, lie free side by side, and 
 show no tendency to run together ; but if placed in human serum 
 and some other fluids, the biconcave discs approach one another in 
 a peculiar orderly fashion, so as to form adherent rolls resembling 
 
 1 I am indebted to some unpublished researches of Dr. Wiltshire's for 
 much interesting information on this subject. Any new fact mentioned is 
 owing to him, unless the contrary is stated. 
 
224 ROULEAUX FORMATION 
 
 piles of money. This phenomenon has attracted a good deal of 
 attention, though less than it deserves, and the exact mechanism 
 by which it is produced is still uncertain. It has obviously some 
 resemblance to agglutination. It is quite conceivable, for example, 
 that an agglutinating serum, when greatly diluted, might act so 
 weakly that the corpuscles might be drawn gradually together, 
 and so approach one another in such a fashion as to form the 
 peculiar rolls, very much as in the orderly deposition of molecules 
 to form a crystal, which occurs when a strong solution of a salt is 
 slowly evaporated, so as to allow the molecular attractions to 
 come into play in a regular fashion. But this is not the case, 
 since, as was shown by Descatello and Sturli, it is not possible to 
 dilute an agglutinating serum with normal saline solution so as to 
 convert it into a rouleaux-inducing one. And according to Lange, 
 an agglutinating serum which has had its agglutinin removed by 
 saturation with corpuscles will still give rise to the phenomenon 
 under discussion. 
 
 Both the corpuscles and the fluid in which they are suspended 
 require consideration in the discussion of the question why 
 rouleaux formation occurs in shed blood, though not in the tissues. 
 Human serum will always cause it, though its potency in this 
 respect varies greatly from time to time in the same person, and 
 also in different individuals (as tested on the same specimen of red 
 corpuscles). This power is quickly destroyed on dilution, being 
 almost always annulled on the addition of an equal quantity of 
 normal saline solution. The roulogenous principle also occurs in 
 human milk, but not or not commonly in that of cows, and is not 
 destroyed by boiling for five minutes. Viscosity appears to play 
 some part of secondary importance, and not clearly defined : 
 solutions of colloids, such as gum or gelatin, may induce it, as 
 was pointed out by Wharton Jones. The roulogenous substance 
 occurs to a very large extent in inflammatory exudates, but not in 
 transudates. 
 
 It is possible that the induction of rouleaux formation may be 
 due to the development of some substance in the serum at the 
 moment the blood is shed, and it is also possible that it may be 
 due to some change in the red corpuscles. It has been shown 
 that biconcave discs suspended in water and smeared with grease 
 will run together in this way, and Brunton has suggested that 
 when the blood is shed some fatty acid is liberated by the action 
 of carbon dioxide. It is also possible that the change which 
 
THE AGGLUTININS 225 
 
 occurs when the blood is shed may be one of shape. Thus 
 Weidenreich and others hold that red corpuscles are normally 
 bell-shaped, and that the moment they leave the vessels they 
 become biconcave discs. If there is an increase in the tension 
 generated at their surface of contact with the serum, the con- 
 ditions for rouleaux formation would appear to be present. 
 Weidenreich's facts have been strongly disputed, and are not 
 generally accepted. 
 
 Rouleaux formation cannot be induced in corpuscles which 
 have been heated to 44 C. 
 
CHAPTER IX 
 THE PRECIPITINS 
 
 AGGLUTININS are antibodies obtained by the injection of particu- 
 late substances, and have the property of causing these substances 
 to collect into clumps. In exactly the same way proteids in solu- 
 tion, when injected into suitable animals, bring about the formation 
 of another class of antibodies, which possess the power of clumping 
 the molecules of the proteid in a solution similar to that injected. 
 This manifests itself by the formation of a precipitate. After the 
 addition of the clear antiserum to the clear proteid solution, the 
 mixture becomes opalescent, and then opaque, and after a time a 
 precipitate is cast down, leaving a clear supernatant fluid. Hence 
 these substances are called precipitins, the substance with which 
 they form a precipitate, and which calls them into existence when 
 injected into a suitable animal (their antigen), being termed pre- 
 cipitable substance, or precipitogen, and the insoluble combination 
 of the two, precipitum. They are in all respects closely allied to 
 \ agglutinins, if not absolutely identical. The fact of their acting in 
 V a clear fluid does not prove that they exert their effect in a true 
 solution, since modern physico-chemical research has shown that 
 proteids do not form solutions, but merely emulsions or suspensions 
 of molecules or of complexes of molecules. The effect of the 
 addition of a precipitin is to cause an agglutination of these 
 molecules, which is entirely analogous with the agglutination of 
 typhoid bacilli. The laws which govern the reactions of the 
 precipitins and agglutinins are entirely similar, and, theoretically, 
 it would probably be more accurate to consider them under one 
 head. The practical applications of the two classes of antibodies 
 are, however, very different, and it is more convenient to treat 
 them as separate substances. 
 
 The first substances of this group to be discovered were the 
 bacterio - precipitins of Kraus, first investigated in 1897, anc ^ 
 referred to elsewhere. Kraus found that, if he took an old 
 
 226 
 
THE PRECIPITINS 227 
 
 culture of typhoid bacilli and filtered it to remove the bodies of 
 the bacteria, and then added some typhoid serum to the clear 
 solution, a precipitate was formed. The same happened with 
 cultures of cholera and plague organisms, after addition of the 
 appropriate sera, so that the reaction is, within limits, specific. 
 The limits of the specificity are not yet thoroughly ascertained, 
 but there are distinct evidences of group reactions similar to those 
 seen in the agglutinins. Thus Norris found a common precipitin 
 for organisms of the cholera group, for some cocci, etc. There 
 were, however, some exceptions ; thus, a rabbit which had been 
 injected with cultures of B. prodigiosus developed a precipitin which 
 acted on filtered cultures of B. coli and V. Metchnikovi, as well as 
 on the filtrate from the organism injected. He thought, however, 
 that the reaction is a more intimate and constant test of group 
 relationships than is agglutination ; thus he prepared an antiserum 
 to a bacillus belonging to the hog-cholera group which did not 
 agglutinate typhoid or colon bacilli, but which gave the precipitin 
 reaction with their filtrates. The bacillus, it may be pointed out, 
 is a member of the same group as the other two, so that (in the 
 case of this particular serum) the agglutination reaction is mis- 
 leading. 
 
 Bacterio-precipitins may be prepared by the injection of cultures 
 of the bacteria, or the filtrates from the old cultures spoken of 
 above ; it is evident, therefore, that they are antibodies to substances 
 in solution. Some authorities, it is true, have thought that the 
 actual antigen and precipitable substance consist in reality of the 
 broken-off flagellae, which are sufficiently fine to pass through a 
 Berkefeld filter ; but this can hardly be the case, since a bacterio- 
 precipitin can be obtained for non -flagellate organisms. It is true 
 that the best-known and most powerful of these antibodies are for 
 organisms which possess flagellae, and it is quite probable that the 
 clumping of these filaments does occur in some cases, and intensi- 
 fies, or may be mistaken for, a true precipitation. 
 
 Bacterio-precipitins cannot be (or have not been) prepared to all 
 organisms. Thus, diphtheria antitoxin does not form a precipitate 
 with diphtheria toxin, a substance prepared on lines exactly similar 
 to those used in procuring the precipitating solution for typhoid 
 and cholera. Diphtheria toxin is an excreted substance, not a 
 substance dissolved out of the bacterial protoplasm. 
 
 The first to observe precipitins to other proteid solutions, and in 
 particular to serum, was apparently Tchistovitch, in 1898. He 
 
 152 
 
228 PRECIPITOID 
 
 investigated the formation of an antitoxin for eel serum by inject- 
 ing that substance into rabbits, and found the serum thus obtained 
 not only neutralized the toxic effects of the eel serum, but formed 
 a precipitate with it. In an exactly similar way, he prepared a 
 precipitating serum to a non-toxic serum that of the horse and 
 investigated its properties. He found that the precipitate formed 
 by the interaction of the antiserum and its antibody was soluble in 
 dilute acids and alkalis, but insoluble in water, alkaline carbon- 
 ates, and neutral salts. These results were corroborated by Bordet, 
 whose classical researches on the haemolysins were made about 
 this time. He found that the injection of denbrinated fowl's blood 
 into rabbits caused the appearance of agglutinins, haemolysins, and 
 of precipitins in the serum of the latter, so that the fowl's red 
 corpuscles would be first clumped and then laked, after the 
 addition of the immune serum, and this substance, when added to 
 fowl serum, led to the formation of a precipitate. Bordet also 
 demonstrated the formation of a precipitating serum for milk 
 (lacto- serum). These researches were corroborated by Myers, 
 Uhlenhuth, Wassermann and Schiitze, Nuttall, and others, and 
 the reaction has now been very fully studied, and found to be of 
 considerable practical importance. 
 
 Precipitins are in all respects closely analogous to the agglutinins. 
 They are formed under the same conditions i.e. t as a reaction of 
 the cells to a foreign material, probably in all cases of a proteid 
 nature and appear themselves to be proteids. They are destroyed 
 by pepsin and other proteolytic enzymes, and by acids and alkalis. 
 They appear to be allied to or carried by the globulins. When 
 heated they appear to undergo a change into precipitoid, a sub- 
 stance which has the power of combining with the molecules of 
 precipitogen, but which does not precipitate them ; this is shown 
 by the fact that a further addition of precipitin does not cause 
 precipitation, indicating that the effect is not due to a mere 
 weakening of the substance or to its partial destruction. Hence 
 we deduce that the precipitin molecule consists of two portions 
 a thermostable haptophore or combining group, and a thermolabile 
 functionating group, the action of which is necessary for agglutina- 
 tion of the molecules to occur. This change takes place at a 
 temperature of 50 to 60 C., but it also occurs slowly when 
 precipitin solutions are kept at ordinary temperatures, so that these 
 become gradually useless as tests for their antigens ; and the 
 change may be hastened by the action of light or of various 
 
THE PRECIPITINS 
 
 22g 
 
 chemical substances. Precipitating sera should, therefore, be 
 kept in a dry state in a cool place, and preserved from light. 
 
 Precipitoids appear to have a stronger affinity for the precipitate 
 substance than has the unaltered precipitin an alteration in 
 affinity similar to that which we have seen to occur sometimes in 
 the case of agglutinin, and which leads to the formation of what 
 we have termed pro-agglutinoid. Thus, if a serum which has 
 been heated until it has lost its precipitating power be mixed 
 with unheated precipitin, the mixture will not form any precipitate 
 after the addition of small amounts of the normal serum ; it is only 
 after enough of the latter has been added to combine with all the 
 precipitoid that a precipitate begins to appear. The same pheno- 
 mena may occur in working with old and degenerated sera, or 
 occasionally even with fresh ones. An example will make this 
 clear. 
 
 
 Normal Serum. 
 
 Antiserum. 
 
 Amount of Precipitate. 
 
 7 
 
 
 nil 
 
 6 
 
 
 nil 
 
 5 
 
 
 nil 
 
 4 
 
 
 nil 
 
 3 
 
 
 nil 
 
 2 
 
 
 0*5 
 
 I 
 
 I 
 
 i 
 
 I 
 
 2 
 
 1-25 
 
 I 
 
 3 
 
 2 
 
 I 
 
 4 
 
 3 
 
 I 
 
 5 
 
 3 '5 
 
 I 
 
 6 
 
 375 
 
 
 7 
 
 4 
 
 
 8 
 
 4 
 
 
 10 
 
 3-25 
 
 
 12 
 
 275 
 
 
 H 
 
 2-25 
 
 
 16 
 
 2 
 
 
 18 
 
 1-5 
 
 
 20 
 
 I 
 
 
 24 
 
 nil 
 
 
 3 
 
 nil 
 
 Here the maximum precipitate was given when 8 parts of anti- 
 serum were mixed with i part of normal serum. When i part of 
 normal serum was mixed with 24 parts of antiserum, there was no 
 
230 PRECIPITOID 
 
 precipitate i.e., the precipitoids were at this point just saturated ; 
 but in a mixture of 20 parts of antiserum with i part of normal 
 serum, it appears that, after all the molecules of precipitoid were 
 saturated, there was still enough precipitable substance present to 
 combine with some of the precipitin, and thus to form a precipitate. 
 In the mixture of 8 and i the precipitoid was all saturated, and a 
 maximum amount of precipitable substance left over to combine 
 with precipitin. 
 
 This theory of the " specific inhibition " of the action of this 
 precipitin was the first explanation to be brought forward, and 
 appears to afford a fairly satisfactory explanation of the facts 
 observed. It is, however, quite probable that future physico- 
 chemical research may show it to be erroneous, and that these 
 zones of inhibition are in reality similar to those observed in the 
 non-specific precipitation of colloids, of which we have already 
 spoken. To this subject we shall return. Be the explanation 
 what it may, the phenomena are of considerable practical impor- 
 tance in the application of the precipitating sera to the diagnosis 
 of the nature of an unknown serum or other solution of proteid. 
 The mere fact of not obtaining a precipitate when a solution of 
 unknown strength (e.g., of a dried blood-stain) is added to an 
 anti-serum is not necessarily of any importance, and to obtain 
 accurate results it is necessary to perform a series of quantitative 
 experiments. 
 
 The experiments of Eisenberg, Michaelis, Fleischmann, and 
 others tend to show that the combination between the two obeys 
 the laws governing the combination of weak acids and bases, 
 which we have already discussed, and that when the two sera are 
 added together both free precipitin and precipitable substance may 
 be present at the same time. This, if true, would not explain 
 relations between the precipitin and precipitable substance similar 
 to those given above ; if the law of the mass reaction applied, 
 an excess of antiserum would tend to give the maximum 
 possible quantity of precipitum, though there would always be 
 some unaltered precipitable substance present in an unaltered 
 state. It is, however, doubtful whether the substances do actually 
 interact as weak acids and bases. Von Dungern, experimenting 
 with antisera obtained to the proteids of cold-blooded animals, 
 found that the reaction was rigorously quantitative, but that a 
 complication was introduced by the presence of two varieties of 
 precipitin, special and partial. The special precipitins are sup- 
 
THE PRECIPITINS 23! 
 
 posed only to act on the proteid which has been used for the 
 immunization, whereas the partial ones act both on it and on 
 other foreign proteids. If these are formed at different rates of 
 speed, it is easy to see that at a certain stage in the process of 
 immunization an animal's serum may contain a precipitin (e.g., 
 the special precipitin) and a precipitable substance (e.g., that which 
 acts as an antigen to the partial precipitin). In most of their 
 reactions the precipitins certainly act as if they had a powerful 
 combining affinity for their antigens, and von Dungern's observa- 
 tions may supply the explanation of the apparent discrepancies. 
 
 If precipitable substance (e.g., egg-albumin) be heated, it under- 
 goes a change comparable to that sustained by precipitin on 
 heating : it loses its power of becoming precipitated with anti- 
 serum, but retains its function of combining therewith. This 
 altered proteid is sometimes called precipitoid, since it is analogous 
 with the precipitoid derived from precipitin by the action of heat. 
 The term, however, is a bad one, since its use leads to confusion 
 between the two substances, and the only word which is at all 
 suitable is "precipitogenoid." Precipitogenoid, therefore, consists 
 of the haptophore portion of the molecule of precipitogen, another 
 portion of this molecule which must be present in order that the 
 reaction may take place being destroyed. From the fact that it 
 retains its haptophore group we should expect it to act as an 
 antigen, just as toxoid does ; and this is the case, for the injection 
 of precipitogenoid calls forth the production of ordinary precipitin. 
 In any complete quantitative study of the interactions of serum 
 and antiserum it is necessary to investigate the question of the 
 presence of precipitogenoid as well as of precipitoid, and the 
 problems thus may become complicated in the extreme. 
 
 Precipitins, like the agglutinins, act most quickly at body 
 temperatures, and show their essential similarity in the fact that 
 salts are necessary for both reactions ; short of absolute absence, 
 the amount of the precipitum form depends on the quantity of 
 salts present (Friedmann). 
 
 There is an interesting analogy between the agglutinins and the 
 precipitins, in that the latter as well as the former are occasionally 
 seen in the serum of unimmunized animals, though the precipitins 
 are much more uncommon in this situation. Thus, Hoke found 
 the presence of bacterio-precipitins to nitrates from B. typhosus 
 and V. cholera fairly frequently present in the serum of the ox, 
 more rarely in that of the goat, pig, and sheep. The presence of 
 
232 SPECIFICITY 
 
 serum precipitins is rarer, but occurs, according to Noguchi, in 
 cold-blooded animals (crustacea, etc.), and Lamb found in normal 
 rabbit serum a precipitin for cobra venom, and Obermayer and 
 Pick one for dysglobulin of egg-white in the same fluid. 
 N^The relation of the precipitins as regards specificity forms a 
 difficult and most important question. It was at first thought 
 that the specificity was a sharp one, and that a serum prepared by 
 the injection of human serum would only precipitate with human 
 serum, and a lacto-serum prepared by the injection of cow's milk 
 would only precipitate with that fluid, and not with the milk of 
 other animals. Thus, Uhlenhuth prepared a precipitin by inject- 
 ing a rabbit with ox serum, and found it gave a precipitate with 
 that fluid, but not with the serum of the horse, donkey, pig, sheep, 
 dog, cat, deer, fallow-deer, hare, guinea-pig, rat, mouse, rabbit, 
 chicken, goose, turkey, or pigeon ; but he also showed that anti- 
 egg serum was not sharply specific, and would coagulate solutions 
 of albumin from eggs other than those of the species used for the 
 injection. Wassermann and Stern also showed that antihuman 
 serum would react, though but slightly, with the serum of the 
 baboon, and Stern confirmed this, and found it reacted with the 
 serum of other monkeys. Hence the idea gradually arose that 
 the precipitin obtained by the injection of a serum from one species 
 of animal is not specific for that species, but will give a precipi- 
 tate, though of less amount, with the sera of other species, provided 
 that they are sufficiently closely allied zoologically. This has been 
 especially studied by Nuttall, who expresses this relationship 
 between species close together in the animal scale as a " blood- 
 relationship." He showed that, provided the serum were powerful 
 enough, it would react with all the bloods of animals in the same 
 great division of the animal kingdom (mammalia, birds, reptiles, 
 amphibia, etc.). Thus, a strong antihuman serum will give a 
 precipitate with human serum, even when highly diluted, with 
 apes, monkeys, etc., but not in such high dilutions, and a slight 
 trace of precipitate after a long period when mixed with the sera 
 of more remote mammalia, but no precipitate with the blood of 
 birds, fishes, etc. Quite similar relationships hold with the 
 lacto-sera and with the precipitating sera for muscle proteids; 
 the anti-sera for egg proteids is apparently less specific. 
 
 Hence we deduce that the precipitins are not specific as regards 
 the animal species from which they are derived, but possess that 
 partial specificity seen in the cytotoxins and in the " group 
 
THE PRECIPITINS 233 
 
 reactions " of the agglutinins ; that is to say, they are specific as 
 regards the antigens which bring them into existence, irrespective 
 of the source from which that antigen was derived. This appears 
 to be substantiated by experiments on purified and recrystallized 
 proteids, the precipitating sera for which show a high degree of 
 specificity. Too frequent recrystallization of proteids, however, 
 appears to injure their power of inducing the formation of 
 precipitins. 
 
 Further, proteids which have been altered in character by 
 chemical processes (iodized, nitrified, or denitrified) will cause 
 the formation of precipitins which are specific for the transformed 
 molecules of proteid, no matter from what source they were 
 derived. These observations are of profound interest, since they 
 appear to show that it is possible by artificial means to alter com- 
 pletely the haptophore group of a proteid molecule. According 
 to Uhlenhuth, there is little or no specificity in the antisera pre- 
 pared against the proteids of the crystalline lens. An antiserum 
 prepared by injections of serum will precipitate all albuminous 
 fluids except a solution of the lens, which in its turn will not pre- 
 cipitate with serum. But an anticrystalline serum prepared by 
 the injection of ox lenses into rabbits will give precipitates with 
 lens solutions from mammals, birds, amphibia, and even fish. 
 
 Hence, too, a practical point in connection with the employment 
 of precipitating sera in the detection of the source of blood. Here 
 it is necessary to use a high dilution of the blood to be tested, and 
 if a reaction is given, to try again with higher dilutions until the 
 limit is reached. In testing dried blood-stains for medico-legal 
 purposes it is, of course, usually impossible to determine the exact 
 amount of serum which has been taken up by the solvent (normal 
 saline solution). It is found, however, that a i : 1,000 dilution 
 of serum in normal saline solution will give a good froth when air 
 is allowed to bubble through them from a pipette, and this will 
 give a rough idea as to the amount of proteid material dissolved 
 out of the clot to be tested. 
 
 The delicacy of the reaction is truly astonishing. Thus Ascoli 
 obtained an anti-egg albumin serum which gave a precipitate with 
 a i : 1,000,000 dilution of egg-albumin, and Stern an antihuman 
 serum which reacted with serum at a dilution of i : 50,000. These 
 are extreme figures, but sera active on solutions diluted i : 5,000 are 
 frequently obtained. 
 
 As regards the substances for which precipitins can be obtained, 
 
234 ISOPRECIPITIN 
 
 we find them limited in all cases to the proteids. All the coagu- 
 lable proteids will give rise to the formation of precipitating sera. 
 As regards the formation of these substances by means of digested 
 proteids (peptone and albumoses), the facts are less certain. 
 Myers obtained a precipitin to Witte's peptone, but according to 
 Obermayer and Pick complete peptic digestion destroys both the 
 power of inducing the formation of antibodies and of being pre- 
 cipitated. The former is first to go, so that a partially digested 
 proteid solution which will no longer precipitate with an antiserum 
 will yet lead to the production of a precipitin when injected into a 
 suitable animal. The results with tryptic digestion appear to be 
 entirely similar. Complete destruction leads to loss of both 
 functions. It appears, therefore, that it is only the giant molecule 
 of proteid that can be agglutinated ; the smaller, diffusible 
 molecule of the products of proteolysis, like the molecules of 
 toxin, though they may, and probably do, unite with their anti- 
 bodies, do not manifest this combination by forming clumps. 
 Proteids which have been coagulated by heat have still the power 
 of forming a precipitin (which, of course, manifests its action on 
 solutions of the unaltered proteid) when injected. 
 
 As regards the species of animal from which the precipitins are 
 to be prepared, it is natural to choose an animal as remote in the 
 zoological scale as possible. In the case of human sera or in- 
 different substances, such as solutions of albumin, etc., the rabbit 
 is usually chosen, since it is easily obtained and handled, and will 
 yield a very considerable amount of serum. Where antisera to 
 animals closely allied to rabbits are to be obtained, fowls or other 
 large birds are most suitable. There are, however, some observa- 
 tions which go to show that there are differences between individual 
 rabbits which are comparable in every respect to those between 
 the different red corpuscles of goats and other animals, as seen in 
 Ehrlich's experiments on the isohaemolysins. Thus Schiitze 
 injected the serum of rabbits into rabbits. In two cases out of 
 ten he obtained a precipitin which reacted with the serum injected. 
 This substance is called isoprecipitin. It may often be noted 
 that a precipitate falls when different samples of diphtheria anti- 
 toxin (in animals in which the earlier stages of the process of 
 immunization have been carried out by means of serum toxin) are 
 mixed together, and this is probably an analogous phenomenon. 
 These precipitins are, however, very feeble as compared with 
 those obtained by the injection of the serum of a certain species 
 
THE PRECIPITINS 235 
 
 into an animal far removed in the zoological scale, and their 
 explanation is obvious in the light of what has been already said 
 with regard to individual differences in receptors. 
 
 According to Ewing, an antihuman serum prepared from the 
 fowl shows a far higher degree of specificity than those obtained 
 by injections into rabbits. 
 
 A short account of the practical application of the precipitins 
 may not be out of place. The chief is, of course, the medico-legal 
 identification of blood-stains, the chief exponents of which are 
 Uhlenhuth and Wassermann. The antiserum is obtained from 
 rabbits, which are treated by intravenous or intraperitoneal in- 
 jections at intervals of three or four days. The material used 
 for the injection may be blood obtained by venesection or vein 
 puncture, or from the placenta or from the cadaver, or pleuritic or 
 ascitic fluid may be used ; in any case strict asepsis is necessary. 
 The amount given rises from i to 3 or 4 c.c. in the case of 
 intravenous injections, or twice as much or even more in the 
 peritoneum. The course of treatment lasts three or four months. 
 Another and simpler method is to give larger doses up to 10 c.c., 
 or even more intraperitoneally at intervals of a week. The 
 animal is then chloroformed, and as much blood as possible 
 collected either from the heart or carotid artery. 
 
 The fluid to be tested is prepared by maceration of the clot, 
 piece of stained linen, etc., with normal saline solution, or with 
 i per cent. NaOH. In the case of a very old stain, Ziemka 
 recommends the use of a strong solution of potassium cyanide, 
 which is subsequently neutralized with tartaric acid. This is 
 examined with the microscope and tested spectroscopically to 
 determine the presence of blood-corpuscles and pigments. The 
 solution is then filtered. Air is then allowed to bubble through 
 the fluid to make sure that proteid material has actually passed 
 into solution ; this is indicated by the production of a stable foam. 
 Three tests are made. In the first tube one part of the fluid under 
 examination is mixed with two of the antiserum, the second 
 contains the fluid alone, and the third antiserum plus normal 
 saline solution. Further controls, in which the antiserum is 
 mixed with diluted serum from animals other than man, may also 
 be made if necessary. The tubes are usually incubated and 
 examined from time to time, and a positive result is obtained if 
 there is a precipitate in the first tube and not in the others. 
 Further tests are then made with greater dilutions, and with a 
 
236 DEVIATION OF COMPLEMENT TEST 
 
 powerful antiserum a reaction can usually be obtained in dilutions 
 so high that proof of the presence of proteids is barely obtainable 
 by ordinary chemical means. The weak point in the method is 
 that it is never possible to say exactly how much of the proteid 
 matter of the clot has been dissolved, and thus to compare the 
 effect of the antiserum on the solution with its action on diluted 
 serum of man and of other animals. Given a sample of unaltered 
 and undried serum, the test can be carried out with almost 
 complete certainty ; but this is rarely, if ever, possible in actual 
 practice. 
 
 Another test, based on Gengou's reaction, has recently been intro- 
 duced by Neisser and Sachs. It is carried out in the following 
 way : A hsemolysin for ox corpuscles is prepared by injecting 
 these bodies into a rabbit. Another rabbit is injected with human 
 blood, so as to lead to the production of a precipitin. When the 
 test is to be made the fresh serum of the latter animal (or if only 
 stale serum is at hand, some fresh normal rabbit's serum must be 
 added to supply complement) is added to the fluid to be tested. 
 If human serum is present, even in an amount so small that no 
 precipitate is formed, the antigen and antibody combine and with- 
 draw the complement from solution. To test this ox corpuscles 
 are sensitized with the heated (decomplemented) serum of the first 
 rabbit and thoroughly washed, and some of the mixture added. 
 If the complement has been withdrawn, of course no haemolysis 
 will occur. Certain obvious controls are employed to demonstrate 
 that the corpuscles were actually sensitized, and that complement 
 was present in the rabbit's serum before the addition of the fluid 
 suspected of containing human blood. 
 
 This test is extraordinarily sensitive, Neisser and Sachs finding 
 that the millionth part of a cubic centimetre of human serum was 
 readily demonstrable. They claim also that it is more specific 
 than the ordinary precipitin test, it being necessary, for instance, 
 to use T^Viy c.c. of ape's serum to get the same result. But the 
 technique is complicated, and it appears, moreover, that comple- 
 ment may be abstracted in an altogether non-specific manner by 
 substances other than the combination of antigen and antibody. 
 Thus Uhlenhuth examined some spots of blood on a sack which a 
 cabdriver (who was found dead) used as a seat, and found it 
 brought about a fixation of complement, though it gave no reaction 
 with the precipitin test. He then tested the material of which 
 the bag was made, and found it also had the power of absorbing 
 
THE PRECIPITINS 
 
 237 
 
 complement. According to Neisser and Sachs, however, this 
 power is destroyed if the serum solution is boiled, but is unaltered 
 in the case of non-specific substances. Another serious objection 
 is that a similar deviation may be brought about by means of 
 sweat, so that if the reaction were obtained in a stain on body- 
 linen it would be of little value. 
 
 The precipitin reaction has also been used for determining the 
 nature of meat (whether fresh, as in the case of beef suspected to 
 be horse-flesh, or prepared, as in sausages, etc.). The serum is 
 prepared by injecting meat-juice or an (unheated) watery extract 
 of the meat, and the test is carried out on lines similar to those 
 described above. It has also been employed to determine the 
 nature of bones, i.e., whether they are human or otherwise. 
 
CHAPTER X 
 PHAGOCYTOSIS 
 
 METCHNIKOFF'S researches on phagocytosis in the lowly organized 
 animals formed a starting-point for an entirely new series of 
 researches on the subject of immunity, and his treatise on the 
 {< Comparative Pathology of Inflammation " must ever remain a 
 great medical classic, as well as a most fascinating work. 
 Metchnikoff was primarily a biologist, and his attention was 
 attracted to the spectacle of amoebae and other unicellular 
 organisms containing bacteria. In these lowly constituted 
 animals the process by which the cell takes in foreign particles 
 can be watched with ease, and the steps of the process traced. 
 The amoeba throws out arm-like processes, which surround the 
 bacterium, close on it, and in a few minutes an organism 
 which was previously lying free is deeply embedded in the 
 animal's protoplasm. Metchnikoff watched its fate, and found 
 it lost its sharp outline and clear appearance, became granular, 
 and in a little while disappeared altogether. He found also that, 
 in many cases at least, a minute vacuole was formed round the 
 ingested organism, and further research showed that this vacuole 
 contained a fluid which was acid in reaction and held in solution 
 a digestive ferment allied to pepsin. Considering this observation 
 more closely, it is obvious, firstly, that the amoeba must be regarded 
 as being immune to the organisms which it ingests and digests, 
 and, secondly, that in this case the processes concerned in im- 
 munity are those concerned in nutrition : the amoeba is immune 
 to the bacterium because it can make use of its protoplasm as 
 nourishment. 
 
 But this process, as Metchnikoff soon found, is not confined 
 to the unicellular organisms. His most beautiful and classical 
 series of researches deal with the properties and action of the 
 leucocytes in a small fresh-water crustacean (daphnia), which, 
 from its transparency and small size, is a very suitable object for 
 
 238 
 
PHAGOCYTOSIS 239 
 
 observation. Leucocytes, it may be pointed out, are cells which, 
 from their isolation from one another, power of independent move- 
 ment, etc., are closely analogous with amoebae and other lowly 
 organized protozoa. 
 
 Metchnikoff found that daphnia is subject to a disease due to 
 the invasion of its body-cavity by the spores of a yeast the 
 monospora and that if these spores gained access in large 
 numbers they multiplied, formed into mature organisms, and 
 finally killed their host. When, however, few spores gained 
 access, he found that the daphnia's leucocytes approached them, 
 
 FIG. 46. AN AMCEBA WHICH HAS INGESTED NUMEROUS SPECIMENS OF 
 MICROSPH^RA. (Metchnikoff.) 
 
 a, #, Vacuoles. 
 
 formed a wall round them, and finally digested and destroyed 
 them. It is obvious, therefore, that the immunity of these 
 animals is relegated, so to speak, to its leucocytes. If these are 
 efficient, the animal is preserved from its invaders; whereas, 
 if they make default, the latter multiply, and bring about the 
 lethal issue. 
 
 MetchnikofFs experiments were by no means confined to the 
 action of the leucocytes on bacteria. They included a careful 
 and exhaustive study of the method of absorption of all manner 
 of particles, organized and unorganized, in the tissues and body- 
 cavities of animals of all positions in the animal world, and they 
 proved to the full the importance of phagocytosis and intra- 
 cellular digestion, and one of the chief if not, indeed, the only 
 
240 
 
 PHAGOCYTOSIS IN THE LOWER ANIMALS 
 
 method by which intruding particles are dealt with in the animal 
 economy. And, further, they correlate in the clearest possible 
 way this " scavenging " of the tissues with normal processes of 
 
 FIG. 47. DAPHNIA CONTAINING LARGE NUMBERS OF MONOSPOR/E. 
 (Metchnikoff.) 
 
 FIG. 48. HIND PART OF DAPHNIA. 
 At a the spores of monospora are shown surrounded by leucocytes. 
 
 digestion and nutrition. Take, for instance, the absorption of 
 the red blood-corpuscles of birds, which are very suitable for 
 research, being readily recognized (they are nucleated), non- 
 poisonous, and easily digested. Metchnikoff showed that these 
 
PHAGOCYTOSIS 241 
 
 substances are absorbed from the alimentary canal of the lower 
 invertebrates by a process of intracellular digestion, and by that 
 alone. Thus, when the alimentary canal of a planarian (Dendro- 
 ccelmn lacteum, an animal resembling the liver-fluke in its general 
 anatomy) is filled with blood, the latter is found to undergo the 
 changes in colour familiar in bruises, and this is due to the fact 
 that the blood has been taken from the alimentary canal by the 
 cells lining it, and there has undergone the digestive changes. 
 These processes can readily be traced under the microscope, and 
 the blood-corpuscles can be seen, at first embedded in protoplasm 
 and of normal contour, and later enclosed in vacuoles and of altered 
 shape. Complete digestion takes several days, and every stage of 
 the process is easy to watch. Absorption of organized food particles 
 takes place from the alimentary canal in exactly the same way 
 in actinians, molluscs, and many other lower animals, and in 
 many of the cases which have been investigated the vacuoles are 
 found to contain a digestive ferment allied to trypsin or pepsin. 
 In higher animals this process of intracellular digestion does not 
 occur, or only to a slight extent (in the case of fats), the animal 
 finding it more advantageous to secrete the digestive juices into 
 the alimentary canal, and to absorb the products of their action 
 therefrom in a state of solution. 
 
 Absorption of particulate substances which have gained access 
 to the tissues takes place, to a very large extent, in a method 
 entirely similar. Thus Metchnikoff showed that when bird's 
 corpuscles are injected into the tissues of the larva of the cock- 
 chafer, or into the snail, earthworm, the peritoneal cavity of the 
 goldfish, etc., the process is also intracellular and entirely similar 
 to those which occur when these corpuscles are injected into the 
 alimentary canal of the planarian. In most cases the corpuscles 
 are absorbed by the leucocytes, in others by the cells of the part 
 (such as the endothelial cells lining the peritoneum) ; but in all 
 cases there is the same vacuolation, the same series of changes 
 in the ingested corpuscles, and the same final result. We shall 
 not be far wrong in associating the absorption of these corpuscles 
 in a very close way with processes of digestion and nutrition. 
 
 The French school have studied these processes of absorption 
 of particulate bodies from the tissues in a very complete manner, 
 and have shown beyond dispute the importance of phagocytosis 
 in this respect. Thus particles of carbon in the lung, the granules 
 of pigment left after interstitial haemorrhages, etc., are all ingested 
 
 16 
 
2^2. METCHNIKOFF'S THEORY OF IMMUNITY 
 
 and removed by phagocytes of one sort or another, and this dis- 
 covery throws a flood of light on the meaning of many of the 
 phenomena of inflammation, more especially on the leucocytic 
 invasion of the injured tissues, which has for its object, in part 
 at least, the removal of particulate substances which are the cause 
 of the injury. Further, the dead or injured tissues, the result of 
 the action of the irritant, are eroded and removed by phagocytic 
 action. If a small volume of tissue, situated in a region to which 
 numerous leucocytes can gain access, be killed, it may be com- 
 pletely removed piecemeal in this way. We may quote as an 
 example the complete absorption of the central slough which 
 often takes place in acne pustules or small boils, especially after 
 treatment with staphylococcic vaccine. Very interesting in this 
 connection is the process of the absorption of the tail of the 
 tadpole, which is removed in an entirely similar way by the action 
 of phagocytes. And it is worthy of notice that the digestive 
 power of the phagocytes is a very powerful one, and substances 
 usually deemed entirely insoluble may be gradually removed by 
 their action. 
 
 We have already referred to the action of leucocytes in ab- 
 sorbing and neutralizing toxins, and have quoted the beautiful 
 experiments of Besredka on trisulphide of arsenic, which, when 
 placed 'in the peritoneal cavity, is absorbed by these cells and 
 prevented from exercising its poisonous action ; whilst, when 
 shielded from their attack by being enclosed in bags which are 
 permeable to fluids, but not to solids, it undergoes gradual 
 solution, and leads to fatal intoxication. 
 
 Metchnikoff applied these researches on phagocytosis to the 
 question of immunity, and formulated a complete and logical 
 theory on the subject, which he illustrated with many striking 
 researches and examples. For him and modern advances tend 
 more and more to corroborate the truth of this view, though in a 
 much more complicated way than he thought the defence of the 
 animal economy is entrusted entirely to the phagocytes, and 
 especially to the leucocytes. If a bacterium enters the tissues 
 these cells may at once make their way to the seat of infection, 
 and proceed to ingest the bacteria and kill them intracellularly. 
 In this case the animal recovers, with or without a transient illness, 
 and we say it is immune. If another species of bacterium enters, 
 the effects may be different : the leucocytes may. perhaps, be 
 repelled instead of being attracted, or, if attracted, may be killed 
 
PHAGOCYTOSIS 243 
 
 by the action of the bacterial toxins, so that no phagocytosis 
 occurs ; or perhaps they may take up the bacteria and then be 
 killed by the toxins. In any case, the result is the same : the 
 bacteria continue to grow and to produce their toxins, and the 
 result is death. The animal is susceptible to the bacterium 
 because its leucocytes are unable to deal with it. Immunity, 
 therefore, is a function of the phagocytes. 
 
 So far we have dealt with natural immunity. The application 
 of Metchnikoff's theory to acquired immunity is equally simple, 
 though much less satisfactory. He argues that the leucocytes, 
 in their contest with a particular species of bacterium, become 
 educated to overcome this bacterium, and are able to deal with it 
 in future with great ease. In the first infection there may be a 
 balanced contest of some severity and duration, but as a result 
 the leucocytes, like war-trained veterans, are readily able to cope 
 with the invader a second time. This theory, though ingenious, 
 cannot be maintained at the present day. Its truth rests, of 
 course, on the truth of Metchnikoff's main thesis, which is only 
 partially true, and which is only one factor in the complicated 
 phenomena of immunity. We may just point out, however, that 
 the life of a leucocyte is, in all probability, a comparatively short 
 one, to be measured by days, or at most weeks, so that acquired 
 immunity due to the education of the leucocytes would be of 
 short duration. Nor is it of any assistance to argue that in the 
 struggle against the invading bacterium the fittest leucocytes 
 would survive, and so lead to the general improvement of the 
 leucocyte species : for leucocytes do not propagate themselves, 
 but are emitted from the bone-marrow, run their course in the 
 blood, degenerate, and die, their place being taken by others from 
 the same source. The education, therefore, must be one of the 
 bone-marrow, and we cannot conceive how this could take place 
 as the result of phagocytosis going on in a distant area. It is 
 possible that something of the sort may occur, but only by the 
 action of toxins and other bacterial products circulating in the 
 blood-stream, and being thus brought into the marrow. Other 
 considerations might be urged, but the theory has now but a 
 historic interest. It has served its purpose : it has been the 
 means of suggesting many researches which have helped greatly 
 in the elucidation of a most difficult subject. 
 
 Before discussing the role of phagocytosis in the infective pro- 
 cesses we must deal briefly with two subjects : firstly, the means 
 
 1 6 2 
 
244 CHEMOTAXIS 
 
 by which the phagocytes are brought into contact with the 
 bacteria in other words, of chemotaxis. This is a phenomenon 
 which is displayed by almost all motile and unicellular organisms, 
 whether animal or vegetable, and by the leucocytes of the higher 
 animals, and manifests itself in a movement of the organism in 
 response to a chemical stimulus. To take an example from the 
 bacteria : if a capillary tube containing a solution of meat-extract 
 be placed in a watery emulsion of B. coli or B. typhosus, the 
 bacteria will be seen to group themselves round the mouth of the 
 tube, which they ultimately enter. This is an example of positive 
 chemotaxis, the bacteria being attracted by the extractives, which, 
 indeed, they utilize as food. On the other hand, bacteria will 
 tend to remove themselves from an area from which alcohol and 
 / similar substances are diffusing : this is negative chemotaxis. As 
 a general rule, we may say that motile organisms tend to be 
 attracted into a region rich in useful and nutritious substances, 
 and are repelled from injurious ones, but this is not invariably 
 true in the artificial conditions of experiment. An organism may 
 be lured by a useful substance into a region where there is a 
 sufficient quantity of an injurious one to kill it. 
 
 In cases where leucocytes make their way into a tissue infected 
 with bacteria it must be, therefore, because the latter give off a 
 substance which has a positive chemotactic action on them. This 
 action may readily be shown by experiment, and the easiest way 
 is to work with the lymph of a cold-blooded animal, since in this 
 way all difficulties connected with a warm stage are avoided. If, 
 for instance, we take a few anthrax bacilli and place them on a 
 slide, add a drop of frog's lymph (from the dorsal lymph sac), and 
 apply a cover-glass, the leucocytes can be easily seen to crawl 
 actively up to the bacilli. To this experiment (Kanthack's) we 
 shall have to recur. 
 
 In nearly all cases we find that leucocytes are attracted in large 
 numbers into the area in which the bacteria are situated i.e.. 
 nearly all bacteria give off substances which are positively 
 chemotactic for leucocytes. In a few cases, however, this appears 
 at first sight not to happen, for when tissues which are infected 
 with very virulent bacteria are examined microscopically, they 
 are often extremely poor in leucocytes. As an example, we may 
 take any example of acute spreading gangrene of bacterial origin. 
 But, as Kanthack pointed out, these are not necessarily examples 
 of negative chemotaxis, and it is quite probable that the paucity 
 
PHAGOCYTOSIS 245 
 
 of the leucocytes is due to their paralysis and destruction by the 
 powerful toxins which are given off. Certainly since the use of 
 methods involving experiments on phagocytosis in vitro no valid 
 example of negative chemotaxis has been adduced, and it is highly 
 probable that leucocytes are attracted by all bacteria, whether 
 their toxins are mild or potent. In the former case the leucocytic 
 infiltration will be very obvious ; in the latter the cells will be first 
 paralyzed and then destroyed as soon as they reach an area in 
 which the toxin is present in a high degree of concentration. 
 
 We must not assume that this property of being attracted by 
 bacteria is of any advantage to the leucocyte itself, arguing from 
 the fact that the free-swimming unicellular organisms are attracted 
 by food and oxygen. The leucocytes are in many cases attracted 
 into the infected area to their own undoing, and it must not be 
 forgotten that even in an inflammatory process which is mild in 
 nature and favourable in result the number of leucocytes which 
 may be killed in the conflict is enormous. The leucocytes are not 
 independent protozoa inhabiting the blood and tissues, but an 
 integral part of the organism. It is to the advantage of the latter 
 that the former should be attracted at once to the seat of invasion, 
 and hence the processes of evolution have led to the development 
 of this function in the nomadic cells of the body. These are 
 extraordinarily susceptible to chemotactic influences ; they seem 
 to be attracted by any deviation from the normal constitution of 
 the tissues and fluid : a slight injury, a haemorrhage, the presence 
 of a poison, of a foreign body of any sort or of any dead or useless 
 tissue, and the leucocytes are immediately attracted into the area 
 affected. The more we regard the process, the more we must 
 regard it as one of the most exquisite examples of means to ends 
 met with in the animal economy. 
 
 Secondly, with regard to the nature of the cells which have the 
 power of acting as phagocytes. Of these the most important are 
 the leucocytes, and especially the polynuclear and large hyaline 
 cells of human blood. All the leucocytes, however, have phago- 
 cytic powers, as is well seen in opsonic estimations : the cells 
 which take up the fewest bacteria are the eosinophiles and the 
 small lymphocytes. The former are very deficient in this direc- 
 tion, and we may be certain that their main function is entirely 
 different. The lymphocytes, too, take up but a small number of 
 bacteria ; but when activated by a suitable haemopsonic serum, they 
 take up red blood-corpuscles in considerable numbers, and a 
 
246 VARIETIES OF PHAGOCYTES 
 
 lymphocyte may then present a remarkable appearance, having 
 ingested two or even three red corpuscles, each as large as itself, 
 the narrow band of protoplasm being extended over the corpuscle 
 in a most extraordinary way. 
 
 It is noticeable, too, that even among the polynuclears there are 
 great differences in phagocytic powers. This is best seen in a film 
 of pus from a case of gonorrhoea, in which certain cells (indis- 
 tinguishable from the others) are packed full of cocci, whereas the 
 vast majority are entirely free. The same fact is brought out 
 
 FIG. 49. RED CORPUSCLES INGESTED BY POLYNUCLEAR LEUCOCYTES 
 AND LYMPHOCYTES. (Original.) 
 
 clearly in opsonic experiments, where some leucocytes are often 
 found to take up very large numbers of bacteria, the general 
 average of the other cells being low. 
 
 Besides the leucocytes, some of the tissue cells, which are 
 either free or have the power of becoming so, are active phago- 
 cytes. Of these, the most important are the endothelial cells. 
 These are only flat plates of protoplasm when under normal 
 conditions. When submitted to the action of almost any irritant 
 they become cuboidal or columnar, and are then most active 
 phagocytes. A good example of this may sometimes be seen in 
 sections of a thrombosed vein at a certain stage : the endothelial 
 cells are columnar and contain much protoplasm, and this latter 
 is packed with pigment granules absorbed from the altered blood 
 in the lumen. But the process goes farther than this, and the 
 endothelial cell (whether of the serous membranes, vessels, or 
 lymph clefts) either breaks loose from its attachments or buds off 
 
PHAGOCYTOSIS 247 
 
 fresh cells, in either case leading to the production of free and 
 motile endothelial cells, which have the closest resemblance to 
 the large hyaline cells of the blood, though they may be much 
 larger. These cells are most active and important phagocytes, 
 especially in the peritoneum. They have also the power of 
 undergoing organization, especially into fibrous tissue, and many, 
 if not all, of the fibroblasts of granulation tissue are endothelial in 
 origin. Further, some at least of the giant cells so familiar in 
 chronic inflammatory processes are derived from the endothelial 
 cells of the lymph clefts and lymph capillaries. This has been 
 proved to demonstration by Bergengriin in the case of the giant 
 cells in leprosy. Our knowledge of the origin of the giant cells of 
 tubercle is less exact, but analogy with those of leprosy would 
 lead us to infer that they are endothelial also. In both diseases 
 the giant cells are most important phagocytes. 
 
 Epithelial cells only exceptionally act as phagocytes. We 
 have already referred to the ingestion of fat by the columnar cells 
 of the intestine, and the other important example is supplied by 
 the epithelial cells lining the alveoli of the lungs. These are 
 flattened plaques under normal conditions, but in the presence of 
 an irritant they become cuboidal or columnar, detach themselves, 
 or bud off similar cells, and are powerful phagocytes. These are 
 the dust cells so frequently seen in sputum. 
 
 The nature of the cells which take part in phagocytosis is 
 determined to some extent by the nature of the irritant. Thus, 
 when the pyogenic bacteria are ingested it is usually by poly- 
 nuclear leucocytes, whereas it is extremely rare to find these 
 cells containing tubercle bacilli in the tissues, though they will 
 take them up readily enough under the artificial conditions of 
 opsonic experiments. MetchnikofF classifies phagocytes into two 
 groups macrophages and microphages. His description of these 
 cells is not absolutely clear, but in general the microphages 
 correspond to the polynuclear leucocytes, and the macrophages to 
 the large hyaline cells of the blood and the endothelial cells of 
 the serous sacs and connective tissues. He claims that the 
 former are especially concerned with the phagocytosis of bacteria, 
 the latter with red blood-corpuscles and similar objects. This 
 distinction is not a valid one, since endothelial cells are ex- 
 tremely active phagocytes for bacteria, and polynuclear cells will 
 ingest red corpuscles with great readiness when provided with 
 a suitable opsonin (see Fig. 49). It would appear that under 
 
248 PHAGOCYTES OF THE PERITONEUM AND LUNG 
 
 suitable conditions any phagocyte can ingest any abnormal body 
 of suitable size. 
 
 Under certain .conditions there may be traced a remarkable 
 sequence in the advent of various phagocytes to an infected area, 
 which almost suggests a symbiosis of the nomadic cells. Thus, 
 when a culture of a bacteria is injected into the peritoneum of the 
 lower animals, a very definite sequence of events takes place. 
 The peritoneal fluid normally contains some small mononuclear 
 cells, probably of endothelial origin, and a few polynuclears. 
 For an hour or so after the injection these cells are diminished in 
 numbers, and the eosinophiles disappear altogether. The exact 
 cause of this diminution is not quite clear. It may be due to the 
 dilution of the peritoneal fluid by the solution injected, or to the 
 destruction of the cells, or to their clumping together on the 
 omentum, but in any case is not due to negative chemotaxis. For 
 the next two hours or so there is a gradual increase -of the 
 polynuclears, at the end of that time an influx of small mono- 
 nuclears, until in about six hours these and the polynuclears are 
 present in approximately equal numbers. After this the mono- 
 nuclears (which are probably budded off from the peritoneal 
 cells, and are thus endothelial in origin) gradually become larger, 
 forming what Metchnikoff calls macrophages ; then the fluid 
 slowly becomes concentrated, and the polynuclears gradually 
 disappear, many being ingested by the large mononuclears. 
 Finally these too disappear, but it takes about a fortnight for the 
 animal to revert to its normal condition. Both varieties of cells 
 endothelial and polynuclear take part in ingesting the cocci. 
 
 Briscoe has shown that a remarkable series of processes also 
 occurs when organisms, etc., are injected into the alveoli of the 
 lungs. It varies according to the substance injected, and we 
 may take the result of the injection of the potato bacillus as an 
 example. In the first hour and a half phagocytosis is very active, 
 the bacilli being taken up exclusively by the pre-existing alveolar 
 epithelial cells. Up to this time practically no polynuclears have 
 made their appearance, but they now commence to be attracted 
 into the alveoli, where they occur in large numbers for about 
 twenty-four hours. They take, however, but a small share in the 
 phagocytosis of the bacteria, and are themselves taken up by the 
 alveolar cells. Some proliferation of these latter cells occurs, 
 and the eosinophiles increase for the first twenty-four hours, and 
 then gradually diminish. We cannot trace the reason for this 
 
PHAGOCYTOSIS 249 
 
 sequence of phenomena, but it is evident that the division of 
 labour is carried to a high pitch amongst the phagocytes, and 
 that there must be some controlling influence which regulates the 
 appearance of the cell when it is required. 
 
 We must now turn to a discussion of the importance of these 
 facts in connection with immunity as it appeared to the patholo- 
 gists before the discovery of the antibodies. Much of this is 
 
 A '^" 
 
 l - & fi > 
 
 FIG. 50. FIG. 51. 
 
 FIG. 52. 
 FIGS. 50 TO 52. FROM SCRAPINGS FROM THE LUNGS HALF AN HOUR, Two 
 
 HOURS, AND TWENTY-FOUR HOURS AFTER THE INJECTION OF POTATO 
 
 BACILLI INTO A BRONCHUS. (From films lent by Dr. Briscoe.) 
 
 The bacilli, which occurred in large numbers in the alveolar cells half an hour 
 after injection, are not shown. 
 
 mainly of historic importance, but it is of extreme interest, and it 
 is to the controversy which occurred between the cellular and 
 cellulo-humoral schools that we owe much of our knowledge of 
 the processes of inflammation and of the functions of the leuco- 
 cytes. This controversy was carried out with great skill on both 
 sides, and was the means of suggesting numerous experiments 
 of much beauty and ingenuity. To begin with, Metchnikoft's 
 
25O OBJECTIONS TO THE THEORY 
 
 position was simple and logical. He pointed out that in mild 
 and non-fatal infections phagocytosis usually occurred, and the 
 bacteria could be readily seen inside the leucocytes, whereas in 
 fatal ones little phagocytosis took place, if any. He therefore 
 enunciated the paramount importance of the process in immunity, 
 and at one time considered it would cover the whole field of the 
 phenomena. 
 
 But his conclusions did not pass unchallenged, and the sup- 
 porters of the humoral school adduced numerous examples of 
 recovery from infection where little phagocytosis could be 
 observed, and went farther, and showed that recovery might 
 occur under conditions in which phagocytosis was impossible. 
 The best experiments of this sort were those of Baumgarten, 
 which were repeated by Sanarelli. These observers placed non- 
 virulent bacteria in the peritoneal cavities of animals enclosed in 
 bags of collodion or other substances which would permit the 
 free diffusion of the peritoneal fluids, but would prevent the access 
 of the leucocytes, and they found under such conditions that the 
 bacteria were completely destroyed. This was, of course, an 
 example of bacteriolysis of a type with which we are now 
 familiar. Other observers, including Metchnikoff himself, failed 
 to get these results ; but in an experiment of this sort a positive 
 result is of more value than a negative one. It is possible, for 
 example, that the walls of the bags which Metchnikoff prepared 
 may have been sufficiently impermeable to prevent the access of 
 the bacteriolytic substances. Then other observers found that 
 bacteria often underwent changes indicative of death and 
 destruction before they were taken up by the phagocytes. Thus 
 Nuttall found that when attenuated anthrax bacilli were placed 
 in a fine tube in the tissues of a rabbit's ear, the organisms showed 
 degeneration forms before they were taken up by the leucocytes, 
 and thought that they were injured by the serum before being 
 ingested. We have already alluded to this experiment as one of 
 the starting-points of the researches on the alexins. As a result 
 of experiments such as this, the humoralists relegated phagocytosis 
 to a part of quite secondary importance. They held that the 
 injury or death of the bacteria by the humours of the body was 
 the important factor, and admitted only that the phagocytes acted 
 as scavengers to remove the dead or disabled organisms. To 
 this Metchnikoff responded by allowing a leucocyte to take up a 
 living and virulent anthrax spore, and then isolating the leucocyte 
 
PHAGOCYTOSIS 
 
 251 
 
 and planting it on a suitable culture medium, on which the cell 
 died ; but the spore survived, showing that it was taken up with- 
 out any previous injury. He also traced in a very clear and full 
 manner the steps by which a tubercle bacillus of absolutely normal 
 appearance, and apparently vigorous and healthy, undergoes 
 
 .d 
 
 F 1G . 53. PROCESS OF ABSORPTION OF TUBERCLE BACILLI IN GIANT CELLS. 
 a, Unaltered bacilli ; b, c, d, and 0, various stages in the process. 
 
 degeneration, death, and absorption in the giant cell. His case 
 was proved to the hilt in the case of certain bacteria, whereas his 
 opponents proved theirs in others. They were dealing with 
 immunity of different types, and the time was not ripe for the 
 solution of the problem. 
 
 The views of another school which sprang up at this point, and 
 which attempted to reconcile these two views, are of more impor- 
 
252 CELLULO-HUMORAL THEORY 
 
 tance, in that they approach more closely to the modern theory of 
 opsonic immunity, and, indeed, are as close an approximation to 
 it as could have been formed in the then state of knowledge. 
 They were as follows : The importance of phagocytosis was 
 recognized, and it was also admitted that bacteria were frequently 
 prepared for ingestion by dissolved substances, but it was thought 
 that these substances emanated from the leucocytes. The phago- 
 cytes were thought to produce an alexin which injured the 
 bacteria, and then to devour them. Baumgarten's collodion-bag 
 experiments were explained by supposing that the leucocytes 
 which collected round the bags in the peritoneum gave off alexin, 
 which diffused through and was sufficient to kill the leucocytes, 
 though more slowly and with more difficulty than if the phago- 
 cytes had been able to give the coup de grace. In dealing with 
 organisms of very low virulence it was admitted that phagocytosis 
 might be all-sufficient. 
 
 Some of the experiments pointing in this direction may be 
 briefly referred to, though many have been alluded to before in 
 the chapter on the complements. Nuttall continued his experi- 
 ments on the destruction of anthrax bacilli by a comparison of 
 the action of blood and serum, and found that the latter was 
 enormously the more powerful ; and this he explained by the 
 assumption that the protective substances are given off in the 
 solution of the leucocytes which occurs in the process of clotting, 
 and many other experiments were forthcoming in support of this 
 view. But the most beautiful researches were those of Kanthack 
 and Hardy, alluded to previously, but now to be described at 
 greater length. When anthrax bacilli are placed in frog's lymph 
 and examined microscopically, the first phenomenon which occurs 
 is the approach of the eosinophile leucocytes to the bacilli. These 
 cells lose their granules, and at the same time the bacilli begin to 
 show signs of degeneration, the inference being that the granules 
 are dissolved, and that the solution acts injuriously on the 
 bacteria i.e., is alexin. The next step is for the hyaline cells 
 to approach the area of conflict, and to fuse with the eosinophiles 
 to form a plasmodium around the bacilli. Then the oxyphile 
 cells separate themselves from the plasmodium and move away, 
 and then the hyaline cells can be seen to have taken up the 
 bacilli, fragments of which can still be seen within them. Lastly, 
 a number of cells with basophile granulations are attracted, but 
 their function is unknown. It is obvious that there is here a 
 
PHAGOCYTOSIS 253 
 
 division of labour, the hyaline cells being the phagocytes and the 
 eosinophiles the mother cells of the defensive substances. The 
 granules may be regarded as a " pro-enzyme " stage of alexin. 
 
 It must not be thought from this experiment that it was held 
 that the eosinophile cells are always the cells which secrete the 
 
 V X X X \ 
 
 \ \ \ \ i 
 
 "ft 
 
 S W ' ft 
 
 FIG. 54. KANTHACK AND HARDY'S EXPERIMENT. (Original.) 
 
 1-6, An oxyphile leucocyte attacking a thread of anthrax bacilli ; the 
 figures were drawn at intervals of one and a half to two minutes, the 
 whole sequence occupying twelve minutes. 7-14, A thread repeatedly 
 attacked by three oxyphile leucocytes, one of which formed a plasmodium 
 with a hyaline cell when observations were commenced ; drawn at 
 intervals during a period of one hour. 15, A thread attacked by a plas- 
 modium, consisting of an oxyphile and a hyaline cell, the former having 
 lost its granules. The alteration in the bacilli, which was quite clear in 
 the specimens, is not shown. 
 
 The whole drawn from one preparation, the first series immediately after it 
 was put up, the second after half an hour, and number 15 after two hours. 
 
 alexin. This is certainly not the case in man, where these cells 
 play a very small part in inflammatory reactions of ordinary type. 
 Here we must assume that, if a similar process occurs at all, the 
 
254 ACTION OF " CYTASE IN PHAGOCYTOSIS 
 
 injurious substance is provided by the polynuclears, which thus 
 play both parts. 
 
 Kanthack's experiment is the best and most direct evidence of 
 the extracellular injury of bacteria by substances derived from 
 the leucocytes occurring as a preparation for phagocytosis. 
 
 Metchnikoff resisted these views for a time, but soon had to 
 admit that phagocytosis is not the only factor in immunity ; and 
 he then altered his theory in an ingenious way, and regarded the 
 extracellular injury or solution of organisms as being essentially 
 the same process as that by which they are digested after being 
 taken in by the phagocytes. He considers that bacteria, red 
 corpuscles, etc., after being taken in, are digested by the action 
 of a proteolytic enzyme which he calls " cytase " a term which 
 has been already alluded to as a synonym for complement or 
 alexin. Of this there are two sorts : macrocytase, which is 
 formed by the macrophages, and which digests corpuscles, cells, 
 etc. ; and microcytase, formed by the polynuclears, and powerful 
 against bacteria. Ordinarily these enzymes are restricted to the 
 cells which form them, and where ingested bodies are contained 
 in vacuoles, these latter contain a solution of the suitable cytase ; 
 but when solution of the phagocytes occurs the cytase is set at 
 liberty, and may then exert its action on cells or bacteria which 
 are lying free. Metchnikoff regards this as a process of much less 
 importance than phagocytosis, and points out that the solution 
 which it brings about is rarely complete : thus, when bird's 
 corpuscles are ingested, they are entirely absorbed, nuclei and 
 all ; whereas when they are acted on by a haemolysin (which 
 Metchnikoff regards as a macrocytase), the nuclei remain. This 
 is certainly true as regards the action of most sera on bacteria, 
 solution being rarely complete, and it is only in the highly potent 
 sera obtained by prolonged immunization to certain bacteria that 
 complete disappearance of the bacteria occurs as a result of the 
 action of serum ; yet when taken up by the leucocytes they are 
 digested altogether, sometimes with great rapidity. 
 
 The difference between these views and those of the cellulo- 
 humoralists is roughly this : Metchnikoff looks upon the protective 
 substance as a digestive enzyme which has for its object the 
 transformation of the foreign cells, etc., into proteids suitable for 
 the nourishment o the phagocyte ; whereas most bacteriologists 
 regard them as being allied to the toxins rather than to the 
 enzymes, and as being specially intended for the defence of the 
 
PHAGOCYTOSIS 
 
 255 
 
 body against invaders. The point is one of theoretical interest 
 rather than of practical importance, and we have already pointed 
 out that the complement is apparently used up in its activity, and not 
 set free to attack other molecules, as is the case with the enzymes. 
 Another minor point is that Metchnikoff seems to regard the 
 setting free of cytase as only occurring when the mother cell is 
 dissolved, whereas most of the bacteriologists who admit the 
 origin of alexin from leucocytes regard it as a product of its 
 secretory activity. The point has been referred to before. 
 Metchnikoff explains the phenomena which occur in immunized 
 
 or. 
 
 C 
 
 FIG. 55. PROCESS OF ABSORPTION OF ANTHRAX BACILLI IN THE LEUCO- 
 CYTES OF THE PIGEON. (Metchnikoff.) 
 
 (Showing various stages of alteration of the bacillus whilst in the protoplasm 
 of the leucocytes.) 
 
 as opposed to normal animals in this way : We will take the 
 absorption of bird's corpuscles from the peritoneum as an 
 example. When the injection takes place into normal animals, 
 there is no extracellular destruction of the corpuscles (haemolysis), 
 because there is no cytase free in the peritoneal fluid, no cor- 
 puscles having been broken down ; the corpuscles are taken up 
 by the phagocytes, but with some difficulty, since they have not 
 been prepared in any way for the process. When a second or 
 third injection is given, some haemolysis occurs, and this is 
 because the cells of the peritoneum are broken down by the 
 brusque introduction of the corpuscles ; this breaking down is 
 termed phagolysis, and is regarded as being a necessary pre- 
 liminary to the liberation of the cytase. The fresh leucocytes 
 which arrive now proceed to ingest the corpuscles with great 
 
256 ANALOGY WITH DIGESTION 
 
 avidity, since they are already partially digested. We should 
 explain the phenomena very differently : the haemolysis is a result 
 of the action of amboceptor and complement, and the phagocytosis 
 of the action of an opsonin. 
 
 The explanation of bacteriolysis and haemolysis by means of 
 complement and amboceptor might appear to be difficult on the 
 theory of the reference of the whole process to the digestive 
 action of the phagocytes, but Metchnikoff has applied the 
 researches of Pawlow in a very ingenious way to show a 
 parallelism between cytolysis and digestion. It will be remem- 
 bered that pancreatic digestion depends upon the action of two 
 substances an enzyme, protease, which occurs in the pancreatic 
 juice, and another substance, enterokinase, which occurs in the 
 succus entericus. Metchnikoff regards the protease as analoerous 
 
 0>wJrV--tX>ft&tr^ 
 
 with cytase, and the enterokinase as analogous with compleitieftt 
 or substance sensibilatrice. Delezenne showed (though I believe 
 his results are not universally accepted by physiologists) that 
 protease has no power of attaching itself to proteids, whereas 
 enterokinase has such a power, and the substance thus sensitized 
 can then be attacked by protease. This, if true, is exactly 
 similar to the action of amboceptor and complement. We may 
 suppose, then, that amboceptor represents some substance used 
 by the phagocyte to assist the action of cytase or alexin on the 
 bacteria, etc., and normally retained in the protoplasm. 
 
 When an organism which is easy to deal with is injected it is 
 taken up by the phagocytes and dealt with in their protoplasm, 
 no preparatory action being necessary. Under other circum- 
 stances, when the infection is a more virulent one, some of the 
 phagocytes are killed and dissolved, and their digestive enzymes 
 escape, and partially digest the bacteria, which are then ready for 
 phagocytosis. When there is a balanced contest of long dura- 
 tion another substance is formed, which, under normal circum- 
 stances, is not necessary for intracellular digestion, but which 
 facilitates it in difficult cases ; this also may escape into the 
 juices, and still further facilitate the preparatory stages of diges- 
 tion. Lastly, as a rarity, enough of these soluble substances may 
 be set free to dissolve the bacteria altogether, and render phago- 
 cytosis unnecessary. To Metchnikoff cellular digestion and . 
 nutrition are the important factors in immunity ; extracellular 
 action is a less important and occasional phenomenon, and occurs 
 mainly or entirely as a preparation for phagocytosis. His theory 
 
PHAGOCYTOSIS 257 
 
 is logical, complete, and well supported by evidence, but it does 
 not take into account the more recent work of Sir Almroth 
 Wright and his followers, and this now calls for discussion before 
 the role of phagocytosis in immunity can be profitably discussed 
 further. 
 
 It may be admitted that Wright did not discover the fact that 
 serum may aid phagocytosis by acting on the bacteria ; this had 
 been already shown by Denys and Leclef in 1895, by Mennes 
 and by Markl. And Neufeld and Rimpau had carefully investi- 
 gated the same property in the serum of animals immunized to 
 streptococci and pneumococci, and had described their bacterio- 
 tropic substances, which are apparently identical with what we 
 now know as thermostable opsonins. This does not detract in 
 the least from the credit due to Wright, who by devising a simple 
 quantitative method of examination, readily applicable in clinical 
 medicine, made a very great advance in our knowledge of the 
 theory of the subject, and has added a most important and useful 
 method of examination of the blood. The credit for the intro- 
 duction of the use of vaccines in the treatment of established 
 disease (as opposed to its prevention) is, of course, due to him 
 alone. 
 
 The name opsonin (opsono = I cater for, I prepare for food) is 
 given to substances which occur in the serum and have the power 
 of preparing bacteria and other cells for ingestion by the leuco- 
 cytes, and which are, or are held to be for there is no absolute 
 proof different from the substances which we have previously 
 considered. We shall discuss this question of identity or non- 
 identity subsequently, and shall be content at present with saying 
 that, whereas bacteria that have been exposed to the action of 
 alexin are, or may be, obviously injured, a bacterium may be 
 saturated with opsonin without being injured in the least, and 
 may still retain its viability and virulence uninjured. 
 
 The fundamental experiments of Wright and Douglas were of 
 this nature, and they are easy to repeat and unimpugnable in 
 accuracy. An emulsion of leucocytes, free from serum, is pre- 
 pared by receiving blood in normal saline solution containing 
 citrate of soda, centrifugalizing, removing the supernatant fluid 
 and replacing it with saline solution, mixing and recentrifugalizing. 
 This process must be repeated until all trace of serum is removed, 
 and the top layer of the deposit is then pipetted off, and will be 
 found to be rich in leucocytes. 
 
 17 
 
258 FUNDAMENTAL EXPERIMENTS ON OPSONINS 
 
 The first experiment is to determine whether leucocytes thus 
 free from serum are able to ingest bacteria. To this end they 
 are mixed with an emulsion of staphylococci or tubercle bacilli, 
 enclosed in a capillary tube and incubated at 37 C. for a quarter 
 of an hour. At the end of that time the emulsion is expelled, and 
 films are prepared and stained in the ordinary way. It will be 
 found that the leucocytes have taken up very few bacteria, if any. 
 It is obvious, therefore, that phagocytosis goes on to a very small 
 extent in the absence of serum. Some species of non-pathogenic 
 
 
 
 
 "'\ 
 
 ' 
 
 " 
 
 FIG. 56. ON THE LEFT, A PORTION OF AN OPSONIN FILM (OF PNEUMOCOCCI) ; 
 ON THE RIGHT, A PORTION OF A SIMILAR FILM, TAKEN FROM A PREPARA- 
 TION IN WHICH NO SERUM WAS USED. (Original.) 
 
 bacteria are taken up well in the absence of serum, and one micro- 
 coccus which I have met with was not ingested under any circum- 
 stances whatever. 
 
 Secondly, a mixture similar to the above is prepared, but with 
 the addition of one volume of serum, so that the mixture consists 
 of an equal volume each of leucocytic emulsion, bacterial emul- 
 sion, and serum. This is incubated and examined as above, and 
 it will be found that many bacteria are taken up ; the number 
 depends on the thickness of the emulsion and on the source of the 
 serum, but if the former be rich and the latter potent there may 
 be an average of twenty or even far more per polynuclear. The 
 bacteria which are not ingested show no signs of digestion, there 
 being no loss of sharpness of contour or of staining activity. 
 
PHAGOCYTOSIS 259 
 
 It is clear, therefore, that serum has a great power in aiding 
 phagocytosis. Is this due to an action on the bacteria or to a 
 stimulation of the leucocytes ? Two experiments show that the 
 former occurs ; there is but little direct evidence for or against 
 the latter. 
 
 In one experiment Wright (having shown that the power of the 
 serum is destroyed by heating it to 55 C. for thirty to sixty 
 minutes, or to 60 to 65 C. for fifteen minutes) allowed serum to act 
 on bacteria, and then heated the mixture until the activity of the 
 serum was removed. He found bacteria thus treated were taken 
 up readily. This must have been due to an action which the 
 serum had exerted on them before it was heated, and any action on 
 the leucocytes is out of question, since they were only acted on by 
 heated and inactive serum. 
 
 Another and even better proof of the same fact may be obtained 
 by acting on bacteria with serum, centrifugalizing and removing 
 all trace of the latter by repeated washings with saline solution. 
 Bacteria thus treated are taken up with great readiness, and here 
 no free serum at all comes into contact with the leucocytes. 1 
 Opsonin, therefore, combines with bacteria, and Bulloch showed 
 that this process goes on at ordinary temperatures and at o C. 2 
 Bacteria which have once been acted on by opsonin ("opsonized") 
 may be heated to 60 C. for five hours, and are still assimilable by 
 leucocytes ; this shows that they are profoundly affected, but they 
 may be absolutely unchanged in appearance. 
 
 There is no method by which an absolute measurement of the 
 amount of opsonin present in a specimen of blood can be made, 
 but comparative measurements can be made easily enough by the 
 process elaborated by Sir Almroth Wright. In order to do this 
 it is necessary to have as a standard either the serum of a normal 
 person, or preferably a mixture of sera from several normal 
 persons, so that slight individual variations or abnormalities may 
 be ruled out. The emulsion of leucocytes (" cream ") is prepared 
 as described above, and the emulsion of bacteria made by stirring 
 a little of a young culture of the organism in question in some 
 saline solution, taking care to remove clumps by sedimentation or 
 centrifugalization. When tubercle bacilli are being used it is 
 most convenient to employ dead and dried bacilli, which are 
 
 1 This experiment was performed by Markl in 1903, using plague bacilli. 
 - This is not altogether confirmed by Ledingham's more recent work, 
 which is discussed subsequently. 
 
 I 7 2 
 
260 
 
 OPSONIC TECHNIQUE 
 
 ground up in a mortar with saline solution before use. The 
 mixtures of leucocytes, bacteria, and serum are made in capillary 
 pipettes mounted with an indiarubber nipple, and furnished with 
 a unit mark about i inch from the free tip, which is drawn to 
 a fine point. The process is as follows : As much cream as will 
 reach to the unit mark is drawn into the pipette, then a little air 
 (to serve as an index), then a unit of the emulsion, another bubble 
 of air, and finally a unit of serum. These are then blown out on 
 to a glass surface, mixed intimately together, sucked into the 
 tube, the end of which is now sealed. The tube is now placed in 
 the incubator and the time accurately noted. Then the process 
 
 FIG. 57. WRIGHT'S CAPILLARY PIPETTES, AS USED IN DETERMINATIONS 
 OF THE OPSONIC INDEX. (Emery's " Clinical Bacteriology and 
 Hsematology.") 
 
 The small figure shows the tip magnified. The middle figure shows the 
 pipette charged with leucocytic cream (in this case two volumes are 
 shown), emulsion of bacteria, and serum. In the lowermost figure these 
 are mixed together and the tip sealed. 
 
 is repeated in exactly the same way, but the control serum is used 
 instead of that which is being investigated. Each pipette is 
 incubated for exactly the same length of time, rerribved from the 
 incubator, the tip broken off, the contents expelled, and films 
 made. These are obtained in a suitable way, examined under the 
 microscope, and the number of bacteria which have been taken 
 up by 50 or 100 poly nuclear leucocytes is counted in each. 
 Thus we may find that in the control specimen (in which 
 healthy blood was used) there are 300 bacteria in 100 leucocytes ; 
 in this case we say the " phagocytic index " is 3. In the other 
 specimen (in which the patient's blood was used) we might find 
 150 bacteria in 100 leucocytes, giving a phagocytic index of 1*5. 
 
PHAGOCYTOSIS 26l 
 
 We see in this case that the patient's blood has but half the 
 opsonic power of normal blood ; this we express by saying that 
 the opsonic index is 0-5. The opsonic index is obtained by dividing 
 the number of bacteria found in a certain number of leucocytes 
 in the films made with the patient's serum by the number of 
 bacteria in the same number of leucocytes in the films with the 
 control serum, and expresses the phagocytic power of the patient's 
 serum as compared with that of a healthy person. It is not 
 necessarily an exact measure of the amount of opsonin, since on 
 dilution of a serum the opsonic index falls at first slowly and then 
 more quickly, forming a flat-topped curve when plotted out in the 
 usual way (see Fig. 59, p. 265). 
 
 Other methods for the estimation of the opsonic index have 
 been suggested, and require some mention. In the earliest 
 method that of Leishman the patient's blood was mixed 
 directly with an emulsion of the bacteria in normal saline solution 
 in equal parts, and a drop of the admixture placed on a slide, 
 covered with a cover-glass, and incubated for a definite time. A 
 control specimen was prepared in a similar way, using normal 
 blood. After the incubation, films were prepared by sliding the 
 cover-glass off the slide, stained, and a count made as in the 
 method now in use. A similar but rather better method is also 
 employed, and is extremely convenient in some cases. The 
 bacterial emulsion is prepared as above, the organisms being 
 suspended in normal saline solution containing sodium citrate. 
 A mixture of this emulsion and of the patient's blood in definite 
 amounts (usually equal parts) is prepared, sucked into the pipette 
 (the tip of which is sealed), and incubated for a quarter of an 
 hour or twenty minutes. The process is the same as Leishman's 
 except that the mixture is incubated in a pipette, and not between 
 slide and cover-glass. A control is also prepared, using the same 
 emulsion and* normal blood, and is also incubated for a quarter 
 of an hour. At the end of this period films are prepared, and 
 the process finished in the ordinary way. 
 
 This method is theoretically more accurate as a test of the 
 phagocytic activity of the patient's blood as compared with normal 
 blood than is the opsonic index as determined in the ordinary 
 way, in which leucocytes from the same source are used in both 
 determinations i.e., in that of the patient's serum and in that 
 of the control. Thus, if in any case the leucocytes were so 
 injured that they had very little phagocytic power, the opsonic 
 
262 OPSONIC TECHNIQUE 
 
 index as determined by Wright's method might nevertheless be 
 normal ; yet this blood might have but little power of destroying 
 bacteria which gain access thereto. There is some experimental 
 evidence that alterations in the power of the leucocytes do 
 actually occur ; thus Shattock and Dudgeon, in some experiments 
 with granules of melanin (which, like bacteria, require to be 
 opsonized before they can be taken up by leucocytes), found that 
 either more or less might be taken up by the patient's leucocytes 
 as compared with normal ones, using the same serum in all cases. 
 The numbers varied between 0-46 and 2*9, taking the normal 
 number as unity. It must be pointed out that this method does 
 not give the opsonic index of the serum, and that in cases, e.g., 
 in which a low result is obtained it affords no information as to 
 whether the leucocytes or the serum is at fault, or both. Further, 
 there is a possible error owing to the possible difference in the 
 number of the leucocytes in the unit volume of the two specimens 
 of blood. Where the patient has a leucocytosis and this is very 
 common in the type of case in which opsonic estimations are 
 required the difference may be very great. The result of this 
 has not been fully elucidated, but it is obvious that where the 
 bacterial emulsion is not very thick the number available per 
 leucocyte is very different in the two cases. This is a point 
 worthy of consideration in the determination of the opsonic index 
 by Wright's method. 1 When the bacterial emulsion is very 
 dilute a large error is introduced, and even if very large numbers 
 of leucocytes are counted the results are untrustworthy. The 
 best results theoretically would be obtained where the emulsion 
 was so thick that every leucocyte would take up as many bacteria 
 as it was capable of doing in the given time. This is impracticable, 
 however, as the labour in counting leucocytes containing very 
 many bacteria is great, and the error in counting is also large. 
 Probably the best results are obtained where the phagocytic 
 index in the control is about 4, and it is a good plan to 
 perform an orientating experiment to determine the appropriate 
 strength of the emulsion before commencing a large series of 
 opsonic determinations. 
 
 1 It has been investigated by Ruth Tunnicliffe, who finds no very great 
 differences in a series of estimations in which the bacteria (diphtheria bacilli) 
 varied from 125,000 to 1,000,000 per cubic millimetre, all the other factors 
 being constant ; and by Walker, who finds that the index rises greatly if a 
 thicker emulsion is employed. 
 
PHAGOCYTOSIS 263 
 
 Another modification of Wright's method, introduced by 
 Simon, concerns the method of counting only. A large number 
 of leucocytes are counted, and are classified simply into those 
 that contain bacteria and those that are free. Of course, the 
 emulsion must not be too thick, or practically all the leucocytes 
 will have taken some up. The process is repeated with the 
 control, and the results compared; thus, if in the control film 
 25 per cent, of leucocytes were empty, and in the patient's film 
 50 per cent., the index would be = 0-5. A comparison of the 
 results obtained by this method and by careful counting show 
 that they are fairly comparable, and the process may be used 
 where it is only necessary to determine whether the index is 
 high or low. 
 
 Another and more important method is that of dilution or 
 extinction, as introduced by Dean and by Klien. It is especially 
 useful in the case of bacteria, such as B. typhosus and V. cholera, 
 which are dissolved by fresh serum when but slightly diluted. 
 Further, when an attempt is made to determine the opsonic index 
 to the former, and the pipette is incubated for but five minutes, 
 numerous shadows and partially digested bacilli are seen within 
 the leucocytes, thus introducing a new and very important error. 
 In order to avoid this, Klien determines the degree of dilution of 
 the serum necessary for the complete extinction of its opsonic 
 action. In preparations in which no serum is used the phagocytic 
 index is usually below 0*5, and the serum to be tested is diluted 
 until the degree of dilution is found, which gives a phagocytic 
 index no higher than this. Working by this method, Klien 
 obtained results very different from those obtained by Wright's 
 method. In the process of immunization of a rabbit the index 
 (by the latter method) remained low, varying only between 0-82 
 and 1-65, whereas by the process of dilution it was seen to be 
 actually greatly raised. Before the commencement of the im- 
 munization the opsonic power of the serum was extinguished 
 when the latter was diluted thirty times, whereas afterwards it 
 did not disappear until diluted 3,072 times. It appears clear that 
 in the case of bacteria like this the results obtained by Wright's 
 method are quite misleading. Klien states that the bacterial 
 emulsion should be a thick one, and should be of about the same 
 density in successive experiments, if these are to be comparable. 
 The main objection to this method is its tediousness: many 
 pipettes have to be prepared, and many films examined. 
 
264 
 
 METHOD OF EXTINCTION 
 
 It has an advantage over Wright's method even in cases in 
 which the bacteria are not dissolved in the serum or leucocytes, 
 in that it provides a definite measure of the amount of opsonin 
 present, which the ordinary method does not do, as is shown by 
 the fact that the opsonic index of a mixture of equal parts of 
 serum and normal saline solution is more than half that of 
 undiluted serum. 
 
 An enormous number of opsonic determinations have been 
 carried out, and the results have been of extreme interest. It is 
 
 3000 
 
 2,800 
 
 2,600 
 
 2,400 
 
 2.200 
 
 2,000 
 
 1.800 
 
 1.600 
 
 1,400 
 
 1,200 
 
 1.000 
 
 800 
 
 600 
 
 400 
 
 200 
 
 IB 17 19 21 23 25 27 29 31 2 4 6 8 10 12 14 
 
 * = Leucocytes per cubic mm. (figures at right of chart). 
 - = Opsonic power of serum. 
 
 = Bacteriolytic power of serum. 
 
 t-- - = Agglutinative power of serum. 
 
 FIG. 58. INFLUENCE OF INOCULATION OF TYPHOID VACCINE ON THE OPSONIC 
 POWER OF THE SERUM OF A RABBIT, AS SHOWN BY THE DILUTION 
 METHOD. (After Klien.) 
 
 found that the indices of healthy persons is approximately the 
 same, and does not vary much from day to day. In the case of 
 tubercle a very large number of determinations of the indices of 
 normal persons have been made, and it is found that, with one 
 or two exceptions, due perhaps to accidental errors in technique, 
 they all lie between o\S and 1-2, taking i as a standard. In reality 
 they agree very much more closely than this, for the great 
 
PHAGOCYTOSIS 
 
 majority lie much nearer to i. When the estimations are carefully 
 carried out, very few will be found below 0*95 or above i'O5- We 
 may regard the opsonic index for a given organism as a definite 
 quantity in a healthy person. Some sera are lower or higher than 
 others, but the difference is but slight, and the index of the same 
 person is found to show but slight daily variations as long as he 
 remains in good health. A few observations go to show that the 
 index is slightly lowered in persons who, without being ill, are in a 
 
 Serum 4: 1 
 
 3:2 2:3 
 
 C4 N.Safine 
 
 FIG. 59. SHOWING EFFECT OF DILUTION OF NORMAL SERUM ON THE 
 NUMBER OF BACTERIA TAKEN UP. (Original.) 
 
 state of lowered vitality, and that the onset of a mild disease, 
 such as a cold, may cause a fall in the index to tubercle or other 
 disease. 
 
 When the patient suffers from a disease due to a given organism, 
 and his index is tested against this organism, the results obtained 
 are of extreme interest. Taking the acute diseases first, we find 
 that as a rule the index is low at the commencement of the illness, 
 and that it rises, either gradually or suddenly, when recovery 
 takes place ; and in some cases there is a definite correlation 
 between the course of the index and that of the disease. For 
 example, Macdonald has shown that in an attack of pneumonia 
 the opsonic index of the patient's serum to the pneumococcus 
 remains at a constant low level until the crisis is reached, when it 
 shows a sudden rise, attaining a point above the normal level. It 
 remains elevated for a short time, and then relapses to normal or 
 below normal. It is difficult to believe that the rise in the opsonic 
 index and the consequent increase in phagocytosis which we 
 
266 
 
 OPSONIC INDEX IN ACUTE INFECTIONS 
 
 should expect to be caused thereby is not the cause of the crisis 
 and the patient's recovery. The short duration of the high level 
 of the index is interesting, as we know that the immunity left 
 after an attack of pneumonia is but temporary. 
 
 A gradual rise of the index often takes place in staphylococcic 
 diseases e.g., boils ; and when the index is traced from day to day, 
 it may be seen that it is low to begin with, during the onset and 
 increase of lesion, but that it rises more or less gradually until it 
 
 Days of disease 
 
 I 2 5 4 6 6 7 8 9 10 II 12 13 14 15 
 
 1-6 ^ 
 
 1-6 /\ 
 
 1-4 / \ 
 
 B / V - 
 
 FIG. 60. TYPES OF REACTION OF THE OPSONIC INDEX IN PNEUMOCOCCIC 
 INFECTION. (After Eyre.) 
 
 (a) Immediate, as seen in mild diseases ; (b) delayed ; and (c) progressive 
 decline, as seen in severe and fatal infections. 
 
 reaches a point well above normal. At the same time the disease 
 begins to improve, suggesting again that the phagocytosis de- 
 pendent on the amount of opsonin in the blood is the actual 
 cause of the recovery. Very many observations of this type have 
 now been made with many organisms, and as a general rule we 
 may say that in acute diseases (excluding tubercle) the index is, as 
 a rule, low during the onset and culmination of the disease, and 
 raised during involution and recovery. Exceptions may be met 
 with, but the sequence of events happens too often to be a mere 
 coincidence (see Figs. 60, 61, 63, and 64). 
 
PHAGOCYTOSIS 
 
 267 
 
 Hence the opsonic school of immunity has formed a theory 
 which may be enunciated as follows : The immunity to certain 
 organisms (not to all) depends on phagocytosis, and this can only 
 take place in virtue of the preparation of the organism by the 
 action of opsonin. Where this substance is present in normal 
 amount the person is sufficiently immune to resist ordinary 
 infections; but if for any reason the amount is lowered or the 
 
 2 
 
 1-8 
 1-8 
 1-7 
 1-6 
 1-5 
 14 
 1-3 
 1-2 
 l-l 
 1-0 
 9 
 8 
 7 
 6 
 5 
 4 
 3 
 2 
 
 FIG. 61. OPSONIC INDEX IN DIPHTHERIA. (Tunnicliffe.) 
 
 infection very virulent, phagocytosis cannot occur, and the disease 
 progresses. There is then a new formation of opsonin, just as 
 there is of other antibodies, and this goes on until there is 
 sufficient to sensitize all the bacteria and render them amenable to 
 phagocytosis, when recovery occurs. When this does not take 
 place the patient's phagocytes cannot ingest the bacteria, and the 
 disease progresses. 
 
 There is one assumption that will require critical consideration 
 subsequently, and that is that opsonin is an antibody. 
 
 The behaviour of the index in chronic infections is different, 
 
268 OPSONIC INDEX IN CHRONIC INFECTIONS 
 
 and is difficult to explain on the opsonic theory of immunity. In 
 a chronic staphylococcic lesion, such as acne, the index may be 
 low, normal, or high, and this is also the case to a most marked 
 extent in tuberculosis. Wright classified the cases of this disease 
 into two groups: (i) strictly localized tubercle, such as lupus, 
 mild glandular cases, tuberculous abscesses, etc. ; (2) cases 
 associated with constitutional disturbances. In the former he 
 found the index uniformly low (from 0-13 to o ! 88), whereas in 
 the latter there was great variation, the index being below normal, 
 or as high as 2 or more. Further researches, however, have 
 not confirmed this, and the indices of patients with lupus will 
 often be found very high. As a rule, however, the patients with 
 localized tubercle, if kept at rest in bed, will be found to have 
 a constant index, whereas in those with a progressive disease it 
 will be found to vary from day to day, being often very high. 
 
 These variations are attributed to auto-inoculation i.e., to the 
 discharge from the lesion of a few bacteria or of a small dose 
 of bacterial toxin, which makes its way into a region suitable 
 for the elaboration of a further amount of opsonin, acting just 
 as an injection of a vaccine, and causing a negative, followed by 
 a positive phase. When the patient is kept absolutely at rest in 
 bed this does not occur, or only to a comparatively slight extent, 
 and the index is more or less steady. If, however, the patient be 
 allowed to exert himself, even slightly, or if the lesions are 
 gently massaged, specific substances are set free, auto-inoculation 
 occurs, and the index exhibits its characteristic oscillations. It 
 is also dependent to some extent on the temperature, as has been 
 shown by Inman and others, tending (in phthisis) to fall with a 
 rise of temperature, and vice vevsz. In general, a fluctuating 
 temperature accompanies a subnormal index, a rise occurring 
 when the oscillations become less. The injection of a bacterial 
 vaccine may cause a rise of temperature, especially if the amount 
 is large, but does not always, and should not, do so. 
 
 In chronic infections a high opsonic index does not necessarily 
 imply that a patient is doing well. In general tuberculosis the 
 index is often normal or elevated, and a rise may occur just 
 before death. This is also the case in acute infections, such as 
 erysipelas, in which a sudden and great elevation may immediately 
 precede the fatal issue (Fig. 63). 
 
 These results are difficult to harmonize with the opsonic theory, 
 but Wright points out that it is not sufficient for there to be 
 
PHAGOCYTOSIS 
 
 269 
 
 enough opsonin in the blood; it must reach the diseased 
 tissue. 
 
 Some observations go to show that it may be unable to do 
 this under certain conditions. Thus Bulloch found the liquor 
 puris from a staphylococcic abscess entirely devoid of opsonin to 
 staphylococci. This might have been due to absorption by the 
 bacteria in the pus, so he cleansed the abscess, and, taking the 
 first few drops which collected, found them also very deficient 
 in opsonin. It appears, therefore, that this substance, though 
 present in the blood, was unable to make its way through the 
 
 DATE 
 
 6 
 
 7 
 
 8 
 
 9 
 
 10 
 
 n 
 
 F* 
 
 104 
 
 103 
 102. 
 101 
 100 
 99 
 98 
 97 
 96 
 
 O.I. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 2 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 2 i 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 2- 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 2- 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 2 : 
 
 
 
 
 
 
 
 
 
 A 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
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 x ^ 
 
 
 
 
 
 
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 ^ 
 
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 fi 
 
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 w 
 
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 N / 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
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 3 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 FIG. 62. SHOWING INVERSE RELATIONSHIP BETWEEN TEMPERATURE AND 
 OPSONIC INDEX IN PHTHISIS. (Inman.) 
 
 The continuous line shows the temperature. 
 
 wall of the abscess to the place where it was wanted. Again, 
 Wright has shown that the serous fluid in cases of tuberculous 
 pleurisy and peritonitis is very low in opsonin as compared with 
 the circulating blood, and has made use of this fact as a means 
 of diagnosis. It must be obvious that in the case of an extra- 
 vascular object like a tubercle, and especially a caseous mass, 
 that a slight alteration in the opsonic index of the blood can 
 have but a slight immediate effect ; any beneficial effect of a 
 high index must be slow in manifesting itself. To remedy this, 
 Wright attempts to flush the morbid tissues with blood or lymph 
 
270 
 
 SPECIFICITY OF OPSONINS 
 
 by diminishing the viscosity of the blood by the exhibition of 
 citrates and other anticoagulants, by the use of hot applications, 
 and by Bier's method of congestion. 
 
 The first point which arises in a discussion of the opsonic 
 theory deals with the specificity of the opsonins themselves. Are 
 we to imagine that there is a specific opsonin to each organism, 
 and that during the process of immunization this increases, 
 whilst the others remain constant ? Unless this is the case, the 
 theory fails, for we know that immunity is specific. 
 
 101 
 100 
 99 
 
 2-5 
 
 1-5 
 
 Day 
 7 8 
 
 $ 10 II 12 13 
 
 FIG. 63. BEHAVIOUR OF THE OPSONIC INDEX IN A MILD (a) AND SEVERE 
 (6) CASE OF ERYSIPELAS. (After Tunnicliffe. ) 
 
 The latter shows the preagonal rise ; the broken line in the first chart 
 indicates the temperature. 
 
 The question may be investigated in two ways by absorption 
 of the opsonins and by comparison of different sera. 
 
 The first method was employed by Bulloch and Western, who 
 added an emulsion of tubercle bacilli to normal serum, and found 
 but a slight reduction of the opsonic index to staphylococci, 
 suggesting the difference of the two opsonins. But these 
 results have not been confirmed by later writers, and it is quite 
 certain that a sufficient amount of tubercle bacilli will remove 
 practically the whole of the opsonin to staphylococci. These 
 experiments tend, therefore, to show that the opsonins are not 
 
PHAGOCYTOSIS 27! 
 
 specific, and that any immunity due to them would be a general 
 one. 
 
 The second method is by a comparison of various sera in their 
 action on various organisms. For example, we may take two 
 sera, and compare them in their action on tubercle bacilli and on 
 staphylococci. If we find uniformly that a serum which is low 
 to one is also low to the other, it will tell strongly against the 
 theory of specificity. This, however, is what we do not find, 
 and it is quite usual to discover that a serum which is very low 
 to the tubercle bacillus as compared with a normal control has 
 a normal index to staphylococci as compared with the same 
 control. Of this there can be no doubt. Further, after the 
 injection of a vaccine composed of the dead bodies of certain 
 organisms, it is usual to find the opsonic index to that organism 
 rise, whereas to others it remains unaltered. This tends very 
 strongly to show that opsonins are specific bodies. 
 
 Quite similar results are seen when the behaviour of the 
 opsonic index to two or more bacteria is followed from day to 
 day in a patient suffering from an infection by one of them. 
 Thus, in a patient who was recovering from a severe furuncle 
 the index to staphylococci and tubercle bacilli was observed, with 
 the result shown in Fig. 64. 
 
 Here we may regard the tubercle opsonin as being normal 
 throughout, the slight variations met with being well within the 
 range of experimental error. The index to staphylococci, on the 
 other hand, ranged between 0*4 and 1*35, and showed a general 
 parallelism with the amelioration in the patient's condition. It is 
 obvious that the two indices are not due to the presence of a 
 single opsonin. 
 
 Reverting to the saturation experiments, we may perhaps 
 explain them as follows : Any opsonin can prepare any bacterium 
 for phagocytosis if it combines with it ; but there are different 
 opsonins, with very different degrees of affinity for different 
 bacteria. 1 Thus we may suppose the tubercle opsonin to have a 
 powerful affinity for the tubercle bacillus, a slight one for the 
 staphylococcus, so that the addition of a few tubercle bacilli will 
 
 1 It now seems fairly clear that the explanation of these experiments is that 
 fixation of complement (which in this case acts as an opsonin) takes place. 
 Normal serum contains an amboceptor ( = thermostable opsonin) to staphylo- 
 cocci, though in small amount ; and this, when combined with staphylococci, 
 will attract all the opsonin to it, the staphylococcus opsonin most powerfully. 
 
272 
 
 SPECIFICITY OF OPSONINS 
 
 remove it from a sample of serum, whereas a large number of 
 staphylococci are required. In this case opsonins will have a 
 sort of modified specificity comparable with that of the agglutinins 
 for the coli group, and this appears to harmonize Bulloch's results 
 with those of later observers. 
 
 An example of this selective absorption of opsonins may be 
 given, chiefly to illustrate the methods employed in this class 
 
 1-5 
 14 
 1-3 
 1-2 
 
 II 
 
 9 
 8 
 7 
 6 
 5 
 4 
 3 
 -2 
 I 
 
 eo to 
 
 I 
 
 FIG. 64. BEHAVIOUR OF OPSONIC INDEX TO STAPHYLOCOCCI AND TUBERCLE 
 BACILLI DURING NATURAL RECOVERY FROM AN ATTACK OF FURUNCU- 
 LOSIS. (Original.) 
 
 of experiment. A specimen of normal serum was mixed with an 
 equal amount of very thick emulsion of staphylococci, kept at 
 37 C. for one hour, and then centrifugalized until all the cocci were 
 removed, leaving the fluid (a). A second amount of serum was 
 treated similarly, but the staphylococci emulsion was diluted 
 100 times (b). It was hoped that the staphylococcic opsonin 
 would be completely removed from the first, and only partially 
 removed from the second specimen. This was tested as follows : 
 
PHAGOCYTOSIS 273 
 
 Four experiments were carried out, in each of which the fluids 
 ( unit of each) were mixed with i unit of leucocyte "cream," 
 and of a fine emulsion of staphylococci, incubated, and films 
 prepared and counted. Thus : 
 
 Staphylococci in ,, l 
 50 Leucocytes. 
 
 1. Normal serum + normal saline ... ... 170 1*0 
 
 2. ,, ,, + supernatant fluid (b) ... 125 o'6g 
 3- ,, ,, -f- ,, ,, (*) ... 55 ' 21 
 4. Normal saline -f normal saline ... ... 24 
 
 These fluids were then tested in exactly the same way with 
 regard to their action on tubercle bacilli. Thus : 
 
 Tubercle Bacilli , , 
 in 50 Leucocytes, 
 
 1. Normal serum + normal saline ... ... 145 ro 
 
 2. ,, ,, -f supernatant fluid (b) ... 132 0*93 
 3- ,, + ,, ,, (a) ... go 0-6 
 4. Normal saline + normal saline ... ... 9 
 
 Here it is obvious that the staphylococci have removed the 
 staphylococcic opsonins more powerfully than the tubercle 
 opsonin. The strong emulsion removed 80 per cent, of the 
 former and only 40 per cent, of the latter. 
 
 A striking example of the fact that there is more than one 
 sort of opsonin is supplied by observations on the haemopsonins. 
 Most specimens of blood-serum are unable to act as opsonins 
 for the red corpuscles, which are not taken up by the leucocytes 
 under the ordinary conditions of opsonin investigation in vitro ; 
 but some specimens do possess the power of opsonizing red 
 corpuscles. It is obvious, therefore, that haemopsonin is not the 
 same as bacteriopsonin. 
 
 We must now discuss the nature of these opsonins. Are they 
 familiar substances (e.g., complements or amboceptors) mas- 
 querading under a new name, or are they essentially different ? 
 And if so, are they antibodies, or are they allied to other protective 
 substances, such as the alexins of the cellulo-humoralists or the 
 cytases of Metchnikoff ? This is an extremely difficult subject, 
 and one which has not yet been satisfactorily solved. 
 
 The main evidence in favour of the view that they are specific 
 
 1 The indices given are corrected by the deduction of the number of bacteria 
 taken up spontaneously (Expt. 4) from each of the totals. This may be 
 termed the corrected opsonic index, and ought to be given where great 
 accuracy is required. 
 
 18 
 
274 
 
 RESULT OF INJECTIONS OF VACCINES 
 
 antibodies is derived from a study of their behaviour when a 
 patient is inoculated with their specific antigens. If, for instance, 
 a patient with a low index for tuberculosis is inoculated with a 
 small dose of new tuberculin (say y^^ milligramme), consisting 
 of the dead bodies of the tubercle bacilli, a very definite train of 
 phenomena, closely comparable to the results of an injection of diph- 
 theria toxin, is produced. In each case there is an immediate fall 
 
 30 
 9 
 8 
 7 
 6 
 5 
 4 
 3 
 2 
 
 I 
 20 
 
 9 
 8 
 7 
 6 
 5 
 * 
 3 
 2 
 
 I 
 1-0 
 
 9 
 8 
 7 
 6 
 5 
 
 3 
 2 
 I 
 
 da.1t. 
 
 -i :>- 
 
 -ri~ 
 
 4 4 i-4--i i -4- 4-4- | -4-4 4-i-4-4-i- 
 
 | i : : I : 
 . j ^ j,...^ j.-.^.-^-.. 
 
 1-fc'l 
 
 ^ JH i M I 1 N Li i i-i l-i-l -i-4-i 
 
 rciij nijiciuiE xruinpi 
 
 "T t~j~ 
 
 ..] I....1.. 
 
 is |fcj n 
 
 lift! Mi :Uu ;I3| 
 
 FIG. 65. EFFECT OF A SINGLE INJECTION OF TUBERCULIN, SHOWING THE 
 "FALSE RISE." (Wright.) 
 
 of variable duration, followed by a rise to a higher level than the 
 initial one in other words, there is a negative followed by a 
 positive phase. (In some cases there is a sharp " false rise " of 
 short duration, which precedes the negative phase, a phenomenon 
 which, as far as I am aware, has not been found with the un- 
 doubted antibodies.) Now this rise, as has been already pointed 
 out, is to some extent at least a specific one ; an injection of 
 uterculin does not cause a rise in the opsonic index to staphy- 
 
 
PHAGOCYTOSIS 
 
 275 
 
 lococci or pneumococci. There is, therefore, one important feature 
 possessed by the opsonins in common with the antibodies : in each 
 case an injection of the specific antigen causes first a diminution 
 and then an increase in the amount present. 
 
 There is, however, an important difference. In the other anti- 
 bodies e.g., in diphtheria antitoxin the amount present in the 
 blood can be raised to a point enormously above that of normal 
 blood by a series of inoculations of suitable doses of toxin at suit- 
 able intervals. Here the effect of repeated injections is a cumu- 
 lative one, the second raising the index above the level which it 
 
 FIG. 66. RESULT OF A SINGLE DOSE OF STAPHYLOCOCCIC VACCINE, 
 SHOWING NEGATIVE PHASE. (Original.) 
 
 reaches after the first, and so on. But in the case of the opsonins 
 to most bacteria there is no such summation of results. An in- 
 jection of tuberculin may raise the opsonic index from 0-5 to 2 or 
 a little higher, but with a second injection it is not possible to 
 start with 2 as a base and raise the index to 3, and so on. The 
 maximum indices are not very much above the normal level. In 
 the case of tubercle it is very unusual to find an index as high as 
 2, whilst with the organisms of suppuration, etc., slightly higher 
 figures may occasionally be found. 1 As we have already shown, 
 this does not prove that the amount of tubercle opsonin present 
 
 J The highest indices of all are met with in the case of the meningococcus. 
 I have seen them exceed 10 in patients treated with vaccine, and higher 
 figures have been recorded. The explanation of these figures will be given 
 subsequently. 
 
 1 8 2 
 
276 DIFFERENCE BETWEEN OPSONINS AND ANTIBODIES 
 
 in blood never exceeds twice the normal and the actual amount 
 may be much more but anything like the enormous amounts 
 which can be obtained when working with antitoxins or agglutinins 
 are never met with in the case of these bacteria at least. 
 
 FIG. 67. SHOWING THE DIFFERENCE BETWEEN THE BEHAVIOUR OF THE 
 TRUE ANTIBODIES (DOTTED LINE) AND OPSONIN TO SUCH ORGANISMS AS 
 TUBERCLE (LOWER LINE) WHEN SUCCESSIVE INJECTIONS ARE GIVEN. 
 (Schematic.) 
 
 FIG. 68. SUMMATION OF NEGATIVE PHASES IN OPSONIN FORMATION AS THE 
 RESULT OF INJECTIONS IN RAPID SUCCESSION. (Schematic ) 
 
 When injections are repeated during the negative phase a 
 phenomenon of summation may be met with, as Wright first 
 pointed out. Here the first injection may lower the index, and 
 the second and third lower it still more, until a very low figure is 
 reached. A phenomenon similar to this may be seen after the 
 injections of toxins (Fig. 68). 
 
PHAGOCYTOSIS 277 
 
 It is on these facts that Wright's vaccine therapy, or, as it is 
 sometimes called, opsonin therapy, is based. The object of the 
 treatment is to bring about an immunization of the patient by 
 means of an increase of the opsonin circulating in his blood, and 
 this is achieved by the injection of a suitable vaccine. This con- 
 sists in all cases of the dead bodies of the bacteria causing the 
 disease. In the case of tubercle Koch's new tuberculin (TR 
 or TE) is used in variable amount, but not usually more than 
 T ^5- milligramme of dry material per dose. In the case of other 
 bacteria the vaccine is prepared by cultivating the organism on a 
 suitable solid culture medium, emulsifying with normal saline 
 solution, and heating to a temperature just sufficient to insure 
 sterility usually 60 C. for one hour is requisite. The emulsion is 
 then inoculated on to a culture medium, incubated in order to test 
 its sterility, and the number of bacteria which it contains is counted, 
 in order to determine the amount to be used as a dose. Suitable 
 dilutions are then made. The dose varies with different bacteria. 
 Thus, with staphylococci 250,000,000 to 1,000,000,000 cocci may 
 be given, whereas with B. coli 25,000,000 is usually enough for 
 the first dose. 
 
 The treatment is controlled by a frequent estimation of the 
 opsonic index, and this is supposed to be advisable for three 
 reasons: (i) It avoids the possibility of a summation of the 
 negative phases, and so a worsening of the patient's condition by 
 lowering his immunity to the infective organism. As a rule, the 
 negative phase is but of short duration, but occasionally it is pro- 
 longed, and this is especially the case when large doses have been 
 given. I have seen it as long as three weeks in a case of tubercle. 
 (2) It enables a suitable dose to be selected. Thus, if we find a 
 certain number of bacteria cause a long negative phase, the next 
 injection should consist of a smaller one, when the negative phase 
 may be reduced and the rise may be greater. With a very small 
 dose the negative phase may be eliminated altogether, or may be 
 reduced so much that it is overlooked. (3) Whilst the index is 
 raised decidedly above normal it is assumed that the patient is 
 benefiting, and another injection is only required when it begins 
 to fall. As a rough general rule, the injections have to be repeated 
 at intervals varying from a week or fortnight, but individual 
 patients show decided differences in this respect. 
 
 Of the practical success of this treatment in certain diseases 
 there can be no doubt, and whatever we may think of its theoretical 
 
278 " OPSONIN-THERAPY " 
 
 aspect, Sir Almroth Wright must receive the greatest credit for 
 its introduction. Before his researches the idea of injecting a 
 vaccine into a patient already suffering from a bacterial disease 
 was unthought of, although, of course, it was well known as a 
 method of producing immunity when disease was feared. The 
 question is often asked, Why inject more staphylococci into a 
 patient who has already too many ? The answer may, perhaps, 
 be as follows : The staphylococci which cause the lesion come 
 into contact with dead and diseased tissues only, and it is easily 
 conceivable that these may be very unsuitable to discharge so 
 vital a function as the formation of antibodies, whereas a few cocci 
 injected into the healthy tissues may cause a large amount. This, 
 however, does not explain the benefit which has been observed in 
 some cases of endocarditis and other haemal infections, for in them 
 the bacteria must be constantly gaining access to the healthy 
 endothelial cells, if to no others. But it is well known that not all 
 the tissues are equally adapted for the production of antibodies ; 
 thus, when diphtheria toxin is injected into the blood-stream little, 
 if any, production of antitoxin takes place. As a general rule, 
 when antibodies are required the blood-stream is the worst place 
 in which to inject the antigen, the serous membranes next, and 
 the connective tissues the best. Dr. Whitfield has suggested to 
 me that the reason may be that the stimulation of the opsonins 
 occurs best when dead bacteria are injected. Thus in the early 
 stage of the disease only living organisms are present, whilst later 
 we must suppose some are killed or die from some cause, and 
 then the stimulation of opsonin formation begins. The idea is 
 worth considering, but the subject is still obscure. 
 
 As regards the nature of these results : In tubercle, speaking 
 from my own experience, I can only report a moderate degree of 
 success, and this only in small lesions, such as tubercle of the iris 
 or cornea and of tuberculous ulcers. I have had but one or two 
 encouraging results and numerous failures with tuberculous glands, 
 bone disease, etc., though others have apparently been more 
 successful. In phthisis there appears to be some slight benefit 
 when combined with other treatment, and tuberculous sinuses 
 sometimes heal very quickly. I should only recommend the 
 treatment myself as an adjunct toother methods, or when surgical 
 interference is impossible or inadvisable. 
 
 With the diseases due to acute infections with staphylococci, 
 pneumococci, B. coli, and some other organisms, however, the 
 
PHAGOCYTOSIS 279 
 
 results are most beneficial. We may often see boils apparently 
 on the point of bursting retrocede in a most striking manner after 
 a single injection of staphylococcic vaccine, and pustular acne is 
 often equally benefited. Localized lesions of pneumococcic origin 
 often clear up quickly under the action of pneumococcic vaccine : 
 thus a case of empyema of the frontal sinus, due to this origin and 
 of four years' duration, was cured in five injections, spread over a 
 period of about twqjnonths. Numerous cases of cure of chronic 
 infections of the urinary tract with B. coli have been recorded, and 
 some cases of gonorrhceal arthritis have been cured in a remark- 
 able manner. In a case of my own a patient, with five large and 
 numerous small joints affected, was completely cured in three 
 months, after having been crippled for over two years. One or two 
 undoubted cases of ulcerative endocarditis have been cured, and 
 others in which there was a haemic infection (with streptococci), 
 though the evidence in favour of a valvular infection is less con- 
 vincing. The results in cases of Malta fever are also very 
 encouraging. As a rule, however, we may say that the special 
 scope of the method is in the treatment of localized infections. 
 
 A point of great practical importance, and one that has some 
 theoretical interest as pointing to a high degree of specificity in 
 the opsonins, is the fact that good results are sometimes obtained 
 only when the vaccine used is from a culture of the organism in 
 question from the patient himself. This is sometimes seen in 
 staphylococcic infections ; acne is occasionally very resistant to 
 stock vaccines, and yields readily to treatment with an emulsion 
 prepared from a culture from the patient's own pus. This 
 phenomenon is specially marked in the case of streptococci and 
 B. coli. 
 
 In admitting the success of vaccine therapy, we do not neces- 
 sarily admit the truth of the theory on which it is based, nor the 
 necessity for the opsonic control of the doses. It is certainly true 
 in general that with acute lesions there is a low opsonic index, 
 and that when amelioration or cure takes place a rise to or 
 above normal occurs, but this is not invariably the case. Thus, 
 occasionally tuberculous patients improve whilst the index 
 remains low, and those with a meningococcal infection often go 
 steadily downhill whilst the index is very high, though in the 
 latter case the symptoms are in general more severe when the 
 index falls. Now it is quite true and perfectly conceivable that 
 the continued existence of a lesion in spite of a very high opsonic 
 
280 OBJECTIONS TO THE OPSONIN THEORY 
 
 index may be due to a failure of the serum or leucocytes to gain 
 access to the lesions which are densely surrounded by inflam- 
 matory material. . We have adduced a similar reason to explain 
 the non-success of certain bactericidal sera. But it is otherwise 
 when we find that a patient improves when there is a low index, 
 for here we must admit that even this deficient amount of opsonin 
 is sufficient ; and, further, it is impossible to explain the formation 
 of- new lesions (e.g., staphylococcic) in patients in whom the 
 opsonic index is high often very high on any such grounds. 
 Again, a great rise in the opsonic index not infrequently occurs 
 just before death, as in the chart of the index in a fatal case of 
 erysipelas already figured. The more carefully the opsonic index 
 is considered, the more certain will it appear that a high index is 
 not an indication of immunity ; it neither proves that the lesion 
 is undergoing cure nor that a fresh infection will not occur. It 
 may, of course, occur concurrently with other properties in the 
 blood or tissues on which immunity does depend indeed, since it 
 is commonly due to the presence of a natural or artificial vaccine, 
 it usually does so but the parallelism between a rise in the 
 amount of opsonins and an increase in the grade of immunity is 
 not absolute. -Nor is a low index any proof of lack of immunity, 
 since patients may improve remarkably during a prolonged 
 negative phase. One of the most striking cases of amelioration 
 of a severe case of tubercle which I have ever seen occurred 
 during a negative phase lasting over three weeks. Allen has 
 noted a similar occurrence in gonorrhreal infections, from which, 
 however, he draws the assumption that the clinical signs are 
 a totally unreliable guide to the appropriate time for a fresh 
 injection a deduction which is logical only if we regard the 
 raising of the opsonic index, and not the cure of the patient, as 
 the object of treatment. 
 
 It seems probable, from a consideration of the phenomena of 
 phagocytosis in vitro, that a very small amount of opsonin even 
 less than that which is present in a serum in which the index is 
 very low is quite sufficient to sensitize any bacteria that are 
 likely to gain access to the tissues or blood. In our laboratory 
 experiments the conditions are certainly much less favourable 
 than they are in the living body ; the leucocytes are certainly not 
 in the same state of functional activity as they are in the body, 
 and there is a limited supply of serum instead of a constant stream 
 thereof. In spite of this, an enormous number of bacteria are 
 
PHAGOCYTOSIS 28l 
 
 taken up, and in some cases digested, within a few minutes. In 
 the body, of course, the action may go on for hours. The opsonin- 
 leucocyte mechanism would appear far stronger than is necessary 
 for the defence of the body. That it is not so indicates some 
 fallacy in the conclusions to be derived from these experiments 
 in vitro. We shall revert to this subject subsequently, and in the 
 meantime be content with pointing out that where a very small 
 amount of opsonin would appear sufficient for the resources of 
 the body, but little importance can be attached to small fluctua- 
 tions, or to a rise, e.g., from 0-8 to i. 
 
 The dread of a low opsonic index appears to have arisen on 
 purely theoretical grounds, and the only direct research on the 
 subject which seems to have been undertaken points rather in the 
 other direction. According to Pfeiffer and Friedberger, guinea- 
 pigs injected with bacterial vaccines (typhoid and cholera) do not 
 thereby become hypersensitive to doses of living cultures given 
 twelve or thirty-six hours afterwards ; on the contrary, they have 
 acquired an increased power of resistance, even after the shorter 
 period. And a very remarkable fact was noticed : this increased 
 resistance was not specific, since animals injected with heated 
 typhoid bacilli survived a lethal dose of cholera as well as of 
 typhoid. They conclude that the fear of a negative phase is 
 exaggerated ; and it must not be forgotten that the essence of 
 the " opsonin therapy " consists in administering a dose of vaccine, 
 in the first instance, while the index is low. 
 
 There is thus no direct proof that the period of the negative 
 phase is coincident with the period of hypersensitiveness to 
 infection. And when we compare it with the period of increased 
 sensitiveness to toxins, we find that, whereas the negative phase 
 comes on almost immediately, the hypersensitiveness to toxins 
 or tuberculin, or anaphylaxis to serum, takes some days to 
 develop. 
 
 Other theoretical interpretations of the undoubted good effects 
 of vaccine therapy are possible. Thus, a very probable explana- 
 tion is that it causes a local reaction in the form of an aseptic 
 inflammatory process in the neighbourhood of the lesion, which, 
 like the similar reaction caused by ultra-violet or X rays, has (in 
 some way not yet understood) a curative effect. The nature of 
 these " reactions " is considered subsequently ; in the meantime 
 it is sufficient to say that in the case of tubercle (and it is 
 probably a general effect) an injection of dead bacilli, or of the 
 
282 ALTERNATIVE EXPLANATIONS 
 
 products thereof, causes a sharp rise in temperature and an 
 inflammatory process around the tuberculous focus. If the dose 
 of tuberculin be greatly reduced the local reaction takes place, 
 but there is no rise of temperature. This is best seen when 
 small doses of TR are used in the treatment of tuberculous 
 iritis, in which the iris can often be seen to become injected after 
 each dose ; and I have observed the same reaction in a very 
 marked form after the use of diluted old tuberculin in von 
 Pirquet's reaction. In this case the dose absorbed must have 
 been infinitesimal, since the temperature did not show the slightest 
 sign of a rise. 
 
 Other possibilities are that the vaccine may cause a general 
 tissue immunity, or that it may produce some degree of immunity 
 on the part of the leucocytes, or may at least alter them in some 
 way so that they are more able to perform their duties as phago- 
 cytes ; and, of course, other antibodies, such as antiendotoxins, 
 may be produced as a result of the injection, and of these the 
 opsonic index affords us no estimate. 
 
 In reverting to the question of the nature and properties of the 
 opsomns, the question of their thermo-stability first claims our 
 attention. The results obtained by various observers are not 
 quite in accord, and indicate very clearly that more than one 
 substance may have the same action. The opsonin present in 
 normal serum is in a high degree thermolabile. It is destroyed 
 by heating to 55 C. for half to one hour ; at 60 C. most disappears 
 in five minutes, the rest more slowly, little being left in fifteen 
 minutes. Wright and Reid, however, found that in cases of 
 tuberculosis some of the opsonin is more thermostable, and 
 whereas in heating a normal control to 60 C. for ten minutes 
 reduces the opsonic index to almost nothing, the same proceeding 
 may only lower the index of a tuberculous serum to 0-4 or so, 
 though the indices of both samples were formerly the same. 
 They suggested this as a means for the diagnosis of tubercle. 
 Other observers have failed to corroborate their results, and they 
 are certainly not true of all cases. Dean showed that in certain 
 sera obtained by the high immunization of animals to certain 
 bacteria (staphylococci, dysentery, and typhoid bacilli) there are 
 substances which act as opsonins, and which are thermostable. 
 His results have been corroborated for pneumococcic serum by 
 Macdonald and Rosenau, by Muir and Martin, and many others. 
 It is evident, therefore, that there is more than one substance 
 
PHAGOCYTOSIS 283 
 
 which can prepare bacteria for phagocytosis. There is a thermo- 
 labile substance which occurs in normal serum, and a thermo- 
 stable one which is found in immune serum ; and this latter also 
 contains a thermolabile substance, since (as a rule) its index is 
 lowered by heat. Thermostable opsonin occurs in minute traces 
 in normal serum, since the index is never reduced quite to the 
 level seen in a control specimen made with normal saline by 
 heating to 60 C , and we need have no hesitation in recognizing it 
 as a specific antibody. It will be convenient to deal with it first, 
 and the question naturally arises, Is it amboceptor ? In other 
 words, Has amboceptor the power of preparing bacteria for 
 phagocytosis in addition to sensitizing them to the action of 
 complement ? The two substances arise under the same con- 
 ditions, and are identical in their power of resisting heat, faculty 
 of combining with bacteria, and in their specificity. The second 
 question arises, Assuming thermostable opsonin is amboceptor, is 
 the action of complement also useful in preparing bacteria for 
 phagocytosis, or does the process go on equally well without it ? 
 Now it is certain that complement is not necessary for the action 
 of thermostable opsonin; otherwise it would only exert its action 
 in a heated serum when subsequently activated by fresh serum, 
 and this is not the case. If thermostable opsonin is amboceptor, 
 therefore, it can exert its effects without the action of com- 
 plement. But some experiments go to show that thermostable 
 opsonin may be more potent when reactivated. Thus Crofton 
 found an antistreptococcic serum might stimulate phagocytosis 
 more when mixed with fresh human serum than with an equal 
 amount of normal saline. 
 
 Similar results have been obtained more recently by Dean, 
 who finds that the opsonic effect obtained by heated serum and 
 normal serum may be greater than the sum of the two effects 
 separately. The subject has been very carefully investigated by 
 Chapin and Cowie, who were able to avoid the possibility of 
 certain errors by performing their saturation experiments in a 
 cold room, kept at o C. throughout the experiment. They found 
 that a normal human serum treated with staphylococci at this 
 temperature might have the whole of its opsonic power removed, 
 and yet would still reactivate a heated serum i.e., the thermo- 
 stable opsonin combines with bacteria at o C., and is probably 
 amboceptor. They found that staphylococci treated with normal 
 serum at o C. and then washed are slightly more susceptible to 
 
284 AMBOCEPTOR AND OPSONIN 
 
 phagocytosis than are normal ones, but the difference is not great. 
 They are, however, much more easily opsonized by normal serum, 
 or by serum that has had its amboceptor removed by treatment 
 with staphylococci in the cold. 
 
 In other cases the conditions are more complex, for when a 
 potent bacteriolytic serum is present, bacteriolysis may occur to 
 such an extent as to diminish . the number of organisms which 
 can be taken up by the leucocytes. We then get the " reversed 
 ratio " phenomenon described by Leishman and Dean. It is as 
 follows : Under ordinary conditions the index falls greatly on 
 heating, as has been shown. This is called the normal ratio. 
 But in some of the potent sera obtained from highly immunized 
 animals the opsoni^ index may apparently rise after heating to 
 two or three times that of the raw serum. This Dean explains 
 and his explanation is an extremely rational one by invoking 
 the bacteriolytic action of the unheated serum. The number of 
 bacteria in the emulsion is reduced, so that there are fewer for the 
 leucocyte to take up ; some that are not completely dissolved 
 may lose their power of retaining stains and become invisible ; 
 bacteria partially acted on may be readily digested within the 
 leucocyte, so that they are not counted ; and, lastly, the dissolved 
 bacteria may have a toxic effect on the leucocytes. The 
 phenomenon of the reversed ratio may be taken as an argument 
 in favour of the equivalence of thermostable opsonin and 
 amboceptor. 
 
 The strongest argument, however, is derived from the experi- 
 ments of Dean, who has shown that in different samples of 
 immune sera there is a distinct parallelism between the two 
 functions : when the serum is powerful as a bacteriolytic agent, 
 when activated with a suitable complement, it is also powerful as 
 an opsonin after heating. It must be admitted, of course, that a 
 serum may be opsonic, but not bacteriolytic ; but this is explicable 
 on the assumption that much less of the substance is required to 
 sensitize the bacterium to the attack of leucocytes than is necessary 
 to render it soluble by complement. This has been confirmed 
 by Neufeld and Bickel, who found that a very minute amount of 
 haemolytic serum, far less than would produce haemolysis, would 
 act as a haemopsonin. 
 
 The opsonic index does not rise pan passu with the bacteriolytic 
 power, but this is partly due to the fact that the criteria are 
 
PHAGOCYTOSIS 285 
 
 different in the two cases. We have already shown that incre- 
 ments in the amounts of opsonin cause smaller and smaller 
 rises in the opsonic index as we proceed. There is, however, 
 a much closer parallelism between the bacteriolytic power and 
 the amount of thermostable opsonin present as shown by the 
 degree of dilution. This is well shown (in the case of typhoid 
 fever) by the chart given by Klien and inserted previously 
 
 (Fig. 58). 
 
 To sum up : Amboceptor appears to have the power of 
 sensitizing bacteria for phagocytosis, and this power appears to 
 be increased by the concurrent action of complement. Further, 
 there appears to be no sufficient evidence for the existence of 
 a thermostable opsonin apart from amboceptor, as has been 
 maintained by Neufeld and Hime. 
 
 (There is an additional possibility that the part of a thermo- 
 stable opsonin may be enacted by agglutinin. 
 
 I believe that in the case of the haemopsonins of normal 
 human serum the substance is a thermostable agglutinin with a 
 second thermolabile zymotoxic group. Natural haemopsonic sera 
 are, as far as I have seen, always powerful agglutinators of the 
 red corpuscles which they opsonize, and when they are heated to 
 60 C. the opsonic power is destroyed, but the agglutinative 
 faculty is unaltered.) 
 
 These facts may serve to explain the discordant results as to 
 the presence of thermostable opsonins in the sera of tuberculous 
 patients. It has been shown by Bruck that antibodies to the 
 tubercle bacillus are not always or usually present in the blood of 
 infected persons, and it is only when they are present that we 
 should find a thermostable opsonin. 
 
 If thermostable opsonins resemble amboceptor in their pro- 
 perties, there is an equally close resemblance between thermo- 
 labile opsonin and complement. Each occurs in normal serum, 
 and is destroyed by a short heating to 55 to 60 C. Are they the 
 same ? 
 
 The main fact against the theory of their identity is the 
 specificity, partial though it may be, of the opsonins ; for there is 
 no reason to think that different bacteria are attacked by different 
 complements, even if we accept the theory of the multiplicity of 
 these bodies to the fullest degree. But we have already seen 
 that the specificity of the opsonins is not complete, and that the 
 
286 COMPLEMENT AND OPSONIN 
 
 whole of the staphylococcic opsonin may be removed by the 
 addition of sufficient amounts of tubercle bacilli. 1 
 
 A second fact, closely allied and perhaps in reality identical 
 with the foregoing, is the rise in a particular opsonin after an 
 injection of a suitable vaccine, the others remaining constant. 
 This rise cannot be accounted for (in my opinion, at least) by 
 the appearance of small quantities of thermostable opsonin, 
 since it may occur when this substance cannot be found in the 
 serum. 
 
 On the other hand, there are very remarkable analogies between 
 the two substances. In each there is the same difference of 
 opinion as to whether it occurs in normal plasma, or is only 
 developed when clotting and destruction of leucocytes occur. 
 Wright and Douglas found the amount of opsonin present in 
 serum and in citrated plasma exactly the same, whereas Briscoe 
 found that very little phagocytosis took place when staphylococci 
 were injected into a surviving heart in which no clotting took 
 place. These divergencies are quite similar to those found by 
 different investigators in the case of complement. 
 
 Again, it has been already shown that when a blood-clot 
 contracts, the first serum which can be collected is poor in com- 
 plement compared with that which follows, and that after a time 
 the amount again diminishes. An exactly similar phenomenon 
 may sometimes, though apparently not always, be demonstrated 
 with opsonin (Henderson Smith). Hence an important practical 
 point : the patient's blood should always be collected at the same 
 time as the control in determinations of the opsonic index. 
 
 Thirdly, it has been shown by Levaditi that the aqueous 
 humour of the rabbit contains no complement and but a trace of 
 opsonin. But when the fluid which recollects after puncture was 
 examined, it was found to be rich in both substances. He found 
 a similar relation between the two substances in redema fluid. 
 As against these results we have to put the researches of Leding- 
 
 1 Since the above was written Muir and Browning have adduced very definite 
 evidence of a partial specificity in the case of the complements. They find 
 that the bactericidal action of normal serum may be due to the direct action 
 of complements, and that, on weakening normal serum by successive additions 
 of dead bacteria, the first effect is a falling off in the bactericidal action as 
 tested on that bacterium. Then the bactericidal action on the other bacteria is 
 diminished, and with a larger addition the haemolytic complement is absorbed. 
 This indicates features exactly like the partial specificity seen in opsonins, 
 and a similar absorption without the intervention of an immune body. 
 
PHAGOCYTOSIS 287 
 
 ham and Bulloch, who found that when the number of leucocytes 
 in the blood was increased by injections of cinnamate of sodium, 
 there was an increase in the complement, but not in the 
 opsonin. 
 
 It may be pointed out that if opsonin and complement are the 
 same, we must suppose that the opsonin test is the most delicate 
 method of demonstrating this substance that we have, since 
 phagocytosis may be facilitated by substancesfwnicnjji} comple- 
 ments cannot be detected by ordinary tests. Further, we must 
 assume that it unites with bacteria direct, and sensitizes them for 
 phagocytosis without the intervention of amboceptor. There is 
 no serious difficulty in accepting both suppositions. 
 
 Lastly, Muir has shown that the substances which have the 
 power of absorbing complement (such as compounds of red blood- 
 corpuscles and their amboceptor) also remove the thermolabile 
 opsonin. We are forced, therefore, to the conclusion that com- 
 plements may play the part of opsonins. But to do this we must 
 necessarily broaden our ideas of the complements, and attribute 
 to them some degree of specificity ; otherwise the opsonic index of 
 any given sample of serum as measured against a given control 
 should be always the same, which, as we have already emphasized, 
 is not the case (see footnote, p. 286). 
 
 These results suggest another train of ideas as to the role of 
 bacteriolysis in immunity. We have already seen reason to 
 believe that this is not of the greatest importance, and have found 
 it difficult to think that so elaborate a mechanism should be of so 
 little apparent use. May it not be that the complements are 
 specially intended for use as opsonins, and that their action in 
 bacteriolysis is a secondary one, and comparatively of less 
 importance ? This, of course, is a close approximation to Metch- 
 nikoff's views, but there is this difference : his cytase is a 
 digestive ferment which, in the case of microcytase, is adapted to 
 attack all sorts of bacterial proteid. But with the opsonins or 
 complements we must assume that different molecules occur 
 which have different combining affinities for the protoplasm of 
 different bacteria, or, in other words, which differ slightly in their 
 haptophore groups. Yet this difference is one in degree and not 
 in kind, for they all have some power of uniting with all bacteria, 
 and a great power of uniting with the bacterium-amboceptor 
 combination. 
 
 On this theory the appearance of amboceptor will take on a new 
 
288 SOURCE OF OPSONIN 
 
 significance, and we must regard this substance as a device for 
 attaching more complements and more varieties of complement to 
 an invading bacteria than can easily combine with it direct. In 
 other words, we must regard the cytophile group of the ambo- 
 ceptor as being specific, whilst the complementophile group has 
 the modified specificity which we attribute to the opsonins. The 
 presence of amboceptor will therefore enable the bacterium to be 
 prepared for phagocytosis by the concurrent action of many com- 
 plements which otherwise would only be able to attack it with 
 great difficulty. 
 
 And many facts, notably the liberation of endotoxin taking 
 place when bacteriolysis occurs, would lead us to believe that 
 this preparation for phagocytosis is the true function of ambo- 
 ceptor and complements, and that the appearance of the latter in 
 excess is a comparatively rare phenomenon in disease, and when 
 it occurs in enormous amounts (such as is seen in highly immunized 
 animals) is an artificial phenomenon comparable with the enormous 
 amounts of antitoxin seen in antitoxin -horses. Recovery from an 
 attack of disease caused by B. coli may occur without the appear- 
 ance of any amboceptor to B. coli demonstrable by ordinary tests ; 
 there may, nevertheless, be quite sufficient to act as a thermo- 
 stable opsonin. We are far from denying that bacteriolysis ever 
 occurs under natural conditions, but when there are plenty of 
 leucocytes of sufficient functional activity, it is difficult to avoid 
 the conclusion that they would ingest the bacteria when these 
 were sensitized by complement alone, or complement and a little 
 amboceptor, and before this latter substance had been developed 
 in amount sufficient to cause bacteriolysis. This latter process 
 may perhaps be the last line of defence, to be used only if the 
 leucocytes are injured by the toxins or by the high temperature, 
 or if they are present in insufficient numbers. 
 
 It has been pointed out already that there is some reason to 
 think that, whilst complement and amboceptor can each sensitize 
 for phagocytosis separately, they exert a more potent action when 
 both are present. 
 
 As regards the source of opsonin, little is definitely known. 
 If we regard the thermolable opsonin as identical with comple- 
 ment, we shall regard it as probably derived from the polynuclear 
 leucocytes, and this is corroborated by Levaditi's observations 
 on the aqueous humour. Eyre has also shown that the amount 
 of opsonin (to pneumococci) in the serum in pneumonia may be 
 
PHAGOCYTOSIS 
 
 289 
 
 roughly parallel with the number of leucocytes per cubic milli- 
 metre (Fig. 69). 
 
 This, however, was not corroborated by Bulloch and Leding- 
 ham in the case of the hyperleucocytosis caused by cinnamate 
 of soda. But it is highly doubtful whether leucocytes hurried 
 prematurely from the bone-marrow, etc., are, as the result of the 
 injection of chemical substances, as active functionally as those 
 occurring normally in that situation ; and this is corroborated 
 
 FIG. 69. RELATION BETWEEN LEUCOCYTES, OPSONIC INDEX, AND TEMPERA- 
 TURE IN A CASE OF PNEUMONIA. (Eyre.) 
 
 Dotted line = number of leucocytes per cubic millimetre ; thick line = opsonic 
 index; thin line = temperature. 
 
 by the fact that these observers found the leucocytes in question 
 deficient in phagocytic powers. The point is one of some im- 
 portance in connection with the lack of benefit which so often 
 follows an artificial leucocytosis brought about for therapeutic 
 purposes. 
 
 A few words on the subject of MetchnikofFs views on the op- 
 sonins may be added. He thinks that when bacteria gain access 
 to the blood or tissues, the presence of opsonins or other pre- 
 paratory substances is unnecessary, and the unaltered organisms 
 can be attacked by the fresh and vigorous leucocytes. The 
 
 19 
 
2QO METCHNIKOFF S VIEWS ON OPSONINS 
 
 absence of phagocytosis in vitro in the absence of serum he 
 attributes to a weakening and injury of the cells, due to the 
 method by which they are prepared, and admits that these 
 weakened and altered leucocytes will ingest bacteria more easily 
 and more quickly if the latter have previously been prepared by 
 the action of serum. He admits, however, that the opsonic index 
 determines the defensive resources of the blood, and in doing so 
 would appear to range himself definitely amongst the adherents 
 of the opsonin theory. But there is no reason to think that 
 washed leucocytes are weakened in respect of their phagocytic 
 powers ; they can take up enormous numbers of (opsonized) 
 bacteria in a very short space of time, and it is difficult to believe 
 that they could take up more in the living body ; and if, as there 
 is reason to think, phagocytosis is a physical process akin to 
 agglutination, the functional activity of the leucocytes is a factor 
 of little importance in phagocytosis, though essential for the 
 other, equally necessary, phenomena of digestion and solution 
 which Jake place subsequently. Metchnikoff finds that washed 
 Bacteria can take up large numbers of bacteria slowly, even in 
 the absence of serum. This, however, proves nothing, since we 
 have seen there is some reason to believe that opsonin may be 
 formed from the leucocytes themselves. But, as a matter of fact, 
 the increase in phagocytosis in preparations incubated for one or 
 two hours as compared with those incubated for fifteen minutes 
 is slight as compared with that consequent on the addition of 
 serum. 
 
 The influence of the source of the leucocytes taking part in 
 phagocytosis is not yet fully investigated, and there are no facts 
 known at present which tend to show that those from an immunized 
 animal have any special powers in this direction. Bulloch showed 
 in a few cases that leucocytes from different sources would take 
 up the same number of bacteria if used with the same opsonic 
 sera. There are also observations tending to show that diseased 
 or abnormal leucocytes e.g., those produced in excess as a result 
 of the injection of certain substances, such as nuclein are deficient 
 in phagocytic activity. 
 
 In a very few cases some phenomena indicating an immunity, 
 and consequent increased phagocytic power of the leucocytes, 
 from an immune or infected person, as compared with the normal, 
 have been noticed. This, of course, is quite in accordance with 
 MetchnikofPs theoretical views. The examples are not numerous, 
 
PHAGOCYTOSIS 2QI 
 
 and the best is, perhaps, that given by Bassett-Smith, who found 
 that in Malta fever the patient's leucocytes may be decidedly 
 more potent than normal ones, when used in conjunction with 
 the patient's serum, though, when normal serum is used, the 
 
 difference may disappear. Thus : 
 
 Cocci per Leucocytes. 
 
 Patient's serum -f patient's leucocytes + emulsion of cocci 23-0 46-0 
 
 ,, ,, + normal leucocytes -f ,, ,, 8'6 25-0 
 
 Normal serum + patient's leucocytes + ,, ,, i6'o 30^0 
 
 + normal leucocytes + ,, ,, 19*0 29-0 
 
 Rosenau has also brought forward evidence to show that 
 leucocytes from cases of pneumonia have greater phagocytic 
 powers than those from healthy persons, and are less easily 
 killed by heat. 
 
 Much attention was attracted of late by Bail's theory of the 
 aggyessins. Bail found that if washed tubercle bacilli were injected 
 in large amount into the peritoneum of guinea-pigs infected with 
 tubercle, the animals died rapidly i.e., in eight hours or so. 1 
 There was a fluid exudate (containing lymphocytes) in the peri- 
 toneal cavity, and this exudate (centrifugalized to get rid of cells 
 and bacteria) was found to have a remarkable action in increasing 
 the virulence of young tubercle bacilli to normal animals. Thus, if 
 a few cubic centimetres were injected together with the bacilli, 
 death occurred in about twenty hours, instead of in some weeks. 
 He found that this virulence was apparently due to an inhibitory 
 effect which the fluid exerted on phagocytosis. When bacilli 
 were injected into a normal animal without the exudate, many 
 polynuclears appeared in the peritoneal fluid and many large 
 mononuclear cells, and many of the bacilli were taken up ; but 
 when bacilli and exudate were injected, few cells other than 
 lymphocytes were seen, and there was no phagocytosis. 
 
 These observations were confirmed and extended by Bail and 
 others, and similar phenomena were found to occur in the case of 
 numerous other organisms, if not in all. A very striking example 
 was given by Weil in the case of the bacillus of chicken cholera, 
 which is extremely virulent to rabbits, so that a millionth of a 
 culture (containing perhaps but one bacillus) is certainly fatal. 
 A minute trace was injected into the pleura, and the animal died 
 in a few hours. Several cubic centimetres of turbid exudate, the 
 
 1 This, of course, is equivalent to the tuberculin reaction in an extreme 
 form. 
 
 192 
 
2Q2 THE AGGRESSINS 
 
 cells of which had not taken up any bacteria, were collected, and 
 were found to have a most potent effect in increasing the lethal 
 action of the organism. This could not be tested on rabbits, 
 since they were loo susceptible, but in guinea-pigs it was found 
 to lower considerably the lethal dose. A most interesting obser- 
 vation was made : A guinea-pig which had received a small dose 
 of a culture of chicken cholera, and had apparently recovered 
 completely, was injected eight days after with some of the exudate, 
 and died of chicken cholera septicaemia, showing that the bacteria 
 were but latent, and had been allowed to become virulent and 
 active in virtue of the action of the exudate. Further, this fluid, 
 when injected into rabbits, was found to immunize them against 
 subsequent injections of the organism, even if mixed with the 
 exudate, and so rendered more virulent. 
 
 To these substances Bail gave the name of aggressins, and 
 considered them to be an entirely new type of specific substances 
 formed by the organism, and having the power of raising its 
 apparent virulence by checking phagocytosis and allowing the 
 invading microbe to flourish without hindrance : thus, by means 
 of the concurrent presence of its specific aggressin, an almost in- 
 nocuous organism, such as B. subtilis, becomes extremely virulent. 
 According to Bail and his followers, aggressins are only formed in 
 vivo ; but this is denied by others, who claim that a watery emulsion 
 of certain bacteria has many at least of their peculiar characters. 
 
 Immunity due to the injection of aggressin is supposed to depend 
 on the formation of a specific antibody, or anti-aggressin. It is 
 produced very rapidly after the injection of the aggressin, and lasts 
 several weeks or more, and is supposed to be due to the immediate 
 neutralization of any aggressin which the bacterium may form in 
 vivo by the anti-aggressin, so that phagocytosis in unchecked. 
 
 Agressins are sharply specific, except perhaps in the case of 
 those for B. typhosis and B. coli i.e., the injection of one aggressin 
 will not prevent the phagocytosis of any species of bacterium 
 other than that by the action of which it was prepared ; hence 
 they are not mere leucocyte poisons : they are thermolabile. 
 
 A substance which prevents phagocytosis may act on the leuco- 
 cyte, the bacterium, or on the serum. The fact just described 
 (that phagocytosis of a bacterium A can go on in the presence of 
 an aggressin B) shows that the action of the aggressins is not on 
 the leucocytes. Further, as Weil and Nikayama have shown, 
 bacteria which have been acted on by their aggressins and the 
 
PHAGOCYTOSIS 2Q3 
 
 latter removed by washing, are readily ingested. The action, 
 therefore, must be on the serum i.e., aggressin must act as an 
 anti-opsonin. 
 
 This leads us to Wassermann and Citron's explanation of the 
 phenomena. They suppose aggressins to be simply solutions of the 
 bacterial protoplasm which have the power of combining with the 
 specific protective substances of the animal, and so disarming its 
 methods of defence. In other words, they are solutions of endo- 
 toxin of feeble toxicity. This view is strongly supported indeed, 
 practically proved by the researches of Doerr, who found that 
 aggressins caused a precipitate when mixed with their specific 
 immune sera, and that their presence might bring about an 
 absorption of the complements, just as if they were free bacterial 
 receptors. There are a few minor differences between aggressins 
 prepared in vivo and those obtained from cultures in vitro, but 
 not more than we might expect from the differences in their mode 
 of production. 
 
 If aggressins are merely free molecules of bacterial protoplasm, 
 we should expect them to combine with opsonins, just as do the 
 bacteria themselves, and hence to act as anti-opsonins. And this 
 supplies a striking proof of the specificity of the opsonins, for, as 
 already stated, an aggressin of one organism (e.g., B. coli) does 
 not prevent the phagocytosis of another organism (e.g., B. subtilis). 
 This must apply to the thermolabile opsonin, or opsonin proper, 
 since these experiments were made on normal animals. 
 
 The relationship between virulence and phagocytosis is an 
 interesting one. As a general rule, it will be found, as shown by 
 the extensive researches of Metchnikoff and his school, that there 
 is an inverse ratio between the two : when an organism is viru- 
 lent for an animal it will be ingested by the leucocytes to a very 
 slight extent, and vice versa. This refers, of course, mainly to 
 natural immunity, since in acquired immunity other factors, such 
 as the action of bacteriolysins or antitoxins, may come in. There 
 are, however, some exceptions. Thus, tubercle bacilli injected 
 into the peritoneum of normal guinea-pigs are readily taken up by 
 the phagocytes. We must assume in this case that an organism 
 may be taken up whilst it is alive and uninjured, that it may be 
 entirely indigestible by the leucocyte, and may continue to grow 
 and multiply in its interior. This is also sometimes seen in acute 
 infections : the common localization of the meningococcus in the 
 polynuclear leucocytes is well known, and Andrewes has described 
 
2Q4 EFFECT OF VIRULENCE ON PHAGOCYTOSIS 
 
 a case of general haemic infection by this organism which ran a 
 rapid fatal course in spite of all the organisms (as far as could be 
 seen) being taken up by the leucocytes. In general, however, the 
 law holds good, and where there is abundant leucocytosis the 
 disease tends to recovery ; when there is little or none, to death. 
 
 As far as we know at present, the failure of phagocytosis which 
 occurs with virulent bacteria is due to their deficient opsonization ; 
 but whether this is because they require a large dose of opsonin 
 before they can be ingested, or whether the opsonin cannot com- 
 bine with them, has not yet been determined quite satisfactorily. 
 It is this resistance which very virulent bacteria exert to phago- 
 cytosis which causes the very high indices seen in meningococcic 
 infections. If the index is determined using the very virulent 
 organisms recently isolated from a case of cerebro-spinal fever, 
 very little, if any, opsonization and phagocytosis take place in 
 the specimen in which normal serum is used, whereas a fair 
 number are taken up when the serum from a patient is employed. 
 If, however, the index be determined using an old laboratory 
 culture, much more phagocytosis will be caused by normal serum, 
 and the index will be nearer unity. The relation between viru- 
 lence and lack of phagocytosis is discussed subsequently in the 
 section on immunity to bacteria. 
 
 Lastly, many bacteria form toxins, of one sort or another, which 
 prevent phagocytosis by a direct action on the leucocytes. It has 
 been shown that tetanus spores and bacilli, when washed per- 
 fectly free of toxin, are quite innocuous to all animals, and are 
 readily taken up by the phagocytes ; the presence of toxin, it may 
 be in small amounts, by killing or injuring the leucocytes, allows 
 the bacilli to grow in the tissues and elaborate more toxin. Similar 
 facts probably occur in the case of diphtheria. We have already 
 referred to the production of leucocidin by streptococci, and it is 
 obvious that when this is formed in the tissues in large amount 
 phagocytosis will be reduced or stopped altogether. 
 
 The nature of phagocytosis requires some discussion. We are, 
 perhaps, rather too apt to be influenced by the readily observed 
 phenomena of ingestion of bacteria, diatoms, etc., by amoebae, 
 and to assume that it is in all cases an active process on the part 
 of the leucocytes, which are usually considered to approach their 
 prey by active movements directed by positive chemotaxis, and 
 to seize them by means of their pseudopodia. Chemotaxis does, 
 of course, occur in the tissues, but it is clear that it does not take 
 
PHAGOCYTOSIS 2Q5 
 
 place in the artificial conditions of opsonin estimations, where the 
 bacteria are uniformly distributed throughout the fluid, and there 
 is no reason why the leucocyte should be attracted in one direc- 
 tion rather than in another ; and movement of leucocytes either 
 does not occur at all or does so only to a very minute extent in 
 saline solution. It takes place much more actively in unheated 
 serum a fact which gives some support to the theory of stimulins, 
 previously mentioned, but not discussed. It is quite possible that 
 all the facts related concerning opsonic action may be due to one 
 or more substances which occur in the serum, and which have 
 the power of stimulating the leucocyte, or of altering it in a 
 manner to be discussed subsequently. The phenomena of the 
 phagocytosis of sensitized bacteria in normal saline solution 
 would, of course, be due to a liberation of this stimulin from its 
 combination with the bacteria. This is known to occur, for 
 sensitized bacteria will yield some opsonin on prolonged soaking 
 in normal saline or heated serum ; the fluid acquires opsonic 
 properties, and the bacteria becomes insensitive to phagocytosis. 
 As Sellards points out, the fact that unorganized bodies, such as 
 carmine, particles of carbon, melanin, etc., are taken up more 
 readily in the presence of fresh serum is somewhat in favour of 
 this view. It is difficult to think that these substances are affected 
 in a way similar to bacteria or other antigens when combined 
 with their specific antibodies. There appears to be no crucial 
 test for determining the point. 
 
 And there is some reason for thinking that the actual process 
 of phagocytosis may be a physical one, akin to agglutination, and 
 entirely independent of any movements or other vital processes 
 on the part of the leucocytes. The chief evidence in favour of 
 this view arises from the fact that phagocytosis may occur under 
 conditions in which no movements of any sort take place. This 
 was first pointed out by Ledingham in a series of important 
 researches on the relation between temperature and opsonization. 
 He showed that when a series of opsonin mixtures were incu- 
 bated at temperatures varying between 18 and 37 C., the 
 latter temperature brought about much more phagocytosis than 
 the former ; and, further, that at the latter point there was very 
 little difference in the index between preparations incubated for 
 fifteen or thirty minutes, while in the former there was a long latent 
 period in which but little phagocytosis occurred. This he showed 
 to be due to the fact that opsonin combines with bacteria but very 
 
2Q6 TEMPERATURE AND OPSONIZATION 
 
 slowly at 1 8 C. and rapidly at 37 C. Provided the bacteria 
 were sensitized at the latter, it mattered little or nothing whether 
 the mixture were incubated at either temperature, and a very 
 considerable amount of phagocytosis took place as low as 10 C. 
 Now at this point no movements of any sort occur, and it is quite 
 easy to satisfy oneself by actual observation under the microscope 
 that bacteria opsonized at 37 C. may be taken up at a low 
 temperature by Bacteria which remain absolutely motionless 
 during the process. This is even more easily observed by using 
 a modification of a method recently introduced by Ponder, and 
 of very great value in the direct observation of phagocytic and 
 other phenomena. If a drop of blood be placed in a glass cell 
 about o'2 millimetre deep (such as is used in mounting diatoms, 
 etc., in fluid), covered with a cover-glass, and incubated for fifteen 
 minutes or so, both slide and cover-glass will be found to be 
 dotted about with leucocytes which adhere so firmly that all the 
 red corpuscles can be washed off with warm normal saline solu- 
 tions, leaving the leucocytes adherent to the glass. If now the 
 cell be filled with serum mixed with bacteria, and incubated at 
 37 C., or with bacteria thus opsonized and thoroughly washed, 
 the process of phagocytosis can be readily watched, and is seen 
 to take place at 18 C. or lower. Under these circumstances, no 
 active movement or protrusion of pseudopodia takes place at all, 
 and it is easy to watch a sensitized coccus being gradually 
 attracted to and absorbed into the body of the leucocytes. The 
 process strongly recalls the agglutination of bacteria. A coccus 
 lying within a certain distance of the cell is seen, like the others, 
 to be in active Brownian movement, and the appearances would 
 suggest that it is slightly more easy for it to move towards the 
 cell than away from it; It oscillates in all directions, but 
 gradually approaches nearer and nearer the leucocyte, and is 
 finally taken in. Similar phenomena can be seen (using a hot 
 stage) when sensitized bacteria in an emulsion in normal saline 
 solution are added to leucocyte films at 37 C. ; and here also 
 no movement, or but little, takes place. If, however, serum be 
 added, there is usually some movement of the pseudopodia, but 
 little or no locomotion from place to place. 
 
 It is not easy to determine whether phagocytosis may take 
 place in dead leucocytes. I have not been able to detect it in 
 leucocytes killed either by heat or cold, but Rosenau states that 
 when leucocytes killed in the former way are mixed with opsonized 
 
PHAGOCYTOSIS 2Q7 
 
 cocci, they collect round the cell, though they are not actually 
 ingested, and this is confirmed by Sellards. Killed leucocytes 
 probably undergo a sort of coagulation equivalent to rigor mortis, 
 which would prevent the ingress of bacteria. 
 
 Sellards has shown that salts are as necessary for phagocytosis 
 as for agglutination. The isotonic solution in which the 
 leucocytes were suspended was 5-5 per cent, of saccharose ; the 
 bacteria were opsonized by fresh serum, washed thoroughly, and 
 suspended in the same sugar solution. Little or no phagocytosis 
 occurred, but it took place if salts were added. This, again, does 
 not look like a vital process, but is quite analogous with agglutina- 
 tion, in which we have every reason to believe that the effect is 
 due to an alteration of surface tension. So also with the action of 
 serum in aiding the phagocytosis of substances such as carmine or 
 carbon. We have only to suppose that some substance is occluded 
 on the surface of the inert substance, the surface tension of which it 
 alters in the same way as opsonin alters that of the bacteria. 
 
 The degree of opsonization is determined to some extent by the 
 amount of salt present, and is found to be least (in the absence of 
 serum) in a 1*2 per cent, solution ; hence this strength of salt is 
 used by some observers in opsonic determinations in order to 
 reduce the amount of spontaneous phagocytosis as low as possible. 
 Hamburger and Hekma have also shown that a minute trace of 
 calcium chloride has a great influence in increasing the opsonic 
 power of the serum (we have already seen that it aids the ag- 
 glutination of cholera vibrios), and that the activity of the serum 
 is increased by alkalis and diminished by acids. Chloride of 
 potassium, unlike chloride of sodium, has also an unfavourable 
 effect on the leucocytes. 
 
 If we push our investigations a little farther, we may perhaps be 
 led to the belief that the amoeboid movements and protrusion of 
 pseudopodia which leucocytes display under suitable circumstances 
 may themselves be effects of surface tension rather than strictly 
 vital phenomena. Consider the case of the film of blood prepared 
 by Ponder's method, or by the use of a glass cell, as recommended 
 above. When this is incubated, large numbers of leucocytes 
 appear both on the lower and upper surfaces. Now in the latter 
 case the effect cannot be due to gravity, for the leucocytes are 
 heavier than the serum. It would be too great a strain on the 
 imagination to suppose the leucocytes capable of actual swimming 
 movements through the blood (and it may be remarked that many 
 
2g8 PHYSICAL EXPLANATION OF PHAGOCYTOSIS 
 
 find their way to the top before coagulation occurs, though the 
 process appears to continue after that), and the only alternative is 
 a physical attraction between the glass and the leucocytes. This 
 is a perfectly feasible explanation, and if it is true the next stage 
 in the process would necessarily follow. This is the flattening 
 out of the leucocytes, so that they form thin plaques of very much 
 larger diameter than the same cells as seen in ordinary wet films. 
 This is very difficult to explain as any vital effect, but it is 
 exactly what we should expect to happen if the leucocyte (which, 
 like all, or almost all, forms of living protoplasm, is to be regarded 
 as a liquid) were pulled out under the influence of surface tension, 
 just as a drop of liquid paraffin is stretched out into an infinites! - 
 mally thin film when dropped on the surface of water. 
 
 The bizarre forms which the leucocytes assume in a preparation 
 made by Ponder's method, with long pseudopodia, are explicable 
 on the assumption that, owing to irregularities in the cover-glass, 
 the surface tension is not uniform in all directions, or 'that the 
 protoplasm of the leucocyte is not of the same degree of viscidity 
 throughout. Similar irregular protuberances can be produced in 
 globules of oil or water by purely physical means, and Pauli goes 
 so far as to say that " since the discovery of the amoeboid move- 
 ments of oil droplets, and the careful physical analysis of this 
 process by Quincke, the formation of pseudopodia has been 
 robbed of the characteristics of a specific life phenomenon, and 
 later investigations have shown that it is governed in all its 
 details by the laws of surface tension. The taking up of food and 
 the process of defalcation in rhizopods can also be explained in 
 the same way." The process of the ingestion of an opsonized 
 bacterium suspended in serum at the body temperature, in which 
 it is occasionally possible to see the protrusion and seizure of the 
 organism by a long, slender, and flexible pseudopodium, is 
 explicable as follows : Owing to the change of surface tension 
 induced by the action of the opsonin on the bacterium, there 
 is generated an attractive force which tends to draw the two 
 together. The leucocyte, being fixed to the cover-glass like a 
 sucker, does not move, but a small portion of its substance, being 
 liquid or semi-liquid in consistency, is drawn out until it meets 
 the bacterium, which is, of course, also attracted. The two meet, 
 and then it will be found that the organism is firmly held in 
 contact with the pseudopodium, so that it is not released even if 
 the latter be carried to and fro by currents in the fluid. 
 
PHAGOCYTOSIS 2QQ 
 
 The effects of surface tension may also be traced in some of 
 the phenomena of inflammation, especially in the adhesion of the 
 leucocytes to the vessel wall. It has been abundantly shown 
 that this is due to an alteration in the latter, and it appears likely 
 that this is simply due to a change in the tension developed at 
 the surface between the endothelial lining and the serum, in 
 virtue of which the former behaves like the glass in Ponder's 
 method, attracting the leucocyte and causing it to adhere and 
 flatten itself out. This extension, so as to offer as large a surface 
 as possible, which is displayed by the leucocytes, and especially 
 of the polynuclears, when they come into contact with a resistant 
 surface, was noted long ago by Massart and Bordet, and in virtue 
 of it they are able to make their way through the finest pores, 
 even in compact bodies like bone and ivory. The remarkable 
 deformation in shape which leucocytes undergo in acutely inflamed 
 tissues is not usually appreciated. It was pointed out to me by 
 Whitfield, and may often be seen at the edge of the sections 
 where the fixation is perfect, provided the material has been 
 placed in the fixing fluid immediately after its excision. The 
 polynuclear leucocytes are often overlooked altogether, being 
 pulled out into long strands of protoplasm containing nuclear 
 filaments, giving the section a remarkable mossy appearance. 
 This change in the surface tension of the vessels, lymph clefts, 
 etc., probably plays a part of great importance in diapedesis. It 
 is somewhat doubtful, however, whether it can afford a complete 
 explanation of the phenomena of chemotaxis, in which a vital 
 and apparently quasi-intelligent action appears probable. 
 
 It must not be imagined that the vitality of the leucocyte is to 
 be regarded as unimportant in the consideration of phagocytosis 
 as a means of defence. Here the process has only begun when 
 the organism is ingested, and unless suitable digestive ferments 
 are secreted, the bacterium dissolved, and the endotoxin absorbed 
 or otherwise dealt with, the process is useless, or, by carrying 
 bacteria out of the lesion to other parts of the body, may even be 
 harmful. 
 
CHAPTER XI 
 "REACTIONS" AND SIMILAR PHENOMENA 
 
 NOT long after the discovery of the tubercle bacillus Koch found 
 that the effects of an inoculation of living cultures of the organism 
 were quite different in normal and in tuberculous animals. If a 
 normal animal is inoculated by scarification of the skin the 
 wound soon heals, and in about a fortnight a hard nodule forms. 
 This ulcerates, and remains an open ulcer until the animal dies. 
 If a second inoculation be made after the first has run its course 
 to the stage of ulceration, the process is profoundly modified. No 
 nodule is formed at the site of the second inoculation, but the 
 tissue round the first becomes hard, dark-coloured, and finally 
 necrotic, and may be shed en masse and the lesion undergo com- 
 plete cure. Koch found, further, that this change might be 
 brought about by injections of dead cultures even after they had 
 been boiled. He found, too, that a large dose of these killed 
 cultures (which would cause nothing but local suppuration in 
 normal animals) would kill a tuberculous guinea-pig in a short 
 time six to forty-eight hours the symptoms being fever, acute 
 inflammation, running on to necrosis, in the region of the tubercu- 
 lous lesions, and in some cases generalization of the bacilli 
 throughout the body. When very minute doses were used he 
 found, on the contrary, that improvement might occur, and the 
 tuberculous ulcer become cicatrized over. 
 
 This was made the basis of a method for the treatment of 
 tubercle in man. But Koch found the use of killed cultures 
 inconvenient, since the bacilli w r ere but slowly absorbed, and 
 might give rise to abscesses. He argued that the effect was 
 evidently due to some soluble substance which diffused out of the 
 bacilli, and after long research prepared the substance which is 
 now so familiar as the old tuberculin. It is a solution in 40 to 50 per 
 cent, glycerin of the soluble products of the tubercle bacillus, and 
 is prepared by cultivating that organism for several weeks in 
 
 300 
 
AND SIMILAR PHENOMENA 3OI 
 
 glycerinated veal broth in a thin layer, so that there is an abundant 
 supply of oxygen. This culture is evaporated to one-tenth of its 
 volume and filtered through a Chamberland filter. There are 
 numerous slight modifications in the process of manufacture, 
 but they are unimportant. 
 
 Old tuberculin is a syrupy brownish-yellow fluid, with a faint 
 aromatic smell. It contains peptones and traces of other proteid 
 bodies, but the nature of the substance on which its extraordinary 
 power depends is quite unknown. It is in a sense to be regarded 
 as a toxin of the tubercle bacillus, but it is not a true toxin, like 
 those of diphtheria and tetanus, since it is practically non-toxic for 
 healthy animals or for man. Its injection in large quantity may 
 cause a slight febrile reaction, but not much more than a similar 
 injection of peptones, etc., from any other source. It differs, also, 
 in a marked degree from the exotoxins in that it is not destroyed 
 by a temperature of 100, or even of 120 C. It is dialyzable. 
 
 When injected into tuberculous animals it causes the same 
 " reaction " as was produced by the living or dead culture, and this 
 in very minute amount. A dose of i milligramme will cause a sharp 
 reaction in a tuberculous patient, and, indeed, one-tenth of that 
 amount will sometimes suffice. When we consider that the 
 material consists mainly of the nutrient ingredients of the broth 
 Koch thought that the active principle might form i per cent, of the 
 whole its extraordinary potency is evident. 
 
 The phenomena of the " reaction " are as follows : There may 
 be, but usually is not, some inflammatory oedema at the seat of 
 injection. The temperature rises precipitously, often reaching 
 105 F. in a few hours, and falls almost as quickly. With this 
 there are the usual symptoms of fever, malaise, shivering, etc. 
 This is the general reaction. The local reaction occurs round the 
 pre-existing tuberculous lesion, and is best seen in lupus, tubercu- 
 lous ulcers, etc. Its severity depends upon the dose given. With 
 a small dose there is a little redness and swelling and some mild 
 inflammatory oedema, the whole lasting but a day or two. W^hen 
 it subsides the lesion often undergoes great improvement. After 
 larger doses the local reaction is more marked, acute inflammation 
 occurs, the tissues in and around the tuberculous foci undergo 
 coagulation necrosis, and are cast off. When this takes place in 
 the skin it may lead to complete cure, but in the internal organs 
 it is a source of grave danger, often leading to dissemination of the 
 bacilli and a consequent general infection. This occurred in the 
 
302 
 
 THE TUBERCULIN REACTION 
 
 early days of the use of the fluid, when it was hailed as a specific 
 cure for the disease, and Koch's limitations of its use ignored. At 
 present it is used as a method of diagnosis, and found to be of 
 great value and devoid of danger if used with proper precautions. 
 And there can be no doubt that the bad results obtained when the 
 
 Normal 
 98 
 
 FIG. 70. SEVERE TUBERCULIN REACTION IN A CASE OF BAZIN'S DISEASE. 
 (Under Dr. Whitfield.) 
 
 potentialities of the substance were so little known have led to its 
 being unjustly abandoned as a method of cure. Properly applied 
 to suitable cases, it has proved of great value. 
 
 The reaction is a specific one, except that it is sometimes given 
 in patients with syphilis, leprosy, or actinomycosis. This is 
 unusual. 
 
 When patients are treated with gradually increasing doses of 
 tuberculin they become partially immunized, so that no febrile 
 
" REACTIONS AND SIMILAR PHENOMENA 303 
 
 reaction is caused by large doses. Thus Wassermann records a 
 case in which 300 milligrammes caused no reaction. The patient 
 had been treated for a year, the dose being gradually increased. 
 It appears, too, that by careful treatment of animals an antituber- 
 culin can be produced which has the power of inhibiting the effects 
 of tuberculin in tuberculous animals. It has, however, little or 
 no effect in the treatment of the disease another proof that tuber- 
 culin is not the specific toxin. 
 
 Quite recently important modifications have been introduced in 
 the diagnostic application of tuberculin. Von Pirquet's reaction, 
 or the cnti-veaction, is elicited by placing a drop of tuberculin 
 (undiluted or a 25 per cent, solution) on the skin, and performing 
 scarification just as for an ordinary Jennerian vaccination. It is 
 advisable to make a similar control scarification without using 
 tuberculin in order that the lesions may be compared. The 
 simple inoculation shows a little redness, which soon disappears. 
 That made with tuberculin does the same if the patient is not 
 tuberculous. If he is, a small red papule is formed, which increases 
 for three or four days and disappears in a week or so. 
 
 Calmette's method, the ophthalmo - reaction, is obtained by 
 instilling one or two drops of diluted tuberculin into the conjunc- 
 tival sac. He recommends the use of old tuberculin which has 
 been precipitated with absolute alcohol and redissolved in distilled 
 water, as being less irritating and less likely to cause a pseudo- 
 reaction in a non-tuberculous patient. The reaction in this case 
 consists of a mild attack of conjunctivitis, lasting twenty- four 
 hours, and accompanied by redness and swelling of the caruncle 
 and a small amount of mucoid secretion. The reaction should be 
 over in twenty-four hours, but in some cases undesirable results 
 have occurred from a secondary infection with organisms capable 
 of causing a more severe conjunctivitis or keratitis. For this 
 reason the test should be used with care, if at all. 
 
 These tests are most applicable in children, since in adults the 
 frequency of cured tubercle, also leading to hypersensitiveness, 
 may lead to reactions where there is no clinical tubercle, or the 
 patients may be more or less immune. 
 
 - Reactions are given in other diseases, the most important being 
 glanders. Mallein is a fluid obtained from cultures of B. mallei 
 exactly as tuberculin is obtained from tubercle bacilli. It is non- 
 toxic to normal animals, but it causes a febrile reaction in those 
 infected by glanders, even if there is but a small latent lesion. 
 
304 OTHER REACTIONS 
 
 There is also a local reaction at the site of the lesion, though this 
 is less marked than in tubercle. There is, however, a very 
 marked production of inflammatory oedema at the site of the 
 inoculation, and this furnishes the most certain test for the 
 disease. A hard raised mass is formed, which increases in size 
 for twenty-four hours or more, becoming as large as the palm of 
 the hand, and persists some days. It often gradually travels 
 down the neck (in the horse), as if under the action of gravity. 
 
 It will be noticed that an essential feature in these reactions is 
 the rapid development of symptoms after an inoculation or 
 injection in a subject already infected with the disease. Von 
 Pirquet has brought forward other examples, which, if less 
 dramatic in their course than Koch's phenomenon, are at least 
 comparable with the cuti-reaction. Thus it has been noticed that 
 the second (Jennerian) vaccination, practised some years after the 
 first, runs a rapid course. Von Pirquet has shown that if a 
 revaccination be made a few months after the first, the reaction 
 takes place in twenty-four hours. It does not, of course, 
 develop, in the same way as a primary vaccination, but a small 
 papule, often surrounded by an areola, makes its appearance, and 
 lasts some two or three days. 
 
 Chantemesse has observed in typhoid fever phenomena similar 
 to those seen in Calmette's ophthalmo-reaction in tubercle. He 
 instils a single drop of typhoid "toxin" (obtained by cultivating 
 typhoid bacilli in extract of spleen digested with pepsin), and 
 finds that in normal persons there is but a little transient redness, 
 whilst in typhoid patients there is redness, lachrymation, and the 
 formation of a sero-fibrinous exudate. The process attains its 
 maximum in six to twelve hours. He makes use of this reaction 
 as a method of diagnosis. 
 
 The phenomena of the " negative phase," seen probably in all 
 antibodies, but specially studied in connection with the opsonins, 
 are probably similar in nature to these reactions, although the 
 doses of vaccines given are usually so small that the clinical 
 manifestations do not appear. Sometimes, however, this does 
 happen. Thus Irons found that a dose of 500,000,000 dead 
 gonococci caused no reaction in healthy persons ; but if given to 
 patients already suffering from a gonococcal infection, it produced 
 fever, pains in the joints, and general malaise. In most cases the 
 difference between the behaviour of a healthy and infected person 
 or animal is traceable solely in the variations of the opsonic 
 
" REACTIONS " AND SIMILAR PHENOMENA 305 
 
 index. When an ordinary dose of any vaccine is given to a 
 healthy person the opsonic index undergoes but slight changes, 
 and in particular there is no fall or negative phase. There may 
 be a slight subsequent rise. When the same dose is given to a 
 person infected with the same organism, the negative phase 
 (perhaps preceded by a " false rise ") is most marked, and is 
 followed by a positive rise, or sometimes by a series of rises and 
 falls, gradually dying away like a wave. Similar phenomena can 
 be produced in a healthy person, but here the dose must be much 
 larger. Evidently, therefore, the presence of an infecting agent 
 other than tubercle causes a condition of unstable equilibrium, in 
 which the tissues react in a different manner to healthy ones. 
 And the same condition of altered sensitiveness may persist for 
 long after the disease or injection of a vaccine, so that a dose of 
 dead bacilli that has but little action in health causes a great 
 output of antibodies. This reaction appears to be a general one, 
 occurring with bacteriolysins, agglutinins, etc. 
 
 Attempts have naturally been made to account for a phe- 
 nomenon so remarkable as the tuberculin reaction, and the large 
 number of explanations suggest that none is altogether satisfactory. 
 Many of them do not call for notice. 
 
 Koch's explanation, which was put forward more as a working 
 hypothesis than as an established fact, was this : The bacillus 
 formed a toxin, which diffused outwards from the colonies in the 
 tissues, and when in a sufficient state of concentration set up a 
 coagulation - necrosis going on to caseation. In the zone of 
 tissues just beyond this region the necrosis-producing substance is 
 present, but not in a sufficient degree of concentration to kill the 
 tissues. The injection of a little more of this substance ?.., of 
 tuberculin is sufficient to turn the scale, and a rapid increase of 
 the necrosis takes place. He explains the beneficial effects of 
 the treatment in this wise : The necrotic tissue does not form a 
 suitable medium of growth for the tubercle bacillus (which is but 
 rarely seen in caseous material), and the extension of the process 
 may lead to the complete enclosure of the bacteria in dead and 
 altered tissues, in which they are incapable of further growth. 
 
 This theory assumes that the substance which produces necrosis 
 is identical with the active principle of tuberculin ; but tuberculin 
 in large doses will not produce necrosis in a healthy animal. It 
 seems also to fail to account for the remarkable rise in the 
 temperature, since it occurs in patients who are not febrile, as 
 
 20 
 
306 EXPLANATIONS OF THE TUBERCULIN REACTION 
 
 we should expect them to be if tuberculin were diffusing from 
 their lesions. 
 
 Ehrlich's views are quite similar to Koch's, and he regards the 
 reaction as due to the effect of the tuberculin on tissues which are 
 injured by it at the time of the injection, and in which a slight 
 extra dose is sufficient to turn the scale. 
 
 Others have thought that the reaction is indicative of a hyper- 
 sensitiveness of the patient to tuberculin, using the term in the 
 sense in which we employed it in dealing with the toxins. This, 
 of course, is true, but it scarcely seems a sufficient explanation in 
 itself. We shall revert to the subject after giving an account of 
 some most remarkable discoveries that have recently been made 
 concerning this subject. 
 
 Marmorek holds in all probability correctly that tuberculin 
 is not to be regarded as in any sense the true toxin of the tubercle 
 bacillus. This is only formed when the organism is living para- 
 sitically in the tissues, or in artificial conditions bearing a very 
 close approximation thereto, not in such a simple medium as plain 
 broth. Tuberculin has this effect on a tuberculous animal : it 
 stimulates the tubercle bacilli to a sudden and energetic production 
 of toxin, which gives rise to the local reaction, and, passing into 
 the vessels, to fever and its concomitant general phenomena. 
 There is nothing inherently improbable in this suggestion, except 
 that no reason is forthcoming as to the way in which tuberculin 
 exerts this very remarkable action, but there is little direct evidence 
 in its favour. The toxin which Marmorek claims to have pro- 
 duced by the application of this principle is so weak as not to 
 be worth calling a toxin. 
 
 Wassermann and Briick point out that the extremely minute 
 amount which must be present in the blood at a given time leads 
 to the supposition that the tuberculin injected must leave the blood- 
 stream and become concentrated in the region of the tuberculous 
 focus. Thus, if a person with 5,000 c.c. of blood reacts to an injec- 
 tion of i milligramme of tuberculin, the dilution will be i : 5,000,000, 
 and they find that this dilution injected directly into a tuberculous 
 lesion gives absolutely no reaction. They then proceed to argue 
 that this attraction of the tuberculin from the blood must be due 
 to the presence in the tuberculous tissue of an antitoxin or anti- 
 tuberculin. They investigated the presence or absence of this 
 substance by means of the method of fixation of the complements. 
 Extracts of tuberculous tissues, when mixed with tuberculin, 
 
AND SIMILAR PHENOMENA 307 
 
 acquired the power of absorbing haemolytic complements from 
 fresh serum, and so of inhibiting the haemolysis of sensitized red 
 corpuscles. Extracts of normal organs had no such power. 
 
 This is made the basis for their theory of the reaction. The 
 injected tuberculin circulates in the blood until it reaches the 
 antituberculin present in the lesions. The two combine, and in 
 doing so attract the complements which we must suppose to be 
 free in the plasma. This fixation is supposed to be followed by 
 cytolysis of the cells of the part. This accounts for the local 
 reaction. In this solution of the tissue cells products of disintegra- 
 tion are set free, pass into the blood, and give rise to fever, causing 
 the ioeal reaction. Thus neither the jbcal nor the general reaction 
 is due to the direct toxic action of the tuberculin itself. In this 
 the theory approaches somewhat to Marmorek's, and is in funda- 
 mental opposition to the older theories of "addition." 
 
 YVassermann and Briick bring forward an important piece of 
 evidence in favour of their theory by finding antituberculin present 
 in the serum of patients who had been treated with increasing 
 doses of tuberculin and had lost their power of reacting. In them 
 the tuberculin injected would be immediately neutralized in the 
 blood, and so never reach the lesion. The theory is ingenious, 
 and may possibly turn out to be the correct one, but there are 
 difficulties. Thus the authors find tuberculin as well as antituber- 
 culin in the diseased tissues, and it is difficult to see why the two 
 do not neutralize one another. And we might also ask why no 
 digestive phenomena should follow the union of the antituberculin 
 and tuberculin in the blood of injected patients, and the subsequent 
 absorption of the complements. Why should not the proteid 
 molecules be digested, liberate their products, and produce fever ? 
 It would seem that the antituberculin present in the lesion must 
 be in a state of fixation to the cells, or it must be carried away in 
 the blood-stream, and this, according to Wassermann and Briick) 
 rarely happens except after injections. But we do not know 
 definitely of any such antitoxin, the nearest approach to it being a 
 superabundance of suitable sessile receptors, which, if they occurred, 
 might very well make their way into the extracts used in the test, 
 and simulate an antitoxin. And if this were the case, there is no 
 explanation why these receptors are not shed in the normal tuber- 
 culous process, but are after the use of tuberculin. It is difficult, 
 too, to see why the presence of these abnormally numerous 
 receptors might not be made the basis for a " theory of addition " 
 
 20 2 
 
308 EXPLANATIONS OF THE TUBERCULIN REACTION 
 
 without invoking the aid of the complements. But the whole 
 subject is theoretical to a degree, and needs, moreover, independent 
 experimental verification. 
 
 Bail's researches on the aggressins have been referred to already. 
 The application of his theory to Koch's phenomenon is obvious. 
 According to him the endotoxins are only set at liberty when 
 bacteriolysis occurs, not after phagocytosis. This is in all prob- 
 ability correct in most cases, though perhaps not in all. When, 
 therefore, tubercle bacilli are injected into the peritoneal cavity of 
 a normal guinea-pig and extensive phagocytosis occurs, there is 
 little or no febrile reaction ; but in the tuberculous animal the 
 bacilli produce aggressins, which paralyze the phagocytes, and, 
 w r hen a second injection is made, the bacteria undergo rapid 
 bacteriolysis, endotoxin is set free, and rapid death follows. Bail 
 compares the results of the solution of large quantities of cholera 
 bacilli in an immunized animal with what is seen after the injec- 
 tion of tubercle bacilli along with a small amount of the peritoneal 
 exudate from a tuberculous animal. In each case there is extra- 
 cellular bacteriolysis, and death in a few hours, obviously from the 
 toxin set free. 
 
 This might account in a satisfactory way for Koch's phenomena 
 when caused by the injection of cultures, but seems to fail utterly 
 when applied to the tuberculin reaction ; for tuberculin is neither 
 an " aggressin " in Bail's sense, nor an endotoxin of the tubercle 
 bacillus, and it cannot undergo bacteriolysis. The theory resembles 
 that of Marmorek. 
 
 Von Pirquet's explanation of the early reaction after Jennerian 
 vaccination calls for some notice, though it is not immediately 
 applicable to Koch's phenomenon. It introduces some new and 
 interesting conceptions. According to this author, the result of 
 an infection is to alter the way in which an animal reacts subse- 
 quently to a second infection with the same organism. This he 
 calls allergia. In some cases this may lead to hypersensitiveness, 
 but in the majority it leads to a temporary immunity, followed by 
 a condition in which the animal is no longer immune, but possesses 
 the power of forming antibodies in the region of inoculation more 
 quickly and easily than a normal one can do. When a second 
 inoculation is made, the bacteriolysins present in the blood may be 
 sufficient to destroy the bacteria introduced, setting free their 
 toxins, which act locally and cause the early reaction. Or this 
 may be delayed until local antibodies are formed. This occurs 
 
"REACTIONS" AND SIMILAR PHENOMENA 309 
 
 more quickly than in the normal person. It leads to an early 
 development of the specific lesion of vaccinia. 
 
 The essential point of this theory is that an infection from which 
 recovery has taken place may lead to an alteration of the facilities 
 with which antibodies may be formed, which alteration persists 
 for a long time. 
 
 It seems desirable here to make a further reference to the 
 subject already mentioned briefly as " hypersensitiveness to 
 toxins," but now more generally termed anaphylaxis i.e., the 
 opposite of prophylaxis. The term was introduced by Richet, 
 who studied especially the poison of the actiniae, which he found 
 to be extremely powerful, the lethal dose being about -009 gramme 
 per kilo of body-weight. He found that a non-lethal dose increased 
 the susceptibility of the animal to a second injection, and that this 
 hypersensitiveness might last as long as six months after the first 
 injection. This, of course, is quite similar to the phenomena we 
 have described in connection with diphtheria and tetanus, which 
 renders it so difficult to immunize small animals to these sub- 
 stances, and which is the cause of much danger in the early stages 
 of antitoxin formation in the higher animals. Richet has also 
 studied the poison formed by the common mussel, which he calls 
 " mytilo-congestine," and finds exactly similar facts; indeed, it is 
 probable that it is a general phenomenon of all the poisons which 
 can act as antigens. In the case of mytilo-congestine the measure 
 of the hypersensitiveness is simple, since one of the most constant 
 symptoms of its action is vomiting, which occur? almost as soon 
 as the injection is made. He finds that in an animal which has 
 previously been injected the emetizing dose is from a tenth to a 
 quarter of the amount originally necessary. Richet has elaborated 
 a theory to account for this phenomenon, and for anaphylaxis in 
 general. He holds that the condition is due to the presence in the 
 blood of a toxogenic substance, which gives rise to a poison after 
 reacting with the mytilo-congestine injected. This toxogenic sub- 
 stance is not formed immediately, for Richet does not find hyper- 
 sensitiveness to come on for five or six days, and it persists for 
 some fifty days, that being the average duration of the state. He 
 holds that the animal produces antitoxin also, but more slowly. 
 When the toxogenic substance has disappeared the antitoxin 
 remains, and the animal is immune. The main evidence in favour 
 of this theory is the fact that the serum of an anaphylactic animal 
 will produce a similar condition in a second animal. Currie has 
 
3IO HYPERSENSITIVENESS OR ANAPHYLAXIS 
 
 enunciated a theory very like this in regard to serum anaphylaxis, 
 to be described shortly. 
 
 Other theories might be cited, but there is only one which gives 
 an explanation which is at all satisfactory without introducing many 
 unproved suggestions. It was introduced quite recently by Good- 
 man, and proceeds on lines somewhat similar to those we followed 
 when dealing with the question of immunity to toxins. The cells 
 of the body maybe classified into three groups: (i) The nerve 
 cells essential to life, and with a high degree of affinity for toxin ; 
 (2) cells not essential to life, but with a higher degree of affinity 
 for toxin than the nerve cells possess ; and (3) inert cells without 
 susceptibility to toxin. If a dose of toxin be injected, the second 
 class of cell will have its receptors satisfied first, and any toxin 
 which is left over will then attack the nerve cells, which we 
 assume to be the only region where it will do harm. A lethal dose 
 of toxin, therefore, is the amount which will satisfy the receptors 
 of the second group of cells and leave enough toxin to injure the 
 nerve cells sufficiently to cause death. Now if a first injection 
 just sufficient to combine with the receptors of Group 2 were 
 given, a very small additional amount would be sufficient to cause 
 death, since it would go straight to the nerve centres. So far the 
 theory is unsatisfactory, since it is simply a theory of summation, 
 and the total amount necessary to cause death, if given in divided 
 doses, should together form the amount necessary if given in one 
 dose, which is very far from being the case. We have seen that 
 T J_ of the " lethal dose " of tetanus toxin may cause death if given 
 in divided doses. To account for this Goodman supposes that the 
 toxin which combines with the non-essential cells may cause a sort 
 of spreading necrosis of the receptors of the latter, or may interfere 
 with their nutrition ; in either case more of these receptors may 
 be destroyed than the toxin actually combines with. If we can 
 imagine one molecule of toxin destroying ten receptors, the animal 
 would become as susceptible as if ten times the dose were given 
 at once. Put in another way, if it takes x molecules of toxin to 
 satisfy the receptors of non-essential cells, and }( molecules to 
 combine with those of the central nervous system and kill, then 
 if in the sensitizing dose each molecule of toxin destroys ten 
 receptors, the lethal amount necessary for a second dose would be 
 
 but + a. x we must suppose much larger than a. 
 
 He compares this process with the injury to the excretory 
 
"REACTIONS" AND SIMILAR PHENOMENA 311 
 
 organs which often follows the action of poison. The kidneys, 
 etc., excrete the substance, but in doing so are injured, and a 
 smaller dose of the poison may now produce a great effect, since 
 it cannot readily be eliminated. 
 
 The main objection to this theory is that it is difficult to 
 imagine such a selective destruction of the receptors as seems 
 necessary to account for the fact that the hypersensitiveness is 
 specific. We should expect the creeping necrosis or interference 
 with nutrition to act more generally, so that an animal highly 
 sensitized to one toxin would show some degree of sensitiveness 
 to others. It seems also inadequate to explain the facts of serum 
 anaphylaxis, which will now be described, since here the animal 
 is sensitized with minute amounts of a substance which causes no 
 toxic symptoms in comparatively enormous doses in a normal 
 animal. Here the anaphylaxis appears to be the production of a 
 new sensitiveness rather than the exaltation of one previously 
 existing. 
 
 There are two of these phenomena of hypersensitiveness to 
 serum Arthus' phenomenon and Theobald Smith's phenomenon, 
 both of which are referred to as " serum anaphylaxis." The latter 
 is the more important. 
 
 Arthus' phenomenon appears when a guinea-pig receives 
 several injections, at intervals of a few days, of normal horse 
 serum, a substance which in itself is scarcely more toxic than 
 normal solution. After a few such inoculations the animal becomes 
 hypersensitive, or anaphylactized, and after another injection an 
 cedematous mass, an aseptic abscess, or an area of necrosis, 
 appears at the site of a new inoculation, which need not be in a 
 region in which a previous injection has been made ; the altera- 
 tion is a general, and not a local, one. After several of these 
 injections the animal becomes cachectic, and dies after several 
 weeks. An animal thus sensitized will die rapidly after the 
 injection of 2 c.c. of serum into the veins. 
 
 It should be noticed that these results are not due to the accu- 
 mulation of the horse serum in the system, since they may be 
 brought about by the injection in divided doses of an amount 
 which an animal can stand with impunity if given in a single 
 dose. 
 
 Theobald Smith's phenomenon occurs when an animal has been 
 sensitized by a very small injection of horse serum ( T J^ c.c., or 
 even as little as T^nro^nnr c>Ct ' an almost inconceivably small 
 
312 THEOBALD SMITH'S PHENOMENON 
 
 amount to produce so great an effect), and kept for a fortnight or 
 more. If then a second injection of a larger amount of the same 
 serum be made (^ c.c. or more, the usual testing dose being 5 c.c.), 
 the animal develops a series of remarkable symptoms, the most 
 noteworthy being respiratory failure, paralysis, and clonic spasms. 
 Symptoms usually appear within ten minutes, and death occurs 
 within an hour. Death does not always follow. The less sensitive 
 the animal, the later the development of symptoms (which in 
 highly sensitive animals come on within ten minutes), and the 
 greater the chance of survival. The process evidently affects the 
 nervous system in a very special way, and the heart may continue 
 to beat for an hour after death. In some cases, but not in all, 
 there are definite haemorrhagic lesions present ; they usually occur 
 in the stomach, less frequently in the caecum, lungs, spleen, 
 adrenals, or other parts. 
 
 The phenomena had often been seen in the process of testing 
 diphtheria and other antitoxins for the presence of free toxin, in 
 which several cubic centimetres of the serum are injected intra- 
 peritoneally into guinea-pigs. Animals that have been previously 
 used for the standardization of the antitoxin are often employed, 
 and as these have received minute doses of the latter substance 
 they may be hypersensitive. The phenomenon is a familiar one, 
 but it is only recently that its true method of origin has been 
 apparent. It has no connection with the antitoxin as such, and 
 the same phenomena of hypersensitiveness may be produced by 
 means of egg-albumin. 
 
 The action is to a certain extent a specific one. An animal 
 sensitized with horse serum is less susceptible to the serum of the 
 cow, pig, sheep, etc., than to that of the horse. It may show 
 symptoms after the injection of one of these heterologous sera, 
 but usually recovers. And the same is true .for an animal sensi- 
 tized by small doses of another sera. Symptoms are not usually 
 produced by horse serum, and if they are, are not fatal. Animals 
 can be sensitized by feeding with horse serum or with horseflesh. 
 Rosenau and Anderson thought that children might be sensitized 
 in this way, and so develop toxic symptoms after the use of anti- 
 toxin, but abandoned the idea. 
 
 Otto and Rosenau and Anderson thought that small doses were 
 necessary for the production of this form of hypersensitiveness, 
 large ones appearing to bring about immunity ; but Gay and 
 Southard show that large doses simply delay the incubation 
 

 " REACTIONS " AND SIMILAR PHENOMENA 313 
 
 period. After an injection of T J^ c.c. or T i )ir c.c. the animal is 
 hypersensitive in a fortnight or less, whereas after a dose of 8 c.c. 
 the sensitiveness does not reach its maximum for some forty-five 
 days. The duration of this anaphylaxis is not exactly determined, 
 but it certainly lasts several months. 
 
 Gay and Southard further found that during the period of in- 
 sensitiveness which follows a large dose the animal actually 
 contains the substance which acts as a sensitizing agent. Thus a 
 guinea-pig which had received (in divided doses) 17 c.c. of normal 
 horse serum was bled fourteen days after the last dose : 1*5 c.c. 
 of its serum was found to sensitize a normal guinea-pig, so that it 
 died in ninety minutes after an injection of normal horse serum. 
 (Rosenau and Anderson had already found that the young of 
 sensitized animals are also sensitive.) Further, Gay and Southard 
 found the sensitizing substance present in the blood of sensitized 
 animals. Thus a guinea-pig received T J<j- c.c. of horse serum, 
 and after twenty-nine days was bled, and 1-5 c.c. found to sensi- 
 tize another animal. The first pig was tested and found to be 
 sensitive. 
 
 These discoveries are sufficiently astonishing, and there appears 
 to be no satisfactory explanation for them. The period of incuba- 
 tion would suggest that we are dealing with antibodies of some 
 sort, but there is no evidence to show that the precipitins play any 
 part in the process. An animal may be highly sensitive when no 
 precipitin can be demonstrated in its serum. 
 
 Gay and Southard think that the hypersensitiveness depends on 
 the presence of a substance which occurs in horse serum, and 
 which they distinguish by the name anaphylactin. 1 This they do 
 not consider to be the same as the toxic ingredient of the serum, 
 for this reason : a sensitized animal will not develop symptoms 
 after the injection of blood from an animal in the refractory stage, 
 though this, as we have seen, was sufficient to sensitize it. (The 
 injections were made into a vein, the sensitive animal reacting to 
 very minute doses administered by this channel.) They failed to 
 find any proof of the formation of antibodies in the animal during 
 the refractory stage. They regard the reaction as being one of 
 the cells of the sensitized animal, and as being due to a heightened 
 power of absorption of the " toxic substance " on the part of cells 
 which have been exposed for a certain period to the action of the 
 anaphylactic substance. The action of this toxic substance 
 1 Analogous to Richet's toxogenic substance. 
 
314 THEOBALD SMITH'S PHENOMENON 
 
 appears to be to make the fatty constituents of the cells flow 
 rapidly together. This occurs especially in the endothelium of 
 the capillaries, and leads to haemorrhages. 
 
 This can hardly be regarded as a full explanation, and fails, 
 moreover, to explain a most remarkable fact discovered by 
 Besredka : that sensitized animals do not react to the injection of 
 horse serum if previously anaesthetized with ether. No theory 
 that has yet been suggested will explain all the extraordinary 
 phenomena connected with this subject, and no good purpose 
 would be served by discussing others that have been brought 
 forward. We seem to be on the eve of a series of discoveries 
 that may prove to have as great an effect on our ideas of immunity 
 and cell nutrition as the discovery of the antibodies themselves, 
 and until the facts are better known hypotheses seem out of 
 place. 
 
 We may, however, be permitted to point out that it is quite 
 possible that the tuberculin reaction may be a phenomenon of 
 exactly the same order as the serum anaphylaxis of the guinea-pig. 
 Tuberculin is in itself non-toxic for a normal animal, just as is 
 serum, but we may imagine that the tubercle bacilli in the lesions 
 produce it in small amounts until the animal becomes anaphy- 
 lactized, in which case it will react violently to a small injection. 
 The wasting and fever which accompany the evolution of the 
 disease may be phenomena akin to the phenomenon of Arthus, 
 and simply indicate the reaction of a hypersensitive animal to 
 repeated small doses of a non-toxic substance. If this is the case 
 we may regard tuberculin as being, after all, the true toxin of the 
 disease, though non-toxic to animals unless the processes of tissue 
 nutrition and metabolism have been profoundly modified by the 
 prolonged action of minute amounts of the same substance. That 
 a reaction does not occur in a normal person after two injections 
 of tuberculin does not surprise us, for the active principle is (as 
 shown by the fact that it dialyzes) of small molecule, and may be 
 eliminated more quickly than the large-moleculed toxic ingredient 
 of horse serum, so that for hypersensitiveness to occur it may be 
 necessary to have a continued stream of minute amounts from a 
 lesion in the tissues. It may be pointed out, however, that occa- 
 sionally a local reaction may be seen round the spots at which 
 tuberculin has been previously injected, suggesting that the tissues 
 in this region may be anaphylactic. 
 
 There appears to be no connection between this phenomenon of 
 
" REACTIONS " AND SIMILAR PHENOMENA 315 
 
 serum anaphylaxis and the few cases that have occurred of sudden 
 death after the injection of antitoxin, since these have usually (not 
 invariably) occurred after the first dose. In some cases, if not 
 in all, they have happened in children the subjects of the lymphatic 
 diathesis, who are subject to sudden death on very slight provoca- 
 tion. And the remarkable lesions recorded by Gay and Southard 
 have never been seen after death from a single dose of antitoxin ; 
 nor do the extraordinary symptoms develop. 
 
 This seems the most suitable place in which to discuss the 
 " serum disease," or series of unpleasant though transient pheno- 
 mena which may occur after the use of antitoxin, though it must 
 not be considered that it is necessarily a phenomenon having any 
 connection with those that have been already described. 
 
 The symptoms have been carefully analyzed by von Pirquet, 
 and the following account is based very largely on his observations. 
 The most constant symptom is fever, which is usually remittent 
 in type. Next in frequency is a rash, which may be general or 
 confined to the vicinity of the region at which the injection was 
 made. The type of the rash varies in different cases, but all 
 forms are associated with the most unpleasant symptom of the 
 serum disease namely, severe itching. The types of rashes are 
 those included under the term "erythema multiforme." The 
 severest, which is associated with the greatest degree of fever, 
 is the morbilliform ; the scarlatiniform is associated with less ; 
 and the urticarial rash, which is the commonest, is usually accom- 
 panied with but little elevation of temperature. 
 
 There is usually enlargement of the lymphatic glands corre- 
 sponding to the region where the injection was administered, and 
 this enlargement may be the first sign of the onset and of the cure 
 of the disease. The pains in the joints often form one of the most 
 unpleasant of the symptoms, and the articulations usually affected 
 are the metacarpo-phalangeal, wrist, and knee, in that order of 
 frequency. Albuminuria, not going on to nephritis, is occasionally 
 present, and slight oedema is common. The severity of the 
 disease is roughly proportioned to the amount of serum injected. 
 
 According to von Pirquet and Schick the leucocytes are 
 increased in number until the symptoms develop, when a rapid 
 fall (due chiefly to a decrease in the polynuclears) takes place. 
 
 The disease is painful, but not dangerous, complete recovery 
 invariably occurring. The few cases of sudden death that have 
 been recorded as taking place after an injection of serum appear 
 

 3*6 THE 
 
 to be quite different in nature, and in many cases were dependent 
 on the " status lymphaticus," or " thymicus," a condition in 
 which a slight disturbance of any kind may cause sudden 
 death. 
 
 In general terms, the severity and frequency of the serum 
 disease are proportionate to the amount of serum given the larger 
 the dose, the greater the likelihood of its development and the more 
 severe the disease. There are, however, some exceptions : 
 (i) Certain samples of serum appear more potent in this respect 
 than others. The purified diphtheria antitoxin prepared by 
 Gibson (which consists of a solution of globulin) appears to 
 reduce the occurrence of the disease to a minimum. (2) The 
 serum disease is more likely to occur when a second injection is 
 given at an interval of three or four weeks or longer after the 
 first. 
 
 Under ordinary circumstances the disease manifests itself after 
 an interval of eight to twelve days, or sometimes longer, after the 
 injections. This period is insignificant, since it approximates 
 closely to the period of maximum development of most of the 
 antibodies after an injection of an antigen. Hence it was 
 suggested (by Hamburger and others) that the symptoms might 
 be due to the development of a precipitin, which, by combining 
 w T ith the unaltered horse serum still present in the patient's blood, 
 might cause the production of small precipitates in the circula- 
 tion. These, being deposited in the minute capillaries of the 
 skin, joints, etc., might act as emboli, and cause the characteristic 
 symptoms. Now the blood of a patient who has been treated 
 with serum is frequently found to contain precipitin after a week 
 or ten days, so that the possibility of this explanation is obvious. 
 The phenomenon of the accelerated reaction also appears to lend 
 it support. A patient who has been injected with horse serum 
 may be presumed to have precipitin persisting in his system for 
 some unknown but possibly lengthy period afterward, and on the 
 injection of more horse serum (its antigen) might cause a precipi- 
 tate which would lead to an immediate or accelerated development 
 of the serum disease. It is found in practice that this immediate 
 reaction does occur, but is comparatively rare. In ninety cases 
 where a second injection was given the disease was developed 
 within six hours in nine ; in thirty-nine others it was produced 
 within a period varying from nineteen hours to five days (Goodall). 
 The " immediate effect" described by Goodall differs somewhat 
 
"REACTIONS" AND SIMILAR PHENOMENA 317 
 
 from the usual " accelerated effect " occurring in patients injected 
 with serum for the second time. The symptoms are rigor, 
 pyrexia, vomiting, rash, and collapse, whereas the effects which 
 develop in a day or two consist of rash, joint pains, and pyrexia. 
 
 The precipitation theory is now generally abandoned, since 
 further investigation shows that there is no close parallelism 
 between the occurrence of the disease and the presence of 
 precipitins in the serum. Thus it is often found that the symptoms 
 are present when not the slightest trace of antihorse precipitin is 
 demonstrable. On the other hand, precipitins may occur when 
 the disease does not develop, though this is a fact of less impor- 
 tance, since it is necessary, on this theory, that the precipitin and 
 the unaltered horse serum should be present simultaneously, and 
 this latter fact is usually impossible of proof. But Widal and 
 Rostane have brought forward very strong negative evidence in 
 showing that the disease does not necessarily occur after the 
 intravenous injection of a powerful antihuman precipitating 
 serum in man. 
 
 These arguments, though strong, cannot be regarded as entirely 
 conclusive. As regards the absence of the precipitin in some 
 cases of serum disease, it may be pointed out (a) that it may have 
 all been removed in the form of precipitum when the sample was 
 collected, and (b) that the demonstration in vitro of a minute 
 amount of precipitin is by no means easy, and requires an appro- 
 priate correlation between the precipitating and normal sera, or 
 the phenomenon may be missed. Further and this applies to the 
 negative experiments of Widal and Rostane we do not know 
 exactly what conditions are necessary to the union of precipitin 
 and antigen to form an insoluble precipitate, and whether these 
 are always present in vivo. Some experiments, it is true, appear 
 to show that the combination never occurs in the animal body, 
 but they are inconclusive, and the supposition is in the highest 
 degree unlikely. It appears probable that precipitation, like 
 agglutination, depends on the presence of certain salts, as well as 
 on that of the antibody and antigen, and it may be that these are 
 sometimes absent or not present in an available form. In this 
 connection we may quote the researches of Netter, which are of 
 great practical importance. He showed that the administration 
 of calcium lactate at the time of the injection, and for a day or 
 two after, caused a great diminution in the number of cases of the 
 serum disease. His results have been generally substantiated, 
 
318 THE 
 
 and, in addition, the drug has been found to be of decided 
 therapeutic value. Hence it appears that a diminished calcium 
 content of the patient's blood at the time of the injection may be 
 of some importance in bringing about the conditions necessary for 
 the development of the disease. If this is the case, it might 
 account for the symptoms in some cases and not in others, 
 although precipitin might be produced in both. And there are 
 probably other factors which are at present unsuspected, or the 
 use of the calcium salt would be efficacious in all cases. 
 
 The alternative theory is, of course, that the patient gradually 
 becomes hypersensitive to the serum, and that the grade of 
 hypersensitiveness necessary for a reaction is reached before the 
 serum is eliminated from the blood, or, in the case of an im- 
 mediate or accelerated reaction, has not passed off before the 
 second injection is given. It would make it a phenomenon 
 analogous to those of Koch or Theobald Smith. 
 
 A good argument in favour of this theory is the long interval 
 which may occur between the first injection and the second in the 
 case of an accelerated reaction. This may be a year or more a 
 very long period for antibodies to persist in the blood after a single 
 injection of antigen. 
 
CHAPTER XII 
 COLLOIDAL THEORY OF ANTIBODIES 
 
 THE fact that antigens and their antibodies are, as far as is known 
 at present, exclusively colloid in chemical character renders it 
 advisable to glance briefly at some of the main facts and theories 
 concerning these bodies and their reactions. They are substances 
 of the greatest possible interest to the biologist, since the living 
 tissues and the fluids of the living animal are, without exception, 
 colloids and colloidal solutions ; protoplasm, cell nuclei, etc., being 
 mixtures of colloids in a jelly-like form, whilst the fluid part of 
 blood-lymph, etc., is a colloidal solution. It is necessary to 
 remember this, since the reactions of colloids and of crystalloids 
 in presence of colloid appear to follow laws which are different 
 from those governing the reactions of crystalloids alone, and we 
 may doubt whether these latter ever take place in the living body. 
 Colloids are not necessarily organic bodies, since metals such as 
 gold and silver may be obtained in the colloidal state, as well as 
 many metallic salts, such as ferric oxide, silicic acid, etc. These 
 simple colloids appear to be governed by laws similar to those 
 concerned in the reactions of the organic colloids formed by the 
 action of vital processes. 
 
 The chief features which distinguish colloids from crystalloids 
 are (i) that they do not undergo dissociation into their ions when 
 dissolved in water ; and (2) that this so-called solution is not a true 
 one, but merely a suspension of unaltered molecules or groups of 
 molecules. These two characters are probably fundamentally the 
 same. A colloid solution, therefore, is merely a very fine emulsion 
 or suspension of particles of the substance, and Siedentopf and 
 Zsigmondy have demonstrated a method by which these particles, 
 though infinitely small as compared with the most minute bacteria, 
 can be rendered visible and their numbers estimated. The method 
 is simple enough in theory, though the actual arrangements are 
 somewhat complicated. A powerful beam of light is passed trans- 
 
 319 
 
32O NATURE OF COLLOIDAL SOLUTIONS 
 
 versely through a true solution placed in the field of a powerful 
 microscope, the ray passing at right angles to the optical axis. 
 No light enters the lens, and the whole field remains in darkness. 
 If, however, a colloid solution be similarly treated, these particles 
 will reflect the light in every direction, and some rays will enter 
 the lens. The result is that the particles are seen as minute 
 luminous points on a dark background. The process is analogous 
 with the examination of the stars with a telescope, w r hich, no 
 matter how powerful, never shows the star as a disc, but, merely 
 by collecting more light, renders it brighter, and shows stars so 
 small as to be invisible to the naked eye. 
 
 The particles in question are so small that they are prevented 
 from falling to the bottom of the fluid by friction ; if, however, 
 they clump together, the particles become heavier, whilst the 
 surface which they expose to the fluid does not increase at so great 
 a rate, until at last aggregates of molecules are formed, which fall 
 more or less rapidly. This is what takes place in coagulation of 
 a proteid, whether brought about by heat or by the addition of 
 electrolytes, etc. The factors which inhibit and which cause this 
 clumping of molecules thus come to be of vital importance. The 
 force which tends to cause this clumping is probably surface 
 tension (Hardy, Bredig, Perrin), which is developed at the junc- 
 tion of the molecule and the water, and which, as explained in the 
 section dealing with agglutination, tends to draw together any 
 particles within a certain distance of one another, so that the 
 surface may become as small as possible. This, of course, is not 
 a peculiarity of colloidal solutions, but occurs in all emulsions of 
 insoluble particles. Surface tension, therefore, is constantly tend- 
 ing to make the particles of colloid in a solution approach one 
 another, form aggregates, and so cause precipitation or coagula- 
 tion. 
 
 This action is counterbalanced in a stable solution by a force 
 of electrical repulsion. The colloid particles all carry a feeble 
 charge of positive or negative electricity, and therefore tend to 
 repel one another. The existence of this charge and its nature 
 can be shown by passing an electrical current through the fluid. 
 Under these circumstances the colloids do not undergo dissocia- 
 tion with passage of the + ions to the - pole, and vice versa, like 
 the electrolytes ; instead, the molecules pass bodily to one or other 
 pole, according to the sign of the electric charge they carry. Thus 
 Field and Teague find that antitoxin is carried towards the cathode, 
 
COLLOIDAL THEORY OF ANTIBODIES 321 
 
 and must therefore carry a positive charge. Further researches 
 show that absolutely pure colloids, free from all traces of electro- 
 lytes, carry no charge at all, and are not conveyed by an electrical 
 current. The charge depends upon the nature of the ions present : 
 traces of acid and of acid salts give it a positive charge, whilst 
 alkalis and alkaline salts do the reverse (Pauli). 
 
 The process of coagulation of proteids, therefore, must depend 
 upon the neutralization of this electrical charge, and this can be 
 accomplished either by electrolytes or by colloids. Non-electro- 
 lytes (i.e., substances which do not split up into electrified ions in 
 solution) do not bring about coagulation in this way. Many of 
 them, such as sugar and urea, are inert ; others, such as alcohol, 
 act in an entirely different manner. The precipitation of an albu- 
 minous solution by means of a strong acid takes place thus : The 
 negatively-charged particles attract to themselves the positively- 
 charged hydrogen ions ; their charge is now neutralized, and the 
 force of attraction due to their surface tension is no longer counter- 
 balanced by an electrical repulsion ; the particles are drawn 
 together, form larger and larger aggregates, and finally cohere into 
 masses so large as to come under the influence of gravity, when 
 precipitation takes place. The coagulation of albumin by alcohol 
 is due to the fact that proteids are not soluble in that fluid, so that 
 when it is added to a watery solution of proteid, the water is with- 
 drawn and the particles brought so close together that surface 
 tension comes into play, and makes them coalesce into aggregates. 
 The truth of this explanation appears from Pauli's observation 
 that when no electrolytes are present alcohol acts very readily as 
 a precipitant there is no electrical repulsion between the particles. 
 But if a little acid or alkali be added, and the molecules be thus 
 given a mutually repellent electrical charge, the precipitation is 
 inhibited or entirely prevented. Colloids can also be precipitated 
 by colloids as well as by electrolytes, but in this case they must 
 be of opposite sign. Thus, in testing for albumin in urine by 
 means of acetic acid and ferrocyanide of potash, the addition of 
 the acid insures that the particles shall acquire a positive charge 
 (if they have it not already), which is then neutralized by the 
 colloidal ferrocyanic acid of negative sign. Numerous other 
 examples might be quoted. 
 
 This taking up of substances by colloidal particles is an example 
 of the phenomenon known as adsorption. This process is difficult 
 of definition, but in general terms we may regard it as a combina- 
 
 21 
 
322 ADSORPTION 
 
 tion between two substances dependent on physical attraction 
 rather than on chemical affinity, and taking place in very variable 
 ratios, rather than in simple ones dependent on combinations of 
 atoms or molecules, such as occurs in true chemical union. But 
 it must be admitted that there is no absolutely sharp line of 
 demarcation between the two classes of phenomena. In most 
 cases the two substances entering into the phenomenon of adsorp- 
 tion exist as such side by side in the compound, which is to be 
 regarded rather as an intimate admixture of the two than as an 
 entirely new substance. The question arises as to whether the 
 union between antigen and antibody is not really a process of 
 adsorption, and in the attempt to solve this problem several very 
 startling analogies with colloidal adsorption have been discovered. 
 It will be convenient to discuss some of these, and then to give 
 an account of the analogies met with in the chemistry of the 
 colloids. 
 
 Bordet has shown that the amount of haemolytic immune body 
 which can be taken up by a given volume of corpuscles varies 
 according to whether the corpuscles be added at the same time or 
 in successive small portions. Thus, in one example 0-4 c.c. of a 
 haemolytic serum dissolved 0-5 c.c. of corpuscles if added at once. 
 But if 0-2 c.c. of corpuscles were added first, and then successive 
 amounts of 0*1 c.c. put in, no solution took place after that of the 
 first portion added. This he explains, as we have already seen, by 
 invoking a physical process of the nature of adsorption, comparing 
 it with the adsorption of a dye by filter-paper. Other explanations 
 may be possible, but an exactly similar phenomenon may be seen 
 in the mutual adsorption of colloids of opposite sign. Thus, if to 
 a given amount of solution of an electro-positive colloid an amount 
 of solution of an electro-negative colloid exactly sufficient for 
 neutralization of the opposite charges be added, the result will be 
 the immediate commencement of the process of agglutination, 
 which will go on until all the colloids are precipitated. But 
 if a small amount of the second colloid be added to the same 
 volume of the other, a different state of affairs is brought about- 
 New aggregates of the two are formed, which are not necessarily 
 neutral in reaction, and experience shows that the conditions are 
 now less favourable to precip tation, which may require much 
 more of the second colloid for its complete accomplishment, the 
 intermediate bodies being apparently less easily precipitated than 
 the unaltered colloid. Similar phenomena may also be seen in the 
 
COLLOIDAL THEORY OF ANTIBODIES 323 
 
 action of agglutinin on bacteria. Here also the solid particles may 
 take up much more antibody than is necessary for agglutination 
 to be induced if the serum be added all at once. It may be pointed 
 out that this has a practical value in the estimation of the degree 
 of agglutinating action as determined by the degree of dilution in 
 which the action will take place. It is not proper to make a strong 
 dilution of the serum in the bacterial emulsion, and to dilute this 
 with further amounts of the latter, unless the subsequent dilutions 
 are made quickly. Otherwise, all the agglutinin may be removed 
 by the bacilli in the first dilutions, and those subsequently added 
 be apparently unaltered. The most accurate method is to prepare 
 all the serum dilutions required of double strength, and to add to 
 each an equal volume of bacterial emulsion. On the other hand, 
 when a bacterial emulsion is added to a serum in successive doses, 
 more agglutinin is taken up than when the whole amount is added 
 at once. 
 
 Next as regards the phenomena in which an excess of antibody 
 has apparently a reversing action, the best-known examples of 
 which are : (i) the deviation of complement, or Neisser-Wechsberg 
 phenomenon ; (2) the presence of zones of inhibition in which no 
 precipitation occurs in mixtures of serum and precipitin ; and 
 (3) the similar phenomena observable in the agglutinins. All 
 these have been discussed previously and explanations suggested. 
 These explanations have the merit of not involving any pheno- 
 mena of nature different to those familiar in immunity reactions, 
 but there are objections to all. Thus, Neisser and Wechsberg's 
 explanation of the deviation of complement would have us believe 
 
 CLfiH^no C C fJ f-p-r- 
 
 that uncombined complement has a greater affinity for alexin than 
 that which has had its cytophile groups combined with cell re- 
 ceptors a phenomenon exactly opposite to that which occurs in 
 haemolysis, where the process can be studied with greater accuracy. 
 We have also seen difficulties in the way of accepting Gay's 
 explanation, the full particulars of which are not yet available. 
 In the same way the inhibiting effects of large doses of precipi- 
 tating or agglutinating serum may apparently be explained on the 
 hypothesis of the existence of precipitoids and agglutinoids of 
 higher combining affinity than their unaltered antibodies, but it is 
 at least doubtful whether this is entirely satisfactory. It is difficult 
 to believe that the effect of moderate heat is to increase the com- 
 bining affinity of the agglutinin, whereas greater heat destroys it 
 entirely, and it is altogether different to what is observed in the 
 
 21 2 
 
324 EXPLANATION OF SPECIFIC INHIBITION 
 
 case of toxin, which is exactly equal to toxoid in this respect, and 
 Dreyer and Jex-Blake bring forward very strong evidence against 
 this view. Some .of the more important of their facts may be 
 summarized thus : If it were true, the more the serum is weakened 
 by heat (and the greater the production of agglutinoid) the greater 
 should be the zone of inhibition, and vice versa. This they found 
 not to be the case, for a serum that had had its agglutinating power 
 very largely destroyed by heat might show a very small zone of 
 inhibition, whilst another which had been hardly injured might 
 have a very large one. Further, a serum might show a zone with 
 an emulsion of bacteria in saline solution, but not in broth. 
 
 The alternative explanation of all these phenomena is based on 
 facts observed in the mutual precipitation of colloids, for here 
 again exactly similar zones of inhibition are seen. For example, 
 as Neisser and Friedemann have shown, a suspension of particles 
 of mastic in water (made by dropping an alcoholic solution into 
 water) takes on a negative charge, and can be precipitated by 
 positive colloids or by positive ions. Thus, if ferric chloride be 
 added precipitation occurs, and if the dose be increased it gradually 
 becomes more and more rapid and abundant until a certain amount 
 of ferric chloride is present, after which it becomes less and less 
 until no reaction takes place at all. And similar facts have been 
 observed in numerous other cases of interaction of colloids. Their 
 explanation is somewhat as follows : When the two colloids are 
 present in such an amount that their electrical charges mutually 
 annul one another, and they are therefore formed into aggregates 
 which tend to run together, the addition of fresh colloid bearing 
 an electric charge disturbs the conditions, and may, by electrifying 
 the masses already formed, cause mutual repulsion, and, if suf- 
 ficient amount of the second colloid be added, a re-solution of all 
 .the masses. Hence solutions, say, of an electro-negative colloid 
 may be dependent on (a) the presence of mutually repellent mole- 
 cules or aggregates of molecules in an inert fluid, or (b) on the 
 presence of these molecules in a fluid also containing a large 
 number of molecules of opposite sign, whereas in the intermediate 
 mixtures the conditions for precipitation are present. Here the 
 precipitate is soluble in excess of both substances, just as pre- 
 cipitum is soluble in excess either of precipitin or of its antigen. 
 
 This theory of the action of agglutinins has been investigated 
 and strongly supported by Biltz, Neisser and Friedemann, Pauli, 
 and others, and Dreyer and Jex-Blake have in particular given 
 
COLLOIDAL THEORY OF ANTIBODIES 325 
 
 strong evidence in its favour. For example, they find definite 
 zones of inhibition in the precipitation of B. coli by orthophosphoric 
 acid, a substance in which the existence of agglutinoids is out of 
 the question. Thus, in a series of tubes each containing 1-5 c.c. 
 of an emulsion of this organism in normal saline solution, agglu- 
 tination occurred in the tubes to which amounts from 118 centi- 
 grammes down to 4 centigrammes were added, and also in those 
 containing from i-i milligrammes to o-oi milligramme, but there 
 was none in which amounts from 4 centigrammes to i'i milli- 
 grammes were added. Further, there is a close analogy between 
 this phenomenon and the precipitation of gum mastic by ferric 
 chloride, for in each case the extent of the zone of inhibition 
 diminishes as the emulsions of the substance precipitated are made 
 stronger. 
 
 The facts at our disposal are not yet sufficient to enable the 
 phenomena of agglutination of bacteria to be fully explained, but 
 the process may take place somewhat as follows : Normal bacteria 
 pass toward the anode when an electric current is passed through 
 the fluid, and are therefore to be regarded as particles carrying a 
 negative charge. The addition of agglutinin removes this charge, 
 and the particles become electrically inert. This is shown by the 
 fact that when placed in an electric current they do not move in 
 either direction. If, however, very large amounts of agglutinin 
 are added, the point of neutrality may be reached and passed, and 
 the bacteria acquire a positive charge, again becoming mutually 
 repellent. Remarkable facts which may have some bearing on 
 this subject have been brought forward by Neisser and Friede- 
 mann. Emulsions of mastic are, as we have seen, precipitated by 
 ferric hydroxide, and it has been shown that the addition of a 
 small amount of organic colloid, whether positive or negative or 
 inert like gelatin, protects these particles, so that they are no longer 
 agglutinated by colloids or electrolytes of opposite sign. A pheno- 
 menon probably similar is seen when salts which form a colloidal 
 precipitate are mixed in a solution of gelatin, when the mixture 
 remains transparent, the colloidal particles being prevented from 
 undergoing flocculation. Silver chromate, formed by the inter- 
 action of potassium chromate and silver nitrate, may be obtained 
 in a transparent and stable form by this process. Particles thus 
 protected are not carried in either direction by an electric current. 
 Neisser and Friedemann have shown that mastic emulsions are 
 " protected " by the addition of a little serum, leech extract, or 
 
326 COLLOIDS AND HAEMOLYSIS 
 
 extract of typhoid bacilli, and they think that normal bacteria 
 may be compared with these particles surrounded by a defensive 
 envelope which prevents the flocculating action of substances of 
 opposite sign. The action of agglutinin they believe to consist in 
 the removal of this layer, so that then the ions of opposite sign 
 can unite with the bacteria and bring about their agglutination. 
 This explanation would explain clearly the role of salts in agglu- 
 tination, and is supported by Kirstein's observation that typhoid 
 bacilli cultivated in medium free from albuminoid material is 
 clumped by salt without the addition of serum. The theory, how- 
 ever, appears at present not to account for the facts already cited 
 with regard to the transport of normal and charged bacteria in an 
 electric current. 
 
 Some remarkable analogies have been brought forward by 
 Landsteiner and Jagic between the process of haemolysis by 
 simple colloids and by specific antibodies. Thus a solution of 
 colloidal silicic acid acts in a manner closely recalling a specific 
 haemolysin. It clumps and dissolves the red corpuscles of rabbits, 
 and agglutinates their spermatozoa, but has no action on typhoid 
 bacilli ; it thus shows signs of selective action, though not of true 
 specificity. Its action is manifested in extremely small doses ; it 
 is rendered inert by heat, and gradually falls off even at the room 
 temperature. Further, phenomena are observable which strongly 
 recall the action of complements and of lecithin in reactivating 
 heated serum or in dissolving sensitized corpuscles, since red 
 corpuscles which have been agglutinated by colloidal silicic acid 
 are dissolved by traces of lecithin or of fresh serum, but not by 
 serum which has been heated to 60 C. ; but if the silicic acid 
 is present in excess, no haemolysis occurs. 
 
 A difficulty arises from the fact that emulsions of red corpuscles 
 are agglutinated by both positive and negative colloids (ferric 
 hydroxide, ferrocyanide of copper). Girard-Mangin and Henri 
 have given an explanation of this, which is briefly as follows : 
 When a red corpuscle is suspended diffusion of salts, especially 
 of sulphates of calcium and magnesium, takes place from their 
 surface, and these facilitate the precipitation of positive and 
 negative colloids respectively, so that the corpuscles come to be 
 surrounded by a layer of precipitated colloid material. It is this 
 zone of precipitated material which actually determines the agglu- 
 tination of the corpuscles. Thus these authors consider three 
 substances as taking part in the process : (a) the corpuscles ; 
 
COLLOIDAL THEORY OF ANTIBODIES 327 
 
 (b) the salts which gradually diffuse therefrom ; and (c) the 
 colloid or agglutinin. They brought forward strong evidence 
 in favour of this theory by showing that agglutination is less 
 powerful when the corpuscles are suspended in normal saline 
 solution (the salts of which inhibit the exosmosis of the salts 
 in the corpuscles) than when they are in isotonic sugar solu- 
 tion (in which exosmosis is unchecked). Further deductions from 
 their theory (for which the original articles must be consulted) 
 were verified experimentally. 
 
 The analogies between colloid adsorption and the interactions 
 of toxin and antitoxin have been studied by Biltz, Pauli, and 
 others, and very remarkable facts adduced. For instance, there 
 appear to be phenomena indicating that the action of some anti- 
 toxins at least is reversible when a large amount is present. Thus, 
 according to Jacoby, the action of crotin (a phytotoxin of simple 
 nature and closely analogous with the bacterial exotoxins) is 
 increased by the addition of minute amounts of antitoxin, large 
 quantities of which neutralize its activity. Phenomena somewhat 
 similar are seen with the true toxins for example, Danysz (cor- 
 roborated by von Dungern and Sachs) has proved that diphtheria 
 antitoxin will neutralize more toxin when added at once than when 
 added in successive portions. Thus if the amount of toxin which 
 is just neutralized by a certain amount of antitoxin be divided into 
 two parts, and added to the antitoxin at an interval of twenty-four 
 hours, the whole may be toxic, although the amounts of toxin and 
 of antitoxin present are exactly the same in the two cases. Both 
 these effects can be explained on Ehrlich's pluralistic conception. 
 Thus Jacobi's results may be explained on the assumption that 
 small amounts of anticrotin combine with the non-poisonous 
 prototoxoid, and that this renders the mixture more toxic, because 
 this prototoxoid would otherwise seize on the receptors of the 
 sensitive cells, to the exclusion of the active toxin. This, however, 
 is very difficult to believe. Taking the action of crotin in pro- 
 ducing haemolysis as a test, it would appear that the neutralization 
 of some of the toxin, even if inert, would render the substance 
 less haemolytic ; this appears to follow from Bordet's proof that 
 red corpuscles can take up much more than their haemolyzing dose 
 of an antibody. The Danysz effect is also explicable on the 
 theory of the presence of a non-toxic epitoxonoid of no toxicity 
 and of feeble combining affinity. This slowly forms a firm com- 
 bination with the antitoxin, and renders it useless for neutralizing 
 
328 ANALOGIES WITH DANYSZ' EFFECT 
 
 more toxin. Explanations are also available on the Arrhenius- 
 Madsen explanation, regarding the combinations as examples of 
 mass reaction. 
 
 Regarding reactions such as these as interactions of colloids, 
 we find them paralleled in simple reactions. Thus in some cases 
 the addition to a colloidal solution of a small amount of a second 
 colloid of opposite sign may render the solution more stable, and 
 protect it from precipitation by an excess of the second substance. 
 The partially neutralized aggregates appear to possess more re- 
 pulsive power than those carrying their full electric charge. 
 Again, the effect of an addition of one colloid to another of 
 opposite sign is often brought about slowly, and the material only 
 gradually attains its permanent form. It is probably this fact 
 that renders solutions of antitoxin so unstable. Interreactions take 
 place between the colloids themselves and the electrolytes in the 
 serum, aggregates are formed, and the antitoxic potency falls off 
 rapidly at first and subsequently more slowly. It is a matter of 
 great difficulty to prepare stable solutions of proteid materials. 
 As we have already pointed out, the amount of colloid necessary 
 to precipitate a constant amount of another of opposite sign is 
 reduced to a minimum if the addition be made at once, and 
 rendered much greater if it is made slowly in small amounts with 
 an interval between each. This is closely analogous with the 
 Danysz effect. And this must also be considered in its bearing 
 on Ehrlich's method of attempting to neutralize a certain dose of 
 toxin by successive addition of small amounts of antitoxin, the 
 method by which the whole of the elaborate pluralistic conception 
 of the structure of toxins has been built up. According to the 
 colloidal theory, this is explicable on the assumption that tran- 
 sitional compounds of toxin and antitoxin of very diverse nature, 
 not necessarily dependent on the proportions of the two substances, 
 are formed. This is practically the view put forward by Bordet 
 from a consideration of the reactions of the antibodies, and especially 
 alexin and anti-alexin, and supported by the workers who have 
 examined the question from the standpoint of colloidal reactions. 
 
 The difference between the L and L+ dose, which is so 
 important a feature in Ehrlich's work, also finds an analogy in 
 the reactions of simple colloidal substances. Thus ferric hydroxide 
 neutralizes arsenious acid (whence its use as an antidote in acute 
 arsenical poisoning), and it was found by Biltz that if one lethal 
 dose of the latter substance was added to a neutral mixture of the 
 
COLLOIDAL THEORY OF ANTIBODIES 32Q 
 
 two, this mixture did not regain toxicity. Several lethal doses 
 must first be added, just as it is necessary to add several lethal 
 doses of diphtheria toxin to the L dose. 
 
 A difficulty arises from the fact that both toxin and antitoxin 
 pass in the same direction in an electric current. These experi- 
 ments are naturally difficult to carry out, since the effect of 
 electrolysis must be eliminated. When this is done, according to 
 Field and Teague, both substances travel towards the cathode, and 
 this whether the solution is acid or alkaline. But we must 
 remember that when toxin and antitoxin interact they always do 
 so in a very complex fluid, containing many other substances, 
 both colloids and electrolytes, and until we can determine the 
 electric charge of these substances in a pure form, these experi- 
 ments can hardly be considered to outweigh the remarkable 
 analogies described above. It is highly probable, judging from 
 analogy with the other antibodies, that free ions are essential to 
 the neutralization of toxin by antitoxin, and the researches of 
 Girard-Mangin and Henri show us how complex the conditions 
 may become. 
 
 We have seen that it is important to know whether the 
 compounds of antibody and antigen are dissociable, and that the 
 latter view explains some difficult phenomena seen in immunity. 
 On Ehrlich's theory the combination between the two is a strong 
 one, and once it has been allowed to take place, the two substances 
 are not dissociated into their components, whereas on Arrhenius's 
 and Madsen's theory the combination is an unstable one, and dis- 
 sociation constantly occurs. The experimental proof in favour of 
 the occurrence of this phenomenon appears on the whole satisfac- 
 tory. Red corpuscles saturated with immune body mixed with 
 normal corpuscles will part with some of their antibody, so that 
 the latter become sensitized. A mixture of tetanus toxin and 
 antitoxin, which causes no symptoms when injected into the leg of 
 a guinea-pig, causes tetanus when adrenalin has previously been 
 injected locally (explicable on the fact that toxin is absorbed by, 
 the nerves and antitoxin by the vessels, which in the second case 
 are constricted), and other examples might be quoted. It appears, 
 however, that this irreversibility only occurs if the two substances 
 are not kept in contact for a sufficiently long time, as was pointed 
 out by Martin and Cherry in their nitration experiments very 
 early in the history of the chemistry of the action of antitoxin. 
 Thus in the latter example, if the tetanus toxin and antitoxin be 
 
 I 
 
330 COLLOIDAL CHEMISTRY 
 
 kept in contact for two hours, no tetanus is produced, in spite of 
 the local action of the adrenalin. This appears to harmonize best 
 with the colloidal theory. The interactions of colloids of opposite 
 sign and the precipitation of colloids by electrolytes may be 
 reversible if the conditions are changed early, but the compounds 
 formed gradually become more and more stable. But if the con- 
 tinuation were that of two substances of strong chemical affinity 
 we should expect it to be stable from the first, and if it followed 
 the laws of mass reaction it would be reversible, no matter how 
 long it had stood. 
 
 There appears to be no crucial experiment by which the truth 
 of these three theories can be tested. The arguments brought 
 forward by the upholders of each involve the study of the exceed- 
 ingly complicated laws connected with the true relations and 
 completeness of the reactions, and involve advanced mathematical 
 consideration. It is, however, doubtful whether the processes are 
 measurable with the accuracy required in the investigation of 
 these complicated formulae, and in some cases very slight altera- 
 tion of the observed results would render the facts explicable on 
 a theory other than that in support of which it was adduced. At 
 the present time the balance of the evidence appears to be decidedly 
 in favour of the colloid hypothesis. 
 
CHAPTER XIII 
 ON IMMUNITY TO BACTERIA 
 
 WE are now in a position to discuss the mechanism by which a 
 bacterial infection is combated. It must be pointed out in the 
 first place, and kept in mind throughout, that different microbes 
 are dealt with in different ways. Some, such as many of the 
 protozoan blood parasites, may be tolerated, since the tissues of 
 their host are immune to the action of their toxins ; others are 
 removed by phagocytosis, after preparation by opsonins, or perhaps 
 by alexins, amboceptors, or other substances, or possibly without 
 previous treatment ; and yet others may be dissolved by the process 
 discussed under the heading of Bacteriolysis. Each of these 
 processes gives rise to a different form of immunity, which we 
 may call (i) immunitas non sterilisans, (2) phagocytic immunity, 
 and (3) bacteriolytic immunity. And the latter two processes 
 (which are the only ones of practical importance) rarely, if ever, 
 exist in a pure state ; almost all invading bacteria are, or may be, 
 dealt with by a combination of the two. 
 
 We must also notice that the defensive processes will be greatly 
 modified by the nature of the region in which the combat between 
 the invaders and the defensive mechanism goes on. There are 
 three main cases : (i) the circulating blood, (2) the tissues, and 
 (3) regions, such as the peritoneum, combining^some of the 
 characters of the other two. We will take them Jh this order. 
 
 In dealing with the processes that take place when bacteria gain 
 access to the circulating blood, our main difficulty at first sight is 
 not to explain the existence of immunity, but the reason why 
 infection ever occurs ; for the defensive mechanism seems more 
 than adequate to combat any dose of bacteria which is likely to 
 gain access to the blood under natural conditions. The processes 
 by which the bacteria are removed are phagocytosis and bacterio- 
 lysis, or the two combined, and in either case the defence would 
 appear to be more than adequate. Thus, in an experiment for the 
 
 331 
 
33 2 DIFFICULTIES OF THE PROBLEM 
 
 determination of the opsonic index in which thick emulsions of 
 bacteria are used the leucocytes are often found absolutely packed 
 with streptococci or tubercle bacilli after a short incubation ; if 
 on the average 100 cocci are taken up, calculation will show that 
 the whole volume of blood might phagocyte 2,000,000,000,000 in 
 a few minutes. Tubercle bacilli are perhaps hardly so easily 
 taken up, but it is difficult to see how the comparatively moderate 
 number of these bacteria which gain access to the blood after the 
 rupture of a small caseous gland into a vein can escape immediate 
 phagocytosis : yet we have no reason to believe that this accident 
 often occurs without causing general tuberculosis. Perhaps a 
 more convincing example is given by determinations of the opsonic 
 index in septicaemia, where the bacteria e.g., streptococci are 
 known to be circulating in the blood. Here the opsonic index is 
 usually low (0-5 or less), but even then it is sufficient to enable the 
 leucocytes to take up an enormously greater number of bacteria 
 than we can discover in the blood. 
 
 Similar phenomena occur in the case of bacteria which are com- 
 bated largely by means of bacteriolysins. Thus, normal blood 
 serum is markedly bactericidal to typhoid bacilli, yet infection 
 occurs, and many observers have noticed relapses when the 
 bacteriolytic power of the blood is extremely high. In these 
 cases, therefore, the bacteria can gain access to the blood (for 
 typhoid fever is always in part a septicaemia) in spite of barriers 
 which would appear unsurmountable. And we have already had 
 occasion to mention that anthrax may occur in animals the blood 
 serum of which is powerfully bactericidal ; the animals, indeed, 
 may be extremely susceptible. 
 
 Before attempting to explain the apparent discrepancy, it must 
 be pointed out that, as a matter of fact, septicaemia is a rare 
 disease in proportion to the opportunities for its occurrence, and 
 that the continued presence of bacteria in the blood without a local 
 lesion from which they are constantly discharged is extremely 
 uncommon. Thus in diphtheria, tetanus, the staphylomycoses, 
 etc., in which we should imagine that there is every chance for 
 bacteriaemia to occur, it is practically unknown. It is common in 
 diseases like ulcerative endocarditis, the early stages of typhoid 
 fever, and in pneumonia, in which we have reason to believe that 
 there is a constant shower of organisms discharged from the local 
 lesion direct into the blood. Here, however, we can feel fairly 
 certain that the bacteria which we find in the blood are only there 
 
ON IMMUNITY TO BACTERIA 333 
 
 temporarily, and are rapidly destroyed : the reasons being, firstly, 
 that they are never present in large numbers, as they would be if 
 they lived and multiplied in the circulation, and, secondly, that 
 they do not as a rule form secondary lesions, as we might expect 
 them to do, when deposited in the tissues. We may fairly assume, 
 therefore, that in a disease like typhoid fever bacteria are constantly 
 passing from the lesions into the blood, but are rapidly destroyed, 
 so that the defensive mechanism of the fluid is sufficient to prevent 
 the occurrence of a true septicaemia, in which bacterial growth 
 continues in the blood. The same holds in pneumonia and in 
 ulcerative endocarditis when not accompanied by secondary 
 infective lesions, and probably most other diseases, and does not 
 militate against the view that the blood is a sufficient defence in 
 the majority of the cases in which the infective material gains 
 access to the interior of the vessels. Excluding these and similar 
 examples, septicaemia is a rare disease, and is commoner in the 
 case of the protozoal than in bacterial infections. 
 
 As regards the failure of the process of phagocytosis, which 
 must occur when an organism which is combated by phagocytosis 
 does gain a foothold in the blood, we may point out that we have 
 yet but little knowledge of the exact state of the bacteriotropic 
 substances in the living plasma. Wright, it is true, has shown 
 that citrated plasma contains the same amount of opsonin as 
 serum ; but the two fluids are not absolutely identical, and the fact 
 that opsonin increases in amount in the successive fractions of 
 serum squeezed out from a clot leads us to believe it probable 
 that it does not exist as such in the plasma ; and if, as we have 
 shown is likely, the naturally existent thermolabile opsonin is the 
 same as alexin, it is, on the whole, probable that there is none in 
 the living blood. 
 
 Briscoe's experiments reproduce the natural conditions more 
 closely. He injected emulsion of bacteria into the ventricle of a 
 heart after excision and clamping of the auriculo-ventricular 
 groove. After a time some of the blood was removed and 
 examined, and practically no phagocytosis was found to have 
 taken place. But many bacteria were taken up when the same 
 animal's leucocytes and serum and the emulsion of bacteria were 
 incubated outside the body in the ordinary way. Further, in 
 some cases a little clotting occurred in the heart, and in these it 
 was found that the leucocytes which were included within the 
 clot took up many more bacilli than those which remained free. 
 
334 FUNCTION OF THE SPLEEN IN PHAGOCYTOSIS 
 
 These results argue strongly in favour of the fact that opsonin 
 does not occur in the circulating plasma as such, and is only set 
 free during the process of coagulation or of phagolysis. This 
 conclusion was corroborated by the fact that opsonized bacteria 
 were taken up freely, showing that the leucocytes were not at 
 fault, 
 
 We can get no information on this point from an examination 
 of the blood in these infections, since a small number of non- 
 virulent bacteria will be digested and completely disappear in a 
 few minutes if taken up by a leucocyte ; but as a matter of fact, it 
 is very uncommon to find ingested bacteria in the leucocytes of 
 the circulating blood in natural diseases. 
 
 It might appear that these explanations go too far, and would 
 lead us to the conclusion that phagocytosis is without value as a 
 defensive process when bacteria reach the blood-stream. This is 
 not the case, and it is probable that the great factor to consider is 
 the spleen. This acts as a sort of filter, and in its pulp the flow 
 of blood is slow, so that there is abundant opportunity for phago- 
 cytosis to occur. The same is true, though in a lesser degree, of 
 all regions of the body in which the circulation is tardy. 1 Hence, 
 when bacteria gain access to the blood they will always be found 
 in considerable numbers in this organ, which Kanthack called 
 years ago " the graveyard of the bacteria." This is especially the 
 case in typhoid fever, and the method formerly in use in the 
 diagnosis of the disease, by making cultures from traces of spleen 
 pulp removed by puncture, almost invariably gave positive results. 
 Films or sections of the spleen pulp will often show bacteria 
 ingested by the leucocytes in many diseases, even although none 
 can be detected in the blood. The role of the spleen in immunity 
 is probably a complex one, and this may explain the divergent 
 views held as to its importance in this respect. Thus it was 
 found long ago by Tizzoni and Cattani that it was impossible to 
 immunize animals to tetanus after splenectomy ; but this was not 
 corroborated by other observers, who found the animals equally 
 immunizable after the results of the operation had completely 
 passed away. But in this connection we must remember that the 
 splenic tissue is very rapidly regenerated, and that in all prob- 
 ability the haemolymph glands and other structures take on its 
 functions vicariously after its ablation. ^ 
 
 1 Leucocytes which have taken up bacteria whilst in the circulation 
 probably accumulate in the lungs also. 
 
ON IMMUNITY TO BACTERIA 335 
 
 The role of the spleen in facilitating phagocytosis is well seen 
 in the spirilloses. Metchnikoff showed many years ago that after 
 death from relapsing fever the cells of this organ are packed with 
 spirilla, such cells being rarely, if ever, seen in the circulating 
 blood. When monkeys are inoculated with blood containing the 
 parasite, a febrile attack occurs, but recovery soon takes place. 
 During the disease free spirilla occur in the blood, but they soon 
 disappear ; but if the animals are killed after this, the organisms 
 may be found within the splenic leucocytes. Further, Souda- 
 kewitch showed that recovery often failed to occur in animals 
 inoculated after splenectomy, and in this case the spirillum 
 appeared in large and increasing numbers in the blood, and no 
 phagocytosis was observed. Very similar facts have been 
 observed by Levaditi and Manouelian in tick-fever. During the 
 height of the disease the spirillum occurs in the blood, and is 
 exclusively extracellular. After the crisis it is found only in the 
 phagocytes of the spleen and liver (Kupffer's cells). 
 
 Besides the spleen, the bone-marrow is also a region where 
 abundant phagocytosis occurs, and the functions of the two tissues, 
 like their structure, are similar. In each case there is a slow flow^ 
 of blood in the pulp, close proximity of the blood to the tissue 
 cells of peculiar type, and an abundant supply of leucocytes. 
 
 The function of these organs in phagocytosis is probably two- 
 fold. It provides a region where the blood-stream is comparatively 
 tranquil, so that there is no mechanical obstacle to the process : 
 this is probably comparatively unimportant, since it can and does 
 take place under certain circumstances in the full torrent of the 
 circulation. And, secondly, if, as seems probable, thermolabile 
 opsonin is really complement, and if complement is formed by the 
 leucocytes, it is readily conceivable that it may be present in the 
 leucocytic organs when only present in small amount in the rest 
 of the blood. It may actually be called into existence by the action 
 of the toxin on the spleen cells or leucocytes, and be immediately 
 absorbed by the bacteria, so that it never occurs in appreciable 
 amount in the plasma. 
 
 This action will not come into play in the case of the bacterio- 
 tropic substances which are true antibodies, for we have every 
 reason to believe that these are present in the plasma as such, and 
 are not set free in the process of clotting ; yet even here we have 
 seen some evidence which suggests that they, in common with 
 other true antibodies, are formed inlymphoid tissue or from lymphoid 
 
336 FUNCTIONS OF OTHER ORGANS 
 
 cells, or, as some recent research seems to prove, from the endo- 
 thelium. 
 
 The liver is also an important region with regard to the deposi- 
 tion of bacteria, and their subsequent destruction by phagocytosis 
 or other means, but in this case the main cells concerned are 
 Kupffer's cells. We must regard these two organs and the lungs 
 as being intercalated in the circulation with the object, amongst 
 others, of arresting bacteria and other foreign particles which gain 
 access to the circulation, and affording a region in which phagocytosis 
 can take place at leisure. It has been shown experimentally that 
 animals will resist a much larger dose of bacteria if the injection 
 be made into the portal vein than if it is thrown into one of the 
 peripheral veins. The importance of these organs is also well 
 shown from a study of the fate of inert particles, such as carbon 
 or carmine, when injected into the circulation or peritoneum. In 
 either case they make their way with great rapidity to the organs, 
 especially the liver and spleen, in which they occur in large 
 numbers within two hours of the injection. They are also found 
 within the bone-marrow, lymph glands, tonsils, and lungs, and, 
 whatever the region in which they occur, are mostly contained 
 within the leucocytes or tissue phagocytes. Here also intracellular 
 absorption may take place, though of course it is much slower 
 than is the case with bacteria, and according to Siebel the leuco- 
 cytes, with their load of unabsorbable particles, may leave the body 
 via the lung, tonsils, or lymphoid structures of the small intestine. 
 
 The lung plays a part of great importance, but one not fully 
 understood, in the process of phagocytosis of bacteria which gain 
 access to the circulation. According to some writers, it is the only 
 internal organ in which phagocytosis by polynuclears can be 
 demonstrated, but this is certainly not correct. It is true, how- 
 ever, that soon after the injection of streptococci (Tchistovitch) or 
 cholera vibrios (Levaditi) into a rabbit, polynuclears containing 
 these organisms may be found in large numbers in the wide 
 capillaries of the lung ; this is probably the reason for the sudden 
 fall in the number of the leucocytes in the circulating blood which 
 occurs soon after injection. The reason for this collection in the 
 lungs is very far from clear : the region, remote as it is from 
 lymphoid tissue, would appear an unfavourable battle-ground 
 against the infection ; nor are the later stages of the process fully 
 understood. 
 
 The question of the importance of the bacteriolysins in haemic 
 
ON IMMUNITY TO BACTERIA 337 
 
 infections, already alluded to, now requires further mention. It 
 is obvious that the role which it plays varies greatly in different 
 diseases. There is, for instance, no evidence for believing that 
 the blood ever develops any substance which is bacteriolytic for 
 the tubercle bacillus. It is true that the researches of Wassermann 
 and his coadjutors show (by means of the method of fixation of 
 complement) that an antibody may occur in tuberculosis or in 
 tuberculous animals, but there is no evidence that this is a 
 bacteriolysin, or, if so, that it ever occurs in amounts sufficient to 
 bring about solution of tubercle bacilli ; in fact, it appears probable 
 that tubercle may run its whole course without leading to the pro- 
 duction of any recognizable antibody. So, too, with staphylococcic 
 infections. Here it is possible by special methods to produce a 
 serum which has a slight bactericidal effect, but normal human 
 serum or the serum of a patient who has been submitted to a 
 course of antistaphylococcic vaccination is quite powerless in this 
 respect. It is obvious, therefore, that neither the high grade of natural 
 immunity to staphylococci of normal persons nor the increased 
 amount present in active immunity is due to any bactericidal or 
 bacteriolytic substances occurring in the blood. It is quite true 
 that in old specimens of staphylococci pus-free cocci in all stages 
 of degeneration can be found, rendering it quite obvious that a 
 process of extracellular death and destruction of bacteria is at 
 work. This phenomenon is probably to be attributed mainly to 
 the action of the proteolytic enzymes formed by the leucocytes of 
 the pus. These are substances which are too often neglected in con- 
 sideration of immunity. They are, of course, non-specific, and will act 
 on any dead proteid, and on some living organisms. Their action may 
 be demonstrated by allowing some liquor puris from an old abscess 
 to act (in presence of thymol) on some staphylococci which have 
 been previously boiled to prevent autolysis. Another process in 
 which these degenerated forms of cocci may be produced is by 
 spontaneous autolysis in organisms killed by lack of suitable 
 nourishment, or atrepsy. 
 
 In sharp distinction from tubercle and the staphylomycoses, we 
 find such diseases as cholera and typhoid fever, in which bacterio- 
 lysins are formed in large amounts, and are readily demonstrable 
 by simple methods. It is in these and a few other diseases that 
 we may expect the role of these substances in recovery and 
 immunity to be most marked. We find, however, that their 
 action is most difficult to understand, and that its importance 
 
 22 
 
ROLE OF THE BACTERIOLYSINS IN RECOVERY 
 
 appears to diminish the more it is investigated. In general terms, 
 there is no doubt as to the fact that the presence of abundant 
 bacteriolytic substances must be regarded as a cause of immunity : 
 thus, animals may be rendered passively immune, e.g., to intra- 
 peritoneal injections of typhoid or cholera by antecedent or 
 simultaneous injections of powerful bactericidal serum. But 
 further investigation shows that even in this phenomenon there 
 are certain features which must lead us to regard the process of 
 bacteriolysis as of subordinate importance, or even as harmful. 
 In the first place, the demonstration of the fact that the opsonic 
 action of a powerful antityphoid serum (as determined by the 
 process of dilution until extinction of the property occurs) is pro- 
 portionate to its bactericidal action, and other facts already noticed, 
 have led us to the belief that immune body or amboceptor can 
 play the part of an opsonin, and, further, that it does so when 
 present in amount so small that its bacteriolytic action is but slight 
 or not apparent. It is, therefore, in the highest degree probable 
 that the passive immunity conferred by the injection of a bacterio- 
 lytic serum is due to the opsonizing action of this serum, manifested 
 even when diluted with the juices of the animal into which it is 
 injected. 
 
 In the second place, there is this main difference between the 
 destruction of bacteria within the leucocyte after phagocytosis 
 has occurred and the extracellular solution by immune body and 
 alexin : in the former case it is unusual for the endotoxins to be 
 set at liberty, so that as soon as a bacterium has been taken up by 
 a leucocyte, we believe that, as a rule, its capacity for harm has 
 been removed. It undergoes solution within the protoplasm of the 
 leucocyte, which, as appears from the studies of the French school, 
 is peculiarly insusceptible to the action of toxins, is destroyed by 
 the digestive juices, or by other means, and is not, at least in the 
 majority of cases, set free to injure the tissue cells. It is other- 
 wise with the solution of bacteria which takes place from the 
 action of bacteriolytic antibodies, for here the endotoxins are set 
 free, so that the presence of these antibodies, though undoubtedly 
 developed as a part of the defensive mechanism, may be a source 
 of added danger to the animal. For example, if an intraperitoneal 
 injection of a large dose of cholera vibrios be made into the peri- 
 toneal cavities of two animals, and in one some anticholera serum 
 be added, it is often found that this animal dies very rapidly with 
 symptoms of acute intoxication, whilst the other survives longer 
 
ON IMMUNITY TO BACTERIA 339 
 
 and dies of septicaemia. If dead vibrios are used the addition of 
 serum may bring about death, whilst an animal injected with the 
 organisms alone may recover with scarcely a symptom of intoxica- 
 tion. Similar phenomena, though less definite, often follow the 
 use of antistreptococcic serum for patients suffering from strepto- 
 coccic disease : the injection may cause a rapid rise of the 
 temperature and exacerbation of the symptoms. Other explana- 
 tions are possible, but it is highly probable that this is due to the 
 solution of some of the cocci and liberation of their endotoxin. 
 It is possible that in some cases this might be a source of danger, 
 and that this liberated toxin might be sufBcient to kill the patient ; 
 but there is no evidence that this actually occurs, and, as a rule, 
 the phenomena mentioned may be taken as proof that the serum 
 is a suitable one, and that its use should be continued. 
 
 We must, however, be careful in arguing from the phenomena 
 occurring in hypervaccinated animals to those suffering from a 
 natural infection. There can be no analogy between the train of 
 events when a massive dose of pathogenic bacteria is suddenly 
 injected into the circulation or peritoneal cavity of an animal in 
 which there is already an abundance of bacteriolytic substances, 
 and those which follow a natural infection. Considering the 
 latter case, and excluding all considerations of phagocytic action, 
 we may trace the sequence of events in the case of a disease such 
 as typhoid fever somewhat as follows : In the first place, since 
 normal human serum is bactericidal to typhoid bacilli, we must 
 regard the normal resistance against the disease as being due, in 
 part at least, to the presence of immune body in the serum, 
 though we have no means of knowing whether its action is mainly 
 opsonic or mainly bacteriolytic when occurring in the plasma. 
 When infection occurs, the amount of this substance must 
 diminish, since it will combine with the typhoid bacilli to which 
 it gains access, and may, indeed, be sufBcient to kill them all, and 
 thus prevent the development of the disease. Where, however, 
 the organisms gain access in sufficient numbers, so that the amount 
 of immune body or of alexin is insufficient to prevent the further 
 growth of some of them, those that escape will continue to grow, 
 and the disease will gradually increase in severity. At the same 
 time, the dissolved products of the organisms which succumb will 
 reach the seats of antibody production and stimulate a fresh pro- 
 duction of immune body. W T e have seen reason to believe that 
 this production takes place mainly in the lymphoid tissue and 
 
 22 2 
 
340 SEQUENCE OF EVENTS IN GENERAL INFECTIONS 
 
 other regions rich in lymphocytes, and we may probably corrobo- 
 rate this fact with the hyperplasia of the abdominal lymph glands, 
 Peyer's patches, and spleen which occurs in the disease, regarding 
 them as conservative, defensive phenomena, rather than the 
 pathological effects of the bacillus. 
 
 After a latent period or negative phase of a few days (the dura- 
 tion being dependent on the severity of the infection), the antibodies 
 begin to make their appearance. At first put out only in small 
 quantities, they will be absorbed at once by the bacilli present, and 
 will not be demonstrable in the serum or plasma in a free state. 
 A race now ensues. On the one hand, the typhoid bacilli multiply 
 as rapidly as their environment will allow ; on the other, the 
 immune body and other defensive antibodies are being gradually 
 elaborated more and more rapidly. In a very severe infection 
 this latter process may perhaps make default altogether, and the 
 patient succumb during the negative phase. This has not been 
 demonstrated in the case of the bacteriolytic substances, but is 
 well known in the case of the agglutinins. In most cases, how- 
 ever, the increase of bacteria and of antibodies takes place at the 
 same time. After a time bacteriolysis occurs, but in small 
 amount, since but little immune body makes its appearance, and 
 that which is formed at first is probably utilized as opsonin. 
 Hence when solution of the bacteria and liberation of the toxins 
 does occur, it does so at first only to a small extent, and we may 
 regard it probable that the minute amount of endotoxin set free 
 does not add appreciably to the symptoms of intoxication already 
 present. Further, we have now the conditions under which 
 immunity to these toxins may be expected to develop : the setting 
 free of small but gradually increasing doses of endotoxin condi- 
 tions, in fact, closely similar to those obtaining in Macfadyen's 
 experiments. Probably, therefore, by the time that much bacterio- 
 lysis occurs the patient's tissues are more or less immunized. It 
 is, however, not unlikely that the rapid oscillations of temperature 
 characteristic of the latter stages of typhoid fever may be due in 
 part to this liberation of endotoxin. 
 
 Some researches by Bail would appear to indicate that there is 
 a sort of automatic mechanism for the prevention of bacteriolysis 
 in the tissues. He found that no bacteriolysis took place when a 
 powerful serum was mixed with emulsions of cells from the liver, 
 spleen, etc, as well as with bacteria. The explanation is doubtless 
 that the complement is absorbed by these cells, as has been pointed 
 
ON IMMUNITY TO BACTERIA 341 
 
 out by Hoke, Muir, and others. Now, if this process goes on in 
 the living body, it can only indicate that complement (and thermo- 
 labile opsonin) can never occur in a free state in the organs, and 
 hence that bacteriolysis does not occur in these regions. It must 
 be pointed out, however, that amboceptor (or thermostable opsonin) 
 is not absorbed in this way, so that there would be but little inter- 
 ference with phagocytosis. It is quite possible that this absorp- 
 tion of complement does take place in order to avoid the liberation 
 of endotoxin consequent on bacteriolysis. 
 
 The mobilization of the patient's defences is usually manifested 
 by an increase in the number of the leucocytes in the circulating 
 blood. This subserves two functions : there is a greater number 
 of leucocytes to act as phagocytes and there is an increase in the 
 possible number of cells which may produce alexin or complement. 
 Hence we find leucocytosis present almost invariably in cases in 
 which bacteria gain access to the blood; the exceptions being, 
 firstly, very severe infections in which the defensive reaction fails, 
 and, secondly, a group of diseases of which typhoid fever is the 
 best example. The meaning of the first exception is tolerably 
 clear, that of the second not at all apparent. 
 
 The mechanism by which this increased output of leucocytes is 
 produced is chemotaxis. The bacteria in the blood-stream produce 
 a toxin which, in comparatively mild infections or in severe 
 infections in an animal possessed of strong natural immunity, 
 attacks the leucocytes. These cells will, therefore, tend to leave 
 the region in which they are formed and in which they are lying 
 dormant and make their way into the blood, the amount of toxin 
 being greater in the latter than the former situation. At the same 
 time, as Muir has so conclusively shown, there is an increase in 
 the functional activity of the bone-marrow, which manifests itself 
 in an increase of the leucocyte (and especially polynuclear 
 leucocyte) producing cells. A double process goes on, leucocytes 
 being formed more rapidly and attracted from the bone-morrow 
 as soon as they are produced. That this is due to chemotaxis is 
 shown by the fact that it follows the injection of bacterial toxins 
 and other products into the blood-stream, if not too virulent or in 
 too large amount. 
 
 When either of these conditions occurs we see the opposite 
 result a leucopaenia, or at least an absence of leucocytosis, and 
 this is always an extremely bad omen when it occurs in those 
 infectious processes where leucocytosis ordinarily takes place. 
 
34 2 VIRULENCE OF BACTERIA 
 
 Thus in pneumonia the number of leucocytes per cubic millimetre 
 has a most important prognostic value, and in almost all cases it 
 will be found that a patient with a high leucocytosis will recover, 
 even if the attack is a severe one and the clinical signs unfavour- 
 able. This leucopaenia may be attributed either to negative 
 chemotaxis, or to the production of paralysis or death of the 
 leucocytes, or to inhibition of the functions of the bone-marrow, 
 or to a combination of these causes. In any case, it argues a high 
 grade of virulence on the part of the bacteria, which, by lessening 
 the action of the main natural protective forces, increases the 
 chance of a lethal issue to the disease. Let us, therefore, discuss 
 some of the main facts (many of which have been referred to 
 before) concerning the nature of the mechanism of an increase in 
 virulence. 
 
 For an organism to be virulent it must produce a toxin which 
 has a profound action on the cells (and, it may be added, on the 
 important cells) of the animal in question. This, of course, is 
 obvious, and without it the organism could only produce pathogenic 
 effects by such means as deprivation of oxygen or food-stuffs from 
 the host or the production of bacterial emboli results which are 
 of no practical importance. Without an active toxin the bacteria 
 would either lie latent, as occurs in typhoid fever, gonorrhoea and 
 tubercle, or circulate in the blood as a harmless parasite, as is the 
 case with many protozoa in the lower animals. 
 
 As regards the special actions of toxins which render the bacteria 
 especially virulent, we may distinguish two (i) a leucocytic or 
 leucolytic action, and (2) negative chemotaxis. The former is a 
 very common function of highly virulent bacteria; its action is 
 best seen in the production of pus and necrosis in local lesions, 
 but its presence may be inferred from the degenerative changes 
 frequently present in leucocytes in general infections. And these 
 leucocytic substances need not be true toxins, for there is 
 reason to believe that the non-specific enzymes and other 
 substances which the bacteria produce have this action, and 
 that the death and destruction of leucocytes which is so marked 
 a feature of abscess production may be due in part to their 
 action. 
 
 We have already referred to the question of negative chemotaxis. 
 It probably actually occurs, but the proof is hardly convincing, 
 and the subject needs investigation by modern methods in vitro. 
 There is no doubt, however, that in virulent infections the regions 
 
ON IMMUNITY TO BACTERIA 343 
 
 containing the bacteria are often singularly free from leucocytes, 
 be the explanation what it may. 
 
 The first necessity for virulence, therefore, is the possession of 
 an active toxin, which, either by lowering the general vitality, 
 or by repelling, paralyzing, injuring, or killing the leucocytes, 
 diminishes the amount of phagocytosis which occurs. 
 
 A second defence against phagocytosis is the production of a 
 defensive envelope. This is well seen in the case of virulent 
 anthrax bacilli, which form thick gelatinous envelopes in the 
 animal body. Such capsulated forms are only taken up with 
 difficulty by the leucocytes, and the greater the power of capsule 
 formation the greater the virulence of the culture. Similar 
 phenomena have been seen in the case of the streptococci, 
 tubercle bacilli, and other organisms. The capsule of the 
 pneumococcus, which is only developed in the animal body or in 
 culture fluids approximating thereto, is probably a case in point. 
 
 Capsule formation is also probably a defence against bacterio- 
 lysins. Thus, according to Eisenberg, virulent typhoid or coli 
 bacilli stain blue by Giemsa's method, and show a pink edge. 
 Organisms which have undergone this modification are suscep- 
 tible to phagocytosis, but show great resistance to bacteriolysis. 
 And it is probable that the capsule of the anthrax bacillus is 
 intended largely as a defence against bacteriolytic substances. The 
 bacilli can be trained to produce it by cultivation in the serum of 
 the rat, which dissolves the unaltered bacilli in large numbers. 
 
 A third and more subtle method in which a bacterium can 
 increase its virulence is by loss of the receptors (which it possesses 
 in the avirulent state), which have the power of anchoring the 
 defensive substances of the blood. Thus the virulent pneumo- 
 cocci which (as shown by Rosenau) are not ingested by leucocytes 
 in presence of serum escape, as a result of the fact that they do 
 not absorb opsonin. Typhoid bacilli which have been cultivated 
 in immune serum lose their power of combining with amboceptor, 
 and also with agglutinin, and it may be taken as a general rule 
 that the more virulent a culture the less its reaction to its agglu- 
 tinin, and the less it absorbs that substance. Thus typhoid bacilli, 
 when isolated from the body, are usually refractory to agglutina- 
 tion, and gain the property only after several generations of culture 
 on artificial media. 
 
 Hence, therefore, we may picture the process which goes on 
 when a dose of pathogenic bacteria gains access to the blood of a 
 
344 SEQUENCE OF EVENTS IN GENERAL INFECTIONS 
 
 susceptible animal somewhat as follows : The bacteria form a 
 toxin which attracts the leucocytes from the bone-marrow into 
 the blood, and which also stimulates their production in larger 
 numbers. It also attracts the leucocytes into the neighbourhood 
 of the bacteria, and this process is more marked in regions, such 
 as the spleen and bone-marrow, in which the flow is slow. As a 
 result some leucocytes are stimulated to produce complement, or, 
 on the other theory, are killed, and their complement set free. In 
 either case the bacteria are prepared for phagocytosis. This 
 process may now take place, the bacteria be destroyed, and the 
 infection come to an end. This is a type of a mild infection. 
 
 Or it may happen that the toxin is so powerful that the leuco- 
 cytes are repelled, or, if attracted, immediately killed. If this 
 occurs the sole resource of the patient is the bacteriolytic property 
 of the blood, which will then come into action provided, of course, 
 that the bacterium is one to which amboceptor occurs in the normal 
 blood because alexin is set free in the solution of the leucocytes. 
 Experiment, however, leads us to believe that this is a slower 
 and less important process than phagocytosis, and that if the latter 
 is in abeyance the outlook for the patient is bad in the extreme. 
 If both processes fail the infection must be rapidly fatal. 
 
 In an infection in which the defensive and offensive mechanisms 
 are nicely balanced, and an illness of some duration and severity 
 occurs, the processes will be much more complex. The early 
 stages will occur as in a mild infection, and the bacteria will be 
 surrounded by the leucocytes, and perhaps even ingested. In this 
 case it may happen that after ingestion solution will take place, 
 and an endotoxin be set free which will kill the phagocyte, and 
 perhaps also those in the immediate neighbourhood, and in this 
 way some of the bacteria may be shielded from further phagocy- 
 tosis for a time. This and similar processes give the bacteria 
 time in which to undergo their defensive modifications, which they 
 do in virtue of the " survival of the fittest " i.e., those which are 
 best adapted to their environment in the host. Now it is clear 
 that in any culture of bacteria the different individuals have 
 marked differences with regard to their power of absorbing anti- 
 bodies. This is obvious when we consider that otherwise the 
 addition of gradually increasing amounts of an agglutinating 
 serum would cause no effect until a certain concentration was 
 reached, when all the bacteria would clump. As it is, those 
 organisms with the greatest affinity take up the agglutinin in 
 
ON IMMUNITY TO BACTERIA 345 
 
 largest quantity, and when the number of molecules reaches a 
 certain amount they clump, whilst those with less affinity have 
 not yet absorbed sufficient. Probably exactly similar phenomena 
 occur in the opsonization of bacteria. Some organisms are avid 
 for opsonin ; others take it up with difficulty, and a large concen- 
 tration of the substance is necessary before they are prepared for 
 phagocytosis. This probably explains the fact that the number of 
 bacteria taken up by the leucocytes does not increase pari passu 
 with the amount of opsonin present. 
 
 The bacteria which remain immune to the defensive mechanisms 
 will be enabled to live, and in their descendants the conditions for. 
 natural selection will occur variations and an adverse environ- 
 ment. The weaker forms i.e., those which, by the absence of a 
 defensive layer or .the presence of numerous receptors on which 
 the opsonins or bacteriolysins of the host can seize, or those 
 which do not elaborate a powerful toxin will be destroyed, and 
 the more virulent forms will survive, so that a gradual selection 
 of the latter will ensue, and the invaders rapidly increase in 
 virulence. And it must not be forgotten that bacteria multiply 
 with great rapidity, division sometimes occurring in a quarter of 
 an hour, so that nearly a hundred generations are passed through 
 in a day, and abundant opportunity for evolutionary selection 
 occurs. From what we know of the virulence of typhoid bacilli 
 and of pneumococci when causing disease and when living 
 parasitically without the body, there is reason to believe that this 
 increase of virulence always takes place in infections. 
 
 Here, then, the standing forces of the body are insufficient to 
 deal with the invader, and the latter increases in virulence and 
 numbers during the early stages of the struggle. This process 
 will continue until the latent reserve forces are mobilized and 
 fresh defensive substances brought into action. These are 
 
 (1) antitoxins, whether to exotoxins or endotoxins, or the toxins 
 may be dealt with in one or other of the methods discussed under 
 Antitoxic Immunity ; but in any case it is probable that the develop- 
 ment of some degree of toxic immunity, especially, perhaps, of 
 the leucocytes, must precede the destruction of the bacteria. 
 
 (2) Increased amounts of alexin-opsonin having a specific action 
 on the organism in question. We have already noted some of 
 the gaps in our knowledge of this subject, and shall, perhaps, 
 again have occasion to refer to the difficulties in explaining 
 its action, but there can be little doubt of its importance in 
 
346 LOCAL LESIONS 
 
 generalized infections. We notice, for example, that in pneumonia 
 it remains at a low level during the attack, and increases rather 
 suddenly at the time of the crisis ; the pneumococci disappear 
 from the blood at this period, though they remain living in the 
 lung until much later. In cases which recover by lysis, on the 
 other hand, there is a gradual increase in the opsonic index, and in 
 acutely fatal pneumococcal septicaemia the index falls gradually 
 and continuously. When dealing with generalized infections the 
 opsonic index (as far as our researches have gone at present) may 
 be taken as a very rough guide to the degree of immunity and 
 chance of recovery. This does not apply in local infections, but 
 here we are probably justified in believing that the higher the 
 index, the less the chance of generalization. It is worthy of 
 notice that the disease cerebro-spinal meningitis in which the 
 highest indices (sometimes as high as ten) are found is one in 
 which bacteriaemia very rarely occurs, though by analogy with 
 other similar diseases we should rather expect it to do so. As 
 regards the source of this increased amount of alexin-opsonin, 
 there is nothing to be added to what has been already stated. 
 (3) In some cases recovery may be brought about by the produc- 
 tion of thermostable opsonins, whether amboceptor (as appears 
 most probable) or agglutinin. This is rarely, if ever, the case in 
 the pneumococcic diseases, but probably occurs constantly in the 
 maladies, like typhoid fever and cholera, in which agglutinins and 
 bacteriolysins are formed in large amount. We may, indeed, 
 classify diseases roughly into two main groups in this respect : 
 (i) those in which the acquired immunity is due (inter alia) to the 
 presence of thermolabile opsonins : in these we may expect the 
 degree of the immunity to be but slight and its duration short; 
 and (2) those in which it is due to true antibodies : here we may 
 expect the opposite conditions to hold. Pneumonia and typhoid 
 fever may be taken as examples. We do not know accurately 
 the duration of the immunity to the latter disease, but the anti- 
 bodies on which we believe it to depend may be traced for long 
 periods, whereas the blood returns to its normal state very shortly 
 after recovery from a pneumococcic infection. And experience 
 with typhoid vaccine leads us to suppose that the immunity to that 
 disease lasts a year or more. The occurrence of relapses may 
 seem to argue that the protection is in reality very evanescent, 
 but other interpretations are possible. 
 
 The processes which occur in local lesions are similar in 
 
ON IMMUNITY TO BACTERIA 347 
 
 nature, but modified by their occurrence in the tissues and not in 
 a fluid medium, and they tend to deviate more from test-tube 
 conditions than do the blood infections. 
 
 At the commencement of the infection the conditions are quite 
 comparable to those of a haemic infection. Leucocytes are soon 
 attracted into the area, and, if the bacteria are not too virulent, 
 may remove them entirely, the infection being thus cut short at 
 its very commencement. Probably myriads of slight wounds are 
 thus dealt with. But when the bacteria resist phagocytosis, and 
 form virulent toxins, a new set of factors are brought into 
 existence. These have been glanced at previously, and will now 
 require further discussion. 
 
 The dilatation of the vessels of the infected region and the 
 consequent acceleration of the blood-flow is, as has been pointed 
 out, altogether a conservative influence, having for its object the 
 removal of toxins and their dilution in the general mass of the 
 blood. But in all cases except the most trivial the local reaction 
 extends further, and the more extensive changes tend, partly at 
 least, to favour the spread of the infection by shielding the 
 bacteria from the full action of the blood. In other words, the 
 middle stages in the evolution of an inflammatory lesion indicates 
 a victory for the bacterium in so far that it has succeeded in 
 altering the tissues to such an extent as to shield itself from the 
 action of the protective forces. At a still later stage (in lesions 
 going on to recovery) the tissues and juices gain the victory, and 
 there is an alteration in the nature of an increase in the local, and 
 usually of the general, immunity. 
 
 Let us trace what we know of this process in the case of a 
 small staphylococcic lesion. The early stages consist of the usual 
 inflammatory reaction. The increased access of blood, increased 
 number of leucocytes, and probably increased amount of opsonin, 
 present in the tissues may suffice to cut short the process, the 
 whole of the bacteria being removed by phagocytosis. In this 
 case the only clinical signs will be those of acute inflammation, 
 resulting in rapid recovery. 
 
 If, however, the bacteria are present in too great numbers, or 
 are too violent, or if the immunity (local or general) is deficient, 
 the toxins formed will kill the tissues in the neighbourhood, and 
 the bacteria will then lie in a smaller or larger mass of necrotic 
 tissue, which is surrounded by an inflammatory zone. The tide 
 has now turned very definitely in favour of the bacteria, for four 
 
348 SEQUENCE OF EVENTS IN LOCAL LESIONS 
 
 reasons : (i) There is a mechanical obstacle to the access of 
 leucocytes to the bacteria, which make their way through the 
 slough with some difficulty. In ordinary healthy tissues, permeated 
 by capillaries, the leucocytes have never very far to go to reach a 
 given area; but when death of these tissues has occurred, the 
 leucocytes have to crawl in from the still patent capillaries of the 
 periphery, a distance, maybe, of several millimetres. (2) The 
 bacteria in the centre of the lesion have time to elaborate a 
 powerful toxin, which is not diluted by the blood or lymph, since 
 the vessels are all thrombosed, and can only escape by diffusion. 
 As a result, there is a central zone where the toxin is present in so 
 high a concentration that it may either repel the leucocytes by 
 negative chemotaxis or kill them outright. The latter process 
 certainly takes place, as the large number of dead bacteria present 
 in pus sufficiently proves. (3) There is an insufficient supply of 
 opsonin, and perhaps of other defensive substances also, in the 
 fluids at the centre of the lesion. This is brought about partly by 
 removal of these substances by the bacteria of the region. In the 
 case under consideration the staphylococcus opsonin is absorbed 
 by the cocci which, in the absence of the leucocytes, it is power- 
 less to injure. Another possible factor in the removal of these 
 substances is the action of proteolytic enzymes, the defensive 
 materials being digested and rendered inert before they reach the 
 bacteria. Lastly, there is reason to think that opsonins make 
 their way with difficulty through the inflamed tissues. Thus 
 Bulloch found that the serum or liquor puris which was collected 
 from an abscess immediately after it had been cleansed was almost 
 devoid of opsonic power, and this is the case with the fluid portion 
 of pus in general. In some cases I have been able to demonstrate 
 the existence of an antiopsonin, perhaps consisting of cast-off 
 receptors of the bacteria. (4) There is an increase in the virulence 
 of the bacteria, due to conditions already discussed. 
 
 The absence of opsonin from the fluid at the centre of the 
 lesion suggests several considerations of some importance. We 
 see, for instance, that it is not sufficient in the cure of a local 
 lesion for there to be an abundant supply of opsonin in the blood : 
 two other factors are required a permeable lesion and a region 
 suited for the activity of leucocytes. This is very well seen in the 
 case of tubercle, where the opsonic index may be greatly raised (to 
 i '8 or more), and the lesion show no indication of recovery. There 
 is, as a rule, little or no tendency of the disease to spread or 
 
ON IMMUNITY TO BACTERIA 349 
 
 become generalized with a high opsonic index, yet even this may 
 occur. We may trace a certain degree of parallelism between the 
 ease of access of opsonin to all parts of the local lesion and the 
 likelihood of a cure following an elevation of the opsonic index. 
 For example, there is, according to TunniclifFe, a definite relation 
 between the rise of the opsonic index and the disappearance of the 
 membrane in diphtheria, the latter clearing rapidly when the index 
 rises above normal. Here the conditions are these : there is a 
 membrane, a few millimetres thick, composed of dead material, in 
 which the specific bacilli are elaborating their toxins ; below this 
 there is a zone of acute inflammation, hyperaemic and infiltrated with 
 phagocytes. Now it is easier for the toxins to diffuse outward and 
 be washed away by the secretions of the mouth than to pass 
 inwards, and probably but a small fraction actually formed reaches 
 the blood-stream. On the other hand, it is comparatively easy for 
 the protective substances in the serum to pass outwards, the con- 
 ditions being quite different from those in a closed abscess, where 
 opsonins, etc., can only reach the centre of the lesion by diffusion, 
 and not by actual transudation. Here there is a constant stream of 
 lymph from the pervious vessels to the free surface. Thus the 
 low concentration of the toxins renders it easy for the leucocytes 
 to gain access to the bacilli, and the latter have abundant oppor- 
 tunities of becoming opsonized ; hence the conditions for successful 
 phagocytosis are produced. 
 
 A similar train of phenomena follows the free drainage of an 
 acute abscess. The toxins escape outwards, and are no longer 
 forced into the tissues, and at the same time there is a flow of 
 protective lymph into the abscess cavity, and consequent sensitiza- 
 tion of the bacteria, and the removal of the toxins allows the 
 phagocytes to act. If, however, the wall of the abscess is very 
 thick and impermeable, it may be, as in Bulloch's experiment, 
 that the lymph which exudes is deprived of its opsonin and other 
 protective substances during filtration, and the conditions are then 
 less favourable. 
 
 Now consider a large staphylococcic lesion completely embedded 
 in the tissues. There is a large core of dead material, in which the 
 cocci constantly produce toxins, and in which they are completely 
 shielded both from fresh lymph or plasma and from leucocytes. 
 Here we should expect the lesion to be progressive, no matter how 
 high the opsonic index, and this is usually the case. Hence, other 
 things being equal, it is in the smaller lesions that we may expect 
 
350 SEQUENCE OF EVENTS IN LOCAL LESIONS 
 
 benefit from vaccine injections which raise the index, and more 
 especially in cases in which there is a free drain for the toxins 
 ulcers, open abscesses, sinuses, etc., or in those in which there is 
 no dead material (slough or caseous matter), and the blood is 
 brought into close contact with the bacteria. Small isolated 
 tubercles in the iris often yield rapidly to tuberculin injections* 
 whereas large caseous glands are most refractory. 1 Chronicity is 
 also an important factor : a long-standing thick-walled abscess is 
 less amenable to treatment than a small freshly formed boil. 
 
 In order to aid this flow of plasma or lymph laden with pro- 
 tective substances through the lesion, Wright has suggested the 
 exhibition of substances such as citric acid or leech extract, which 
 have the power of diminishing the coagulability of the blood. In 
 certain cases the beneficial effects of these remedies may be most 
 striking. And the local use of hot fomentations, etc., which dilate 
 the vessels, has a similar effect ; in addition to which they probably 
 aid phagocytosis by raising the temperature of the part. 
 
 Next as regards the other pre-existing defensive substances of 
 the plasma the bacteriolysins. In the case of the staphylococci 
 there is no reason to think that they are of any importance, since 
 most authorities hold that there is no proof that serum has any 
 bactericidal action on these organisms under any circumstances. 2 
 And when the blood does normally contain the amboceptor- 
 complement apparatus, the establishment of the lesion sufficiently 
 demonstrates the insufficiency of this apparatus for defence. 
 
 At a later stage, supposing the pre-existing defences opsonin 
 and leucocytes, etc. fail to bring about cure, a second series of 
 factors come into action. These are the antibodies, the chief 
 being antitoxin, amboceptor, and thermostable opsonin, the latter 
 being possibly either agglutinin or amboceptor, as we have seen 
 already. These usually take about a week to be produced, and 
 may be looked upon as the second line of defence. 
 
 As regards their place of production, this may be remote or 
 local. The sites of general production of the antibodies have 
 
 1 In saying this I do not wish to imply that the cure in these cases, and after 
 the use of vaccines in general, is brought about solely by an increase in the 
 opsonins of the blood, 
 
 2 I must point out, however, that in old staphylococcic abscesses it is 
 common to find cocci which have lost their power of retaining Gram's stain, 
 an invariable proof (where applicable) of the early stages of bacteriolysis. 
 This is probably due to the action of the peptic enzyme formed by the 
 leucocytes. 
 
ON IMMUNITY TO BACTERIA 351 
 
 been discussed already, and we have seen reason to believe that 
 the chief regions in which it takes place are the lymphoid organs, 
 the lymph glands, bone-marrow, and spleen especially. These 
 organs appear to have as one of their functions the elaboration of 
 defensive antibodies as an internal secretion in response to toxins 
 and other bacterial products (free receptors, etc.) circulating in the 
 blood. We have also seen that the phagocytes, and especially the 
 polynuclear leucocytes, may act as sources of these bodies, but 
 that the evidence is less convincing than in the case of the 
 lymphoid organs. 1 
 
 The local production of antibodies is probably more important, 
 since it takes place at the spot at which these substances are 
 required. As an example we may take Romer's demonstration of 
 antitoxin in the conjunctiva as a result of installations of abrin, 
 when no antitoxin had yet appeared in the general circulation. 
 The source of these antibodies, as first suggested by Whitfield, is 
 probably the lymphocytes, which form so important a factor in 
 chronic inflammatory lesions, and which occur after a few days in 
 the periphery of acute lesions. These cells do not act as phago- 
 cytes in vivo, though they may do so under experimental condi- 
 tions, and on any other hypothesis their presence in inflammatory 
 lesions is entirely inexplicable. We may feel certain, however, 
 that they are attracted there for some good purpose, and it is in 
 the highest degree probable that this purpose is the elaboration of 
 protective antibodies. It seems fairly certain that the source of 
 the haemic antibodies is the lymphocyte cells of the adenoid 
 tissues, and the cells of a chronic inflammatory lesion are exactly 
 similar. And in many cases if we examine the region in which 
 inflammation has taken place some time previously, we shall find 
 that the cells which were attracted thither have become organized 
 into definite adenoid tissue. 
 
 It seems probable, too, that these small round cells are the basis 
 of the local acquired immunity, concerning which so little is 
 definitely known. After the bacteria have been destroyed the 
 polynuclear leucocytes remove the debris of dead tissues, etc., and 
 then retire, but the lymphocytes persist in the region for long 
 periods. During this time they are perhaps continuously elaborat- 
 ing small amounts of antibodies, and are certainly on the spot and 
 ready for action should a fresh infection occur. It is probable, 
 
 1 More recent researches tend to point to the endothelial cells as the more 
 probable source of origin of the antibodies. 
 
35 2 SEQUENCE OF EVENTS IN LOCAL LESIONS 
 
 too, that they may have become so altered in virtue of having 
 once been stimulated to produce antibodies that they can do so 
 more quickly and easily than normal cells. Thus Cole has shown 
 that an animal which has once been inoculated e.g., with typhoid 
 bacilli will produce on a second injection a much greater output 
 of antibodies than will a normal animal, and that this increased 
 defensive reaction persists for months, long after the animal has 
 apparently become normal. This observation is probably of the 
 highest importance, both as regards general and local acquired 
 immunity. 
 
 Thus in the case of a local lesion we may distinguish two 
 stages : ( i ) That in which the natural resources of the body alone 
 come into action, and in which the main mechanism for com- 
 bating the bacteria is phagocytosis, the main cell the polynuclear 
 leucocytes, and the main defensive substance the thermolabile 
 opsonin. In this stage there is in general a decline of the local 
 immunity due to the action of toxins on the tissues, and the only 
 rise of the general immunity, if present at all, is due to an increase 
 of this opsonin. (2) The second stage is that of the antibodies. 
 The factors taking part in the first stage persist, but there is a 
 new defensive cell, the lymphocyte, and new defensive substances, 
 the antibodies. Here the immunity, both local and general, tends 
 to be raised. We may also distinguish a third stage, in which the 
 polynuclears have retired and the lymphocytes remain a stage in 
 which the natural immunity of the part is reinforced by the actual 
 or potential opsonins due to the lymphoid cells, and in which 
 these are better equipped (in virtue of their previous training) to 
 produce a large amount of antibody in response to a slight 
 stimulus. 
 
 Lastly, we must study briefly the defensive reactions of the 
 peritoneum, a region which has been subjected to a full investiga- 
 tion and which resembles the tissues in some points and the blood 
 in others. In all probability the process of absorption from the 
 other serous sacs is, in general, similar. We have already glanced 
 at the subject, and in what follows much use has been made of 
 the admirable researches of Buxton and Torrey. The case of an 
 intraperitoneal injection of a pathogenic organism of moderate 
 virulence into an animal possessed of a certain amount of natural 
 immunity will be considered e.g., of a laboratory culture of 
 B. typhosus into the peritoneal cavity of the rabbit. 
 
 Under these circumstances, one or other of two trains of events 
 
ON IMMUNITY TO BACTERIA 353 
 
 may take place. In the first the immunity appears to depend 
 mainly on bacteriolysis, in the second on phagocytosis. 
 
 In the former case an enormous number of bacilli are killed in 
 the space of a few minutes, very few being recoverable from the 
 peritoneum by washings with normal saline solution, and when 
 this takes place it is found that few or no bacilli make their 
 way into the blood or organs. In such cases the typhoid bacilli 
 which are recovered show marked signs of degeneration, similar, 
 though less marked, to those seen in cholera vibrios in the classical 
 PfeifFer's reaction; the staining material becomes collected into 
 the centre of the sheath of the bacillus, which subsequently breaks 
 down into minute granules. This process is usually entirely 
 extracellular, but after a time some of the altered bacilli may be 
 taken up by the phagocytes. It would appear at first sight, there- 
 fore, that in this case the complement -amboceptor mechanism 
 is present in the normal peritoneal fluid, ready to bring about 
 immediate bacteriolysis. This, however, is by no means certain, 
 and it is highly probable that the complement does not exist in 
 this fluid, and that its appearance follows the injection, and is due 
 in some way to the leucopaenia which has been already noted. 
 The nature of this leucopaenia has been much discussed, and is of 
 some importance in regard to this question of the nature of the 
 complement. Metchnikoff and the French school generally 
 maintain that the diminution is due to the actual destruction of 
 the leucocytes phagolysis and that during this destruction or 
 solution of these cells alexin or complement, or, as Metchnikoff 
 calls it, microcytase, is set free. As a matter of fact, there is no 
 evidence that this phagolysis does occur, and more recent researches 
 appear to point to another solution of the leucopaenia. It is true 
 that the fluid withdrawn from the peritoneum by means of a 
 pipette is markedly deficient in cells, but those that are present 
 do not show the marked degenerative changes we should expect 
 if they were undergoing rapid solution. And in any case, it is 
 difficult to believe that an injection of substances such as normal 
 saline, which we know preserves the leucocytes in a lively condition 
 for many hours, should have such a profound destructive effect on 
 them in the peritoneum. Normal saline, it may be pointed out, is 
 capable of producing a most marked leucopaenia when injected 
 into the peritoneal cavity. 
 
 The most probable explanation of the phenomenon is that first 
 suggested by Pierallini, and corroborated by Buxton and Torrey. 
 
 23 
 
354 INFECTIVE PROCESSES IN THE PERITONEUM 
 
 According to them, there is a deposition of masses of fibrin on 
 the omentum, and in and upon these masses there are numerous 
 polynuclear leucocytes. 
 
 The formation of fibrin, it need hardly be pointed out, is 
 universally regarded as being due to the liberation of fibrin 
 ferment by the leucocytes. It is not yet settled beyond controversy 
 whether this is to be regarded as a process of secretion, a vital 
 phenomenon, or a process occurring only during the solution 
 phagolysis of the leucocytes. The point is of no importance : 
 the remarkable feature is that, according to the opinions we have 
 considered as most probably correct, complement is set free at the 
 same time as fibrin ferment. The deposition of fibrin on the 
 surface of the omentum may be taken as sufficient proof of the 
 liberation of this substance, so that the rapid destruction of the 
 bacteria which occurs in the peritoneum in some cases cannot be 
 regarded as constituting definite proof that it occurs preformed in 
 that situation. 
 
 In some cases, therefore, bacteria injected into the peritoneum 
 are killed rapidly, almost instantaneously, by a process resembling 
 bacteriolysis, and doubtless due to amboceptor existing in the 
 peritoneal fluid as such at the time of the injection, together with 
 complement which is probably set free from the leucocytes as a 
 result of the presence of the foreign body, but which may possibly 
 also be present in the normal state. As an example of this process, 
 Buxton and Torrey quote the case of a rabbit which received an 
 intraperitoneal injection of about 4,000,000,000 typhoid bacilli, and 
 was then immediately killed, the peritoneum washed out with 
 normal saline solution and plated out. The fluid contained only 
 about 1,000 bacilli, and there were none in the blood or internal 
 organs. In a case which was allowed to survive for two hours no 
 bacilli were found in any part of the body. It is most remarkable 
 that, with all this sudden, almost explosive, solution of bacilli there 
 were no symptoms indicative of the liberation of endotoxins, the 
 temperature remaining constant. The culture was one of very 
 moderate virulence. 
 
 In other cases the whole process differs, and the defence of the 
 body appears to be entrusted to the phagocytes rather than to the 
 bacteriolytic substances, and in these a most interesting train of 
 phenomena occurs. Some destruction of the bacilli may occur in 
 the peritoneum, showing that the bacteriolytic properties do not 
 fail entirely, but comparatively few organisms are destroyed by 
 
ON IMMUNITY TO BACTERIA 355 
 
 this process ; instead, large numbers of bacilli make their way 
 into the circulation by a route not fully known. These rapidly 
 decrease in number, the diminution being quite noticeable within 
 half an hour, and in six hours or less the blood may be entirely 
 sterile. There is, further, a remarkable amount of deposition of 
 the bacilli in the organs, especially the liver, spleen, lung, and 
 bone-marrow ; and in these situations the numbers of organisms 
 decrease for about two or three hours, and then show a very 
 definite rise, which lasts for two or three hours more, and the 
 organisms may attain numbers approximating to those present in 
 these organs immediately after the injection. The numbers then 
 gradually decrease for the next two days or so. Here we may see 
 in a very clear manner an example of the mobilization of the 
 defensive forces discussed previously. The diminution which 
 takes place immediately after the access of the bacilli to the 
 organs is doubtless due to the bacteriolytic substances, either 
 present in the blood as such, or immediately available after 
 phagolysis or secretion of alexin. After a time these substances 
 are exhausted, and it is only after some hours that a fresh supply 
 is elaborated, and destruction of bacilli continues. Further, the 
 process that now takes place is mainly phagocytosis, whereas the 
 earlier defensive process was bacteriolysis. This sequence of 
 events is probably to be interpreted as follows : As soon as infec- 
 tion takes place the small amount of immune body naturally 
 present in the blood combines with the bacilli, and, since some 
 complement is present, bacteriolysis occurs. In this way all the 
 immune body is removed in combination with the bacilli, and all 
 the complement is also absorbed, since the conditions for the 
 Bordet-Gengou phenomenon are present, and complement not 
 actually required for solution of bacilli will be taken up, as well 
 as that which is actually used in the process. Hence all the 
 defensive substances are removed, and the bacilli can flourish 
 unchecked for a time. Soon, however, more complement is 
 produced. This has little or no power of producing bacteriolysis in 
 the absence of amboceptor, but it can, and does, act as opsonin, 
 and abundant phagocytosis occurs. In most diseases this stage 
 would be marked by leucocytosis, but in typhoid fever this does 
 not occur. But we shall shortly see that a polynuclear reaction 
 does take place in the peritoneum, if not in the other regions. 
 
 In the series of researches we are discussing the development 
 of the last line of research the true antibodies was not studied. 
 
 23-2 
 
35^ INFECTIVE PROCESSES IN THE PERITONEUM 
 
 These only come into action later. In the case of a mild infection 
 the whole process of cure may occur without their appearance, and 
 they are only of importance in so far as they are the cause of the 
 subsequent immunity. 
 
 Let us now turn to the sequence of events in the peritoneum in 
 these animals in which explosive bacteriolysis does not occur, or 
 only to a small extent, in that region. Here some phagocytosis is 
 found to take place in the cells lying free in the peritoneal fluid, 
 (the changes in which have been noted previously), but the greater 
 part of the process and this is significant in view of the theories 
 which we have adopted with regard to the liberation of comple- 
 ment-opsonin during the process of coagulation takes place in 
 the masses of fibrin found on the omentum. At first the bacilli lie 
 free in this deposit, often lying more or less parallel ; but in a 
 short time vast numbers are taken up by the macrophages, and 
 very few free organisms may be seen in an hour or two after the 
 injection. Then one of two series of phenomena may occur. The 
 animal may recover ; and in this case the bacilli which are con- 
 tained in the macrophages become pale and granular, and soon 
 disappear altogether, and the fibrin becomes infiltrated and eroded 
 by polynuclear leucocytes. Or the animal may die ; and in this 
 case the bacilli, both those which have been ingested and those 
 which have not, commence to grow rapidly, and the organisms, 
 after diminishing in numbers for a few hours, proliferate so quickly 
 that the animal dies in some twenty- four hours. In these cases 
 the bacilli which have been ingested are not destroyed, and retain 
 their normal appearance and staining reactions throughout. 
 Here it is obvious that the first line of defence, the rapid initial 
 phagocytosis, has been unsuccessful, and that the secondary 
 reserve forces have not had time to come into action. It is 
 specially pointed out by Buxton and Torrey that in these cases the 
 invasion of the fibrin by the polynuclear leucocytes does not occur, 
 a fact strongly confirmatory of the view that it is these cells that 
 give rise to the opsonin or complement, the deficiency in which 
 brings about the lethal issue. 
 
 Here we must conclude a short and altogether inadequate con- 
 sideration of a subject of the highest importance. Much more 
 might be written on the subject, but the very fact that the 
 phenomena do not lend themselves to a brief discussion only 
 shows how very imperfect is our present knowledge of the events 
 which actually take place in the juices and tissues during the 
 
ON IMMUNITY TO BACTERIA 357 
 
 process of recovery from an infective process. The mechanisms 
 themselves are now fairly well known, the nature of the substances 
 concerned therein thoroughly investigated, and their sources ascer- 
 tained with some degree of probability; but when we come to 
 apply our knowledge to the actual events of the living body, our 
 difficulties begin in earnest. It is in this field of research that 
 future advances are most to be expected. 
 
 In conclusion, let us emphasize the enormous importance which 
 we have been led to attach to the leucocytes in the struggle against 
 the infective diseases. In all probability the substance we call 
 alexin, thermolabile opsonin, or complement, and which, in one 
 or other of its actions, constitutes the first line of defence, is 
 formed by the polynuclear leucocytes. In itself its action is but 
 slight, and it requires to be supplemented either by the leucocyte 
 itself, in case the reaction is mainly phagocytic, or by the action 
 of amboceptor, which we believe to be derived from the masses of 
 leucocytes known as lymphoid tissue. Again, the bacterium may 
 give off toxin, especially if it has escaped being taken up by the 
 leucocyte, and in this case these cells exhibit another phase of 
 their protean activities, for that they can actually absorb toxin in 
 solution appears quite certain. If this action fails the last resource 
 is the neutralization of the toxin by antitoxin ; and although this 
 cannot be regarded as definitely proved, there is at least some 
 reason to believe that this may be formed in the lymphoid tissues. 
 Lastly, certain facts would seem to show that it is quite likely 
 that even the compound of toxin and antitoxin is by no means 
 inert unless it has been taken up by the leucocytes, and that the 
 action of the latter substance is simply to prepare the former so 
 that these cells may attack it. In every phase of the struggle 
 against the bacteria the leucocyte appears, on certain or presump- 
 tive evidence, as the particular cell to which the defence of the 
 body is entrusted. 
 
CHAPTER XIV 
 
 PRACTICAL APPLICATIONS 
 Staphylococcic Infections. 
 
 COMMON as is disease due to the staphylococcus (boils, carbuncles, 
 pustular acne, osteomyelitis, etc.), our knowledge of the inner 
 mechanism of the pathogenicity of this organism is still somewhat 
 scanty. The only toxins definitely known to exist are a staphylo- 
 lysin and a leucocidin. The former is produced in three or four 
 days in faintly acid broth, and reaches its maximum in about 
 fourteen days. It is destroyed by a temperature of 56 C., and in 
 other respects resembles the true toxins. Many animals, and 
 notably the horse, contain a natural antistaphylolysin, which 
 neutralizes the action of this haemolysin, and is apparently a true 
 antibody. It is present in human serum, and may perhaps 
 account for the fact that anaemia is not a striking feature of 
 Staphylococcic diseases. Animals from which it is absent, such 
 as the rabbit or goat, can be made to produce it by immuniza- 
 tion with solutions of the lysin. Leucocidin is produced under 
 the same conditions as the haemolysin, and the two are usually 
 produced side by side ; but they are distinct substances, the former 
 being the more easily destroyed by heat. Its action is not 
 entirely specific : it kills the leucocytes and dissolves them, but 
 has also some action on the ganglionic and other cells. As in the 
 case of the hsemolysin, a natural antibody exists in the serum of 
 man, the horse, etc., and can be prepared from other animals. 
 
 These substances, especially perhaps the latter, may play some 
 role in the production of disease, in that they may inhibit phago- 
 cytosis by a poisonous action on the leucocytes. But there 
 is little doubt that there is some other toxic body, probably an 
 endotoxin, since young cultures killed by heat, as used for vaccines, 
 are decidedly toxic, causing local irritation, and, if used in large 
 doses, a rise of temperature. These vaccines contain no haemo- 
 lysin or leucocidin. 
 
 358 
 
PRACTICAL APPLICATIONS 359 
 
 Immunity. The resistance against staphylococci appears to be 
 almost entirely dependent on phagocytic action. Ordinary 
 methods fail to demonstrate any bactericidal action, either in 
 normal or immune serum. Andrewes and Gordon, however, by 
 the use of special methods, have succeeded in showing that such 
 an action, though very slight, occurs even in the serum of normal 
 animals, and to a greater extent, though still but slight, when the 
 animal has been immunized. Perhaps the antibodies to the soluble 
 toxins mentioned above may have some protective action. 
 
 The staphylococcus is very susceptible to phagocytic action, 
 and there is a general correlation between the opsonic index and 
 the stage of evolution of the lesion, as shown in Fig. 64. The 
 opsonic index is greatly raised in the serum of immunized animals, 
 and it would appear that injections of dead staphylococci have the 
 power of increasing both the thermolabile and thermostable 
 opsonin. The duration of the immunity is not definitely known, 
 but is certainly short. 
 
 Diagnosis. This is made entirely by the demonstration of the 
 infective organism an easy task. In a few cases the opsonic 
 index may afford some help, being usually low during the acute 
 stage. 
 
 Agglutinating sera can be prepared, but their action is not 
 powerful, and this reaction is useless in diagnosis. 
 
 Treatment. Antistaphylococcic sera have been prepared, but 
 the results of its use are not encouraging, and the only valid 
 method is the use of vaccines. These should be prepared either 
 from virulent cultures derived from severe boils or carbuncles 
 or from the lesion it is desired to treat. In practice it is a good 
 plan to commence with a stock vaccine prepared from a virulent 
 culture, and to prepare an autochthonous vaccine for use if the 
 first does not succeed. In general opsonic control is not necessary, 
 and the doses may vary between 100 and 1,000 millions, repeated 
 every ten or fourteen days. 
 
 Streptococcic Infections. 
 
 The toxin of the Streptococcus pyogenes is still not definitely 
 known. We have already referred to the haemolysin which it 
 produces. There is some reason for believing that this substance 
 has some clinical action in acute infections, and that it is produced 
 to a greater extent by virulent than by non-virulent cultures. 
 
360 STREPTOCOCCIC INFECTIONS 
 
 Apart from this, the existence of a soluble toxin is doubtful. 
 Filtered broth cultures of the organism are poisonous, but 
 most observers have been^able to produce immunity therewith, 
 and the toxic substances are probably merely simple metabolic 
 products. Parascandalo, it is true, claimed to have been more 
 successful, but his results have not been corroborated, and there 
 is some reason to believe that the nitrate which he used was not 
 free from bacteria, living or dead, and that his animals were 
 really vaccinated with a vaccine. The toxin is probably an 
 endotoxin, though of this but little is known. The bodies of the 
 bacteria are highly irritating, and very small doses of vaccines 
 have to be given at the commencement of the treatment. 
 
 Diagnosis. This is made by the demonstration of the micro- 
 organism in all cases. Agglutinating sera may be prepared, 
 but the appearance of the property in the serum in human 
 disease is not sufficiently marked or constant to be of value. 
 
 Immunity. Human blood appears to contain an antilysin which 
 neutralizes the action of streptocolysin, and it is quite possible 
 that this substance may play some part in the defence of the body 
 against infections. There is also a little evidence for the forma- 
 tion of a true antitoxin against the actual and efficient toxin of the 
 streptococcus, whatever this may be. This is the fact that in 
 some cases, though unfortunately not in all, the injection of anti- 
 streptococcic serum is followed by a marked immediate benefit in 
 cases of septicaemia, etc. The temperature may fall, the delirium 
 and headache pass off, and the pulse improve in a striking manner 
 within an hour or two of the injection. Now Wright has pointed 
 out that but little is known of the mode of action of the serum, 
 and that it may contain a toxic ingredient and really be a vaccine 
 in disguise. If this is the case we should not expect it to produce 
 good effects so early as actually happens in favourable cases, in 
 which its action resembles that of diphtheria antitoxin, and 
 certainly suggests that some poison is neutralized. Probably, 
 therefore, acquired immunity to streptococci is partly antitoxic or 
 anti-endotoxic. 
 
 Bacterial immunity is probably mixed, partly bactericidal or 
 bacteriolytic and partly phagocytic. The serum of normal 
 animals has usually some bactericidal action, and this can be 
 raised by immunization to a high figure ; further, the serum of a 
 immunized animal is a powerful agent in conferring passive 
 immunity, a property not belonging (apparently) to sera of high 
 
PRACTICAL APPLICATIONS 361 
 
 opsonic value. But probably phagocytosis is of the main value 
 in the struggle against the streptococcus in the body. The 
 organisms are very readily taken up by the leucocytes in presence 
 of serum, and are frequently seen inside the pus cells in suppura- 
 tive diseases- In general the opsonic index may be taken as a 
 fair index of the degree of immunity, since it is usually low during 
 the course of the infection and rises as cure is brought about. 
 This has been already shown in the case of erysipelas : the index 
 returns to normal in one to three days after the rise, indicating 
 that the immunity in this case is but transient (see Fig. 63). This 
 is of interest in view of the frequency with which this disease is 
 recurrent. I have seen a case in which attacks occurred every 
 three weeks for a year. But the solid immunity conferred by 
 vaccination and the use of massive cultures is more lasting, due 
 probably to the formation of immune bodies and thermostable 
 opsonins. 
 
 Local immunity probably also plays a part of great importance 
 in streptococcic infections, as shown by the fact that the ery- 
 sipelatous lesion usually spreads at one margin and heals at 
 another simultaneously. Yet even here the spread may be due to 
 failure of access of the blood to the bacteria. This is suggested 
 by the frequent success of the local use of iodine and other 
 rubefacients in arresting its spread. A similar effect may some- 
 times be seen in chronic cases by the use of citrates or one or 
 other of the means suggested by Wright for lowering the coagu- 
 lability of the blood. A case in which the disease had been 
 present for five weeks recovered in two days when treated in 
 this way. 
 
 Treatment. Protective treatment is not employed. It is, how- 
 ever, interesting to. note a fact pointed out by Sir James Paget 
 many years ago. Pathologists are as a rule more or less immune 
 to septic infections, and these comparatively rarely occur in those 
 who are in the daily habit of performing post-mortem examina- 
 tions. The attack which proved so nearly fatal to him occurred 
 after a long absence from the post-mortem room. Probably most 
 pathologists are vaccinated against the disease. 
 
 The curative treatment (apart, of course, from that of the 
 local lesion, if any, and the use of general toxic and sustaining 
 measures) resolves itself into the question whether a serum or 
 vaccine should be used. This question cannot be definitely 
 settled at the present time, and the recommendations which are 
 
362 STREPTOCOCCIC INFECTIONS 
 
 given here may require great alteration in the future. In several 
 and rapidly progressing septic cases, such as those due to post- 
 mortem wounds, etc., the use of serum is indicated, and offers the 
 best chance of success. In view of the very great benefit which 
 is frequently derived from this measure, it appears highly im- 
 proper to wait until a vaccine is prepared. This should be taken 
 in hand at once, so that a homologous vaccine may be ready if 
 subsequent events suggest its use, but in the meantime serum 
 should be employed. The use of minute doses of a stock vaccine 
 (5 to 10 millions per dose) may be considered, and there 
 appears to be no reason why they should not be given along with 
 the serum. If practicable, opsonic estimations should be taken, 
 but the streptococcus is not as a rule a very satisfactory organism 
 to work with. In the absence of such observations, doses of the 
 size mentioned above may be given every three or four days. 
 
 In chronic septicaemia or ulcerative endocarditis of streptococcic 
 origin the use of vaccines offers more prospect of success, though 
 even here cures have been brought about by means of the serum, 
 which should be used whilst cultures are being taken from the 
 blood and a vaccine prepared. At present it seems desirable to 
 use careful opsonic control in cases of this class. An excellent 
 illustrative case treated by Douglas should be referred to for 
 details. Here the doses were 5 tc 12 millions, and the 
 injections were given each time the index fell. The case was 
 certainly one of septicaemia, but the evidence for the existence of 
 ulcerative endocarditis is not conclusive. 
 
 In chronic local inflammatory disease of streptococcic origin 
 vaccine treatment is probably the best, but here ordinary methods 
 (especially Bier's) may be of more advantage. In the absence of 
 opsonic control, the doses may begin at 10 millions and rise to 
 100 millions, and be given once a week. 
 
 Erysipelas does not usually require specific treatment, and in 
 severe cases serum often answers well. The use of vaccines has 
 not been tried on a sufficiently large scale to allow us to judge of 
 its value. A case under my care of recurrent erysipelas, in which 
 attacks had occurred about every three weeks with some degree 
 of regularity for a year, was apparently cured by a few doses of 
 vaccine, commencing at 25 millions and rising to 50 millions, and 
 administered once every ten days. In some chronic cases of 
 erysipelas it is probable that there is a considerable amount of 
 haemic immunity, but the protective substances are unable to 
 
PRACTICAL APPLICATIONS 363 
 
 reach the bacteria. In these the use of citrates or citric acid (as 
 recommended by Wright) is occasionally of great value. 
 
 The mode of action of antistreptococcic serum is still quite 
 uncertain, and its use is purely empirical. Probably in most 
 cases it acts as a bacteriolytic substance. Wright has suggested 
 that it may contain free toxin, and so act as a vaccine ; but the 
 fact that it will passively immunize animals treated with it is 
 adverse to this supposition. It is prepared by gradually immuniz- 
 ing horses and other animals until they can withstand large doses 
 of the living organisms. The differences between the various 
 brands mainly arise from the nature of the culture used for the 
 purpose. The earlier sera to be prepared were procured by the 
 injection of a single strain of streptococcus isolated from a case 
 of erysipelas, puerperal fever, etc. It was found, however, that 
 this serum was not successful in every case, and attempts were 
 made to improve it. Starting from the supposition that the 
 reason for the non-success was the fact that the streptococcus 
 used was not exactly the same variety as that causing the infec- 
 tion in the patient, polyvalent sera were prepared by treating horses 
 with cultures from many various sources, and it is sera of this 
 nature that are now in general use. Another explanation was 
 that the cultures used were not of sufficient virulence, and as 
 antibodies to an attenuated organism may not act against the 
 same culture when the virulence is exalted, strains of great 
 potency were obtained by means of passage through rabbits. Yet 
 a third method has been adopted, based on the fact that an 
 organism which is highly virulent for one animal may be harmless 
 to another species. Here passage is avoided, and the cultures 
 used in the immunization of the horse are taken direct from 
 human disease, and are used soon after isolation '.., before 
 virulence has been lost by prolonged cultivation in vitro. There 
 is no very clear evidence that any special advantage attaches to 
 any of the sera thus prepared ; in any given case it is a matter of 
 chance which sample will prove successful. The criterion as to 
 the suitability of a given sample of serum is purely an empirical 
 one. The patient's pulse, temperature, nervous condition, etc., 
 should be watched with the closest attention, and any definite 
 improvement occurring within the first twenty-four (and usually 
 within the first four) hours of the first doses should be an indica- 
 tion to continue with the use of the same brand of serum. In 
 some cases each injection causes a slight but definite rise of 
 
364 PNEUMOCOCCIC INFECTIONS 
 
 temperature, and the symptoms undergo a brief exacerbation, 
 followed by an improvement in the general condition. The 
 meaning of this phenomenon is somewhat doubtful, Wright hold- 
 ing that the rise is due to the presence of toxin in the serum. It 
 seems, however, more probable that it is due to a solution of some 
 of the streptococci and consequent liberation of endotoxin. It is 
 not a contra-indication' b to the use of the serum, though it may 
 suggest care in its use. If no obvious result follows the use of 
 the serum, the brand should be changed, another specimen being 
 administered forthwith. 
 
 Antistreptococcic serum is, as a rule, not standardized, and the 
 initial dose should not be less than 10 c.c., whilst 20 to 25 c.c. is 
 not too much. Subsequently 10 c.c. may be given each day as 
 long as it appears to be of benefit. In a generalized infection it 
 seems reasonable to give the injections intravenously, but there is 
 no proof that the method is of more value than the ordinary 
 process of injecting it into the cellular tissue of the flank. 
 
 Good results have been obtained in severe cases of scarlet fever 
 by the use of serum obtained by the use of streptococci obtained 
 from scarlatinal throats (S. conglomerate} . It appears tolerably 
 clear in this disease that a homologous serum is of advantage, the 
 results being better than those got by the use of ordinary anti- 
 streptococcic serum. 
 
 Pneumococcic Infections. 
 
 The marked remote toxic symptoms frequently met with in 
 pneumonia would suggest that the pneumococcus forms an 
 exotoxin, and there is a certain amount of experimental support 
 for this supposition. The Klemperers made use of nitrates in 
 their experiments in the production of an antitoxic serum, and 
 found them fairly toxic, and possessed of undoubted immunizing 
 powers ; and similar results have been obtained by Washbourn, 
 Isaeff, and others. But the potency of these filtrates is slight 
 compared with that of a true exotoxin, and the results obtained 
 are quite consistent with the supposition that they are due to 
 slight autolysis and liberation of an endotoxin. The fact that 
 lesions occur remote from the lung cannot be taken to suggest 
 that an exotoxin is produced, since we know that in pneumonia 
 considerable numbers of cocci make their way into, and are 
 destroyed in, the blood-stream. Vaccines of the dead cocci are 
 but slightly toxic. 
 
PRACTICAL APPLICATIONS 365 
 
 According to Casagrandi, non-pathogenic varieties of the 
 pneumococcus form a haemolysin, whilst virulent ones do not. 
 The same observer states that certain cultures produce a leuco- 
 cidin. 
 
 The nature of the immunity to the pneumococcus, and in 
 especial the causation of the crisis (when present), has been much 
 discussed, and now seems to be fairly well elucidated. It is 
 doubtful whether any. bactericidal substances are formed in man 
 after an attack of pneumonia ; they may be produced in rabbits as 
 a result of artificial immunization, but are not invariably present, 
 and it is useless to attempt to explain the characteristic crisis by 
 their agency. Anti-endotoxin may perhaps be formed artificially, 
 but its presence in human disease is very doubtful. It is, how- 
 ever, now tolerably clear that recovery in pneumococcic infections 
 is brought about mainly by the action of opsonins, and more 
 especially thermolabile opsonins. This was well shown by 
 Mac Donald, whose investigations have been referred to already. 
 The opsonic index is low during the course of the attack, and rises 
 suddenly to normal at or near the crisis. Eyre distinguishes three 
 types of opsonic index in different forms of pneumococcic infec- 
 tions. The first is the pneumonic type, his results precisely 
 corroborating Mac Donald ; the second is the form in which 
 recovery takes place by lysis, in which the rise is gradual ; and 
 the third, in which the disease progresses to a fatal termination, is 
 characterized by a steady fall in the index. Eyre holds that the 
 resistance of the individual may be measured by his opsonic 
 response, as represented by these three curves. 
 
 The opsonin present in natural disease is, as mentioned above, 
 mostly thermootablc ; but in the immunized sera of hypervaccinated 
 animals thermostable opsonin, the bacteriotropin of Neufeld and 
 Rimpau, is present. In all probability it is to these substances 
 that antipneumococcic serum owes its effects. These are not 
 great according to Eyre, i c.c. of the most powerful serum yet 
 obtained will only protect against some 300 lethal doses as we 
 should expect to be the case if the immunity conveyed were due 
 to a rise in the opsonic value of the blood. 
 
 Agglutinins are usually found in cases of pneumonia, but they 
 are not present in large amount (the serum rarely clumps at a 
 greater dilution than i : 60), though a much more powerful action 
 may be obtained by artificial immune serum. The reaction is of 
 little value in diagnosis, since it may be absent even in pneumonia, 
 
366 PNEUMOCOCCIC INFECTIONS 
 
 and is often absent in the more localized infections. The disease 
 is recognized by the demonstration of the diplococcus. 
 
 In acute pneumococcic infections (pneumonia, septicaemia, 
 peritonitis, etc.) attended with severe constitutional disturbance 
 the use of serum may be tried. Several have been prepared, the 
 methods used differing somewhat in the different cases ; but, with 
 the exception of that prepared by the Klemperers, the animals 
 used are immunized by the use of dead, and subsequently of living, 
 cultures. The best known are Pane's and Romer's. The latter is 
 markedly polyvalent, an important point, since various strains of 
 pneumococci probably differ largely inter se. This was very well 
 shown by Washbourn and Eyre, who found Pane's serum 
 protective against four cultures from different sources, but power- 
 less against a fifth. 
 
 The results of the use of antipneumococcic serum have not 
 been such as to lead to its general use, although several observers 
 have reported very good effects. According to Tauber, the tem- 
 perature falls to normal after one to three injections of 20 c.c., 
 and there is marked mental and general improvement. Stress 
 is laid on this by other physicians, and it may perhaps be due to 
 the cutting off of the supply of endotoxins brought about by the 
 ingestion by the leucocytes of the pneumococci after opsonization 
 by the added serum. 
 
 The results are sufficiently encouraging to lead us to hope that 
 the serum may become of more value in the future, possibly as a 
 result of the discovery of a method by which the dosage and 
 spacing of the injections and the time for bleeding may be more 
 scientifically determined. 
 
 In general, the use of serum for localized lesions does not 
 appear to be of much value ; but great benefit has been claimed to 
 follow the use of Romer's serum in ulcus serpens of the cornea, 
 used either alone or, as Axenfeld recommends, in conjunction 
 with a vaccine. The serum is to be dropped into the conjunctiva 
 or injected beneath it. 
 
 Vaccines have also been used in pneumonia and in pneumo- 
 coccal septicaemia with good results. Thus Eyre has reported 
 a case of the latter disease in which there were metastases 
 (purulent) in the subcutaneous tissues, the hip, the gluteal 
 muscle, etc., and which recovered after five injections. In another 
 case Eyre notes, what is of great importance, that no benefit 
 whatever was derived from a stock vaccine, and it was only when 
 
PRACTICAL APPLICATIONS 367 
 
 the patient's own culture was employed that improvement began. 
 It is in general advisable to treat the case with a stock vaccine, 
 and to commence to prepare a special one if this should fail. In 
 prolonged cases it is sometimes of benefit to prepare a fresh 
 vaccine after a time, in order to counteract any possible change of 
 type of the organism in the body. 
 
 As a rule, the first dose should not exceed 50 millions for an 
 adult and 10 millions for an infant, and, if the disease is febrile 
 and the symptoms acute, may be decidedly less. Subsequent 
 doses may be larger, but as a rule it is not necessary to exceed 
 200 or 250 millions. Probably the opsonic control is of more 
 value here than in most diseases, but in the absence of this doses 
 may be given every week or ten days. The opsonic control, it 
 may be noted, is of more value in acute cases than in chronic 
 ones, since in the latter the index may be persistently normal or 
 high, and yet the disease yields readily to treatment. Thus a case 
 of pneumococcic empyema of the frontal sinus of four years' 
 duration, which had been twice submitted to operation, had an 
 index well above normal, and was completely and permanently 
 cured by four injections of a homologous vaccine at intervals of a 
 fortnight. As a general rule, for the treatment of a small lesion 
 in an adult, such as an unhealed sinus from an empyema, the 
 injections may be 25, 50, 100, and 200 millions, with an interval 
 of a week between the first two and ten days between the 
 remainder, of course subject to any indications which may be 
 derived from clinical observation. It need scarcely be pointed 
 out that but little benefit can be expected in cases in which there 
 is a mechanical obstacle to the escape of pus or the closure of an 
 abscess. A case of very chronic pulmonary abscess, due to 
 pneumococci, in which the X rays showed a considerable amount 
 of thickening, derived no benefit from a long and careful course 
 of vaccine treatment, with and without opsonic control. 
 
 Vaccine treatment has been used in acute pneumonia (according 
 to Allen, routine injections of 25 millions may be given), and is of 
 especial value in unresolved consolidation, which often clears up 
 after its use in a most satisfactory manner, the moist sounds 
 clearing up in a very short time, and the general condition 
 improving rapidly. 
 
 Space forbids mention of all the conditions in which the 
 pneumococcus, perhaps the most protean of all bacteria in its 
 pathogenic effects, has been combated by vaccine - therapy. 
 
368 GONOCOCCIC INFECTIONS 
 
 Special mention should, however, be made of such serious con- 
 ditions as ulcus serpens and other diseases of the eye, in which 
 good results have -been obtained by Allen and others. In general, 
 all localized pneumococcic diseases should be subjected to vaccine 
 treatment, if not easily amenable to surgical operation. 
 
 Gonococcic Infections. 
 
 Here again we have to deal with an organism which exerts its 
 pathogenic effects mainly or entirely by means of an endotoxin. 
 The lower animals are entirely refractory to infection with living 
 cultures, whereas the dead bodies of the cocci produce local 
 inflammatory changes (peritonitis, etc., according to the region 
 into which the inoculation is made), or death if the dose is suffi- 
 ciently large. The toxicity, however, is but slight, and that of 
 the filtrate from the cultures is extremely small. Several 
 observers claim to have produced a soluble exotoxin, but there is 
 considerable doubt as to the interpretation of their results, and in 
 all probability the substances present in these fluids are simply 
 traces of endotoxin, or free receptors let loose by the autolysis of 
 a few of the cocci. Analogy with other diseases would rather 
 suggest the absence of a powerful exotoxin. In the great majority 
 of cases the disease is a purely local one, and the symptoms of a 
 general intoxication very slight. The endotoxin is said to be very 
 stable, resisting the temperature of boiling water for some hours. 
 
 The hgemic indications of immunity are but slight. Bacterio- 
 lysis has not been demonstrated, 1 but there is some evidence to 
 think that immunization may be due at least, in some cases to a 
 rise in the opsonic index, and since some part of the newly-formed 
 opsonin is, or may be, thermostable, it is possible that some small 
 amount of immune body may be produced. According to Allen, 
 the opsonic index in acute gonorrhceal infections is somewhat 
 below normal 0-6 or 07; and when spontaneous cure takes place 
 it rises gradually, going as high as 1-6. If, however, it remains 
 subnormal, the case passes on into one of intractable gleet. But 
 in acute gonorrhceal conjunctivitis in adults the index may be as 
 high as 2-5, showing, as has been already seen in other diseases, 
 that a high index is no proof of immunity. Agglutinins also occur 
 in the serum of immunized animals (Torrey treated a rabbit the 
 
 1 Torrey has recently shown that an antigonococcic serum which he has 
 prepared possesses powerful bactericidal properties. 
 
PRACTICAL APPLICATIONS 369 
 
 serum of which clumped at a dilution of i : 700,000), but this does 
 not occur in man, and the reaction is useless in diagnosis. An 
 interesting point brought out by Torrey's studies is that gonococcic 
 cultures exhibit well-marked differences inter se. The serum of the 
 rabbit alluded to above, which clumped its homologous culture at 
 i : 700,000, clumped another culture only when diluted fifty times 
 or less. This is of importance in connection with the prepara- 
 tions of vaccines and sera. Torrey also showed that antigono- 
 coccic serum contained a bacterio-precipitin. 
 
 Clinical facts would suggest that the immunity to the gonococcus 
 is of very peculiar nature. Thus, as Ricketts well points out, 
 recovery is not due to a loss of virulence of the cocci, for they 
 remain potent to produce infection during all stages of the disease. 
 Nor is it due to the production of acquired local immunity, unless, 
 indeed, this is of such a nature that it can be very easily broken 
 down ; for the patient can be reinfected immediately after an 
 attack, or whilst the disease is in a chronic stage in some part of 
 the urethra or its diverticula. It is conceivable that the gono- 
 coccus is very easily modified by passage through other human 
 beings, and so altered that it is able to reinfect a person who has 
 just recovered from an attack caused by it in its unaltered form ; 
 but this would hardly explain the recrudescence of an apparently 
 cured discharge after excessive indulgence in alcohol. Even the 
 relationship between phagocytosis and recovery is not easy to 
 make out. Unlike other organisms, the gonococci do not appear 
 to undergo the usual morphological changes indicative of intra- 
 cellular digestion (loss of staining power and of sharp outline) 
 usually seen in bacteria after phagocytosis ; nor do the leucocytes 
 which have taken them up show degenerative changes (Ricketts). 
 And the fact that the gonococci are practically all intracellular 
 from a very early period in the disease indeed, whilst it is in 
 active progress would seem to indicate that it is a most inefficient 
 protective mechanism. Further, there is no obvious difference 
 in the phagocytosis according to the height of the opsonic 
 index, which would lead us to believe that, even when the index 
 is low, there is sufficient opsonin to enable all the available cocci 
 to be taken up. As far as it goes, the evidence leads us .to think 
 that the process of cure is due to some local change possibly to 
 some exhaustion of a necessary nutrient material and not to a 
 general haemic reaction. 
 
 Natural local immunity is very marked in the case of this 
 
 24 
 
370 GONOCOCCIC INFECTIONS 
 
 organism. A spread of the disease beyond the mucous membrane 
 of the urethra is extremely rare. Less rare, though still un- 
 common, is extension to the bladder. Haemic infections are 
 also rare, but do occur ; in them, as in other general infections, it 
 is difficult to see how the cocci can run the gauntlet of the leuco- 
 cytes. 
 
 Diagnosis. In most cases, of course, this is made by the 
 demonstration of the specific coccus usually an easy task. If no 
 material is forthcoming (as in gonorrhceal arthritis, etc.), two 
 methods are available the opsonic index and the absorption of 
 complement. According to Allen, the normal index ranges 
 between 0-8 and 1-2, and in chronic infections it may be low 
 (down to o ! 2) or high (up to 2 or more). Further, a dose of 
 75 millions of dead cocci causes but little disturbance in a non- 
 gonorrhoeal patient, but a marked rise if the patient is infected. 
 The method of absorption of complement has been employed 
 with marked success by Meakins, and appears to be of great 
 value. The difficulty of the technique, however, renders it much 
 less easily available than the opsonic method. 
 
 Treatment. The use of serum need hardly be discussed. Some 
 cases, it is true, have apparently been benefited, but it is always 
 possible that the serum may have contained specific toxic bodies 
 which acted as a vaccine. 
 
 The vaccine treatment, on the other hand, is of the greatest 
 possible value. The dose varies between 5 and 500 millions. 
 In general, large amounts are not well tolerated, especially early 
 in the treatment. The doses may, of course, be regulated by the 
 opsonic index, but this is probably not necessary. In acute cases 
 two or three doses of 50 millions may be given ; but it is in the 
 chronic infections, especially, perhaps, in gonorrhceal arthritis, 
 that the method is of especial value. Here the dose may begin 
 with 50 millions, rising to 500 millions, or even twice this amount, 
 and the intervals may be seven to ten days. If no benefit is 
 obtained a further course of treatment under opsonic control may 
 be administered. The results are usually beneficial in the 
 extreme. 
 
 Gonorrhceal conjunctivitis is to be treated on the same lines as 
 acute arthritis, but here, of course, ordinary local antiseptic 
 treatment is all-important. Gonorrhceal iritis is treated like 
 gonorrhceal arthritis, and the results are usually excellent. 
 
PRACTICAL APPLICATIONS 371 
 
 Meningococcic Infections. 
 
 Very little is known concerning the toxin of the meningococcus. 
 Cultures are of very feeble toxicity for animals, and large doses 
 of the vaccine are usually (though not invariably) well tolerated 
 by human patients. It is probably an endotoxin which is only 
 produced under certain conditions. Its effect in the human 
 subject is mainly a local one, manifested chiefly on the tissues in 
 or near which the cocci are localized. The disease is in most 
 cases a local one, the organism being rarely found in the blood 
 or organs other than the brain and cord. 
 
 The frequency with which the organism is found within the 
 polynuclear leucocytes would lead us to believe that the organism 
 is combated in the main by phagocytosis ; and this is confirmed 
 on the whole by the results obtained by a study of the disease by 
 Wright's method. When opsonic determinations are made using 
 a virulent culture, such as one derived from the patient himself, 
 it is found as a rule to be taken up very badly in preparations 
 made with normal serum and leucocytes, and very well when 
 some of the patient's serum is present, so that very high indices 
 are obtained. This is obviously because the cocci have become 
 animalized, like the virulent pneumococci studied by Rosenow 
 and others. They require a large dose of opsonin, such as is 
 present in serum from a patient who is attempting to combat the 
 disease, and not in that from a normal person. The result is 
 that the opsonic index of these patients is often extremely high, 
 figures of 10 or more being common, and 40 has been recorded. 
 Houston has pointed out that old laboratory cultures which have 
 lost their virulence are phagocyted more easily in presence of 
 normal serum, so that the opsonic index of meningitis patients, 
 as determined by the use of these non-virulent cultures, is com- 
 paratively low. Houston has proposed this test for distinguishing 
 between the " true " meningococcus and allied cocci of similar 
 morphological characters. He determines the opsonic index of a 
 patient suffering from true cerebro-spinal meningitis against a 
 normal control, using both a known meningococcus culture and 
 that under consideration. In what he terms the positive reaction 
 there is, in the case of the normal blood, very little phagocytosis, 
 and no agglutination of the cocci which are not ingested, whilst 
 with the blood from the cerebro-spinal case there is much phago- 
 cytosis and marked clumping of the free cocci. It would seem, 
 
 242 
 
37 2 MENINGOCOCCIC INFECTIONS 
 
 however, that the test is simply one of the virulence of the 
 culture, rather than that of its specific nature. As regards 
 agglutination, Houston and Rankin point out that this property 
 also is lost on prolonged cultivation on artificial media ; and as 
 regards the behaviour of the coccus in opsonic estimations, exactly 
 the same phenomena are seen when cultures isolated from what 
 are apparently ordinary cases of basic meningitis are tested against 
 the blood of the patient and a normal control. It seems more 
 reasonable to suppose that the coccus from the basic meningitis 
 cases are either of lower virulence, or that their virulence has 
 developed along different lines from a common non-virulent 
 stock, rather than a different species. 
 
 The opsonin present in the serum in meningitis is to a large 
 extent thermolabile. Some thermostable opsonin is present, the 
 amount being roughly proportionate to the height of the index. 
 
 Agglutination is somewhat difficult of study in the case of this 
 organism, owing to the variations presented by different cultures 
 in this respect and the difficulty in procuring a homogeneous 
 emulsion. Tested by ordinary methods, the blood of meningitis 
 cases does not clump in high dilutions : according to Davis, i : 50 
 is about the average. Kutscher states that the phenomenon is 
 much more marked at 55 C., and that a culture which was not 
 agglutinated at all by a specific serum at 37 C. was clumped in 
 twenty-four hours at 55 C. in dilutions of i : 500, or even i : 1,000. 
 If this is correct it may prove important in the clinical diagnosis 
 of the disease, which at present is based mainly on the characters 
 of the cerebro-spinal fluid, and especially on the presence of the 
 coccus. The cytological and chemical examination of the fluid 
 affords definite evidence of the presence of a meningitis, but the 
 cocci are not always discoverable, especially in cases of internal 
 meningitis, or those in which the foramina at the base of the brain 
 are closed. The opsonic index may be of great value, especially 
 if a virulent culture is at hand. According to Houston and 
 Rankin, the positive " reaction " described above is not usually 
 present before the fifth or sixth day in the epidemic form. 
 
 The serum of immunized animals contains a substance which 
 gives the phenomenon of fixation of complement when com- 
 bined with meningococci, and this fact is used by Kolle in the 
 standardization of his therapeutic serum ; but whether this is a 
 bacteriolysin or bactericidal substance is not definitely known. 
 A cording to Davis, normal blood-serum is bactericidal to menin- 
 
PRACTICAL APPLICATIONS 373 
 
 gococci, and this power is increased in meningitis. He states, 
 however, that the opsonic power of the blood was not altered 
 during the disease. 
 
 The treatment differs in the acute and chronic stages. In the 
 very acute cases the only hope appears to be in the use of 
 a serum, coupled of course with the more ordinary remedial 
 measures, such as repeated lumbar puncture. This probably acts by 
 removing the inert cerebro-spinal fluid (which is almost deficient 
 both in opsonin and in complement), and causing the exudation 
 of fluid containing a larger amount of protective substances. In 
 more chronic cases, and especially in posterior basic meningitis, 
 the use of vaccines, either alone or in conjunction with serum, is 
 more promising. 
 
 Several sera have been prepared Kolle and Wassermann's, 
 Jochmann's, Ruppel's, Flexner's,and Burroughs and Wellcome's 
 and very diverse reports have been published concerning their 
 value. Small differences exist in the methods of preparation, 
 but in all cases large doses of organisms, dead or living, are 
 injected, either subcutaneously or into the veins. The potency of 
 the serum is- tested either by agglutination or by the absorption of 
 complement. According to Houston and Rankin, some of the 
 therapeutic sera in common use have very slight opsonic 
 power and are devoid of agglutinating properties. It seems quite 
 clear that the serum is useless if given hypodermically, and the 
 observers who have obtained beneficial results (Flexner, Levy, 
 and others) have injected the serum into the spinal canal after 
 removing an equal amount of cerebro-spinal fluid by lumbar 
 puncture. The dose is 20 to 30 c.c., repeated several times at 
 intervals of twenty-four hours. Some patients (adults) have 
 received as much as 340 c.c. The injections may cause vomiting 
 and unconsciousness, but no permanent inconvenience ; they are 
 painful, and this is attributed to the use of carbolic acid as a 
 preservative agent. The treatment must be commenced early, 
 and in view of the results of Levy, in whose practice the deaths fell 
 from 78-57 per cent, to 6-25 per cent., and Flexner, who reduced 
 the mortality to 20 per cent., seems to be of decided value. Other 
 observers have, however, been less successful. 
 
 The use of serum from convalescent cases of meningitis (which 
 they find to possess marked bactericidal properties) has been 
 suggested by MacKenzie and Martin. The blood is collected by 
 venesection, whipped, and centrifugalized, and the serum used 
 
374 MALTA FEVER 
 
 for intraspinous injection. In some cases the blood used was 
 taken from the patient himself, and they obtained encouraging 
 results after the use of both methods. The method is perfectly 
 rational, and we might expect on a priori grounds that fresh serum, 
 containing its full amount of complement and thermolabile opsonin, 
 would be of more benefit than stale immune serum. With the 
 idea of causing an increased outflow of preventive substances 
 from the patient's blood by producing a mild aseptic inflammatory 
 process, Briscoe has injected dilute carbolic acid solutions and 
 other fluids into the cerebro-spinal canal, but without success. 
 
 The treatment by vaccines has not attracted much attention, 
 but in the subacute and chronic cases it is well worthy of a trial. 
 I have used it in four cases, with three recoveries and one death. 
 These figures are not sufficiently great to argue about, but what 
 was most obvious was the very decided clinical improvement 
 which followed almost every dose. This occurred too frequently 
 to be mere coincidences, and as these patients were all young 
 children the question of their being due to a mental effect need 
 not be considered. The dose has been 250 millions, increasing to 
 500 millions, and 1,000 millions, and no bad effects have been 
 noticed. They have been usually regulated by opsonic control, 
 and the improvement in the general condition as the index rises 
 appears to me to be more definite in this disease than in most 
 others. It need hardly be pointed out that no form of specific 
 treatment will cure the obliteration of the foramina in the roof of 
 the fourth ventricle and consequent hydrocephalus, which is so 
 frequently present in these chronic cases. 
 
 In some cases the index shows a very marked rise (to 10 or 
 more) as a result of a single injection of a homologous vaccine, 
 whilst in others the reaction is much less, and the level does not 
 rise much above unity. It is probable that these two types of 
 reaction correspond to infections with the cerebro-spinal and basic 
 meningitis types of organism, but the cases show no obvious 
 clinical difference. 
 
 Malta Fever. 
 
 The toxin of Malta fever appears to be an endotoxin. Killed 
 cultures are decidedly toxic for animals, and their prophylactic 
 use in moderate doses has been followed in some cases by the 
 development of chronic aseptic abscesses (Eyre, Bousfield). 
 The type of immunity is not known ; no bactericidal substance is 
 
PRACTICAL APPLICATIONS 375 
 
 known to be developed in the blood, and Eyre's attempts to 
 produce a powerful curative or preventive serum were unsuccessful. 
 There is more evidence pointing to opsonic action in the cure of 
 the disease and the subsequent immunity : the index early in the 
 disease is as a rule low, and it rises during the progress of the 
 case. In some points the immunity reactions of the disease 
 resemble tubercle (slow development of the immunity, frequency 
 of relapses, absence of known bacteriolytic properties in the 
 serum, local toxicity of the cultures), but there is this marked 
 difference, that Malta fever is essentially a septicaemia, the cocci 
 being present in the blood in small or large numbers in practically 
 all cases. 
 
 The agglutinative reaction, first studied by Wright and Smith 
 is of enormous importance in the diagnosis of the disease, and 
 it was Zammit's discovery of clumping powers in the blood of 
 the goat that led to the discovery of the fact that the milk of 
 these animals was frequently infective even when the animal had 
 no signs of disease. Remedial measures based on this phenomenon 
 have been of enormous advantage in checking the disease. 
 
 Various criteria are adopted in the application of the reaction 
 to diagnosis of disease in man. Critien uses a dilution of i : 10, 
 and a time-limit of half an hour, and finds results thus obtained 
 as conclusive as if higher dilutions were used. Bassett-Smith 
 recommends i : 30, with a time-limit of four hours, or twenty- 
 four if the macroscopic method be used. Eyre, however, points 
 out that the blood not infrequently clumps in a high dilution, but 
 not when more concentrated e.g., giving no reaction between 
 i : 10 and i : 100, but clumping strongly at i : 200. It 
 appears, therefore, that a series of dilutions should be made if 
 negative results are obtained with the test adopted as a standard. 
 Agglutination may be manifested in very high dilutions, even as 
 high as i : 500,000. It usually appears about the fifth day. 
 The only other certain method of diagnosis is the isolation of the 
 organism from the blood, usually a fairly easy matter. 
 
 The treatment of the disease by means of vaccines has been 
 studied by Reid, and more recently by Bassett-Smith. The 
 latter recommends a ten-day-old agar culture, emulsified in dis- 
 tilled water, sterilized at 60 C. for an hour, and sufficient carbolic 
 acid added to bring the strength to 0-5 per cent. It is standardized 
 by drying 20 c.c. (of course, before the addition of the carbolic 
 acid), and weighing the solid residue. The results thus obtained 
 
376 TUBERCULOSIS 
 
 are used to determine the degree of dilution necessary, so that 
 20 c.c. of the vaccine should contain 4 milligrammes of solid 
 substance. The doses given were between 0*25 and 0*5 c.c. They 
 were checked by opsonic control twice weekly, and were in 
 general given once a week or fortnight. Bassett-Smith does not 
 approve of the method in acute cases, but his chronic ones 
 seemed decidedly benefited. 
 
 The prophylactic use of the vaccine has not been tried on a 
 scale sufficiently large to enable any very definite inferences to 
 be drawn therefrom. The doses may be 200 to 400 million 
 cocci, and in general two are given, the results being judged by 
 their effect on the agglutination reaction. As noted above, some 
 bad local results have been noticed, and there is in all cases a 
 good deal of fever, malaise, and inflammation at the site of injection 
 and corresponding lymph glands. 
 
 Tubercle. 
 
 The actual toxin and mode of action of the tubercle bacillus 
 remain unknown. The most interesting substance which has 
 been derived from cultures of the organism is tuberculin, which 
 has been discussed previously. That it is not the true toxin 
 appears from the fact that animals immunized to it are still 
 susceptible to the tubercle bacillus, and a serum can be prepared 
 which appears to be an antitoxin for tuberculin, but it has no 
 curative or preventive effects in tubercle. The fatty substances 
 which confer on the bacillus its peculiar staining properties are 
 not devoid of toxicity. They cause chronic inflammatory and 
 caseous changes in the tissues, and may perhaps play a larger 
 role in the evolution of the lesion than is generally thought. And 
 it must be pointed out that tuberculosis may be an afebrile 
 disease throughout. The temperature of phthisis is mainly due to 
 other organisms, notably to the pyogenic organisms which form 
 such frequent secondary contaminations of the vomicae. The 
 chief pure tuberculous affection in which fever is a marked and 
 constant symptom is tubercle of the meninges, and here the local 
 processes are in close proximity to the cerebral cortex. In general 
 tuberculosis there is usually fever, but there is also usually a 
 focus exposed to secondary infections, or a broncho-pneumonia in 
 which other organisms play a part. The main toxic effect which 
 we recognize as being due to the tubercle bacillus is exerted on 
 the surrounding tissues, and there is no disease which is more 
 
PRACTICAL APPLICATIONS 377 
 
 truly a local one ; meaning thereby that in pure tuberculosis the 
 general symptoms indicative of a spread of the toxins into the 
 general circulation are but slight, and the local lesion constitutes 
 almost the whole of the disease. Note, for instance, the good 
 general health which often occurs in patients with tuberculous 
 glands in the neck, even if of some size. And where the general 
 health is impaired, it will often be found to be a predisposing 
 cause rather than a result of the tuberculous process. 
 
 Immunity. These facts would tend to show, what I believe to 
 be the case, that the immunity to tubercle is local rather than 
 general. That general immunity exists there can be no doubt, 
 but we must distinguish between general immunity due to a 
 condition of the blood and that due to a resisting power inherent 
 in all the tissues of the body. Thus to take two patients, one 
 strong and robust and the other of enfeebled vitality : the latter 
 may fall a ready victim to tubercle, whilst the former resists 
 exposure to the most virulent infection : yet there may be no 
 demonstrable difference in the serum of the two persons. There 
 may, it is true, be a slight difference in the opsonic index, but this 
 is not invariably the case. 
 
 The importance of local immunity in tubercle appears clearly 
 from the fact that areas of advance and of cure may occur in the 
 same patient, or even in different parts of the same lesion. This 
 is often very well seen in cases of lupus, but is even more 
 marked in sections from chronic tuberculosis e.g., of a synovial 
 membrane, in which areas of cure (as indicated by fibrosis and 
 organization of the giant-cell systems) and of extension (as in- 
 dicated by caseation and the formation of new tubercles) may 
 almost always be found in the same lesion. In general, tubercle 
 tends to spontaneous cure, and when a lesion continues to spread 
 it will often be found that some second influence (such as a 
 secondary infection) is at work ; even in debilitated subjects there 
 is usually an effort at spontaneous cure in some parts of the 
 lesion. 
 
 As regards the nature of this process of cure, we can say but 
 little. There can be but little doubt that the main curative 
 agency is phagocytosis, but there is no reason to think that this 
 is dependent on the same mechanism of opsonic action that we 
 reproduce so readily in vitro. Polynuclear leucocytes are con- 
 spicuous by their absence from tubercles, and the only cells in 
 which tubercle bacilli have actually been demonstrated in the 
 
37^ TUBERCULOSIS 
 
 living body are the giant and endothelial cells. In these, as 
 Metchnikoff pointed out many years ago, tubercle bacilli can be 
 seen in all stages, of degeneration, from bacilli possessed of normal 
 staining properties to mere " ghosts." Either the endothelial 
 and giant cells actually engulf the bacilli, or, what is perhaps more 
 likely, they grow round them, being stimulated to growth and 
 proliferation by the action of the same toxin which, when more 
 concentrated, leads to caseation and death. Whether any 
 opsonin is necessary for this process to occur, or whether this 
 opsonin is produced locally or makes its way from the blood, we 
 do not know. What we do know, however, is that a high opsonic 
 index does not necessarily imply that the local lesions are under- 
 going cure ; chronic tubercle, and especially lupus of some stand- 
 ing, is often associated with a very high index. But Bulloch 
 found that the cases of lupus which did well on X-ray treatment 
 (which, amongst other effects, causes a permeation of the lesions 
 with plasma) were those that had a high index, and that in the 
 cases in which it was low originally better results could be 
 obtained if it were raised by means of tuberculin injections. It 
 is, therefore, not improbable that opsonin may actually soak 
 through the lymphoid zone and sensitize the bacilli, thus aiding 
 phagocytosis by the giant cells. But this is by no means certain. 
 The results of an injection of tuberculin are probably very 
 complex, and it is at least possible that the curative effect is 
 mainly or entirely due to the production of a local reaction in the 
 neighbourhood of the lesion, as a result of which it is flushed 
 with blood, and perhaps simply increased as regards nutrition. 
 Very small doses of tuberculin are sufficient to cause a slight but 
 definite reaction. This may be quite inappreciable in most 
 regions, but perfectly obvious in the case of tubercles of the iris, 
 which may be seen to become surrounded by a hyperaemic zone 
 after injections of T ^-^ milligramme of new .tuberculin, or even 
 less. Reactions of this nature are, of course, entirely harmless, 
 and are unaccompanied by u !he slightest rise of temperature, if any. 
 The only role which we can assign with any degree of probability 
 to the polynuclear leucocyte in the struggle against tubercle has 
 regard to its action on bacilli which have made their way into the 
 blood. Here the conditions are much more like those which 
 occur in our opsonin experiments, and it is quite likely that 
 opsonization and phagocytosis occur. Even this is not certain, 
 however, for we do not yet know definitely whether opsonin exists 
 
PRACTICAL APPLICATIONS 379 
 
 in the circulating blood-plasma, and that the mechanism is of 
 comparatively little value appears from the fact that rupture of a 
 caseous gland into a vein is so often (as far as we know, always) 
 followed by general tuberculosis. It is possible that living and 
 virulent bacilli may be taken up by the polynuclear leucocytes, 
 carried to distant organs, such as the lymph glands or spleen, and 
 there continue to grow, producing anatomical tubercles. 
 
 As regards the nature of tuberculo-opsonin, the normal opsonin 
 is completely thermolabile, and the rise in the index due to 
 injections or the natural disease is mainly of that nature also. 
 Occasionally the presence of thermostable opsonin is demonstrable, 
 but it never becomes abundant. 
 
 We know but little concerning any other antibody or defensive 
 mechanism. No bacteriolytic or bactericidal action is demon- 
 strable. This may possibly be due to the technical difficulties 
 incidental to investigations of this nature on a bacillus which 
 owes its staining properties to the presence of fats (which we 
 cannot expect a serum to dissolve), and which grows so slowly. 
 Wassermann and others have shown that antibodies occur in 
 patients immunized with tuberculin, but we do not know their 
 nature. Nothing really definite is known concerning an antitoxin. 
 An agglutinin is often formed, but it may be absent throughout 
 the whole course of the disease, so that we cannot regard it as of 
 importance. 
 
 Diagnosis. Where possible this should be made by the recogni- 
 tion of the bacillus, an achievement which modern methods 
 have made possible in many cases in which it would formerly 
 have been regarded as out of the question. Into this and into 
 questions of cytology, etc., we need not enter. The main methods 
 to be considered are (i) the tuberculin reaction, and (2) the 
 opsonic index. 
 
 i. The Tuberculin Reaction. The old tuberculin is used, and is best 
 bought ready prepared, as issued with the German Government 
 stamp. It may be standardized, but this process is uncertain, 
 animals differing markedly in susceptibility. Several methods 
 have been introduced, but are not in general use. Behring's 
 method is to determine the lethal dose for guinea-pigs on sub- 
 cutaneous injection, whilst Von Lingelsheim injects directly into 
 the brain, the susceptibility of which is much greater. As a rule, 
 a normal guinea-pig will stand a dose of i c.c. with impunity. It 
 should be diluted before use with sterile normal saline solution 
 
380 TUBERCULOSIS 
 
 containing 0-5 per cent, carbolic acid or lysol, and kept in sealed 
 " ampoules," each containing i c.c. A convenient method to 
 adopt is to take 99 c.c. of diluent and add the whole of a i c.c. 
 bottle of tuberculin. Each cubic centimetre of this contains 10 milli- 
 grammes of fluid, or yj^ c.c. ; several ampoules are prepared, and the 
 remainder of the fluid diluted with an equal amount of the diluent. 
 Each cubic centimetre now contains 5 milligrammes, or -^^ c.c., and 
 several ampoules are filled with this dilution. Lastly, what remains, 
 or part of it, is diluted with four times its volume of diluent, so that 
 each cubic centimetre contains i milligramme, or y^^- c.c., of tuber- 
 culin. It will be noted that the fraction of a milligramme refers to the 
 tuberculin as sold, and not to any active constituent it is supposed to 
 contain. It is recommended to err on the side of caution, and to com- 
 mence with y^ 1 ^ c.c., and then to go on to ^J^ and finally y^-; but 
 with proper selection of cases it is probable that the risk with ^ J^ is 
 infinitesimal. The rise in temperature which constitutes a reaction 
 should be at least i F., and is usually more. It usually com- 
 mences in eight to twelve hours, rises for another two hours, 
 remains up for a few hours longer, and then falls rapidly. There 
 is often a considerable amount of general malaise. 
 
 It should be used only in cases in which the diagnosis cannot 
 be made by other methods with ease and certainty, and in which 
 it is of great importance that it should be made definitely and 
 rapidly. Thus, a patient living under favourable conditions who 
 developed ambiguous indications of phthisis might fairly be 
 watched for a time, his weight recorded, an attempt made to 
 obtain sputum, etc. But the same signs occurring in a person 
 who was about to get married, or a medical man thinking of 
 taking a resident appointment in a hospital, would be a strong 
 indication for the diagnostic use of tuberculin. Perhaps its main 
 value is that it enables a negative diagnosis to be made with some 
 degree of confidence, and is the only agent which will do so. No 
 reaction after two injections of yj^ c.c. may be taken as definite 
 proof that the disease is absent. 
 
 It should not be used (i) when the temperature is irregular 
 obviously, since the rise might not be due to the tuberculin and 
 (2) when there are secondary infections. 
 
 Some cases of syphilis, leprosy, and actinomycosis are said to 
 react, but this is unusual, and the concomitant presence of tubercle 
 has not always been ruled out. 
 
 In cattle the doses are larger. The tuberculin is usually diluted 
 
PRACTICAL APPLICATIONS 381 
 
 ten times, and the dose varies from 4 c.c. for a large bull to i c.c. 
 for a calf under one year. The temperature is taken for a day or 
 two before the injection, and at the ninth, twelfth, fifteenth, and 
 eighteenth hours afterwards. The rise is gradual, reaching its 
 maximum in twelve to fifteen hours, and then falling gradually to 
 normal. According to Nocard, anything over 2*5 F. is diagnostic, 
 from i -4 to 2-5 F. suspicious, and under i'4 F. unimportant. The 
 temperature of the animal should not exceed 103 F. when the 
 injection is made. 
 
 Von Pirquefs reaction or the cutireaction : This was introduced 
 as a method of avoiding the dangers supposed to be incidental to 
 the use of tuberculin sub cute. A very gentle scarification of the 
 skin is made, just as for Jennerian vaccination, avoiding drawing 
 blood, if possible, and the abraded surface covered with diluted 
 tuberculin (i part with 3 of normal saline containing 0-25 per cent, 
 carbolic acid). A control scarification is made, preferably on the 
 other arm, and covered with the carbolized normal saline solution. 
 The reaction takes the form of a red papule of varying size, 
 sometimes extending for ^ inch or more in all directions from the 
 site of the original scarification. I have seen the skin so sensitive 
 that a vivid reaction was obtained where the diluted tuberculin 
 had accidentally run down the skin. The redness increases for a 
 day or two, and may remain for four or five days, but in general 
 disappears earlier. It is to be compared with the control side, 
 which has also been treated with dilute carbolic acid, but no 
 tuberculin. Von Pirquet now uses undiluted tuberculin, the 
 control scarification not being treated at all. This is probably 
 the better method, and seems devoid of danger. In Moro's 
 method an ointment containing tuberculin is rubbed into the skin. 
 
 The process is probably quite devoid of danger. Some tuber- 
 culin is certainly absorbed, since I have seen a quite definite local 
 reaction round a tubercle in the iris; the temperature, however, 
 does not usually rise, but may do so ; this is a sign that the 
 scarification has been too deep. No reaction is given in advanced 
 cases of the disease, probably because the resisting powers are 
 too low for the production of the necessary antibodies. The 
 same, it may be noted, is true for the ordinary tuberculin test, 
 if it were ever justifiable to use it in such cases. 
 
 Of the value of the test there is no doubt, and a well-marked 
 reaction is conclusive. A good deal of difficulty arises from 
 slight and doubtful reactions, and it sometimes appears to fail in 
 
382 TUBERCULOSIS 
 
 cases definitely tuberculous. A positive reaction is here of more 
 value than a negative one. 
 
 Calmette's reaction was introduced soon after Von Pirquet's, and 
 consists in a slight conjunctivitis and oedema of the caruncle, with 
 sero-fibrinous discharge, which occurs in tuberculous subjects after 
 the instillation of a drop of well-diluted tuberculin into the eye. 
 Since glycerin has an irritant action, a special preparation devoid 
 of this substance is sold for the test. It is put up in a dry form 
 (being precipitated by alcohol), and when dissolved in water 
 according to the instructions gives a dilution corresponding to 
 i : 100 or i : 200 of the original tuberculin. The strength has 
 been gradually decreased owing to calamitous results having been 
 obtained when the stronger dilutions were used. It should 
 certainly never be used when the eyes are affected, and even 
 when they are healthy I personally do not consider the risk worth 
 running unless the diagnosis is of great importance, and all other 
 methods have been tried and have failed. 
 
 2. The Opsonic Index. Numerous observations by different 
 observers have shown that in health the opsonic index to tubercle 
 lies between 0-8 and 1*2, the exceptions being very rare and due 
 perhaps to slips in the technique. As a matter of fact, these are 
 outside figures, and in the great majority of cases the index will 
 be found to lie between 0*95 and 1*05. If, therefore, the diagnosis 
 lies between health and tubercle, an index below 0-8 or above 1-2 
 is very strong evidence in favour of the latter, becoming more 
 convincing the remoter it is from these figures, either above or 
 below. Indices between 0*8 and 1*2 are not of much value, yet a 
 figure of, say, 0-85 on several occasions is very suggestive. 
 
 There is not sufficient evidence as yet to show how other 
 diseases influence the tuberculo-opsonic index. In the majority 
 of cases the figures lie within the normal limits, but there are 
 occasional exceptions. In general, therefore, the results given 
 above will apply, but the conclusions are less certain. 
 
 A rapid variation of the index, either (a) spontaneous, or 
 apparently so, or (b) due to auto-inoculation from exercise or 
 massage of the lesion, or (c] caused by an injection of a small 
 dose of tuberculin, is much more suggestive indeed, practically 
 diagnostic, supposing no errors are made in the determination. 
 And it must be emphasized that when the opsonic index is 
 required for diagnostic purposes, the most careful technique and 
 attention to every detail is absolutely necessary. 
 
PRACTICAL APPLICATIONS 383 
 
 The agglutination reaction has been recommended as a means of 
 diagnosis, but it is not always present at any stage of the disease, 
 and there are practical difficulties in the way of its determination. 
 It does not appear to have come into general use even in France, 
 where much attention has been paid to it. 
 
 Treatment. The essays at a serum treatment have been many, 
 and no method has attained any appreciable degree of success. 
 Good results have been obtained, it is true, by several, especially 
 in the hands of their inventors, but recent work by Hort and 
 others has shown that normal horse serum has some value as a 
 curative agent. Maragliano's serum appears to be prepared by 
 injecting animals with various extracts of tubercle bacilli, and 
 is supposed to be both bactericidal and antitoxic. It is given 
 either alone or in conjunction with a vaccine. McFarland 
 prepared an antituberculin by immunizing donkeys for long 
 periods with tuberculin, and found that it annulled the effects of 
 tuberculin on tuberculous animals, but had no protective or 
 curative powers as tested on guinea-pigs. Clinical evidence 
 appeared to show that it was of some value. 
 
 Marmorek's toxin is quite different from any of the others, 
 which are various extracts of tubercle bacilli, or of their soluble 
 products. It is prepared by cultivating young bacilli (prepared 
 in such a way that they form homogeneous emulsions) in a 
 medium containing leucotoxic serum, prepared by injecting calves 
 with guinea-pig leucocytes. It is supposed to be the actual 
 toxin which is developed in vivo. This substance (which is of 
 feeble toxicity) is used to immunize horses. Some good results 
 have been obtained, but perhaps we may attribute them to minute 
 amounts of tuberculin which may be formed in the cultures and 
 remain unabsorbed in the blood of the horse when it is bled. The 
 experience of most observers has not been favourable to its use. 
 
 The other sera which have been introduced do not call for 
 further notice. 
 
 The most hopeful method of combating tubercle other than 
 the all-important use of fresh air, good food, and careful regimen 
 consists in the use of a vaccine. There are many to choose from 
 old tuberculin, TR, BE, bovine tuberculin, oxytuberculin, 
 antiphthisin, etc.; but of these, Koch's preparations (old tuber- 
 culin, TR, and BE) are the only ones in general use, and appear 
 at least as good as any. 
 
 TR (tuber culinum residmtm) is prepared by triturating living, dry, 
 
384 TUBERCULOSIS 
 
 virulent tubercle bacilli by a mechanical process in an agate 
 mortar. The powdered bacilli are suspended in distilled water 
 and centrifugalized. The supernatant fluid has the properties of 
 the old tuberculin, and is called TO (O = obey, = upper). The 
 residue is dried, and again powdered, emulsified, and centri- 
 fugalized, and the bacilli are now found to have been reduced to 
 a uniform mass. This constitutes TR, which thus corresponds 
 somewhat to an endotoxin. It is diluted with 20 per cent, 
 glycerin, and when diluted it is advised that no carbolic acid 
 should be added. There has been a good deal of confusion with 
 regard to the dosage. It arose from the fact that i gramme of the 
 dried tubercle bacilli is used in the preparation of 100 c.c. of the 
 remedy, so that each i c.c. of the latter contains the material 
 derived from 10 milligrammes of bacilli, and is supposed to be 
 equal in immunizing power thereto. But the amount of dry 
 residue which it contains is only one-fifth of this amount, the fluid 
 being standardized before issue, so that i c.c. contains 2 milli- 
 grammes of solid substance. The dosage adopted in this book 
 concerns this dry residue only. 
 
 Tuberculins are also prepared from bovine tubercle (perlsucht) 
 bacilli, and its use mixed with the human form (as recommended 
 by Allen) appears rational and quite worthy of a trial. The 
 majority of cases of human tubercle are due to bacilli of the 
 human type, but at the worst the material derived from the bovine 
 bacilli will be inert and do no harm. 
 
 Another substance used as a vaccine is Koch's Bazillen- 
 emulsion, or Neutuberculin, usually known as BE. It consists 
 of a suspension of dried and ground up bacilli in equal parts of 
 glycerin and water. When used as a vaccine it is diluted with 
 normal saline solution and heated to 60 C. to insure sterility. 
 This may also be done with TR. It contains 5 milligrammes 
 solid substance per c.c. 
 
 As regards the use of these substances : We may recognize 
 two, or perhaps three, methods (i) the intensive ; (2) the opsonic, 
 and (3) the uncontrolled use of small doses. 
 
 i. In the intensive method the idea is to bring about as 
 much immunity as possible as quickly as possible, by the use of 
 rapidly increasing doses, taking care always to avoid a reaction. 
 The commencing dose is very small, about T \j- milligramme 
 ( = TTrijoo c - c -) f ld tuberculin, or T -^^ milligramme (of solid 
 substance) of TR. The dose is repeated two or three times a 
 
PRACTICAL APPLICATIONS 385 
 
 week, and is gradually increased until 500 milligrammes ( = J c.c.) 
 of old or 2 milligrammes of TR is given. If a reaction occurs 
 an interval of a week is given, and the treatment recommenced 
 with a smaller dose. The dose of BE may be the same as that 
 of TR. 
 
 There are numerous slight modifications in detail, but the fore- 
 going outline will serve as a general description of the process as 
 it is used in many of the Continental clinics. As a rule no 
 attempt is made to estimate the degree of immunity, but Koch 
 suggests the agglutination reaction for this purpose. It usually 
 rises to i : 100, or thereabouts, and may go much higher. 
 
 As a result of the treatment, the patient is certainly immunized 
 to tuberculin, since he fails to react to fairly large doses. The 
 process is then stopped for a time and recommenced. The 
 beneficial effects as recorded (as I have seen on a small scale) are 
 undoubted, and the risks in careful hands and in properly selected 
 cases appear to be slight. 
 
 2. The opsonic method consists in the use of such doses at such 
 intervals as will cause the maximum increase in the opsonic 
 index. The doses here are very much smaller than in the last 
 method. TR is usually employed, and the amounts range 
 between yo^Vc^ anc ^ IWQV milligramme. The idea, of course, is 
 to avoid the possibility of a summation of negative phases, and 
 to call forth the greatest possible formation of protective sub- 
 stances. It is extremely difficult to carry out, since the index 
 alters so quickly in most cases of progressive tubercle that very 
 frequent determinations of the index are necessary. Perhaps it 
 is the ideal method, and all who undertake tuberculin treatment 
 on a large scale ought to acquire some experience of it, but con- 
 siderations of time will prevent its ever coming into general use 
 as a routine treatment. In practice a sort of modified opsonic 
 method is sometimes used. The first few doses are controlled by 
 the index, and the optimum dose and interval determined. These 
 are then preserved throughout the treatment, with perhaps 
 occasional determinations of the index at times. 
 
 It must be pointed out that the theoretical necessity for the 
 opsonic control depends on the acceptance of the fact that 
 tuberculin exerts its beneficial influence by causing an increase 
 in the amount of opsonins present in the blood. But this is not 
 certain. Admitting that the immunity is due to antibodies, these 
 may be bactericidal substances or anti-endotoxins, and we have 
 
386 TUBERCULOSIS 
 
 no reason whatever for thinking that the opsonic index affords 
 any clue to the amounts of these substances formed. And, of 
 course, as is held by many, if the beneficial effects are due to 
 repeated slight local reactions, acting in a way as yet not under- 
 stood, the opsonic index is of little value. It should not be 
 forgotten that a polynuclear leucocyte will engulf an enormous 
 number of tubercle bacilli in a short time even when the opsonic 
 index is low, and it seems improbable that it ever falls so low 
 (even when the negative phase is most marked) as to prevent 
 these leucocytes from doing their work, presuming the bacilli are 
 accessible. A high opsonic index may have some action in 
 preventing generalization of the bacilli e.g., at a surgical opera- 
 tion but even this is not certain. 
 
 It must be noted that the beneficial effects of vaccine treatment 
 by the opsonic control are not denied, but that the opinion is 
 expressed that the same benefit^ may be obtained by simpler 
 methods. 
 
 Space forbids more than a reference to the use of auto- inocula- 
 tion by graduated exercise, massage, etc. This is supposed to 
 expel some bacilli or products thereof into the lymph spaces, etc., 
 where they act as a vaccine. The benefits of these measures is 
 undeniable, but it may be doubted whether it is due entirely or 
 in part to auto-inoculation, and it would appear preferable to 
 employ vaccines in known doses. 
 
 3. The use of small doses without opsonic or other control. This 
 combines the advantages of practicability and safety, and often 
 gives good results. How they compare with those obtained 
 by the other methods cannot be accurately known. Probably 
 no harm has ever resulted from TT ^Ty milligramme of TR, and if 
 this amount is given once in every ten days, or even once a week, 
 many cases improve in a most striking manner. My experience 
 has been mainly confined to its use in surgical cases, and though 
 I have met with numerous disappointments (which I believe are 
 not unknown with the most strict opsonic control), I have seen 
 others in which rapid cure e.g., of a chronic sinus or tuberculous 
 ulcer has been obtained. I should not recommend it or any 
 other method of vaccine treatment in place of surgical or 
 sanatorium treatment, where applicable ; in conjunction with 
 other methods, it has its place. 
 
 Preventive Treatment. This is at present of but little importance 
 in human pathology. A person who is in danger of becoming 
 
PRACTICAL APPLICATIONS 387 
 
 tuberculous should be removed from the infection and placed 
 under good hygienic conditions. A few injections of TR may be 
 given, however, and analogy with a similar procedure in cattle 
 would lead us to believe that it may be of some value. 
 
 Attempts to immunize lower animals to tubercle have been 
 made from a very early period ; it was, indeed, the discovery of 
 the fact that dead bacilli are absorbed with great difficulty that 
 led Koch to devise his TR. The vaccines that have been employed 
 are numerous cultures of low virulence, dead cultures, avian 
 and reptilian bacilli, etc. but the subject entered oh a new and 
 most important phase in 1901, when von Behring published his 
 method, which is of great theoretical interest; and from which 
 important practical results in the stamping out of bovine tubercle 
 (so great a pest to infants, from the prevalence of bacilli in milk) 
 have been esteemed possible. Here the vaccine consists of living 
 tubercle bacilli of human origin. This is of feeble virulence for 
 cattle. It is prepared by being dried in vacuo, emulsified with 
 glycerin in a mortar, and diluted with normal saline solution 
 containing 0*15 per cent, of sodium carbonate. It is standardized 
 in such a way that i c.c. = 2 milligrammes of dried bacilli. The 
 injections are made intravenously; two are given the first of 
 i c.c., and the second, about twelve weeks later, of five times this 
 amount. 
 
 There is no doubt that this process confers a certain amount 
 of immunity, or rather of increased resistance. The process is 
 harmless to calves, but sometimes kills older animals from 
 pulmonary oedema. The immunity lasts for a year or so. 
 
 The method has been carefully examined at Melun by Rossignol 
 and Vallee, and their results were quite favourable. The 
 vaccinated animals were tested (along with controls) by sub- 
 cutaneous injection of bovine tubercle, by injections of these 
 bacilli into the veins, and by keeping them in a shed with animals 
 known to be tuberculous, and in all cases a greatly increased 
 degree of resistance was found. But Lignieres pointed out a 
 remarkable fact, that animals which have been treated in this 
 way and which do not react to tuberculin, and are apparently 
 normal at the autopsy, nevertheless may contain living tubercle 
 bacilli in their lymphatic glands. This is of extreme interest in 
 connection with the question of the latency of bacilli in the tissues. 
 
 Von Behring's latest vaccine is known as tuberculase, or tulase. 
 It appears to be prepared by the action of chloral on tubercle 
 
 252 
 
388 TYPHOID FEVER 
 
 bacilli, but the exact details are not available, and the preparation 
 is not yet obtainable. It seems not to contain living bacilli. 
 
 Attempts have been made to vaccinate against tubercle (and to 
 treat it in human subjects) by ingestion. Some degree of success 
 appears to have been obtained by feeding calves with living 
 human tubercle bacilli, and some physicians have claimed good 
 results by administering a minute dose of TR by the mouth ; but 
 in what way these methods, so uncertain as to the dose which is 
 actually absorbed, are preferable to the use of the svringe is 
 not very clear. 
 
 This is a very brief epitome of a field of research which would 
 require many volumes for its adequate treatment. I have 
 attempted to give the main essentials only. 
 
 Typhoid Fever. 
 
 The pathogenic action of the typhoid bacillus appears to be due 
 entirely to the action of an endotoxin which is set free when the 
 bacillus undergoes solution in the body. This toxin has a local 
 and a general action. The former is marked in the regions which 
 harbour the bacillus, and in which it undergoes solution, either 
 by autolysis or by the action of bacteriolysis the Peyer's patches, 
 abdominal lymph glands, spleen, etc. In these regions it produces 
 hyperaemia, followed by inflammatory hyperplasia of the lymphoid 
 and endothelial cells, which diminish the blood-supply and may 
 lead to necrobiosis of the inflamed tissues. In the case of the 
 Peyer's patches this commonly occurs, the lymphoid tissue being 
 cast off as a slough, and the wound, becoming infected by intestinal 
 organisms, may extend deep into the bowel wall and cause haemor- 
 rhage or perforation. The process is less likely to occur in the 
 solid organs, which are nourished by blood from all sides and in 
 which secondary infections are less frequent. The general effects 
 of the toxin fever, degenerations of the kidney, liver, etc. are 
 not characteristic. The failure of a leucocytic reaction of the 
 bone-marrow, and consequent leucopaenia, is worthy of notice. 
 
 The fact that typhoid fever is a septicaemia and that the living 
 organisms circulate in the blood must not be forgotten. 
 
 Immunity. A short time ago typhoid fever was regarded as a 
 disease in which the immunity was purely bacteriolytic. During 
 an attack of the disease or the process of artificial immunization the 
 amount of immune body (which occurs in small amounts in 
 health) shows a steady rise, and the blood soon becomes extremely 
 
PRACTICAL APPLICATIONS 389 
 
 potent in this respect. The bacteriolytic power of the blood as 
 tested by Wright's method does not necessarily show a great 
 increase ; this is due probably to the lack of complement, and it is 
 possible that relapses may be due to the same cause, deviation of 
 complement occurring owing to the excess of immune body. 
 
 There is, however, no doubt that phagocytosis plays a part of the 
 utmost importance in the natural cure of the disease, and that the 
 opsonic content of the blood (as tested by one of the dilution 
 methods) undergoes a steady rise during the process of immuniza- 
 tion. We may regard the mechanism by which the infection is 
 combated as a double one, bacteriolysis and phagocytosis being 
 jointly concerned. That the latter is of chief importance appears 
 probable, from the fact that typhoid bacilli set free their endotoxin 
 when dissolved by means of bacteriolytic sera. 
 
 The antitoxin or anti-endotoxin of typhoid bacilli can be readily 
 prepared from animals by a process (somewhat prolonged) of 
 immunization with the endotoxin, prepared either by grinding the 
 bacilli, by dissolving them in bacteriolytic sera, or by allowing 
 them to undergo aseptic autolysis. There is, however, no proof 
 for the belief that this substance is developed during the natural 
 process of cure, or that it is a cause of the subsequent immunity. 
 It is quite as possible that the cessation of the febrile process 
 is due to the destruction of the bacilli, and consequent cessation 
 of the supply of toxin. 
 
 The effect of the true typhoid toxin is seen during the earlier 
 stages of the disease, when the temperature is continuous. The 
 intermittent temperature of the later stages is probably due in 
 part to the absorption of other toxins from the ulcerated surfaces 
 of the Peyer's patches, and in part to the liberation of endotoxins 
 which occurs when bacilli are dissolved before being ingested. 
 
 The duration of the immunity after a natural attack of the 
 disease is not accurately determined, but it is certainly long 
 perhaps several years. That due to preventive inoculation is 
 thought to be six months at least, but here again exact figures 
 cannot be obtained. The duration of the antibodies produced in 
 typhoid fever varies. The agglutinins usually disappear within 
 one or two years, but they may persist for seven or more. The 
 bacteriolysin is thought to go sooner, but the observations on 
 which this idea is based were mostly on the bactericidal powers 
 of the blood, and open to fallacy. 
 
 Diagnosis. Usually the W 7 idal reaction is all that is required. 
 
39 TYPHOID FEVER 
 
 The standard that is adopted varies in different laboratories, but 
 in the great majority of cases it will be found that the serum at 
 the end of the first week will clump in a dilution of i : 30 in one 
 hour at the body temperature, and that this is very seldom the 
 case in health or in other diseases. The amount of agglutinin 
 soon increases, and may reach a degree at which it will agglu- 
 tinate at i : 1,000 or more. The naked-eye reaction as carried out 
 by a modification of Wright's method renders it very easy to 
 perform exact quantitative work, and it is an advantage to do so in 
 all cases in which the agglutination does not reach i : 50 on the 
 first examination, since a rise in the power of the serum is 
 practically diagnostic. My routine method is as follows : A unit 
 mark is made about i inch from the end of a rather wide 
 Wright's pipette, and with this 9, 2, 4 and 9 units of a watery 
 emulsion of a young agar culture of typhoid bacilli are placed in 
 four depressions on a porcelain slab ; dead cultures may also be 
 used in the same way. A unit of serum is now sucked into the 
 pipette and mixed with the first pool of 9 units of emulsion. 
 One unit of this is then mixed with each of the remaining three 
 pools, the process being carried out quickly, so as to avoid the 
 absorption of agglutinin from the powerful serum. This gives a 
 series of i : 10, i : 30, i : 50, and i : 100. Each pool is now sucked 
 into a Wright's pipette sealed at the end, and incubated at 37 C. 
 A control of emulsion without serum is also prepared and in- 
 cubated. The tubes are examined at the end of one hour, and 
 agglutination, if present, is most obvious. 
 
 The diagnosis might also be made from a determination of the 
 opsonic index by the dilution method, or of the amount of immune 
 body, but these processes are much more tedious. 
 
 Where a very early diagnosis is required (i.e., before the 
 appearance of the agglutination reaction) the bacilli may be 
 sought for in the stools or blood. This was formerly difficult, but 
 it has been greatly facilitated by the introduction of new methods 
 involving the use of special culture media, in which the bacilli 
 grow rapidly and form characteristic colonies. These are beyond 
 the scope of this work, and a description of them will be found in 
 Hewlett's " Bacteriology," second edition. 
 
 Treatment. The curative treatment is not satisfactory. The 
 use of a simple bacteriolytic serum is useless or even dangerous, 
 for the reasons given earlier. The best hope for the future is 
 in the use of an anti-endotoxic serum, such as was prepared by 
 
PRACTICAL APPLICATIONS 3QI 
 
 Allan Macfadyen and others, and is now put on the market by 
 Messrs. Burroughs and Wellcome, under the supervision of 
 Professor Hewlett. Some promising results have already been 
 obtained by its use, and it seems to be quite harmless. It seems 
 only to be of advantage when used in the early stages of the 
 disease, as is naturally the case with any serum which acts by 
 neutralizing a soluble toxin. 
 
 Chantemesse has apparently attained a very high degree of 
 success by the use of a serum obtained by injecting horses with a 
 soluble toxin, prepared by cultivating the bacillus in an extract of 
 spleen digested with pepsin and subsequently neutralized. It is 
 very doubtful that this is the genuine toxin, since it is feeble in 
 action and not destroyed at 100 C. The serum thus obtained is 
 used in very small doses, and appears to act (as pointed out by 
 Wright) rather as a vaccine than as a means of conferring passive 
 immunity. The results, however, appear to have been excellent, 
 but the serum is not yet obtainable commercially. 
 
 Typhoid fever has also been treated by the use of small doses 
 of vaccine, and some promising results obtained ; but the method 
 is still on its trial. 
 
 Preventive Treatment. The earliest method was that of Wright, 
 and it has probably not been surpassed. It consists in the use of 
 vaccines of typhoid bacilli cultivated in broth for twenty-four 
 hours, killed at 60 C., counted by admixture with red corpuscles, 
 and preserved by means of lysol or carbolic acid. Two doses are 
 given, the first being 750 to 1,000 millions, the second twice this 
 amount, the injection being usually made deep in the subcutaneous 
 tissue of the flank. The injections are given at intervals of one 
 or two weeks. Local and general symptoms of some severity 
 follow : redness and swelling at the site of inoculation, lymphan- 
 gitis, etc., with fever, headache, and general malaise. These last 
 but a short time, and no evil consequences have been recorded. 
 It is necessary, however, for the patient to be prepared to stay 
 in bed during the day of inoculation and the next day : the 
 unpleasant effects may be reduced to a minimum by the simul- 
 taneous administration of chloride of calcium by the month 
 (15 to 45 grains). 
 
 As a result of these injections, protective substances, and espe- 
 cially agglutinins, make their appearance in the blood, and may 
 persist for several years. These may be taken as affording some 
 test as to the degree of immunity conferred, and as to its duration. 
 
39 2 TYPHOID FEVER 
 
 Space does not permit us to discuss at length the evidence in 
 favour of this method of inoculation. It has been strongly, and 
 perhaps unfairly, opposed in certain quarters, but a careful and 
 unprejudiced study of the statistics of the British Army in South 
 Africa and elsewhere seems to render it quite clear that the process 
 develops a real though not absolute protection against an attack 
 of the disease, and is still more valuable in diminishing the 
 mortality rate amongst those attacked. The analysis of these 
 figures is by no means easy, but certain isolated cases are in 
 themselves very convincing. As an example (one out of many) 
 we may quote the Manchester Regiment, in which, out of 
 200 men inoculated there were 3 cases, without a death, whilst of 
 the 517 uninoculated 23 were attacked and 3 died. In general 
 terms we may say that the mortality from an attack fell from 
 over 12 per cent, in the uninoculated to below 6 per cent, in the 
 inoculated, taking the results from the whole of the Transvaal 
 and Natal. One caution is necessary : the first result of the in- 
 jection is the production of a negative phase okjncreased suscep- 
 tibility, and injections should not be practised, if avoidable, when 
 the patient is already exposed to the infection. 
 
 The vaccine is prepared by the Lister Institute, and can be 
 bought ready for use. 
 
 Numerous modifications of the process have been adopted. 
 Pfeiffer and Kolle prepare their vaccine from agar cultures. 
 Bassenge and Rimpau do the same, but use very small doses 
 TJ to f milligramme. Friedberger and Moreschi give intra- 
 venous injections of infinitesimally small doses of bacilli killed at 
 120 C. Wassermann's vaccine is prepared by killing a culture at 
 60 C., allowing it to undergo autolysis at 37 C. for five days, 
 filtering, and desiccating in vacua at 35 C. It forms a yellowish - 
 white powder, of which the dose is 0*0017 gramme, equal to 
 12 milligrammes of the culture. Numerous other methods might 
 be enumerated, but they have mostly been investigated on a small 
 scale only, and Wright's vaccine is of proved efficacy and easy to 
 prepare. 
 
 Antityphoid serum has often been employed, and in all proba- 
 bility is of value in that it affords a rapid (or practically instan- 
 taneous) means by which immunity can be produced, and eliminates 
 the negative phase altogether. The serum is prepared from the 
 horse, which is injected with gradually increasing doses of dead 
 bacilli, and subsequently of living ones. It can be obtained of 
 
PRACTICAL APPLICATIONS 393 
 
 great potency, as determined by its power of agglutination, which 
 may be manifested when it is diluted a million times. But the 
 immunity it confers (judging from laboratory experiments and 
 analogy with other diseases) is but temporary, and the method 
 can only be of occasional value. 
 
 The plan of injecting the serum and vaccine in combination 
 suggested by Besredka is more promising. It is carried out as 
 follows : A twenty-four-hour agar culture of bacilli is mixed with 
 typhoid serum and incubated at 37 C. for twenty-four hours. 
 The bacilli are, of course, agglutinated, and the mass is washed 
 free from serum by repeated centrifugalizations with sterile 
 normal saline solution. The bacilli are then suspended in this 
 solution and heated to 60 C. for one hour. This vaccine is said 
 to produce very rapid immunity, with practically no negative 
 phase, and the local and general reactions it produces are but 
 slight. It has not yet been tested on a large scale. 
 
 Simultaneous injections of serum and vaccine have also been 
 employed by Calrpette and others. The results do not appear to 
 be as satisfactory as those obtained from the use of the vaccine 
 alone. 
 
 Bacillus Coli. 
 
 The Bacillus coli, in addition to its haemolysin (colilysin) already 
 mentioned, produces an endotoxin, on which its pathogenic action 
 doubtless depends. Its vaccine is moderately toxic, causing 
 severe local irritation and general febrile reaction if the initial 
 dose be a large one. Filtrates from broth cultures are practically 
 devoid of toxicity. 
 
 In regard to its immunity reactions B. coli closely resembles 
 B. typhosus. Bacteriolytic and opsonic substances are developed 
 during the course of the disease, and in both cases the opsonic 
 index must be estimated by the dilution method, Wright's original 
 process giving inaccurate results. There is some difference with 
 regard to the production of the agglutination reaction, which is 
 invariably present in typhoid infections, except in those acute 
 cases of the disease in which the patient dies before it has time to 
 develop. In infections with B. coli this is not the case, and in 
 many of the local infections, such as cystitis, pyelitis, etc,, no 
 appreciable agglutinative properties are developed. When animals 
 are immunized with massive doses extremely powerful clumping 
 sera are obtained, but this is not always observed in the treatment 
 of the human patient with vaccines. 
 
394 BACILLUS COLI 
 
 The main difference, pathologically, between the two organisms 
 is that, whereas the disease due to the typhoid bacillus is usually 
 a septicaemia, localized infections (typhoid osteitis, etc.) only 
 occur as sequelae, the diseases due to B. coli are almost in- 
 variably local inflammations, blood infections, except, perhaps, as 
 mere terminal phenomena, being rare in the extreme. These 
 inflammatory lesions are extremely common and important, and 
 of most diverse nature. The majority are in connection with 
 the urinary (cystitis, pyelitis, etc.) and alimentary systems 
 (cholangitis, cholecystitis, appendicitis, etc.). It also causes 
 acute peritonitis, but usually in association with other bacteria. 
 It may affect almost any part of the body, causing broncho- 
 pneumonia, otitis media, endometritis, metritis, and a host of 
 other diseases. 
 
 The only specific treatment of any avail is the use of a vaccine. 
 A serum has been prepared, but appears to be quite useless. 
 The vaccine should always be prepared, if possible, from the 
 patient's own culture, since various strains classified as B. coli 
 differ slightly the one from the other. In addition to this, there 
 are very marked differences in virulence, cultures isolated from 
 the stools being in general less virulent than those derived from 
 the diseased tissues. In cases in which treatment has to be pro- 
 longed it is sometimes an advantage to prepare fresh vaccines 
 from time to time, since the organism may change its type to 
 accommodate itself to the immune substances produced. 
 
 The initial doses should be small (10 to 40 millions), and it will 
 be rarely found advisable to exceed 250 millions. Too large an 
 initial dose may be badly tolerated, causing rise of temperature 
 and a good deal of local reaction, but no permanent bad results 
 seem to have been observed. On the other hand, it may some- 
 times be noted that in severe febrile cases the vaccine acts more 
 like an antitoxin, causing a sudden drop in the temperature and 
 a rapid amelioration of the symptoms, which may or may not be 
 associated with a great improvement in the local lesion. This is 
 shown in the accompanying chart, from a severe case of cystitis 
 under the care of Mr. Burghard at King's College Hospital. As 
 far as his general condition was concerned, he was practically well 
 within ten days of the commencement of the treatment. The 
 amount of pus in the urine fell to less than an eighth of its 
 original volume, but had not disappeared entirely when he was 
 discharged. This has been my usual experience of cystitis due 
 
PRACTICAL APPLICATIONS 
 
 395 
 
 to B. coli vaccine treatment cures all the symptoms and reduces 
 the pus and bacilli present in the urine to a small fraction of its 
 original amount, but fails to remove them entirely. Other 
 observers have been more fortunate (Wright, Western, Norton, 
 and others), but I believe my experience is the general one, and 
 that complete cures are unusual. There is no doubt, however, 
 that the treatment is the best available, the disease being 
 notoriously resistant to simple methods of treatment. If the 
 injections are made with opsonic control, the dilution method of 
 
 FIG. 71. CHART FROM A SEVERE CASE OF CYSTITIS DUE TO B. COLI. 
 
 The continuous line shows the temperature, the dotted line the amount of 
 
 pus in the urine. 
 
 estimating the index should be used. In default of this, the 
 injections may be given every eight to twelve days, doubling the 
 dose until the upper limit is reached. 
 
 The treatment of inflammatory lesions of other regions (chole- 
 cystitis, persistent sinuses, etc.) appears to be more satisfactory, 
 remarkable cures having been obtained. Butler Harris has 
 obtained good results in endometritis, cervical catarrh, and 
 mucous colitis. In the former disease he gives a small dose a 
 week before and after each menstrual period. 
 
 It is in infections due to B. coli that some of the most striking 
 
3Q6 DYSENTERY 
 
 successes of vaccine therapy have been achieved. It has been 
 used in some extremely severe conditions with profound toxaemia, 
 and in no case did it render the patient's condition worse. Small 
 doses should, of course, be used in such cases, but the severity of 
 the disease need be no bar to the use of the remedy. 
 
 Dysentery. 
 
 Bacillary dysentery, under which heading we may include some 
 at least of the cases known as ulcerative colitis, asylum dysentery 
 and infantile diarrhoea, is in its general pathology closely akin to 
 typhoid fever. Its toxin is an endotoxin which is only set free 
 when the bacilli undergo solution, and its cure is associated with, 
 and perhaps due to, the development of a large amount of bacterio- 
 lysin. The main difference between the two diseases is that, 
 whereas typhoid fever is a self-limited disease, in which the patient 
 either dies or immunizes himself in a fairly constant period after 
 infection, in dysentery the disease has a tendency to become 
 chronic, the degree of immunity produced being sufficient to slow 
 the course of the disease, but insufficient to arrest it altogether. 
 The bacilli are in the main limited to the intestinal lesions, but 
 the fact that they have been found in the heart-blood of a new- 
 born foetus whose mother was suffering from the disease leads us 
 to believe that they may circulate in the blood ; in most cases 
 they are probably quickly destroyed, for normal serum has some 
 bacteriolytic action. 
 
 The toxin is contained in the bodies of the organisms, which 
 are much more poisonous than those of typhoid bacilli. It may 
 be obtained by Macfadyen's method of grinding at the temperature 
 of liquid air, by aseptic autolysis at 37 C., or simply by filtering 
 old broth cultures in which some of the bacilli have undergone 
 solution. Conradi's toxin, prepared by submitting emulsions to 
 autolysis for forty-eight hours, was fatal to rabbits in doses of 
 i c.c., causing diarrhoea, subnormal temperature, etc. According 
 to Todd, the toxin is developed best in alkaline broth, and attains 
 its maximum in about six weeks, after which time it begins to 
 diminish in potency. It is more thermostable than the exotoxins, 
 resisting heating to 70 C. for one hour, and only being destroyed 
 slowly at the boiling-point. It is possible to obtain an anti-endo- 
 toxin, but the treatment of the animals has to be conducted with 
 great care. It is probable also that the serum of animals 
 immunized to the bacilli themselves contains some antitoxic 
 
PRACTICAL APPLICATIONS 397 
 
 properties, especially if the bacilli are injected into the veins 
 (Besredka). In preparing this serum it is a great advantage to 
 follow Besredka's process, and make use of sensitized bacilli. If 
 the unaltered bacilli are used the process presents some difficulties, 
 especially in the case of small animals. 
 
 The serum thus prepared possesses powerful agglutinating and 
 bacteriolytic properties, and is extremely powerful as a prophylactic 
 agent. Kruse's serum protected a guinea-pig against a lethal 
 dose of the bacilli in doses of ginn^ g ramme > an d ToiiRr c - c - f 
 Shiga's serum (activated with a suitable amount of complement) 
 would sterilize i c.c. of a twenty-four-hour broth culture of the 
 bacilli. 
 
 Antidysentery serum has now fully proved its value in the 
 treatment of acute dysentery. According to Shiga, it reduces the 
 mortality of the disease by nearly 50 per cent. Kruse claims that 
 his serum causes a rapid diminution in the number of the stools, 
 such as is effected by no other agent, a general improvement in 
 the patient's condition, a shortening of the convalescence, and a 
 diminution of the mortality. Large doses, frequently repeated, 
 are required. 
 
 It has probably a complex action, being at the same time 
 antitoxic and bacteriolytic, and contains opsonins and bacterio- 
 precipitins. 
 
 In chronic cases it is of much less value, and in these forms 
 reliance must be placed on vaccine therapy. This has been care- 
 fully studied by Captain Forster and others. In Forster's patients 
 the mortality fell from 6-3 per cent, to 0-9 per cent., several cases 
 of the extremely chronic type which defies all ordinary treatment 
 for years being completely cured. He uses no opsonic control, 
 and standardizes his vaccines by determining the minimal lethal 
 dose ; this is necessary, since the various strains differ greatly in 
 toxicity. If the minimal lethal dose for a rabbit is about 0*4 c.c., 
 doses of o'i, 0-2, 0-3, and 0-4 c.c. are given at intervals of about ten 
 days. If symptoms of an overdose are produced, the amount given 
 at the next injection is reduced. 
 
 Prophylactic treatment by injections of killed cultures, either 
 as they are or after autolysis or sensitization with an immune 
 serum, or injected in conjunction with immune serum, have been 
 used on a large scale by Shiga and others, with apparent good 
 results as far as the case mortality is concerned, though with less 
 obvious effect on the prevalence of the disease. This phenomenon 
 
3Q8 CHOLERA 
 
 is readily intelligible if we regard the subsequent immunity as 
 being a condition in which the body, having been once trained to 
 do so, readily manufactures antibodies and other protective sub- 
 stances when infection occurs. Since no protective substances 
 are present at the time, infection occurs as in a normal person, 
 but the defensive substances are very quickly produced. Shiga's 
 vaccine was prepared by emulsifying a twenty-four-hour agar 
 culture of the bacillus in 5 c.c. of normal saline, heating to 60 C. 
 for one hour, and submitting the dead bacilli to autolysis at 37 C. 
 for two days. It is then filtered and used in doses of 0^05 to 0-5 c.c. 
 The serum becomes strongly agglutinating and bactericidal. 
 Other methods have been proposed. 
 
 Immunity reactions, especially that of agglutination, are of 
 great value in the diagnosis of dysentery, and especially of the 
 type of the bacillus present. In acute cases this is hardly 
 necessary, since modern methods have rendered the task of 
 isolating the bacilli from the stools an easy one. In the chronic 
 forms this is extremely difficult, and recourse must be had to the 
 agglutination test. The reaction is not as strong as in typhoid 
 fever, a positive result at a dilution of i : 50 being diagnostic. 
 The blood should be tested against any strains of dysentery 
 bacilli which may be available, especially if vaccine treatment is 
 to be used. 
 
 The method of absorption of complement has also been used. 
 
 Cholera. 
 
 In cholera the living organisms are strictly limited to the 
 intestinal contents, and the disease appears to be a pure intoxica- 
 tion, without access of living bacteria to the tissues. It is, how- 
 ever, probable that this is not the case, and that the vibrios enter 
 the blood and there suffer rapid and complete bacteriolysis, their 
 endotoxins being liberated in the process. But there is nothing 
 that can be called a local lesion, and the disease is not a septi- 
 caemia in the ordinary sense of the word. 
 
 The toxin of cholera is a typical endotoxin. The nitrates from 
 broth cultures are of very feeble toxicity, though they possess 
 immunizing properties, due doubtless to some degree of autolysis 
 which has taken place, and to the presence of free receptors. 
 Bacilli killed with agents such as chloroform or thymol are highly 
 toxic, especially if injected along with an immune serum, so that 
 they can be rapidly dissolved. The endotoxin can be prepared 
 
PRACTICAL APPLICATIONS 399 
 
 by aseptic autolysis, or by the freezing and grinding method of 
 Macfadyen. In either case it is thermolabile, being largely 
 destroyed at 60 C., so that cultures from which the toxin is to be 
 prepared must not be killed by heat. Metchnikoff and others 
 claim to have produced a soluble exotoxin by the use of very 
 virulent cultures in broth : it is thermostable and not very 
 potent. An antitoxin to it was prepared, but only very low grades 
 of potency were obtainable. Macfadyen's toxin was much more 
 toxic, and an anti-endotoxin of high potency was obtainable. 
 There is no demonstrable antitoxin in the ordinary bacteriolytic 
 serum obtained by the immunization of animals to the bodies of 
 the bacilli, or in the serum of cholera convalescents. 
 
 Cholera presents the best example of an apparently pure 
 bacteriolytic immunity, and presents a good example of the 
 difficulties inherent in the explanation of this subject. The 
 serum of an immunized animal or that of a person who has 
 recently recovered from cholera is powerfully bacteriolytic, giving 
 Pfeiffer's phenomenon in its earliest discovered and most marked 
 form : it is almost the only organism which is completely dissolved 
 in vitro under suitable conditions. Such a serum is also strongly 
 protective, shielding animals against several times the lethal dose 
 of living vibrios, and it seems difficult to avoid the conclusion 
 that its preventive properties are due to its bacteriolytic action. 
 But this is very difficult to maintain in view of the fact that the 
 serum increases the toxic effect of dead vibrios (and under some 
 circumstances of living ones), owing to the liberation of endotoxin. 
 It seems rather as if the presence of bacteriolytic substances is 
 actually harmful to the animal, allowing the organisms to set free 
 their toxin, instead of being taken up by the phagocytes and 
 remaining harmless. I am not aware that any opsonic experi- 
 ments by the dilution method (which alone would be of value) 
 have been carried out. 
 
 Diagnosis. In dealing with cases of supposed sporadic cholera 
 the main problem is the recognition of the vibrio isolated from 
 the stools, usually an easy matter. The morphological and 
 cultural characters will of course afford great help, but they take 
 some time to work out, and more reliance is to be placed on the 
 immunity tests, which are quicker and more conclusive. The 
 agglutination reaction is most convenient, and can be carried out 
 on the dejecta themselves, if the suspected organisms are present 
 in large numbers. Some of the mucus is broken up in a little 
 
400 CHOLERA 
 
 peptone solution, and two hanging-drop preparations are made, 
 one with the addition of normal serum in i : 50 dilution, the 
 other with a i : 500 dilution of a powerful anticholera serum, 
 such as can be obtained commercially. Cholera vibrios become 
 paralyzed and agglutinated in the second specimen, not in the 
 first. When smaller numbers are present a culture (probably 
 impure, but with the vibrios in sufficient abundance to serve for 
 the test) may be made by incubating peptone-water inoculated 
 with a flake of mucus for eight to twelve hours. This is to be 
 tested with the serum in the ordinary way, and should agglutinate 
 at nearly the same dilution as a known cholera culture. The 
 serum should be a powerful one, clumping at i : 10,000 or 
 more. 
 
 The Pfeiffer's reaction is perhaps more conclusive, and is 
 carried out as follows : The test immune-serum is diluted with 
 broth or normal saline, so that i c.c. contains o-ooi c.c. of serum; 
 i c.c. of this fluid is used to emulsify a loopful of a young agar 
 culture of the suspected organism, and the emulsion injected 
 intraperitoneally into a young guinea-pig. After a few minutes a 
 little peritoneal fluid is withdrawn by means of a capillary tube, 
 and the vibrios will be seen to have become non-motile, and to be 
 undergoing the characteristic change into slightly refractile 
 rounded masses. After a short time more they will be found 
 to have disappeared altogether. A control experiment with 
 normal serum may be made. This test is of great value, many 
 closely allied organisms failing to react. But no test is absolutely 
 conclusive, since a few cultures (notably the El Tor vibrio) have 
 been found to give all or most of them, and yet have been isolated 
 in a region in which cholera is not known to occur. The subject 
 is not yet settled, but in the meantime the probability that any 
 organism which reacts positively to the agglutination and 
 Pfeiffer's tests is true cholera is enormous. 
 
 The serum of persons convalescent from cholera agglutinates 
 the vibrios at dilutions of i : 100 or more for some months after 
 the attack, a fact which may be of some value in determining 
 the nature of a previous disease and a possible immunity to 
 cholera. 
 
 As regards treatment, the ordinary bacteriolytic serum is quite 
 useless, and, as far as I am aware, no potent anti-endotoxic serum 
 has been tried. The prophylactic treatment is on a sounder 
 footing. It was introduced by Ferran, of Barcelona, as early as 
 
PRACTICAL APPLICATIONS 40! 
 
 1884, ver y soon a ft er the discovery of the V. cholera by Koch. 
 His results were of doubtful value, his vaccines being made of 
 cultures of feeble virulence, and perhaps impure. The method 
 was placed on a scientific basis by Haffkine, who showed the 
 necessity for the use of cultures of great virulence. These are 
 prepared by passage through guinea-pigs. A more than lethal 
 dose of a laboratory culture is injected into the peritoneum of a 
 guinea-pig, and the peritoneal fluid (rich in vibrios) is collected 
 after death. This fluid is incubated in a thin layer, so as to allow 
 of thorough aeration, for fifteen hours, and is then administered 
 intraperitoneally into a second animal. After about twenty or 
 thirty passages the culture will have attained its maximum 
 virulence, the lethal dose being some ^ of the original. Its 
 potency falls off in some ten days, and a few further passages are 
 required to restore it. 
 
 The treatment is commenced by a dose of attenuated virus. 
 This is prepared by cultivating an ordinary laboratory stock in 
 broth at 39 C. in conditions of complete aeration. An inocula- 
 tion on agar is made every day, until (after a few days) the fluid 
 is found to be sterile. The process is now recommenced, using 
 the last agar culture that grew, and after several generations a 
 culture of very feeble virulence is obtained. It causes oedema, 
 but no necrosis, when injected under the skin. The vaccines are 
 prepared by cultivating the organisms on agar slants of definite 
 size (10 centimetres long) for twenty-four hours, and emulsify- 
 ing with 8 c.c. of broth, or 6 c.c. of 0-5 per cent, solution of 
 carbolic acid. The dose is i c.c. One or two injections of the 
 attenuated virus, followed by one of the exalted, all at intervals 
 of three to five days, may be given, or the exalted virus only may 
 be used. The injections cause moderate fever, headache, and 
 general malaise, and local tenderness, swelling and enlargement of 
 the corresponding lymph glands, all of which pass off in a few days. 
 
 This method (with various slight modifications with regard to 
 dosage) has now been used on a very large scale in India, with 
 strikingly good results. The immunity lasts for at least a year, 
 and probably decidedly longer if large doses of strong vaccines 
 are used, and, what is somewhat unusual, it manifests itself more 
 in a reduction of the incidence of the disease than in the 
 case- mortality. The value of the method is best seen from 
 statistics from isolated regions in which some persons were 
 vaccinated and others not, all living under the same conditions. 
 
 26 
 
4O2 PLAGUE 
 
 Thus, in the tea-plantations at Catchar, in 6,549 persons who were 
 not vaccinated, there were 198 cases, with 124 deaths, whilst in 
 5,778 vaccinated, there were 27 cases, with 14 deaths i.e., the 
 incidence fell from 3 to under 0-5 per cent, the case-mortality 
 being 62 and 51 per cent, respectively. Numerous other 
 examples might be quoted, and the value of the method is now 
 proved to the full. 
 
 Plague. 
 
 The plague bacillus produces a powerful endotoxin, cultures 
 killed by heat being markedly irritating. There is some evidence 
 that a true exotoxin may be produced, though in small amounts. 
 Filtrates from young cultures are devoid of toxicity, whereas 
 those from older ones may be fairly potent. The fluid portion 
 of culture (in broth grown at 20 C. and kept well aerated) 
 two months old was found by Markl to kill rats in doses of 
 c'i c.c. This might, of course, be due to an autolysis of the bacilli, 
 but this seems improbable from the fact that the toxicity of the 
 filtrate is very easily destroyed by heat, whereas the endotoxin is 
 thermostable. These filtrates have slight immunizing properties, 
 but the plague anti-endotoxin has not been closely studied. 
 
 Immunity appears to be due to the production of bacteriolytic 
 substances : antiplague serum, prepared by immunizing horses 
 first with dead and then with living bacilli, is powerfully bacteri- 
 cidal. According to Wright, the plague bacillus is quite insensible 
 to the bactericidal action of human blood, and recovery is due to 
 opsonization followed by phagocytosis. 
 
 The agglutination reaction is well marked in artificially 
 prepared immune serum, which may clump at i : 1,000 or more 
 and may be of use in the identification of a doubtful bacillus. It 
 s not usually marked, and may be absent in human cases of the 
 disease, and the diagnosis is most frequently made by the 
 identification of the bacillus in fluid from a lesion or from the 
 blood or sputum. According to Cairns, the blood does not usually 
 clump until the disease has been in progress for about a week. 
 The strength of the reaction is not great, rarely rising above i : 50, 
 and is sometimes as low as 1:3 or 1:5. The macroscopic 
 method is advisable. 
 
 The curative treatment of the disease by specific methods 
 resolves itself into the use of a serum, vaccines not having been 
 tried, as far as I am aware. Several sera are prepared, but not all 
 
PRACTICAL APPLICATIONS 403 
 
 have had an extensive trial. Yersin's serum is prepared at the 
 Pasteur Institute, the process being to immunize horses for long 
 periods up to a year and a half by weekly intravenous injections 
 (which do not cause abscesses, as is the case if the injections are 
 given subcutaneously). For the first three months or so dead 
 bacilli are used, afterwards living ones, and a very high degree of 
 immunity is attained. The potency of the serum is estimated by 
 finding the smallest amount which, given twenty -four hours 
 previously, will save a mouse from a lethal dose of living bacilli : 
 this may be as low as 0-02 c.c. Lustig's serum is supposed to be 
 antitoxic as well as bactericidal. It is prepared by the immuniza- 
 tion of horses with a "toxin" prepared by dissolving plague bacilli 
 in i per cent, caustic soda solution, filtering and precipitating with 
 dilute hydrochloric acid. (This has also been suggested as a 
 vaccine.) The precipitate is dissolved in 0*5 per cent, sodium 
 carbonate before use. 
 
 All observers are not agreed as to the efficacy of these sera, but 
 there is a decided preponderance of opinion in their favour. 
 Yersin's serum is most used, and is probably of the greater value. 
 A most important point in connection with its use is that large 
 doses are necessary, and those observers who have not obtained 
 good results have in some cases used quantities which were far 
 too small. Cairns used Yersin's serum in the Glasgow epidemic, 
 and in severe cases gave 150 to 200 c.c., part in the region draining 
 into the affected glands and part intravenously. Choksy, as the 
 result of large experience, urges the importance of a very early 
 use of the remedy, and gives 60 to 100 c.c. for adults and 10 c.c. 
 for infants, giving fresh injections of gradually diminishing amounts 
 every twenty-four hours, until six or eight have been given in all 
 150 to 300 c.c. He used Lustig's serum. In any case the effect 
 of serum is not a great one, a lowering of the case-mortality by 
 about i o to 20 per cent, being apparently the utmost to be hoped 
 for at present. It appears, however, that no other treatment 
 available is so successful. 
 
 The question of the preventive treatment is much more im- 
 portant. In some cases the serum may be used, and is probably 
 most efficacious ; but its effects are but transitory, and its only 
 legitimate use is to tide the person over the time until vaccination 
 can be performed and active immunity acquired. 
 
 Haftkine's plague prophylactic consists of a virulent broth 
 culture of the bacillus, killed by heat and preserved by the 
 
 26 2 
 
44 
 
 PLAGUE 
 
 addition of 0*5 per cent, carbolic acid. Cultures are made in 
 peptonized broth to which a small amount of oil is added. This 
 floats on the surface, and serves as a point of attachment for the 
 characteristic " stalactites." The flasks are kept at the ordinary 
 temperature (of Bombay about 27 C.) and shaken occasionally, 
 to break up the stalactites. Incubation lasts five to six weeks. 
 The vaccine is sterilized at 65 C. for one hour. The dose is 
 2-5 c.c. Constitutional and local symptoms of moderate severity, 
 and lasting for a few days, are produced, but the patient is as a 
 rule able to follow his ordinary occupation. The immunity 
 seems to be developed quite quickly, so that there is no reason to 
 fear any ill-effects from the injections when the patient is actually 
 exposed to plague, and perhaps even infected. According to 
 Bannerman, the protection is developed in twenty-four hours, and 
 asts about eighteen months. 
 
 Of the value of the method there can be no doubt, and statistics, 
 both those on a large scale and those dealing with communities, 
 some of whom are vaccinated and some not, prove clearly that 
 the treatment lowers the likelihood of infection, and also the case- 
 mortality. Thus, in twelve districts in the Punjab in which 
 plague was raging in the winter of 1902-03 the following results 
 were obtained : 
 
 
 Total. 
 
 Cases. 
 
 Per 
 Cent. 
 
 Deaths. 
 
 Per Case- 
 Cent. Mortality. 
 
 Uninoculated (average 
 
 
 
 
 
 l 
 
 population of district) 
 
 639,630 
 
 49,433 
 
 77 
 
 29,733 
 
 47 60- 1 
 
 Inoculated (average 
 
 
 
 
 
 
 population of district) 
 
 186,797 
 
 3-399 
 
 1-8 
 
 814 
 
 0-4 23-9 
 
 With regard to the second group of statistics, the experience in 
 Umarkadi Gaol may be quoted, as one out of many. Half the 
 prisoners, selected purely by chance, were inoculated, and all 
 lived together under exactly the same conditions. Some of each 
 group were liberated, and of the remainder there were 127 non- 
 inoculated, with 10 cases and 6 deaths, and 147 vaccinated, with 
 3 cases and no death. 
 
 The German Commission recommended the use of vaccines 
 prepared from two-day-old agar cultures, sterilized by heat. This 
 is more easily and quickly prepared than Haftkine's fluid. 
 
 The combined method (use of vaccine and serum) has been 
 
PRACTICAL APPLICATIONS 405 
 
 recommended by Calmette, by Besredka, and by Shiga ; the last- 
 named obtained very good results by its use in an epidemic in 
 Kobe. 
 
 Anthrax. 
 
 The nature of the toxin of anthrax is quite unknown, although 
 it has been the subject of much experimental investigation. No 
 exotoxin is formed in ordinary media. If coagulable or coagulated 
 proteids are present in the medium, they will be broken down into 
 peptones, etc., which have some toxic action, but no true toxin is 
 produced. Some observers have found that the filtrate from broth 
 cultures of anthrax, though devoid of toxicity, may have some 
 immunizing powers, a result which we should now attribute to 
 the presence of free receptors. The only importance attaching to 
 these facts is that they may explain the results obtained by some 
 investigators, who obtained albumoses and other bodies of very 
 feeble toxicity from various culture media, and considered them 
 to be the true toxin because they served to immunize animals. 
 And, according to Conradi, there is no evidence in favour of the 
 existence of an endotoxin. Bacilli killed by various methods and 
 disintegrated by Buchner's process yielded a non-toxic fluid. 
 The clinical nature of the disease in some of its manifestations 
 (especially pulmonary anthrax) would rather lead us to believe 
 that a powerful toxin is produced, but of this there is not the 
 slightest shred of experimental verification. 
 
 The process of recovery and the subsequent immunity are also 
 very difficult to understand. Local immunity is very marked, 
 the skin being highly resistant in comparison with the lungs, an 
 infection of which region forms one of the most rapid and intract- 
 able diseases known in man. There are very marked differences 
 with regard to the immunity of different animals. The fowl is 
 highly immune, as are cold-blooded animals. The rat and dog 
 are partially immune, whereas sheep, cattle, and the small animals 
 of the laboratory are very susceptible. 
 
 It is especially noteworthy in the case of anthrax that the 
 presence of bactericidal substances in the blood is no indication 
 whatever as to the degree of immunity* The serum of the rabbit, 
 a highly susceptible animal, has an extremely powerful bactericidal 
 effect, whereas that of the dog and rat have but little. The 
 classical Pfeiffer's phenomenon is not seen in the case of this 
 bacillus, but the altered bacteria may be readily recognized from 
 
406 ANTHRAX 
 
 the fact that they fail to stain by Gram's method. This change 
 is brought about very quickly by a suitable serum, the change 
 being often complete in ten minutes at 37 C. 
 
 There have been numerous attempts to explain the apparent 
 anomalies of the reaction in question. Bail found that dog serum 
 (normally a good culture medium for the anthrax bacillus) 
 becomes highly bactericidal after the addition of a small amount 
 of rabbit serum, even when this is only present in amount so 
 small that it is devoid of bactericidal action per se. This appears 
 to be due to the presence of immune body in the dog's blood, but 
 no complement. If the action of the rabbit's serum is due to the 
 presence of complement, this must be thermostable, for the effect 
 is not annulled by heating to 50 C. Bail and Petterson found 
 that many other sera could be reactivated with rabbit serum 
 (man, ox, calf, pig, etc.), and that extracts of leucocytes or of 
 organs (liver, bone-marrow) might be equally effective. Malvoz 
 also investigated the presence of immune body by means of the 
 Bordet-Gengou reaction (absorption of complement), and found 
 that the amount in the serum was some index as to the degree of 
 immunity. Thus the blood of the ox and guinea-pig contain 
 none, as is the case with the newly-born puppy, an animal 
 susceptible to anthrax, whereas the adult dog contains a large 
 amount. Remy has also studied the question of the reactivation 
 of sera of various species by complements from others, and 
 notably that of the fowl. Thus the serum of the white rat (an 
 immune animal) contains an immune body, for after heating to 
 55 C. it can be reactivated with fowl serum. On the other hand, 
 the serum of the goat after heating cannot be rendered bacteri- 
 cidal in this way. He holds that there is an absolute concordance 
 between the bactericidal power of the blood, the presence of 
 immune body, and the resistance of the animal to infection with 
 this organism. 
 
 Sobernheim and others have explained the susceptibility of the 
 rabbit by supposing that the immune body has a greater affinity 
 for the cells of the animal than for the anthrax bacillus, and is 
 thus absorbed and rendered useless. 
 
 On the other hand, Metchnikoff holds that the immunity is 
 entirely due to phagocytosis, and finds that the extent to which 
 the bacteria are taken up by the leucocytes is proportional to the 
 degree of resisting power. Anthrax bacilli (and especially the 
 second vaccine, which forms a very good emulsion) are very 
 
PRACTICAL APPLICATIONS 407 
 
 suitable objects for the study of phagocytosis. They are taken up 
 with great rapidity, and quickly undergo solution within the 
 leucocyte, first losing their sharp outline and power of retaining 
 Gram's stain, and disappearing altogether in ten minutes or less. 
 This makes the study of the opsonic index a matter of some 
 difficulty, which can be overcome by using isolated spores in test- 
 tube experiments. When no serum is used very few bacilli or 
 spores are taken up, and before the discovery of the opsonins 
 Metchnikoff noted that when rats are injected on the one side 
 with anthrax bacilli and on the other with the same organisms 
 mixed with blood-serum, oedema occurs only at the former place, 
 and it is from this that generalization occurs. Sawtchenko also 
 found that when the injection of the needle causes haemorrhage 
 the rat survives. The very careful and full researches of Metch- 
 nikofF on the degree of phagocytosis in susceptible and non- 
 susceptible animals are probably sufficient to lead us to believe 
 that the ingestion of the bacilli by the leucocytes is the all-im- 
 portant process in the cure of the disease, and the discovery of 
 the opsonins supplies the missing link necessary for us to account 
 for all the facts in a fairly satisfactory manner. We can only 
 conclude that the bactericidal effect of the serum plays a part of 
 comparatively small importance in combating the disease the 
 elaborate researches of Bail, Petterson, Sobernheim, etc., to the 
 contrary possibly, but by no means certainly, owing to the 
 absence of complement. 
 
 The facts of passive immunity are not so fully explained. 
 There are, however, some reasons for thinking that the active 
 substance is an opsonin, perhaps a thermostable one. Thus 
 Sclavo's serum (according to Cler) will render bacilli fit for 
 ingestion after five hours' contact, and it does not lose its efficiency 
 on keeping. On the other hand, the remarkably rapid improve- 
 ment sometimes seen after the -fise of Bandi's serum rather 
 
 suggests the presence of an antitoxin. 
 
 Diagnosis. This is made in all cases by the demonstration of 
 the bacillus. 
 
 Treatment. The preventive treatment is used for animals only. 
 Pasteur's method has already been noticed : it has been largely 
 used, and the results have, on the whole, been good. The 
 mortality from the inoculation is about \ per cent, of all cases, 
 but in some herds the number of deaths is much higher, and 
 serious loss is caused. The immunity is supposed to last for less 
 
408 ANTHRAX 
 
 than a year, when a reinoculation is necessary. The method is 
 not free from objections, but its use in regions of France where 
 anthrax was very prevalent proved of enormous value, and areas 
 in which raising cattle and sheep was rapidly becoming im- 
 possible were practically cleared of the disease. The weak point 
 of the process is that the immunity to infection through the 
 alimentary canal, if it exists, is extremely feeble. 
 
 To remedy the defects of Pasteur's system, Sobernheim has 
 introduced a method of conferring mixed immunity. An anti- 
 anthrax serum and a culture resembling Pasteur's second vaccine 
 are injected simultaneously into different parts of the body, and 
 no second inoculation is given. The doses are 5 to 15 c.c. of the 
 serum and 0^5 to i c.c. of culture. This method of treatment is 
 said to be free from danger, to protect against infection via the 
 intestinal tract ; it has also the advantage of requiring only a 
 single visit. The serum is also curative. 
 
 Curative Treatment. Here the use of serum is indicated. Sclavo's 
 serum is most used in this country. It is obtained by immuniz- 
 ing the animals with Pasteur's vaccines, and then by giving large 
 doses of virulent bacilli mixed with gelatin, which seems to 
 prevent the formation of abscesses. The dose is 20 to 40 c.c., 
 repeated in twenty-four hours if necessary, or four or five doses 
 of 20 c.c. each : the first injection may advantageously be 
 intravenous. It is usually followed by improvement within 
 twenty-four hours, and often causes sweating and a rise of 
 temperature. Sobernheim's serum is obtained by a somewhat 
 different method, and appears to be equally efficacious. The dose 
 recommended is 20 c.c. 
 
 The results of the use of serum in malignant pustule (which is 
 not so dangerous a disease as was once thought, even if untreated 
 by serum, the knife, cautery, etc.) have been very satisfactory: 
 there do not seem to be any observations on its use in the 
 far more serious woolsorter's disease or pulmonary anthrax. 
 Malignant pustule is also treated by the use of very hot fomenta- 
 tions, the idea being to bring about the attenuation of the bacillus. 
 There is little doubt that vaccines might be used if thought 
 desirable in the absence of serum. 
 
 Diphtheria. 
 
 Diphtheria presents a close approach to our idea of a disease the 
 immunity to which is antitoxic, but it is erroneous to imagine that 
 
PRACTICAL APPLICATIONS 409 
 
 the neutralization of the toxin or its destruction or elimination 
 constitutes the whole process of cure. There is a little evidence 
 in favour of the formation of bacteriolytic substances, though 
 experimental evidence on this point is not unanimous. Bandi, it 
 is true, claimed to have been able to immunize animals to the 
 bacilli themselves, and prepared a serum which was supposed to 
 have bactericidal properties ; it has been prepared by others, and 
 can be obtained commercially. It is supposed to be used locally, 
 either in the form of a powder or of lozenges, and is intended to 
 supplement the action of antitoxin. Rist, however, failed to 
 immunize animals to the bodies of the bacilli, and though Lipstein 
 was more successful, his serum was apparently inert as a protective 
 agent. It contained, however, an agglutinin, and the interesting 
 fact was noticed that it only clumps bacilli of the culture used 
 for the injection. This is of some interest, since the Klebs-Loffler 
 bacillus has always been looked upon as a very definite bacterial 
 species; the toxins it produces are always neutralized by the 
 same antitoxin, and though they may be produced in larger or 
 smaller amounts and may contain varying proportions of proto- 
 toxoids, etc. (on Ehrlich's theory), appear to be the same substance 
 in all cases. These experiments would tend to show that, though 
 the bacilli of various types agree in their metabolic products, they 
 may differ in the constitution of their protoplasm. 
 
 The observations referred to previously, show clearly that the 
 process of cure of the local lesion is assisted by the produc- 
 tion of an opsonin. And there is every reason to believe that 
 it is by phagocytosis that the bacilli are combated, bacteriolysis 
 being very doubtful and of comparatively small importance. 
 The cure of the disease therefore is accomplished partly by one 
 or more of the methods discussed in Chapter VI., and partly 
 by phagocytosis. 
 
 Diagnosis. This is made by the demonstration of the bacillus. 
 If necessary, the opsonic test might be used, and Bordet and 
 Gengou have shown by their method of fixation of complement 
 that " sensibilatrices " circulate in the blood. These methods are 
 quite unnecessary. The absolute recognition of diphtheria 
 bacillus in cultures can best be made by an application of an 
 immunity reaction. A pure culture in broth is divided into two 
 parts, and each injected into a guinea-pig. One of the animals 
 receives a large dose of antitoxin, and should this remain unaffected 
 whilst the other dies, the culture is certainly diphtheria. The 
 
410 TETANUS 
 
 method is usually only required in cases where a healthy person 
 contains diphtheroid bacilli in his mouth, nose, skin, etc., and 
 considerations of public health render a determination of their 
 exact nature necessary. 
 
 Treatment. This consists in the early use of antitoxin and the 
 treatment of the local lesion with antiseptics, and the only question 
 of importance concerns the dosage of the former remedy. As a 
 rule, 4,000 to 8,000 units should be given at once, and a second 
 injection at the end of twelve or twenty-four hours ; subsequent 
 doses are given if required. Unless a case is seen very early, a 
 part at least of the first dose may be given intravenously, and 
 this is always advisable in severe cases not seen until the disease 
 has been present for two or three days. Larger doses may be 
 given, but are of doubtful advantage ; a smaller amount should 
 not be given, except perhaps in mild cases. 
 
 The sole preventive treatment in actual use consists in the use 
 of comparatively small doses of antitoxin. The protection which 
 is conferred is usually a strong one, but exceptions have been 
 recorded. It lasts about a month. Essays in vaccination have 
 been made, but not on a large scale. 
 
 Tetanus. 
 
 The pathology of tetanus is akin to that of diphtheria in that it 
 is a local disease with remote symptoms due to the action of a 
 soluble exotoxin on distant structures. It differs from diphtheria 
 mainly in two points : the bacilli are strictly localized to the region 
 inoculated and the immediate neighbourhood, and the toxin, which 
 acts entirely on the central nervous system, reaches it entirely, or 
 almost so, by ascending the nerves from the region in which 
 infection occurs, and not by circulating in the blood-stream. This, 
 at least, is the usual course of events, and when, as occasionally 
 happens, the toxin actually gains access to the blood, it seems 
 likely that even then it does not act on the brain direct, but enters 
 the peripheral nerves at their distal endings and then ascends 
 them to their origin. 
 
 The diagnosis is made entirely by the recognition of the 
 organism in the wound, no agglutination or other tests being used. 
 If (as usually happens) the culture obtained from the w ? ound is 
 impure, it is divided into two parts, the one of which is injected 
 alone, the other in conjunction with tetanus antitoxin. If no 
 other pathogenic bacteria are present the animal that has received 
 
PRACTICAL APPLICATIONS 4! I 
 
 the mixture will survive, whilst the other will develop tetanic 
 symptoms and die. Even if other pathogenic bacteria are present 
 the indications are usually clear, since spasms will commonly 
 develop (in the animal which has received no antitoxin) before the 
 lethal issue. It is best to use a broth culture for this test, so that 
 there may be a good development of toxins. 
 
 The nature of the toxins of tetanus have been already mentioned. 
 There are two, both exotoxins the real poison, tetanospasmin, and 
 tetanolysin. Tetanospasmin is readily prepared by cultivation of 
 the organism in pure culture in almost any medium under anaerobic 
 conditions. It is even more fragile than diphtheria toxin, being 
 rapidly rendered inert in a few days if exposed to air at ordinary 
 temperatures. It is destroyed in eight to eighteen hours by 
 sunlight, by a temperature of 55 C. in one and a half hours, and by 
 exposure to agents such as alcohol, potassium permanganate, and 
 trichloride of iodine. It can be preserved by means of dilute 
 carbolic acid (0-6 per cent.) or chloroform without much loss. 
 Inert solutions have in general powerful immunizing properties, 
 the toxin being converted into toxoids, and not absolutely 
 destroyed. 
 
 It can be prepared so as not to give the reactions for proteid, 
 and is formed when the bacillus is grown on Uschinsky's proteid- 
 free medium. Its potency is enormous. Thus Vaillard prepared 
 a toxin of which the lethal dose for a guinea-pig was 0*001 c.c., 
 containing about 0*000025 gramme of solid matter, only a small 
 portion of which was pure toxin. Brieger and Cohn calculated 
 that the lethal dose of an (impure) toxin for a man was 0-00023 
 gramme. 
 
 The effect of tetanus toxin is manifested almost solely on 
 the central nervous system, and the post-mortem lesions are 
 practically confined to the ganglionic cells, especially of the 
 anterior cornua. It appears probable that there is no direct 
 action on the nerves themselves, but the toxin, like the virus of 
 rabies, reaches the central nervous system mainly, if not entirely, 
 by ascending the nerves leading from the area of inoculation. 
 According to Meyer and Ransom, toxin which gains access to the 
 blood only affects the brain by entering the peripheral nerves via 
 the nerve endings, especially the end-plates, but this is not 
 universally accepted. As in the case of rabies, the richer the area 
 of inoculation in nerves, the more powerful the action of the toxin 
 and the shorter the period of incubation. The brain and cord are 
 
412 TETANUS 
 
 the most susceptible regions, the peripheral nerves next, then 
 regions with an abundant nerve supply, such as the face ; and 
 lastly, regions poorly supplied, such as the subcutaneous and 
 peritoneal tissues. The incubation period of tetanus is thus seen 
 to be composed of : (i) the time necessary for the production of the 
 toxin in the tissues ; (2) for its ascent of the nerves to the brain 
 being longer, other things being equal, if infection takes place at 
 a long distance therefrom ; and (3) the latent period which elapses 
 after the toxin has united with the ganglion cells of the central 
 nervous system, and before the development of symptoms i.e., that 
 in which the enzyme-like action of the zymophore group is being 
 gradually exerted on the protoplasm. The fixation of tetanus 
 toxin in the system is extremely rapid: in rabbits it may dis- 
 appear entirely from the blood in one minute, whilst in other 
 susceptible animals it circulates for slightly longer periods. The 
 importance of this arises from the fact that toxin which has once 
 entered the nerves is thereby shielded from the action of antitoxin. 
 The dose of antitoxin necessary to save the life of an animal which 
 has received a few lethal doses of toxin rises enormously if the 
 injection of the former is delayed more than a few minutes. 
 
 Tetanolysin is even more fragile than tetanospasmin, being 
 converted into toxoids in a few hours at the room temperature. 
 It can be preserved in a dry state. The role which it plays in 
 natural infections, if any, is unknown. 
 
 As regards immunity, there is but little to add to what has been 
 discussed previously. The bacilli are not powerful parasites, 
 being readily ingested by the leucocytes, and destroyed if the 
 conditions are favourable for phagocytosis. In most of the cases 
 which develop tetanus there is a contused or lacerated wound, 
 with much killed and bruised tissues and an abundant con- 
 comitant infection with other bacteria, which still further paralyze 
 the natural resistance of the part. These organisms may have 
 an additional influence in securing a condition of anaerobiosis : 
 tetanus bacilli grown in symbiosis with certain other bacteria 
 which have powerful oxygen-absorbing properties will develop 
 vigorously, and develop toxin in spite of the free access of air. 
 No observations with regard to the opsonic index in tetanus 
 appear to have been recorded. The question of immunity to 
 tetanus toxin has been dealt with already, but we may add that 
 in all probability much of the toxin is destroyed in loco by the 
 unspecific action of the peptic enzyme formed by the leucocytes 
 
PRACTICAL APPLICATIONS 413 
 
 Antitoxin is rarely, if ever, found in human patients who have 
 survived an attack of the disease. 
 
 Treatment. The main question, of course, concerns the use of 
 antitoxin, and two general rules may be laid down : (i) It is of 
 great value as a prophylactic agent, and (2) it is of some value in 
 chronic tetanus i.e., the form with mild symptoms developing after 
 a long period of incubation. 
 
 Its preventive application is indicated in the treatment of all 
 wounds which experience has shown to be followed by tetanus 
 i.e., lacerated and contused wounds, especially if contaminated 
 with garden soil, road debris, etc. Gunshot wounds are especially 
 dangerous, and tetanus is usually extremely prevalent in warfare. 
 It is, of course, somewhat difficult to estimate precisely the value 
 of the treatment, inasmuch as tetanus is not a common disease ; 
 but experience derived from horses, which animals are extremely 
 prone to it, is more conclusive. In some veterinary practices it 
 was so common as to counterindicate any operative measure, and 
 has now been completely eradicated. The duration of the 
 immunity conferred by a single dose is about three weeks, and 
 in the prophylactic treatment of wounds, whether accidental or 
 due to operation, two doses should be given, at intervals of ten 
 to fourteen days. The prophylactic treatment of dirty wounds by 
 means of antitoxin is now a routine method in several Continental 
 clinics, and, as far as I am aware, there has been no case 
 recorded in which it has been followed by the development of the 
 disease, excepting those in which the injection has been given 
 some days after the injury, when the toxin has already gained 
 access to the nerves. One case (under Mr. Lenthal Cheatle) 
 from which I isolated a bacillus identical in cultural and morpho- 
 logical characters with that of tetanus, and in which the organisms 
 occurred in great abundance, was treated with antitoxin at the 
 outset, and healed without a symptom of the disease : the culture 
 was unfortunately not tested by inoculation. 
 
 Calmette prepares a powder of dry antitetanus serum to be used 
 as a dressing for wounds, but its use is very doubtful. Anti- 
 toxin is a good culture medium for bacteria, and unless the wound 
 is fairly clean may decompose and become offensive. The most 
 scrupulous antiseptic technique should be adopted, and it seems 
 probable that the dry dressing presents no advantage over the 
 subcutaneous administration of the serum when this is done. 
 
 The doses should be 5 to 10 c.c. for a man, and 10 to 20 c.c. for 
 
4*4 TETANUS 
 
 a horse. The best method of standardization is that of Roux, who 
 determines the amount of serum necessary to protect a guinea-pig 
 weighing 500 grammes against ten lethal doses of toxin. The result 
 is expressed in terms of the weight of guinea-pig protected against 
 one lethal dose of antitoxin by i c.c. of serum e.g., if ^ c.c. pro- 
 tected a guinea-pig weighing 500 grammes against ten lethal doses, 
 the potency would be 50,000. A potency of 1,000,000 is the least 
 that should be employed. 
 
 The use of tetanus antitoxin in the developed disease is less 
 satisfactory, a fact readily explicable now r that the pathology of 
 the disease is more fully understood. In acute tetanus it is 
 practically worthless, though a few cures have been reported. In 
 many cases of chronic tetanus it is without action ; in a few, 
 however, it is decidedly beneficial, each injection greatly alle- 
 viating the patient's suffering. It is always worthy of trial, but 
 it is hardly necessary to say that the non-specific treatment 
 should not be neglected. If the patient has a sufficient degree of 
 immunity to resist the toxin which has already gained access to 
 his nervous system, the antitoxin will be of value in preventing any 
 more from doing so, inasmuch as it will neutralize it as soon as it 
 is formed. 
 
 The doses should be large 20 c.c. or more at first, and 10 c.c. 
 every day, or every alternate day, subsequently. The site of 
 inoculation is of some importance. The injections may be given 
 subcutaneously in a distant region, as in the use of diphtheria 
 antitoxin ; but, in view of the fact that it takes an appreciable 
 time for it to be absorbed and time is of the utmost value if the 
 remedy is to be of any use it seems advisable to give the first 
 dose either in the region of the wound or intravenously. 
 
 Various methods have been proposed by which the antitoxin 
 can be brought into closer relation with the nerve elements. 
 The intracerebral injection has most to recommend it on theoretical 
 grounds, and several very decided successes have been recorded 
 in severe cases of the disease. The method is as follows : A 
 small flap of the scalp (with its base downwards) is reflected so 
 as to expose the skull a little to one side of the middle line, and 
 just in front of the fronto-parietal suture. A small trephine hole 
 is made through the skull, and an exploring needle is inserted 
 until the lateral ventricle is reached, and cerebro-spinal fluid 
 escapes through the needle. Ten c.c. or more of the serum are in- 
 jected. This- passes down the ventricular system, and bathes the 
 
PRACTICAL APPLICATIONS 415 
 
 respiratory and cardiac centres at the floor of the fourth ventricle. 
 Another method is to inject small quantities of the fluid directly 
 into the spinal cord by means of a needle introduced between the 
 sixth and seventh cervical vertebrae. This procedure would 
 appear to be dangerous, but this is said not to be the case. 
 Lastly, the simplest method of all is to perform lumbar puncture, 
 draw off some of the cerebro-spinal fluid, and replace it with 
 serum, just as in the process of spinal anaesthesia. 
 
 Ransom and Meyer have advocated the direct application of 
 antitoxin to the nerves supplying the region in which the wound 
 is situated, the idea being, of course, to intercept any further 
 access of toxin to the brain and cord. The nerves are exposed 
 by operation as near to their origin as possible, and infiltrated 
 with serum by means of a hypodermic syringe. 
 
 Analogy with other diseases would fully justify the use of 
 vaccine in chronic tetanus. Its preparation would present some 
 difficulties, owing to the heat-resisting power of the spores. 
 
 Syphilis. 
 
 Little is known definitely concerning the mode of cure or of the 
 type of immunity of syphilis. It used to be regarded as one of 
 the diseases which are followed by practically complete immunity 
 of long duration, but .Neisser has brought forward some evidence 
 for thinking that this is not the case, and that it only lasts as 
 long as the disease itself i.e., as soon as it is completely eradicated 
 the patient is again susceptible. Nothing is known as to the 
 toxins of syphilis, and, as regards the method of cure, the only 
 point worth mentioning is the fact that spirochaetes which have 
 been ingested by the leucocytes can be ma^e out occasionally. 
 They stain badly, and are doubtless on the way to complete 
 absorption. The fact that the organism cannot be obtained in 
 pure culture renders researches with regard to the opsonic and 
 bacteriolytic action of the serum very difficult. Indirect researches 
 by means of the deviation of complement constituting the 
 Wassermann reaction, a special method of application of the 
 Bordet-Gengou reaction have led to results of great interest 
 which have recently attracted much attention. 
 
 The first necessity was, of course, the preparation of an antigen, 
 and for this purpose Wassermann made use of the internal organs 
 of a syphilitic foetus, which were swarming with spirochaetes. In 
 
416 SYPHILIS 
 
 its main outlines the technique is exactly the same as that already 
 described. The serum to be tested is heated, to remove comple- 
 ment, and diluted with sterile normal saline solution. A dilution 
 of i : 20 or i : 40 is generally correct, but the point may be deter- 
 mined by preliminary tests with a known syphilitic serum ; and in 
 any case it is an advantage to perform a series of tests with 
 different dilutions, so that a rough idea of the amount of antibody 
 present in the serum may be obtained. This is mixed with an 
 extract of the syphilitic organ (antigen), and some fresh guinea- 
 pig serum (complement) added. The proportions may be i c.c. 
 of diluted serum, 0*1 or 0-2 c.c. of organ extract, and 0*2 c.c. of fresh 
 serum. The whole is incubated for one hour, at the end of which 
 time all the complement will be removed from the fluid if 
 syphilitic antibody is present. Next, corpuscles (e.g., of a sheep or 
 pigeon) are added, together with heated serum from a rabbit 
 which has been injected with the corpuscles in question ; or the 
 corpuscles may previously be sensitized with the inactivated 
 serum, washed, and then added. The whole mixture is then 
 incubated for two hours, with occasional stirring or shaking, 
 and kept some hours in the ice-chest. A positive reaction is 
 shown by the absence of haemolysis. Control tests are also 
 advisable e.g., the corpuscles must be completely dissolved by 
 the heated immune serum and the guinea-pig's serum if the other 
 two ingredients are not added, and there should be no haemolysis 
 if all the substances except the guinea-pig's serum are used. 
 
 Ledingham and Hartoch have shown independently that 
 opsonin is absorbed as well as complement, and this fact may 
 be used as a test of the presence of the reaction. In this case the 
 first part of the test is performed as beforehand the fluid used as 
 the serum in an opsonin estimation, using staphylococci or any 
 other organism, and using as a control guinea-pig serum diluted 
 with normal saline to the same extent as it was in the mixture of 
 organ extract, human serum, and guinea-pig serum. In a positive 
 reaction the phagocytic index in the first preparation will be 
 much below that in the second ; in a negative one they will be 
 equal. 
 
 The exact value of the test is not yet quite definitely settled. 
 It is very rarely present in health, and not common in diseases 
 other than syphilis ; but it does occur, especially in diseases which 
 (like syphilis) are due to animal parasites, such as malaria or 
 trypanosomiasis, and is not uncommon in leprosy and scarlet 
 
PRACTICAL APPLICATIONS 417 
 
 fever. It is rarer in other diseases, but isolated examples have 
 been met with in systematic investigations in a great many 
 maladies ; but here it is the exception, whereas in syphilis it is 
 the rule. In primary and secondary cases it occurs in 90 per 
 cent, or more, and is present in the majority of patients suffering 
 from tertiary syphilis and " metasyphilitic " affections. It is very 
 frequently found in the cerebro-spinal fluid of general paralytics 
 (80 per cent, 90 per cent., or more), even when it is absent from 
 the blood. It is not so common in tabes, and is extremely rare 
 (if it ever occurs) in the cerebro-spinal fluid in non-syphilitic 
 diseases, with the curious exception of scarlet fever, in which it 
 is almost constant. 
 
 So far there is no theoretical difficulty in the interpretation of 
 the phenomenon, but a new fact discovered by Landsteiner, 
 Miiller and Potzl seems to show that the reaction is of a nature 
 entirely different from the ordinary Bordet-Gengou phenomenon. 
 They found that an alcoholic extract of a normal organ (e.g., of a 
 guinea-pig's heart muscle) might be used instead of a tissue rich 
 in spirochsetes ; and further researches have shown that the lipoid 
 substances isolated therefrom, or even comparatively simple 
 substances, as lecithin and taurocholate and glycocholate of soda 
 (Levaditi and Yamanouchi) give the reaction, although apparently 
 not so frequently, as when an extract from a syphilitic organ is used. 
 The "antigen" is soluble in hot alcohol, and this fact alone removes 
 it from the group of true antigens, which, as we have seen, are 
 apparently all proteid in nature. According to Levaditi, the sub- 
 stance occurring in the blood or cerebro-spinal fluid is not an anti- 
 body at all, but either lipoid substances or salts, or the two in 
 combination, and they are set free when tissues are broken down in 
 a certain way, which occurs most frequently in syphilis, but may 
 take place in other diseases. Under ordinary circumstances they 
 are present in a colloid state/but form a precipitate with the lecithin 
 and allied substances extracted from normal organs by hot alcohol, 
 and to this precipitate the complement attaches itself. According 
 to Forges, the serum of syphilitics has the power of precipitating an 
 emulsion of lecithin (0*5 gramme, shaken up with 0*5 per cent, 
 solution of carbolic acid in normal saline) when mixed therewith 
 in equal parts. This he proposed as a test for syphilis, and Nobl 
 and Arzt found it successful in 80 per cent, of cases. Subsequently, 
 Forges replaced the lecithin (which as usually bought is not con- 
 stant in composition) by a recently prepared I per cent, solution 
 
 2? 
 
418 RABIES 
 
 of glycocholate of soda. A mixture of this with an equal amount 
 of serum is incubated for five hours, and observed after it has 
 stood sixteen to twenty hours at the room temperature. The 
 precipitate is specially obvious near the surface. 
 
 Fornet, Schereschewsky, Eisenzimmer, and Rosenfeld find 
 that the sera of syphilitics in the early stages of the disease 
 contain a precipitogen which forms an insoluble compound with 
 a substance or precipitin present in the serum of tabetics or 
 general paralytics. When the one is floated on the other, a 
 characteristic ring appears at the area of contact. They say that 
 normal serum rarely contains the precipitin, but not the 
 precipitogen. What relation this has to any immunity reaction 
 is unknown. 
 
 Rabies. 
 
 The actual causal agent of rabies is not yet definitely ascertained. 
 The peculiar structures known as the corpuscles of Negri which 
 occur in the brain, and especially in the hippocampus major, of 
 rabid animals appear to be quite characteristic of the condition, 
 and may possibly be the actual parasite, although this is not yet 
 universally accepted. It seems, however, fairly certain that their 
 recognition constitutes a sufficient proof of the presence of the 
 disease ; and this is of great importance in view of the necessity 
 for the early commencement of the treatment, which is entirely 
 preventive, and not curative. If the dog by which the patient has 
 been bitten is forthcoming, the corpuscles of Negri can be demon- 
 strated in a short time by simple methods, and the need for 
 Pasteur's treatment ascertained ; apart from this the only method 
 is by animal inoculation, an emulsion of brain substance being 
 injected into the brains of rabbits after trephining. 
 
 Rabies presents one of the most striking examples of local 
 immunity ; the action of the virus is manifested almost entirely 
 on the central nervous system, and in whatever part of the body 
 the inoculation is made the effects are only caused when it has 
 reached the brain and spinal cord ; and in doing so it does not 
 gain access to the blood, but ascends the peripheral nerves. 
 Hence the central nervous system is extremely susceptible to 
 injection, and the other tissues in proportion to their richness in 
 nerves. Subcutaneous (unless into a region like the paw), intra- 
 venous, or intraperitoneal injections only convey the disease if a 
 large amount of extremely potent virus is used. Hence it seems 
 
PRACTICAL APPLICATIONS 419 
 
 reasonable to suppose that there is a fair amount of immunity 
 inherent in all the tissues except in the nervous structures, and 
 that the living virus deposited elsewhere may be entirely destroyed 
 by bacteriolysis or phagocytosis. 
 
 We have already glanced briefly at Pasteur's earlier work on 
 antirabic inoculations, and the method by which immunity is 
 produced. Numerous modifications of the process have been 
 introduced since Pasteur's time. Thus Hogyes of Budapest 
 makes use of fully virulent cords, but given in extremely small 
 doses ; and there is some reason to think that his process does 
 not really differ from that of Pasteur, and that in drying the cords 
 the virus is gradually destroyed and not really attenuated, so that 
 a dose of a fourteen-day cord really contains a small trace of 
 virus of full virulence. A true vaccine i.e., a virus of mitigated 
 virulence can be obtained by passage through monkeys or birds. 
 Further, though the fixed virus is so potent for rabbits, it is quite 
 possible that its virulence for man is slight or nil. Nitsch was so 
 sure of this that he injected 4 to 5 milligrammes of the fresh cord 
 subcutaneously unto himself (in the abdominal region, a part 
 comparatively poor in nerves) without evil results. 
 
 Another method, introduced by Marie, consists in the use of injec- 
 tions of a mixture of virus and serum from an immunized animal. 
 This serum is prepared in a variety of ways, the simplest being to 
 give the virus intravenously. The animal usually employed is the 
 sheep, and the injection consists of rabid brains, hcatcclf up into a 
 fine emulsion with normal saline solution, and filtered through 
 linen. The serum prepared from animals treated in this way 
 possesses powerful ancirabic properties : when mixed with a 
 potent virus it removes entirely all harmful properties, so that it 
 is quite innocuous even on intracerebral injection. It can be 
 titrated against an emulsion of fixed virus of definite strength, 
 and by appropriate treatment a very potent serum can be obtained. 
 It is apparently quite useless in the treatment of the developed 
 disease or of an infected animal, even before the development of 
 symptoms. If it is mixed in excess with fixed virus and injected 
 into animals these do not develop rabies ; on the other hand, but 
 little immunity is produced, and this is supposed to be due to the 
 fact that the virus is so quickly absorbed that it does not act as 
 an antigen. But if the mixture be allowed to stand for some 
 time, and the virus then recovered by centrifugalization and 
 washing with normal saline solution, the clot thus obtained has 
 
 272 
 
42O RABIES 
 
 powerful immunizing properties. In the preventive treatment of 
 rabies on Marie's system the fresh fixed virus, made into a fine 
 emulsion with normal saline solution, is partially neutralized with 
 immune serum, and a dose of 6 c.c. (2 c.c. of i : 10 emulsion of 
 virus and 4 c.c of serum) is given in two places under the skin of 
 the abdomen. This is done for four days, and then injections of 
 dried cord, beginning at that of the sixth day, are commenced. 
 
 Other methods involve the use of heat, of chemical methods 
 (e.g., partial digestion with gastric juice, as practised by Centanni), 
 in order to bring about attenuation or partial destruction of the 
 virus. 
 
 Whatever the method, it appears necessary that the patient 
 should undergo a course of active immunization, various causes 
 (e.g., the long incubation period and the localization of the virus 
 in the nerves) rendering passive immunity an unsafe method of 
 protection. 
 
 Of the value of the process there cannot be the slightest doubt. 
 
 The incidence of hydrophobia after the bite of a rabid animal 
 is variously estimated, the figures usually given being about 
 15 per cent, in the case of dog-bites, and 40 to 80 per cent, 
 in bites from wolves. The probability of the patient's developing 
 the disease depends on the severity of the bite, its position 
 (i.e., whether in regions rich in nerves or the reverse), and 
 on whether the bite is through the clothing, so that some of 
 the virus is wiped from the teeth. In the twenty-two years (down to 
 1907 inclusive, the last year of which the figures are available), 
 ^0,359 patients have been treated at the Pasteur Institute in 
 Paris, with 126 deaths a death-rate of 0-31 per cent. (The 
 patients dying within fifteen days of the commencement of the 
 treatment a small number are excluded from the figures, since 
 in them the disease was too far advanced for a preventive treat- 
 ment to be of value.) 
 
BIBLIOGRAPHY 
 
 [/4 complete bibliography of the subject being obviously impossible in any reasonable 
 space, an attempt has been made to include important articles, and especially 
 those referred to in the text, and articles dealing with the subjects in a complete 
 manner, especially those with a good account of the literature.'} 
 
 CHAPTER I 
 
 GOOD accounts of the general phenomena of immunity may be found in 
 Metchnikoff's " L'Immunite dans les Maladies Infectieuses " (English trans- 
 lation by F. G. Binnie, University Press, Cambridge) ; Ricketts' " Infection, 
 Immunity, and Serum Therapy " (American Medical Associated Press, 
 Chicago) ; in Clifford Allbutt's " System of Medicine," vol. ii., part i. ; in 
 Muir and Ritchie's " Bacteriology." Also Levaditi's " La Nutrition dans 
 ses Rapports avec I'lmmunity " (Masson et Cie.) ; discussion on Immunity, 
 Brit. Med. Assoc., 1904 (" B. M. J.," September 10, 1904). The admirable 
 abstracts and collected articles in the " Central blatt f. Bakteriologie " 
 (Referate), in the " Bulletin de 1'Institut Pasteur," and in " Folia Haemato- 
 logica," will be found invaluable. 
 
 Cold and Wet. See Trommsdorf, Arch. f. Hyg., vol. lix., p. i, and 
 Vincent, Bull. Acad. Med., 1908. Ciuca, Comptes Rendus Soc. Biol., 
 vol. Ixii., pp. 858, 883. Alcohol. See Friedberger, Congres internat. 
 d'Hyg. and Demog. (Brux.), 1903. Rubin, Journ. Inf. Dis., 1904, p. 424. 
 Trommsdorf (vide supra}. Laitinen, Zeit. f. Hyg., vol. Iviii., 1907, p. 139. 
 
 Anesthesia. Snell, Berlin. Klin. Woch., 1903, p. 212. Rubin, Journ. 
 Inf. Dis., vol. i., p. 424. 
 
 Ehrlich (Trypanosomiasis, Atreptic Immunity, etc.), Harben Lectures 
 (H. K. Lewis). 
 
 Walker, Ainley, Journ. Hyg., vol. iii., p. 52 ; Cent. f. Bak., vol. xxxiii., 
 p. 297 ; Journ. Path. Bact., 1903, p. 34. 
 
 Papers on The Early Work on Immunity against Anthrax, etc., will be 
 found in Microparasites in Disease, New Sydenham Soc., 1886. See also 
 Pasteur, Comptes Rendus de 1'Acad. des Sci., 1880. Pasteur, Roux, 
 and Chamberland, ibid., 1883, xcvii. Rabies. See Sims Woodhead's 
 article in Clifford Allbutt's System of Medicine, with a full bibliography 
 up to 1906. See also Chapter XIV. 
 
 A useful account of the Use of Vaccines, etc., in Veterinary Practice is 
 given in Jowett's Blood-Serum Therapy (Bailliere, Tindall and Cox, 1907). 
 
 CHAPTER II 
 
 A full account of the subject will be found in Oppenheimer's " Toxine und 
 Antitoxine " (English translation by Ainsworth Mitchell : Charles Griffin 
 and Co.). This contains a most useful bibliography extending to 1904. 
 
 Antagonism of B. pyocyaneus and B. anthracis. Woodhead and Wood, 
 Edin. Med. Journ., 1890. Nasik vibrio. Kraus, R., Centr. f. Bakt. I. O., 
 
 421 
 
422 BIBLIOGRAPHY 
 
 vol. xxxiv., 1903, p. 488, and Rothberger, ibid., vol. xxxviii., 1905, p. 165. 
 Absorption of Toxins by Tissue. Ignowtowsky, Cent. f. Bakt. T. O., 
 vol. xxxv., p. 4. Vaillard, quoted by Metchnikoff (L'Immunite). 
 
 Wassermanris Experiment. See Chapter IV. 
 
 Combining Reactions of Tetanolysin. Ehrlich, Berlin. Klin. Woch., 
 1898, p. 273. Madsen, Zeit. f. Hyg., 1899, xxxii., 214. 
 
 Action of Tetanus Toxin on Frogs at Different Temperatures. Courmont 
 and Doyon, Comptes Rendus de la Soc. Biol., 1893, p. 618. Morgenroth, 
 Archives Int. de Pharmacodyn, 1900, p. 265. Constitution of Toxin 
 Molecule. Ehrlich, Croonian Lecture, Proc. Roy. Soc., 1900. Ibid., 
 Congres internat. de Med., Paris, 1900, Klin. Jahrbuch, vol. vi. (Die Werth- 
 bemessung des Diphthericheilserums). 
 
 Leucolysins or Leucotoxins. Neisser and Wechsberg, Zeit. f. Hyg., 
 vol. xxxvi., 1901, p. 300. Kerner, Julius, Cent. f. Bakt. I. O., vol. xxxviii., 
 p. 223. Christian, H. A., Deut. Arch. f. Klin. Med., vol. Ixxx., p. 333. 
 Denys and van de Velde, La Cellule, vol. xi., p. 359. 
 
 Bacterial Hcemolysins in Relation to Toxicity. Besredka, Ann. de 1'Inst. 
 Past., vol. xv. Ruedinger, Journ. Amer. Med. .Assoc., 1903. Breton, 
 Comptes Rendus de la Soc. Biol., vol. lv., p. 886. Schlesinger, Zeit. f. Hyg., 
 vol. xliv., p. 428. Bacterial H&molysins. A full bibliography will be 
 found in Oppenheimer, and a good general account of the subject by 
 Besredka, Bull, de 1'Inst. Pasteur, vol. i., 1903, pp. 547, 579. 
 
 Ricin. Ehrlich, Deut. Med. Woch., 1891. Fortschr. d. Med., 1897. 
 Stillmarck, Arb. pharm. Inst. Dorpat (quoted by Oppenheimer). Jacoby, 
 Arch. exp. Path., xlvi., p. 28. Osborne and Mandel, Amer. Journ. Phys., 
 vol. x., p. 36. 
 
 Serum Toxin. Cartwright Wood. See Chapter II. 
 
 Endotoxins (Pyocyaneus}. Wassermann, Zeit. f. Hyg., xxii., p. 263. 
 Endotoxins in general: Macfadyen, Proc. Roy. Soc., 1903, p. 76; 1903, 
 p. 351. Macfadyen and Rowland, Cent. f. Bakt. I. O., vol. xxxiv., p. 618. 
 Macfadyen, Lancet, 1904, p. 494. Macfadyen and Rowland, Journ. Phys., 
 vol. xxiii. Macfadyen, B. M. J., 1906, p. 776. Also Vaughan and Wheeler, 
 Journ. Amer. Med. Assos., 1905. Ransom, Deut. Med. Woch., 1895, P- 457- 
 Petterson, Cent. f. Bakt., vol. xlvi., p. 405. Pfeiffer and Friedberger, Cent, 
 f. Bakt. I. O., vol. xlvi., p. 98. Metchnikoff, Roux and Taurelli-Salembeni, 
 Ann. de 1'Inst. Past., vol. x., p. 257. Besredka, Ann. Inst. Past., 1906, 
 pp. 81, 304. 
 
 See also the discussion on Endotoxins (Kraus especially), Cent. f. Bakt. 
 (Ref.), vol. xlii. 
 
 CHAPTER III 
 
 Methods of Preparing Antitoxin, etc. Levaditi, in Kraus and Levaditi's 
 Handbuch, vol. ii., p. 62. Woodhead, Report of M. A. B., 1901. Hewlett's 
 Serumtherapy (Churchill, 1903). Dean, Trans. Path. Soc., vol. 1L, p. 15. 
 Madsen, Zeit. f. Hyg., vol. xxiv., p. 425. Hibbert, Journ. Exp. Med\, 
 vol. vii., p. 176. Martin, Ann. Inst. Past., vol. xii,, p. 26. Park and 
 Williams, Journ. Exp. Med., 1896, No. i. Atkinson, Journ. Med. Res., 
 vol. ix., p. 173. Salomonsen and Madsen, Ann. Inst. Past., vol. xi., 
 p. 315, and vol. xii., p. 763. Hibbert, Journ. Exp. Med., vol. vii., p. 176. 
 
 Serum-toxin. Cartwright Wood, Proc. Roy. Soc., vol. lix., p. 290 ; 
 Cent. f. Bakt., vol. xxxi., p. 241. 
 
 CHAPTER IV 
 
 Action of Ricin on Red Blood-Corpuscles. Ehrlich, Fortsch. der Med., 
 1897, P- 4 1 - Of Snake Venom. Stephens and Myers, B. M. J., 1898, 
 vol. Ixiii., p. 20. Of Eel Serum. Kossel, Berlin. Klin. Woch., 1898, p. 152. 
 Camus and Gley, Ann. Inst. Past., 1899, P- 779- Tchistovitch, ibid., 1899. 
 Action of Leucocidin. Neisser and Wechsberg, Zeit. f. Hyg., 1901, p. 299. 
 
BIBLIOGRAPHY 423 
 
 Filtration Experiments. Martin and Cherry, B. M. J., 1898, p. 1120. 
 Brodie, Journ. Path, and Bact., 1897, p. 460. Action of Heat on Snake 
 Venom, etc. Calmette, Ann. Inst. Past., 1895, p. 225. Martin and 
 Cherry, Proc. Roy. Soc., 1898. Wassermann, Zeit. f. Hyg., vol. xxii., 
 p. 263. Marenghi. Cent. f. Bakt. I. O., vol. xxii., p. 521. 
 
 Constitution of Diphtheria Toxin. Ehrlich, Die Wertbemessung des 
 Diphtherieheilserums, and numerous other papers, some of the more 
 important of which are in his Collected Papers. Madsen, B. M. J., 1904, 
 p. 567. Oppenheim, Toxin and Antitoxin. Gruber and Pirquet, Munch, 
 med. Woch., vol. 1., pp. 1193, 1259. 
 
 Arrhenius and Madsen' s Theories are discussed at great length in the 
 former's Immuno-Chemistry (Macmillan), where full references are given. 
 See also Madsen, B. M. J., 1904, September 10. Myers, Lancet, 1898, vol. ii., 
 p. 23, and Journ. of Path, and Bact., 1900. Mouton, Bull, de 1'Inst. Past., 
 vol. v., 1907, p. 449 (with bibliography). Arrhenius and Madsen, Zeit. 
 f. Phys. Chem., vol. xliv., p. 6. Gruber and v. Pirquet, Miinch. Med. 
 Woch., vol. 1., p. 1193. Arrhenius, Bull. Inst. Past., vol. ii., 1904, p. 553 
 (good general account). Madsen and Walbum, Cent. f. Bakt. I. O. ( vol. 
 xxxvi., p. 242. Mainwaring, W. H., Journ. Inf. Dis., vol. iii., p. 638. 
 Morgenroth and Pane, Biochem. Zeit., vol. i., p. 354. Nernst, Zeit. f. 
 Electrochemie, x., p. 177. Ehrlich's Reply to Arrhenius Theory, Berlin. 
 Klin. Woch., 1903, Nos. 35 and 37 (XXXVII. in Collected Studies). 
 
 Bordet's Views. Bordet, Ann. Inst. Past., vol. xvii., p. 161. Eisenberg, 
 Cent. f. Bakt., xxxiv., p. 259. Biltz, Zeit. f. Phys. Chem., 1904, p. 615. 
 See also Chapter XII. 
 
 CHAPTER V 
 
 Action of Electricity on Toxins. Kruger, Deut. Med. Woch., 1895, p. 331. 
 D'Arsonval and Charrin, Comptes Rendus de la Soc. de Biol., 1896, pp. 122, 
 280. Marmier, Ann. de 1'Inst. Past., vol. x., p. 468. Knorr, Miinch. 
 Med. Woch., 1898, p. 321. 
 
 Antibodies in Normal Blood. Metchnikoff, L'lmmunite, p. 598. Neisser, 
 Deut. Med. Woch., 1900, p. 791. Cobbett, Lancet, 1899, p. 332. Ibid., 
 Cent. f. Bakt., vol. xxvi., p. 548. Meade, Bolton, Journ. Exp. Med., 
 vol. i., p. 543. 
 
 Regeneration of Antitoxin after Bleedings. Roux and Vaillard, Ann. Inst. 
 Past., vol. vii., p. 64. Salom onsen and Madsen, ibid., vol. xii., p. 763. 
 Action of Pilocarpin. Salomonsen and Madsen, Comptes Rendus de 
 1'Acad. des Sciences, vol. cxxv., p. 122. 
 
 Side-Chain Theory. Ehrlich, Croonian Lecture, Proc. Roy. Soc., vol. 
 Ixvi., p. 424.; Ver. f. Innere Med. Berlin, 1901. Numerous articles in 
 Collected Studies. See also Aschoffs Ehrlich's Seitenkettentheorie 
 (Fischer, 1902), with a very full bibliography. Levaditi, La Nutrition dans 
 ses rapports avec I'lmmunite (Masson and Cie.), which also gives numerous 
 references. Wassermann, Berlin. Klin. Woch., 1898. Plimmer, Journ. 
 Path, and Bact., 1898, p. 489. Figs. 22, 23, 24 are from Emery, The 
 Specific Antibodies, St. Bart.'s Hosp. Journ., 1902. Weigert, Verhandlung 
 der ges. Deutscher Naturforscher und Aerzte, 1896. Bruck, Zeit. f. Hyg., 
 vol. xlvi., p. 176. 
 
 Union of Tetanus Toxin with Brain Substance. Wassermann and 
 Takaki, Berlin. Klin. Woch., 1898. Metchnikoff, Ann. Inst. Past., vol. xii., 
 pp. 81, 263. Marie, ibid., p. 91. Courmont and Doyon, Comptes Rendus 
 de la Soc. Biol., 1898, p. 602. Joukowsky, Ann. Inst. Past., vol. xiii., 
 p. 464. Morax and Marie, ibid., vol. xvii., p. 335, and Comptes Rendus 
 Soc. Biol., vol. liv., p. 1535. Dmitrevsky, Ann, Inst. Past., vol. xvii., 
 p. 148. Besredka, ibid, p. 138. Muller, Cent. f. Bakt. I. O., vol. xxxiv., 
 p. 567. Landsteiner and Boteri, ibid., vol. xlii., p. 562. Wolff-Eisner and 
 Rosenbaum, Berlin. Klin. Woch., 1906, p. 945. Takaki, Beitr. z. Chem. 
 
424 BIBLIOGRAPHY 
 
 Phys. und Path., vol. xi., p. 238. Morax and Tiffaneau, Comptes Rendus 
 de la Soc. Biol., vol. Ixii., p. 15. Noon, Journ. Hyg., vol. vii., p. 101. 
 
 Romer's Experiments. Arch. f. Opthal., vol. lii., p. 72. 
 
 Antispermotoxin. Metchnikoff, L'Immunite, p. 130. Blum, Beit. z. 
 Chem. Phys., 1904. Vaillard and Vincent, Ann. Inst. Past., vol. v., p. i. 
 Besredka, Ann. Inst. Past., vol. xiii., pp. 49, 209. 
 
 CHAPTER VI 
 
 Non-Specific Processes. Herter, Lectures on Chemical Pathology 
 (Smith, Elder, 1902). 
 
 Function of Liver. Brunton, Sir L., and Bokenham, Journ. Path. Bact., 
 1905, p. 50. 
 
 Antitoxin in Blood after Diphtheria, etc. Wassermann, Zeit. f. Hyg., 
 vol. xix., p. 408. Abel, Deut. Med. Woch., 1894, pp. 899, 936. Vincenzi, 
 ibid., 1898, p. 247. Knorr, Munch. Med. Woch., 1898, p. 363. 
 
 Absence of Correlation between Immunity and Antitoxin. Roux and 
 Vaillard, Ann. Inst. Past., vol. vii., p. 64. Behring and Kitashima, Berlin. 
 Klin. Woch, 1901, p. 157. Metchnikoff, L'Immunite, pp. 386 et seq. 
 Behring, Allgemeine Therapie der Infectionskrankheiten, in Eulenberg 
 and Samuel's Lehrbuch der Allg. Therapie. 
 
 Leucocytes in Intoxications. Metchnikoff, loc. cit., p. 413, where nu- 
 merous references are given. Besredka, Ann. Inst. Past., vol. xiii., pp. 49, 
 205 and 465. Dean, Journ. Path. Bact., 1908, p. 154. Ewing, Clinical 
 Pathology of Blood (Kimpton, 1904), p. 292. Vincent, Ann. Inst. Past, 
 vol. xviii., p. 450. 
 
 Stimulins. Metchnikoff, loc. cit., p. 284. 
 
 Wassermann, N. Y. Med. Journ., 1904. 
 
 Immunization to Eel Serum. Tchistovitch, Ann. Inst. Past., vol. xiii., 
 p. 406. 
 
 Specific Processes. See Ehrlich's Croonian Lecture, Harben Lectures, 
 and various papers in his Collected Studies. Wassermann and Bruck, 
 Deut. Med. Woch., 1904, p. 764. Jacoby, Beit. z. Chem. Phys., vol. vi., 
 p. 113. Bruck, Zeit. f. Hyg., vol. xlix, p. 282. Ricketts, Trans. Chicago 
 Path. Soc., vol. vi. Metchnikoff, L'Immunite, Chapters XI, XII. Leva- 
 diti's L'Immunite dans ses Rapports avec la Nutrition. 
 
 Passive Immunity. McClintock and King, Journ. Inf. Dis., vol. iii. 
 p. 701. Goodman, ibid., vol. v., p. 184. Bulloch, Journ. Path. Bact., 
 1898, p. 274. Schiitze, Koch's Festschrift, p. 657. Pfeiffer and Fried - 
 berger, Cent. f. Bakt. I. O., vol. xxxvii., p. 131. Wassermann and Bruck, 
 Zeit. f. Hyg., vol. 1., p. 309. Weil-Halle and Lemaire, Comptes Rendus 
 Soc. Biol., 1906, p. 114. Henderson Smith, Journ. Hyg., vol. vii., p. 205 
 Goodman, Journ. Inf. Dis., vol. v., p. 184. 
 
 Susceptibility to Tetanus Toxins. Knorr, Munch. Med. Woch., 1898, 
 pp. 321, 362. Behring, Fortschr. der Med., vol. xvii., p. 501. Behring's 
 Beitrage, August, 1903. See also Chapters V., XIV. 
 
 CHAPTER VII 
 
 Alexins. Nuttall, Zeit. f. Hyg., vol. iv., 1888, p. 353. Behring, Cent, 
 f. Klin. Med., 1888, No. 32. Behring and Nissen, Zeit. f. Hyg., 
 vol. viii., p. 412. Buchner, Cent. f. Bakt. I. O., vol. v., p. 817. Ibid., 
 Arch. f. Hyg., vol. x., p. 84. Ibid., Arch. f. Hyg., vol. xvii., p. 112. Ibid., 
 Munch. Med. Woch., vol. xlvii., p. 277. Lubarsch, Cent. f. Bakt., vol. vi., 
 p. 481, 529. Pfeiffer, Zeit. f. Hyg., vol. xviii. ; Deut. Med. Woch., 1896, 
 pp. 97, 119. Bordet, Ann. Inst. Past., vol. ix., p. 462, and ibid., vol. xii., 
 p. 688. Landsteiner, Cent. f. Bakt. I. O., vol. xxv., p. 546. 
 
 Ehrlich's Researches are given in his Collected Studies, the main chapters 
 
BIBLIOGRAPHY 425 
 
 being I. -VIII., XVII., XIX., XXI., XXII., XXXII., XXXIII., and XL. 
 
 Marino, Ann. Inst. Past., vol. xvii., p. 321. 
 
 A full account of the subject of Hcemolysins, with an excellent biblio- 
 graphy, is given by Sachs in Kraus and Levaditi, vol. i. ( and the work of 
 Muir and his school has just been published in collected form (The Oxford 
 Press, 1909). See also Flexner and Noguchi, Journ. Exp. Med., vol. vi. 
 Kyes, Berlin. Klin. Woch., 1902 (reprinted in Ehrlich's Studies). Kyes 
 and Sachs, Berlin. Klin. Woch., 1903, p. 21, 57, 82. Kyes, Berlin. Klin. 
 Woch, 1903, p. 956, 982. Bordet's Views. Bordet, Ann. Inst. Past., 
 vol. xiii., 1899, PP- 22 5> 2 73 > v l- xiv., p. 257 ; 1906, p. 467. Muir and 
 Browning, Proc. Roy. Soc., vol. Ixxiv., p. 298 ; Journ. Path, and Bact.^. 
 vol. xiii., p. 76. Muir, Lancet, vol. clxv., p. 446, and B. M. J., Septem- 
 ber 10, 1904. Muir and Ferguson, Journ. Path, and Bact., 1906, p. 84. 
 Metchnikoff, L'Immunite, Chapters VII., VIII. 
 
 Bordet and Gengou' s Phenomenon (Fixation of Complement). Bordet, 
 Ann. Inst. Past., vol. xv., p. 289. Bordet and Gengou, C. R. Acad. Sci., 
 vol. cxxxvii., p. 351. Gengou, Berlin. Klin. Woch., 1906, p. 1532. Muir 
 and Martin, Journ. of Hyg., vol. vi., p. 265. Heller and Tomarkin, Deut. 
 Med. Woch., 1907, p. 795. Cruveilhier, Comptes Rendus Soc. Bio., 
 vol. Ixii., p. 1027. Schutze, Berlin. Klin. Woch., 1907, p. 800. Seligmann, 
 ibid., 1907, p. 1013. Widal and le Sourd, Comptes Rendus de la Soc. 
 Biol., 1901, pp. 673, 841. Wassermann and Bruck, Deut. Med. Woch., 
 1906, p. 449. Bruck, Deut. Med. Woch., 1906, June, p. 945. Also Camus 
 and Pagnier, Comptes Rendus Soc. Biol., 1901, July. 
 
 For References re Gengou' s Phenomena see also Chapter IX. 
 
 Deviation by Toxins, etc. Armand-Delille, Comptes Rendus Soc. Biol., 
 1908. Poyerski, ibid., 1908, p. 896. Weinberg and Parvu, ibid., Novem- 
 ber, 1908. Laubry and Parvu, Soc. Med. des Hop., December, 1908. 
 
 In Explanation of Complementoid. Moreschi, Berlin. Klin. Woch., 
 vol. xiii., September, 1905, p. 1181. Gay, Cent. f. Bakt., vol. xxxix., 1905, 
 pp. 172, 603 ; also vol. xl., p. 695. Pfeiffer and Friedberger, Deut. 
 Med. Woch., 1905, p. 6. Besredka, Ann. Inst. Past., 1905. Sachs, Deut. 
 Med. Woch., May, 1908. Pfeiffer and Friedberger, Deut. Med. Woch., 
 1905, p. 1145. Bordet, Ann. Inst. Past., vol. xv., p. 289. Sachs, Cent. f. 
 Bakt. I. O., vol. xl., p. 125. Bordet, Berlin. Klin. Woch., 1906, p. 17. 
 Pfeiffer and Moreschi, Berlin. Klin. Woch., 1906, p. 33. 
 
 Deviation of Complement. Loffler and Abel, Cent. f. Bakt. I. O. ( vol. xix., 
 p. 51. Pfeiffer, Zeit. f. Hyg., vol. xx., p. 198. Neisser and Wechsberg, 
 Munch. Med. Woch., 1901. Lipstein, Cent. f. Bakt. I. O., vol. xxxi., p. 460. 
 Morgenroth, ibid., vol. xxxv., p. 501. Myers and Stephens, Journ. Path. 
 Bact., vol. v. Kyes, Berlin. Klin. Woch., 1902, and Kyes and Sachs, ibid., 
 
 1903 (both these are in Ehrlich's Collected Studies). Meakins, Johns 
 Hopkins Bull., 1907, p. 259. 
 
 Origin of Complement, Alexin, etc. Hankin, Cent. f. Bakt. I. O., xii. 
 and xiv., p. 853. Denys and Havet, La Cellule, 1894, vol. x., p. 7. 
 Havet, ibid., 1894, vol. x. Denys, Cent. f. Bakt. I. O., vol. xvi., p. 781. 
 Buchner, Munch. Med. Woch., 1894, p. 589; Metchnikoff, L'lmmunite. 
 Bulloch, Trans. Path. Soc., 1901, p. 208; ibid., B. M. J., September 10, 
 
 1904 (with full bibliography). Longcope, Journ. of Hyg., vol. iii., 
 p. 28. Guseff, quoted by " Petrie, loc. cit. Briscoe, Orth Festschrift, 
 1903. Levaditi, Ann. Inst. Past., xvii., p. 187. Korschun and Morgenroth, 
 Berlin. Klin. Woch., 1902. Marino, Comptes Rendus Soc. Biol., vol. lv., 
 p. 689. Schattenfroh, Arch. f. Hyg., vol. xxxi., p. i ; ibid., xxvii., 
 p. 234; ibid., Munch. Med. Woch., 1897, P- 4 T 4 '> ibid., 1898, p. 1109 ; 
 ibid., 1897, P- 4' ibid., Arch. f. Hyg., vol. xxxv., p. 135, Petrie, 
 Journ. Path. Bact., vol. ix., p. 130. Lastschenko, Munch. Med. 
 Woch., 1899, p. 475 ; ibid., Arch. f. Hyg., xxxvii., p. 290. Lambotte, Cent, 
 f. Bakt. I. O., vol. xxxiv., p. 453. Lambotte and Stienon, Cent. f. Bakt. 
 I. O., vol. xl., p. 224. Donath and Landsteiner, Zeit. f. Hyg., vol. xliii., 
 p. 552. Lowit and Schwarz, Zeit. f. Heilk., vol. xxiv., pp. 205, 301. 
 
426 BIBLIOGRAPHY 
 
 Gengou, Ann. Inst. Past., vol. xv., p. 68 ; ibid., vol. xv., p. 232. Falloise, 
 Comptes Rendus Soc. Biol., vol. Ivi., p. 324. Falloise, Bull, de 1'Acad. 
 Royale de Belgique, 1903. Levaditi, Ann. Inst. Past., 1901, vol. xv., 
 p. 894, and vol. xvi., p. 233, 1902. Ainley Walker, Journ. Hyg., 
 vol. iii., p. 52, and Cent. f. Bakt., vol. xxxiii., p. 297. See also Hahn, 
 Arch. f. Hyg., vol. xxv., p. 105. Wauters, Arch, de Med. Exp., vol. x., 
 p. 751. Moxter, Deut. Med. Woch., 1899, p. 687. Tromsdorff, 
 Arch. f. Hyg., vol. xl., p. 382. Van de Velde, Cent. f. Bakt. I. O., 
 vol. xxiii., p. 692. Bail, Hyg. Rundschau, vol. viii., p. 1066. Sweet, 
 Cent. f. Bakt. I. O., vol. xxxiii., p. 208. Malvoz, Ann. Inst. Past., vol. xvi., 
 p. 623. Lazar, Wien. Klin. Woch., 1904, p. 439. Kanthack, vide 
 Chapter X. Steinhardt, Journ. Med. Research, vol. xix., p. 161. 
 Gousseff, abstract in Bull. Inst. Past., vol. i., p. 175. Longcope, Journ. 
 Hyg., vol. iii., p. 28. 
 
 Origin of Immune Bodies, etc. Pfeiffer and Marx, Deut. Med. Woch., 
 
 1898, p. 47 ; Deutsch. Ann. Inst. Past., vol. xiii., p. 689 ; and Cent. f. 
 Bakt. I. O., vol. xxviii., p. 45. Kraus and Schiffmann, Ann. Inst. Past., 
 vol. xx., p. 226. Bulloch, vide ante. Wassermann, Deut. Med. Woch., 
 
 1899, p. 141. Wassermann and Citron, Zeit. f. Hyg., vol. 1., p. 331. 
 Pfeiffer and Marx, Zeit. f. Hyg., vol. xxvii., p. 272. Donath and Land 
 steiner, Zeit. f. Hyg., vol. xliii., p. 552. Kraus and Schiffmann, Ann. Inst. 
 Past., vol. xx., p. 226. Kraus and Levaditi, Comptes Rendus Acad. Sci., 
 vol. cxxxviii. Emden, Zeit. f. Hyg., vol. xxx., p. 19. 
 
 Methods (Haemolysis}. Papers in Ehrlich's Collected Studies, especially 
 Chapter XXIX. (Morgenroth) . Sachs in Kraus and Levaditi's Handbuch 
 der Technik and Methodik der Immunitatsforschung (Fischer, Jena, 
 1907). Moro, Munch. Med. Woch., vol. liv., p. 1026. Gay and Ayer, 
 Journ. Med. Research, vol. xvii., p. 341. Longcope, Univ. of Penn. Med. 
 Bull., xv., p. 331. 
 
 Methods (Bacteriolysis}. Ehrlich's Studies, Chapters IX., XXX. (Neisser 
 and Wechsberg). Klien, Johns Hopkins Bull., 1907, p. 245. Stern and 
 Korte, Berlin. Klin. Woch., 1904, p. 213. Wright, Lancet, December, 
 1900 ; ibid., 1901, March 2 and September 14 ; ibid., Proc. Roy. Soc., 
 Ixxi., p. 54. Gay and Ayer, loc. cit. Andrewes and Gordon, Report of 
 L.G.B. (supplement), 1906-7, p. 141. Goodwin, Proc. N. Y. Path. Soc.. 
 vol. v. 
 
 Cytolysins, etc. ; Leucolysins. Metchnikoff, L'lmmunite. Funk, Cent, 
 f. Bakt. I. O., vol. xxvii., p. 670. Flexner, Univ. of Penns. Med. Bull., 
 vol. xv. Bunting, ibid., vol. xvi., p. 200. Goodman, Journ. Inf. Dis., 
 vol. v., p. 173. Christian, Deut. Arch. f. Klin. Med., vol. Ixxx., p. 333. 
 
 Spermotoxin. Metchnikoff, Ann. Inst. Past., vol. xiv., p. i, 369. 
 Metalnikoff, ibid., p. 577. Moxter, Deut. Med. Woch, 1900, p. 61. 
 Landsteiner, Cent. f. Bakt. I. O., vol. xxv., p. 546. London, Arch, de 
 Sci. Biol. St. Petersburg, vol. ix. Weichardt, Ann. Inst. Past., vol. xv., 
 p. 8^3. 
 
 3 ^'specificity and General. Sachs, Biochem, Cent., 1903. Pearce, Journ. 
 /.^ Exp. Med., vol. viii. ; ibid., Journ. Med. Res., vol. xii., pp. i, 329. Beebe, 
 Journ. Exp. Med., vol. vii., p. 730. Armand-Delille and Leenhardt, C. R. 
 Soc. Biol., vol. Ixii., p. 31. Woltmann, Journ. Exp. Med., vol. vii., p. 119. 
 Forsner, Munch. Med.' Woch., vol. Hi., p. 892. Flexner and Noguchi, 
 Journ. Med. Res., vol. ix., p. 257. Bierry and Pettit, C. R. Soc. Biol. 
 vol. Ivi., p. 238. Dudgeon, Panton, and Ross, Proc. Roy. Soc. Med., 
 vol. ii., No. 2. 
 
 Trichotoxin. Von Dungern, Munch. Med. Woch., 1899. Hoyton, 
 B. M. J., 1902. 
 
 Nephrotoxin. Nefedieff, Ann. Inst. Past., vol. xv., p. 17. Ascoli and 
 Figari, Berlin. Klin. Woch., 1902. Lindemann, Cent. f. Allg. Path., 
 vol. vi., p. 184. Pearce, Univ. Penns. Med. Bull., vol. xvi., p. 217. 
 Bierry, C. R. Acad. Sci., vol. cxxxii. Bierry, C. R. Soc. Biol., vol. 
 lv., p. 496. Le Play and Corpechot, ibid., p. 206. Sheldon, Amos, 
 
BIBLIOGRAPHY 427 
 
 Reports of Med. Staff, Egyptian San. Council, 1906. Albarran and 
 Bernard, Arch, de Med. Exp., vol. xv., p. 13. Woltmann, Journ. Exp. 
 Med., vol. vii., p. 119. 
 
 Gastrotoxin. Bolton, Proc. Roy. Soc., vol. Ixxvii., p. 426, and Ixxix., 
 p. 533 ; ibid., Proc. Roy. Soc. Med., vol. ii., No. 2. Theobary and Bates, 
 Comptes Rendus Soc. Biol., 1903, p. 459. 
 
 Anti-intestinal Serum. Belonowski, Comptes Rendus Soc. Biol., 1907, 
 P- 9- 
 
 Syncytiolysin. Liepmann, Deut. Med. Woch., 1902, p. 911. Weichardt, 
 ibid., 1902, p. 624. Ascoli, Cent. f. Gynekol., 1902. Wormser, Munch. 
 Med. Woch., 1904, p. 7. 
 
 Neurotoxin. Delezenne, Ann. Inst. Past., vol. xiv., p. 686 ; ibid., Comptes 
 Rendus Soc. Biol., 1901, p. 1161. Armand-Delille, Ann. Inst. Past., 
 vol. xx., p. 838 ; ibid., Enriquer and Sicard, Comptes Rendus Soc. Biol., 
 
 1900. Pirone, Arch. Sci. Biol., vol. x., p. 75. 
 
 For Peripheral Nerves. Schmidt, Ann. Inst. Past., vol. xx., p. 601. 
 
 Ophthalmotoxin. Bram Pusey, quoted by Ricketts. Le Play and 
 Corpechot, Comptes Rendus Soc. Biol., 1904, p. 1021. Golovine, Russie 
 Vratch, 1904, abstracted in Bull. Inst. Pasteur, vol. ii., p. 1009. 
 
 Hepatotoxin. Delezenne, Comptes Rendus Acad. Sciences, vol. cxxxi., 
 p. 427. Pease and Pearce, Journ. Inf. Dis., vol. iii., p. 619. Bolton, 
 Proc. Roy. Soc., vol. Ixxiv., p. 135. Bierry and Mayer, Comptes Rendus 
 Soc. Biol., vol. Ivi., p. 1016. 
 
 Adrenotoxic Serum,. Bigart and Bernard, Comptes Rendus Soc. Biol., 
 
 1901, p. 161. Yates, Univ. Penns. Med. Bull., vol. xvi., p. 195. 
 Thyrotoxic Serum. Gontscharnkow, Cent. f. Allg. Path., vol. lix., p. 76. 
 
 Portis, Journ. Inf. Dis., vol. i., p. 127. 
 
 CHAPTER VIII 
 
 Gruber and Durham, Munch. Med. Woch., 1896, p. 285 ; ibid., 1899, 
 p. 1829. Charrin and Roger, Comptes Rendus Soc. Biol., 1889, p. 667. 
 Metchnikoff, Ann. Inst. Past., vol. v., p. 473. Durham, Journ. Path. 
 Bact., vol. iv., p. 13, and vol. vii., p. 240. Grunbaum, Lancet, Septem- 
 ber 19, 1896; ibid., Munch. Med. Woch., 1897, No - T 3- 
 
 Group Reactions. Pfaundler, Munch. Med. Woch., 1899, November 15, 
 p. 472. Posselt and Sagasser, Wien. Klin. Woch., 1903, p. 691. Park, 
 Journ. Inf. Dis., 1906, February, p. i. Frouin, Comptes Rendus Soc. 
 Biol., vol. Ixii., p. 154. Crendiropoulo and Amos, Reports of Egyptian 
 Sanitary Council, 1906. Bordet, Ann. Inst. Past., vol. xiii., p. 225. 
 
 Bacterio-precipitins. Kraus, Wien. Klin. Woch., 1897, August 12. 
 Norris, Journ. Inf. Dis., vol. i., p. 463. See Chapter IX. 
 
 Agglutination of Flagella. Smith and Reagh, Journ. Med. Res., vol. x., 
 p. 89. Buxton and Torrey, Journ. Med. Res., vol. xiv. 
 
 Theories as to the Mechanism of the Process. Nicolle, Ann. Inst. Past., 
 vol. xii., p. 161. Paltauf, Wien. Klin. Woch., 1897. Dineur, Bull. Acad. 
 Med. Belg., 1898, p. 652. Bordet, Ann. Inst. Past., vol. x., p. 195, and 
 vol. xiii., p. 225 (the latter especially). Lowit, Cent. f. Bakt. I. O., vol. 
 xxxiv., pp. 156, 251. Kraus and Joachim, ibid., vol. xxxvi., p. 662, and 
 xxxvii., p. 71. 
 
 Site of Origin of Agglutinin. Pfeiffer and Marx, Deut. Med. Woch., 
 1898, p. 47. Emden, Zeit. f. Hyg., vol. xxx. Wassermann, Deut. Med. 
 Woch., 1899, p. 141. Deutsch, Cent. f. Bakt., vol. xxviii., p, 45. Ruffer 
 and Crendiropoulo, vide ante. 
 
 Colloid Chemistry. Biltz, Zeit. f. Phys. Chem., vol. xlviii., p. 615. 
 Neisser and Friedemann, Munch. Med. Woch., 1904, p. 827. Bechhokl, 
 Zeit. f. Phys. Chem., vol. xlviii., p. 385. See also Chapter XII. 
 
 Absorption Test. Castellani, Zeit. f. Hyg., vol. xl., p. i. 
 
 Park, Journ. Med. Res., vol. vii. Hirschbruch, Arch. f. Hyg., vol. Ivi., 
 
428 BIBLIOGRAPHY 
 
 p. 280. Ballner, Arch. f. Hyg., vol. li., p. 245. Lowit, Cent. f. Bakt. I. O. 
 vol. xxxiv., pp. 156, 251. 
 
 Constitution of Agglutinins, A gglutinoids , etc. Wassermann, Zeit. f. 
 Hyg., vol. xlii., p. 267. Buxton and Vaughan, Journ. Med. Res., vol. xii., 
 p. 115. Eisenberg and Volk, Zeit. f. Hyg., vol. xl. Shibayama, Cent. f. 
 Bakt. I. O., vol. xlii., pp. 68, 144. Joos, Cent. f. Bakt. I. O., vol. xxxiii., 
 p. 762 ; ibid., Zeit. f. Hyg., vol. xxxvi., p. 422. Scheller, Cent. f. Bakt. I. O., 
 vol. xxxvi., p. 694. Smith and Reagh, Journ. Med. Res., vol. x., p. 89. 
 Buxton and Torrey, Journ. Med. Res., vol. xiv., April. Dreyer and 
 Jex-Blake, vide Dreyer, B. M. J., September 10, 1904, p. 564 ; Journ. 
 Path. Bact., vol. xi., p. i. 
 
 Modifications of Bacteria grown in Agglutinating Serum. Ainley Walker, 
 Journ. Path. Bact., vol. viii., p. 34. Welch, Johns Hopkins Bull., vol. 
 xiii., p. 291. Muller, Munch. Med. Woch., 1903, p. 56. Bail, Arch. f. 
 Hyg., vol. xlii., p. 307. Landsteiner, Wien. Klin. Woch., 1897, P- 439- 
 Marshall and Knox, Journ. Med. Res., vol. xv., p. 325. See also 
 Chapter XIII. 
 
 Htzmagglutinins. Landsteiner, Cent. f. Bakt. I. O., vol. xxvii., p. 357. 
 Landsteiner and Leiner, ibid., vol. xxxviii., p. 548. Hektoen, Journ.' Inf. 
 Dis., vol. iv., p. 297. Gay, Journ. Med. Res., vol. xvii., p. 321. Peskind, 
 Amer. Journ. Phys., 1903. Biffi, Ann. d'Ig. Sperim., vol. xiii., abstracted 
 in Bull. Inst. Past., vol. i., p. 526. Shattock, Journ. Path. Bact., vol. vi., 
 p. 303. Ford and Halsey, Journ. Med. Res., vol. xi., p. 403. Eisenberg, 
 Wien. Klin. Woch., 1901, p. 1020. Griinbaum, B. M. J., 1900, p. 1089. 
 
 CHAPTER IX 
 
 Precipitins in Normal Sera. Hoke, Wien. Klin. Woch., vol. xx., p. 347 ; 
 Rodet, Comptes Rendus de la Soc. Biol., vol. Iv., p. 1626. Noguchi, Bull. 
 Univ. Penns., vol. xv., p. 301. Ascoli, abstracted in Bull. Inst. Past., 
 vol. i., p. 343. 
 
 Specificity of Serum Precipitins. Vide Nuttall, loc. cit., in which the 
 main references are given. Uhlenhuth, Deut. Med. Woch., 1901, pp.- 82, 
 499. Wassermann and Schiitze, Berlin. Klin. Woch., 1901, p. 187 ; ibid., 
 I 93> P- J92. Ewing and Strauss, Proc. N. Y. Path. Soc., vol. ii., p. 152. 
 
 Ewing, ibid., vol. iii., p. 14. Deutsch, Cent. f. Bakt. I. O., vol. xxix., 
 
 E. 661. Stern, Deut. Med. Woch., 1901, p. 135. Wassermann, Congr. f. 
 in. Med., 1900. Strube, Deut. Med. Woch., 1902, p. 425. Lenossier and 
 
 Lemoine, Sem. Med., 1901, No. 4. Stern, Deut. Med. Woch., 1901, 
 
 P- 135- 
 
 Precipitoids, etc. Michaelis, Beit. z. Chem. Phys., vol. iv., p. 59. Ober- 
 mayer and Pick, Wien. Klin. Woch., 1903, No. 22, and 1904, p. 265. 
 Von Dungern, Cent. f. Bakt. I. O., vol. xxxiv., p. 355. 
 
 Kraus's Reaction. Wien. Klin. Woch., 1897, P- 73^ ; ibid., 1901, p. 693. 
 Panichi, Cent. f. Bakt. I. O., vol. xliii., p. 188. Norris, Journ. Inf. Dis., 
 vol. i., p. 463 (with bibliography). Hoke, Wien. Klin. Woch., vol. xx., 
 p. 347. Eisler, Wien. Klin. Woch, vol. xx., p. 377. Dopter, Comptes 
 Rendus de la Soc. Biol., vol. lix., p. 69. Smith and Reagh, Journ. Med. 
 Res., vol. x., p. 89. 
 
 Serum Precipitins. Tchistovitch, Ann. Inst. Past., vol. xiii., p. 406. 
 Bordet, ibid., p. 225. Myers, Cent. f. Bakt. I. O., vol. xxviii., p. 237. 
 Wassermann and Schutze, Berlin. Klin. Woch., 1901, p. 187. Nuttall, 
 Blood Immunity and Blood Relationship (Cambridge, 1904), in which 
 there is a full bibliography to the date of issue. Uhlenhuth, Deut. Med. 
 Woch., 1900, p. 734. Michaelis and Fleischmann, Zeit. f. Exp. Path, 
 and Ther., vol. i., p. 537. Von Dungern, Cent. f. Bakt. I. O., vol. xxxiv., 
 p. 355. Obermayer and Pick, Wien. Klin. Woch., 1903, No. 22 ; ibid., 
 I 93. P- 265. Oppenheimer, Beit. z. Chem. Phys., vol. iv., p. 259. 
 
BIBLIOGRAPHY 429 
 
 Precipitins for Crystalline Lens. Uhlenhuth, Deut. Med. Woch., 1906, 
 p. 1244 ; also Koch's Festschrift. 
 
 Practical Application. An excellent account of the technique is given 
 by Welsh and Chapman, Australian Medical Gazette, January 21, 1907- 
 See also Ewing, Clinical Pathology of the Blood, seond edition (Kimpton, 
 London). Graham-Smith and Sanger, Journ. Hyg., vol. iii., pp. 258, 354. 
 Buckmaster, Morphology of Blood (Murray, 1906). Bruck, Berlin. Klin. 
 Woch., 1907, pp. 793, 1510. Zebrowski, C. R. Soc. Biol., vol. Ixii., p. 603. 
 Uhlenhuth Deut. Med. Woch., 1906, p. 1244. Ziemke, Deut. Med. Woch., 
 1 90 1, pp. 424, 731. 
 
 Deviation of Complement. Neisser and Sachs, Berlin. Klin. Woch., 1905. 
 Uhlenhuth, Deut. Med. Woch., 1906, p. 1244. Muir and Martin, Journ. of 
 Hyg., 1906, July, p. 265. Friedberger, Deut. Med. Woch., 1906, p. 578. 
 
 Recognition of Foods. Pniiger, Arch. f. Phys., 1906, pp. 465, 540. 
 Schmidt, Bioch. Zeit., vol. v., p. 422. Uhlenhuth, Deut. Med. Woch., 
 1901, p. 780. Schutze, Zeit. f. Hyg., vol. xlvii., p. 144. 
 
 Recognition of Bones. Schutze, Deut. Med. Woch., 1903, p. 62. 
 
 CHAPTER X 
 
 Metchnikoff's views and experiments are fully set forth in his " L'lm- 
 munite dans les Maladies Infecteuses " (English translation by Binnie, 
 Cambridge University Press, 1905), with numerous references, and his 
 " Comparative Pathology of Inflammation " (translated by F. A. and E. H. 
 Starling, Kegan Paul, Trench and Co., 1893). Buchner, vol. xvii., p. 138; 
 Marchand, Arch. Med. Exp., vol. x., p. 253 ; Massart, Ann. Inst. Past., 
 vol. vi., p. 321 ; Petersson, Cent. f. Bakt. I. O., vol. xxxix., p. 423 ; 
 Savtschenko, Ann. Inst. Past., vol. xvi., p. 106 ; and numerous articles 
 from the French School published in the Annales de 1'Institute Pasteur, 
 Comptes Rendus de la Soc. Biol., etc. An excellent account of the main 
 phenomena is given in Adami's article on Inflammation in Clifford Allbutt's 
 " System of Medicine." 
 
 A bsorption of Tail of Tadpole. Mercier, Arch. Zool. Exper. , vol. v. , p. 151. 
 
 Cells in Peritoneal Fluid. Metchnikoff, loc. cit. Buxton and Torrey, 
 Journ. Med. Res., vol. xv., p. i. Kanthack and Hardy, Journ. Phys., 
 vol. xvii., p. 81. Durham, Journ. Path, and Bact., vol. iv., p. 338. 
 
 Phagocytosis in the Lungs. Briscoe, Journ. Path, and Bakt., 1907. 
 Baumgarten, Cent. f. Inn. Med., 1888, Zeigler's Beit., 1889, and Berlin. 
 Klin. Woch., 1884. Sanarelli, Cent. f. Bakt. I. O., vol. x., p. 514. Kant- 
 hack and Hardy, Phil. Trans., 1894, Journ. of Phys., 1894. 
 
 Enterokinase, etc. Delezenne, vide Levaditi, L'Immunite. 
 
 Opsonins. Sir Almroth Wright's researches have recently been pub- 
 lished in book-form (Studies in Immunization, Constable, 1909), to which 
 the reader is referred for a full account of the main researches on the 
 subject. See also the Practitioner, special number, May, 1908, and the 
 
 discussion on Phagocytosis, B. M. J., November 16, 1907. See also 
 
 Rimpau, Deut. Med. Woch., 1904, p. 
 B. M. J., 1902. Dean, Proc. Roy. Soc., 1905, vol. Ixxvi., p. 506, and 
 
 Neufeld and Rimpau, Deut. Med. Woch., 1904, p. 1458. Leishman, 
 
 May 30, 1907. Muir and Martin, B. M. J., 1907, p. 1783. Noguchi, 
 Journ. Exp. Med., vol. ix., p. 455. Rosenow, Journ. Inf. Dis., vol. iv., 
 p. 285. Gruber and Futaki, Munch. Med. Woch., vol. liii., p. 249. Hektoen 
 and Ruediger, Journ. Inf. Dis., vol. ii., p. 128. Bulloch and Atkin, Proc. 
 Roy. Soc., vol. Ixxiv., p. 379. Neufeld, Arb. der Kais. Gesundh., vol. xxv., 
 p. 164, and Berlin. Klin. Woch., 1908, p. 993. Lohlein, Ann. Inst. Past., 
 vol. xix., p. 647, and vol. xxx., p. 939. Weil, Cent. f. Bakt. (Ref.), 1908, 
 
 P- 337- 
 
 Technique of Opsonin Estimations, etc. Leishman, B. M. J., January n, 
 1902. Wright and Douglas, Proc. Roy. Soc., vols. Ixxii., Ixxxiii. Fleming, 
 
430 BIBLIOGRAPHY 
 
 Practitioner, May, 1908. Walker, R. E., Journ. Med. Res., vol. xix., 
 p. 237. Klien, Bull. Johns Hopkins Hosp., 1907, p. 245. Simon, Journ. 
 Amer. Med. Assoc., 1907, p. 139. Hektoen, Journ. Inf. Dis., vol. iii., 
 p. 434. Veitch, Journ. Path, and Bact., January, 1908. Brown, Journ. 
 Amer. Med. Assoc., 1908. Morland, Inaugural Dissertation (Bern, 1908). 
 Emery, Clinical Pathology and Bacteriology, third edition (H. K. Lewis, 
 1908). 
 
 Opsonic Index in Health. Bulloch, Trans. Path. Soc., vol. Ivi. Fleming, 
 Practitioner, May, 1908. Hollister, quoted by Bergey, Monthly Cyclop, 
 of Prac. Med., August, 1907. Urwick, B. M. J., 1905, July 22. Frazer, 
 Glas. Med. Journ., March, April, etc. 
 
 Opsonic Indices in Diseases. See under the appropriate headings below. 
 Accuracy of Opsonic Determinations. Greenwood, Proc. Roy. Soc. Med., 
 vol. ii., No. 5, where a full bibliography is given. 
 
 Nature of Opsonins. Crofton, Journ. Hyg., vol. v., p. 949. Chapin and 
 Cowie, Journ. Med. Res., vol. xvii., p. 213. Dean, Proc. Roy. Soc., 1907, 
 p. 399. Levaditi and Inman, Arb. Kais. Gesund., vol. xxv., p. 164. 
 Ledingham, Proc. Roy. Soc., 1907. McFarlane, Journ. Amer. Med. Assoc., 
 vol. xlix., p. 1178. Noguchi, Journ. Exp. Med., vol. ix., p. 455. Simon, 
 Journ. Exp. Med., vol. ix., p. 487. Eggers, Journ. Inf. Dis., vol. v., p. 268. 
 Graham, ibid., p. 273. Bohme, Munch. Med. Woch., 1908, p. 1475. 
 Neufeld and Bickel, Ar.b. Kais. Gesund., vol. xxvii., p. 310. Levaditi and 
 Inman, C. R. Soc. Biol., vol. Ixii., p. 683. Eggers, Journ. Inf. Dis., vol. v., 
 p. 263. Hektoen and Ruediger, Journ. Inf. Dis., vol. ii., p. 128. Hektoen, 
 Journ. Inf. Dis., vol. iii., p. 434. Browning, Journ. Med. Res., vol. xix., p. 201. 
 Specificity of Opsonins. Bulloch and Western, Proc. Roy. Soc., vol. 
 Ixxvi. Simon, Journ. Exp. Med., 1906, p. 651. Muir and Martin (W. B. M.), 
 B. M. J., 1906, vol. ii., p. 1783. Potter, Ditman, and Bradley, Journ. 
 Amer. Med. Assoc., vol. xlvii., p. 1793. Russell, Bull. Johns Hopkins 
 Hosp., 1907, p. 252. Hektoen, Journ. Inf. Dis., vol. v., p. 249. McFarland 
 and L'Engle, Journ. Amer. Med. Assoc., vol. xlix., p. 1178. 
 
 Thermolability of Opsonins. Wright and Douglas, Proc. Roy. Soc., 
 vol. Ixxii. Wright and Reid, ibid., vol. Ixxvii. Macdonald, Studies in 
 Path. Aberd. Uni., 1906. Rosenow, Journ. Inf. Dis., vol. iii., p. 683. 
 Muir and Martin, B. M. J., 1906, vol. ii., p. 1783 ; and Proc. Roy. Soc., 
 vol. Ixxix., p. 187. Neufeld and Hime, Arb. Kais. Gesund., vol. xxv., 
 p. 164. Dean, B. M. J., Nov. 16, 1907 (with an excellent general account 
 of the subject to date). See also under Nature of Opsonins. 
 
 Influence of Temperature. Bulloch and Atkins, Proc. Roy. Soc., vols. 
 Ixxii. and Ixxiii. Ledingham, ibid., 1908. 
 
 Influence of Source of Leucocytes. Wright and Douglas, Proc. Roy. Soc., 
 vol. Ixxiv. Bulloch and Ledingham, Studies in Path. Univ. Aberdeen, 
 1906. Fleming, Practitioner, May, 1908. Rosenow, Journ. Inf. Dis., 
 vol. iii., p. 683. Lowenstein, Zeit. f. Hyg., vol. lv., p. 429. Bassett- 
 Smith, Journ. Hyg., 1907, p. 115. Shattock and Dudgeon, Proc. Roy. 
 Soc. Med., vol. i., No. 6. 
 
 Virulence. See Chapter XIII. 
 
 Influence of Salts, etc. Wright and Reid, Proc. Roy. Soc., vol. Ixxvii. 
 Hamburger and Hekma, Biochem. Zeit., vol. ix., pp. 275, 512. Sellards, 
 Journ. Inf. Dis., 1908, June. Noguchi, Journ. Exp. Med., vol. ix., p. 455. 
 Influence of Dilution of Serum. Wright and Douglas, Proc. Roy. Soc., 
 vol. Ixxii. Emery, Trans. Med. Chi. Soc., vol. Ixxxix. Marshall, Journ. 
 Path. Bact., 1908, p. 378. 
 
 Influence of Thickness of Bacterial Emulsion. Tunnicliffe, Journ. Inf. 
 Dis., 1908, January. Walker, Journ. Med. Res., vol. xvi., p. 521. 
 
 Hcemopsonins. Neufeld and Bickel, Arb. Kais. Gesund., vol. xxvii., 
 p. 310. Neufeld and Topfer, Cent. f. Bakt. I. O., vol. xxxviii., p. 456. 
 Barratt, Wakelin, .Proc. Roy. Soc., 1905, p. 524. Keith, Proc. Roy. 
 Soc., 1906. 
 
 Aggressins. Bail, O., Wien. Klin. Woch., vol. xvii., p. 846; ibid., 
 
BIBLIOGRAPHY 43! 
 
 vol. 
 Woch 
 
 xviii., p. 428. Munch. Med. Woch., 1905, pp. 1212, 1865 ; Deut. Med. 
 h., 1905, p. 1788. Bail and Weil, Cent. f. Bakt. I. O., vol. xl., p. 371. 
 sermann and Citron, Cent. f. Bakt. I. O., vol. xliii., p. 373 ; and Deut. 
 
 Wassermann and Citron, Uent. l. .tsakt. l. u., vol. xlni., p. 373 ; 
 
 Hyg., vol. liii., p. 
 Cent. f. Bakt, vol. xli., p. 230. Weil, Deut. Med. Woch., 1906, p. 382 ; 
 
 Klin. Woch., 1905, p. 1102. Citron, Zeit. f. Hyg., vol. liii., p. 515 ; ibid., 
 
 ibid., Wien. Klin. Woch., 1905, p. 406; ibid., Arch. f. Hyg., vol. liv., p. 297 ; 
 and Berlin. Klin. Woch., 1905, p. 430. Salus, Arch. f. Hyg., vol. lv., 
 P- 335 >' ibid., Wien. Klin. Woch., vol. xviii., p. 660. Especially Lancet, 
 August 17 and 24, 1907 (Collected Studies, p. 317). 
 
 Vaccine Treatment. Wright's Collected Studies. Especially Lancet, 
 August 17 and 24, 1907 (Collected Studies, p. 327). Practitioner, May, 
 1908. Allen's Vaccine-Therapy (H. K. Lewis, 1908). Pfeiffer and Fried- 
 berger, Cent. f. Bakt. I. O., vol. xlvii., p. 503. See also under the separate 
 headings. 
 
 CHAPTER XI 
 
 Tuberculin Reaction. Koch, Deut. Med. Woch., 1890 and 1891. Wasser- 
 mann and Bruck, Deut. Med. Woch., 1906, p. 449 (in which there is a good 
 account of the earlier theories). (See also Chapter XIV.) 
 
 Modifications of Tuberculin Reaction. See under Tubercle. 
 
 Mallein Reaction. Vide Jowett's Blood-Serum Therapy, p. 156. Kraus 
 and Levaditi, vol. i. 
 
 Reactions in Gonococcal Infections. Irons, Arch. Int. Med., vol. i., p. 433. 
 Difference in Reactions between Healthy and Infected Persons. Lawson and 
 Stewart, Proc. Med. Chi. Soc., 1905. See also Allen's Vaccine Therapy. 
 
 Anaphylaxis to Toxins. Richet, Comptes Rendus Soc. Biol., vol. Iviii., 
 p. 109 ; Ann. Inst. Past., vol. xxi., p. 497 ; and Comptes Rendus Soc. Biol., 
 vol. Ixii., pp. 358, 643. Goodman, Journ. Inf. Dis., vol. iv., p. 509. 
 
 Hyper sensitiveness to Serum. Arthus, Comptes Rendus Soc. Biol., 
 vol. lv., p. 817. Nicolle, Ann. Inst. Past., vol. xxi., p. 128. Remlinger, 
 Comptes Rendus Soc. Biol., vol. Ixii., p. 23. 
 
 Theobald Smith's Phenomenon. Rosenau and Anderson, Journ. Med. 
 Res., vol. xv., p. 179 ; ibid., vol. xvi., p. 381 ; and Journ. Amer. Med. 
 Assoc., 1906, p. 1007. Besredka and Steinhardt, Ann. Inst. Past., vol. xxi., 
 p. 117. Besredka, Comptes Rendus Soc. Biol., vol. Ixii., p. 477; ibid., 
 vol. Ixiii., p. 294 ; ibid., Ann. Inst. Past., vol. xxi., p. 950 ; and Bull. 
 Inst. Past., vol. vi., p. 841. Gay and Southard, Journ. Med. Res., vol. xv., 
 p. 143. Vaughan and Wheeler, Journ. Inf. Dis., 1907, p. 476. Otto, 
 Munch. Med. Woch., 1907. Doerr, Wien. Klin. Woch., 1908. Gay and 
 Southard, Journ. Med. Res., vol. xviii., p. 407. Weil-Halle and Lemaire, 
 Comptes Rendus Soc. Biol., vol. Ixiii., p. 748. Lewis, Journ. Exp. Med., 
 vol. x. 
 
 Serum Disease. Von Pirquet and Schick, Die Serum-Krankheit (Leipzic 
 and Wien, 1905). Currie, Journ. Hyg., vol. vii., p. 35. Goodall, Journ. 
 
 Hyg., vol. vii. Hamburger and Moro, Wien. Klin. Woch., vol. xvi., p. 445. 
 
 vii., p. 807, and xx., p. 817. Wic 
 and Rostane, Bull. Soc. Med. des Hop. de Paris, 1905, p. 424. Marfan 
 
 Hamburger and Dehne, ibid., vol. xvii., p. 807, and xx., p. 817. Widal 
 
 and Le Play, ibid., p. 274. Netter, Comptes Rendus Soc. Biol., vol. lx., 
 p. 279. Park and Throne, Trans. Assoc. Amer. Phys., vol. xxi., p. 259. 
 Saunders, Interstate Med. Journ., 1908, p. 576. 
 
 CHAPTER XII 
 
 A good general outline of the subject may be found in Pauli's " Physical 
 Chemistry in the Service of Medicine," 1907, translated by Fischer (Chap- 
 man and Hall). See also Findlay's " Physical Chemistry in Medical and 
 Biological Science " (Longmans, Green and Co., 1905). 
 
 Biltz, Zeit. f. Phys. Chem., vol. xlviii., p. 615. Biltz and Siebert, Beitr. 
 z. Exp. Therap., 1905, p. 30. Field and Teague, Journ. Exp. Med., vol. 
 
43 2 BIBLIOGRAPHY 
 
 viii., p. 222 ; and vol. ix., p. 86. Teague and Buxton, Journ. Exp. Med., 
 vol. ix., p. 254. Craw, Proc. Roy. Soc., vol. Ixxvi., p. 179 ; and vol. Ixxvii., 
 p. 311, and other articles. Bordet, Ann. Inst. Past., vol. xvii., p. 161. 
 Nernst, Zeit. f. Electrochemie, vol. x., p. 377. Girard-Mangin and Henri, 
 Comptes Rendus Soc. Biol., vol. Ivi., p. 541, and numerous other articles 
 in the same periodical and in Comptes Rendus Acad. Sci. Landsteiner 
 and Stancovic, Cent. f. Bakt. I. O., vol. xli., p. 108. Landsteiner and 
 Urlirz, Cent. f. Bakt. I. O., vol. xl., p. 265. Flexner and Noguchi, Journ. 
 Exp. Med., 1906, p. 547. Bechhold, Zeit. f. Phys. Chem., vol. xlviii., 
 p. 385. Neisser, Cent. f. Bakt. I. O., vol. xxxvi., p. 671. Neisser and 
 Friedemann, Munch. Med. Woch., 1904, p. 465. Michaelis and Fleisch- 
 mann, Zeit. f. Exp. Path. u. Ther., vol. i., p. 547. Gengou, Ann. Inst. 
 Past., vol. xviii., p. 678. Dreyer, B. M. J., September 10, 1904. 
 
 Danysz Effect. Danysz, Ann. Inst. Past., vol. xvi., p. 331. Jacoby, 
 Hoffm. Beit., vol. iv., p. 212. Sachs, Cent. f. Bakt. I. O., vol. xxxvii., 
 p. 251. Craw, Proc. Roy. Soc., 1905. 
 
 Precipitation of Colloids. Spiro, Beit. z. Chem. Phys., vol. iv., p. 300. 
 Perrin, Comptes Rendus Acad. Sci., vol. cxxxvi., p. 564. 
 
 Hcemolysis by Silicic Acid. Landsteiner and Jagic, Wien. Klin. Woch., 
 vol. xvii., p. 63. 
 
 CHAPTER XIII 
 
 Phagocytosis in Peritoneum. Buxton and Torrey, Journ. Med. Res., 
 vol. xv., p. 5. Petterson, Cent. f. Bakt. I. O., vol. xl., p. 537. Weil, ibid., 
 vol. xliii., p. 190, and vol. xliv., p. 164 ; and Arch. f. Hyg., vol. Ixi., p. 293 ; 
 Journ. Inf. Dis., vol. iv., p. 582. Metchnikoff, L'lmrrhinite. Pierallini, Ann. 
 Inst. Past., vol. xi., p. 308. Wolff, Berlin. Klin. Woch., 1903, Nos. 17-20. 
 
 Bacterial Immunity in General. Metchnikoff, L'Immunite, especially 
 chapters vi. to x. Sauerbeck, Die Krise in der Immunitatsforschung, 
 Folia Serologica, vol. ii., p. i, with full bibliography. Hahn, Kolle, and 
 Wassermann's Handbuch, Fasc. xviii. and xix. Cole, Rufus, Zeit. f. 
 Hyg., vol. xlvi., p. 371. Kisskalt, Zeit. f. Hyg., vol. xlv., p. i. Hoke, Zeit. 
 f. Hyg., vol. xxv., p. 197. Bail, Arch. f. Hyg., vol. Hi., p. 272. Neufeld, 
 Arb. a. d. Kais. Gesundh., vol. xxviii., p. 125. W'erigo, Ann. Inst. Past., 
 vol. viii. Bail, Arch. f. Hyg., vol. liii., p. 272. Hoke, Cent. f. Bakt. I. O., 
 vol. xxxiv., p. 693. Sir Watson Cheyne, Lancet, June 27, 1908. 
 
 In Tick Fever. Levaditi and Manouelian, Comptes Rendus Soc. Biol., 
 vol. Ixi., p. 566, and vol. Ixii., pp. 619, 815. 
 
 Virulence. Walker, Ainley, Cent. f. Bakt. I. O., vol. xxxiii., p. 297. 
 Shaw, B. M. J., 1903, May 9, p. 1074. Cohn, Zeit. f. Hyg., vol. xlv., p. 61. 
 Pfeiffer, Koch's Festschrift, 1903. Stiirtz, Zeit. f. Klin. Med., vol. Hi., 
 p. 422. Bail, Wien. Klin. Woch., vol. xvii., p. 846. Petterson, Cent. f. 
 Bakt. I. O., vol. xxxviii., p. 73. Steinhardt, Proc. N.Y. Path. Soc., vol. iv. 
 Day, Journ. Inf. Dis., 1905, p. 569. Marshall and Knox, Journ. Med. 
 Res., vol. xv., p. 325. Friedberger, Cent. f. Bakt. I. O., vol. xliv., p. 32. 
 Rosenow, Journ. Inf. Dis., vol. iv., p. 285. 
 
 Formation of Envelope, etc. Metchnikoff, L'Immunite, chapter i. 
 Danysz, Ann. Inst. Past., vol. xiv., p. 641. Bordet, ibid., vol. xi., p. 177. 
 Gruber and Futaki, Munch. Med. Woch., 1906, p. 249. Preis, Cent. f. 
 Bakt. I. O., vol. xliv., p. 209. Bail, Wien. Klin. Woch., vol. xix., p. 1278. 
 Bail and Rubritius, Cent, f. Bakt. I. O., vol. xliii., p. 641. Stienon, 
 Comptes Rendus Soc* Biol., vol. xii., pp. 604, 841. 
 
 CHAPTER XIV 
 
 Staphylococci ; Staphylolysin. Van de Velde, Ann. Inst. Past., vol. xv., 
 p. 580. Kraus and Clairmont, Wien. Klin. Woch., 1900. Neisser and 
 Wechsberg, Zeit. f. Hyg., vol. xxxvi., p. 299. 
 
 Leucocidine. Van de Velde, loc. cit. Bail, Arch. f. Hyg., vol. xxxii., 
 p. 133. Neisser and Wechsberg, Munch. Med. Woch., 1902, p. 1261. 
 
BIBLIOGRAPHY 433 
 
 Immunity. Nuttall, Zeit. f. Hyg., vol. iv., p. 353. Wright and Windsor, 
 Journ. of Hyg., vol. ii., p. 397. Andrewes and Gordon, Suppl. Report 
 Med. Officer L.G.B., 1906, p. 141. Wright and Douglas, Proc. Roy. Soc., 
 vol. Ixxxii., and other articles in Wright's Collected Studies. 
 
 Vaccine Treatment, Opsonins, etc. Wright, Lancet, March 29, 1902 ; 
 B. M. J., May 7, 1904, etc. Allen's Vaccine Therapy. Chapman and 
 Cowie, Journ. Med. Res., vol. xvii., p. i. 
 
 Streptococcic Infections; Streptocolysin. Besredka, Ann. Inst. Past., 
 vol. x., p. 880. Casagrandi, quoted by Oppenheimer. 
 
 Toxins. Parascandalo, Wien. Klin., Woch. 1897, P- 86 1. Marmorek, 
 Ann. Inst. Past., vol. ix., p. 593. Roger, Comptes Rendus Soc. Biol., 
 vol. xliii., p. 538. Schenk, Wien. Klin. Woch., 1897, P- 937- Breton, 
 Comptes Rendus Soc. Biol., vol. Iv., p. 886. Simon, Cent. f. Bakt. I. O., 
 1903, pp. 308, 440. Schlesinger, Zeit. f. Hyg., vol. xliv., p. 428. 
 
 Serum Treatment. Marmorek, Ann. Inst. Past., vol. ix., p. 593 ; and 
 Berlin. Klin. Woch., 1902, No. 14. Besredka, Ann. Inst. Past., vol. xviii., 
 
 p. 363. Aronson, Deut. Med. Woch., 1903, p. 439. Tavel, Cent. f. Bakt. 
 I. O., vol. xxxiii., p. 212, and vol. xxxv., p. 513. Neufeld, Zeit. f. Hyg., 
 vol. xliv., p. 161. Simon, Cent. f. Bakt. I. O., vol. xxxv., pp. 308, 440. 
 
 Bordet, Ann. Inst. Past., 1897, p. 177. Wright, Clin. Journ., 1906, p. 78. 
 Neufeld, Zeit. f. Hyg., vol. xliv., p. 161. Sommerfeld, Cent. f. Bakt. I. O., 
 vol. xxxiii., p. 722. 
 
 Vaccine Treatment. Wright, Practitioner, May, 1908 ; and Lancet, 
 August 24, 1907. Douglas, Lancet, February 23, 1907. Crowe and 
 Wynn, B. M. J., August 8, 1908, p. 303. Sutcliffe and Bayley, Lancet, 
 August 10, 1907. Tunnicliffe, Journ. Inf. Dis., vol. v., p. 268. Banks, 
 Journ. Path. Bact., 1908, p. 113. 
 
 Pneumococcic Infections: Toxin. Klemperer, Berlin. Klin. Woch, 1891, 
 and Zeit. f. Klin. Med., vol. xx., p. 165. Washbourn, Journ. Path. Bact., 
 vol. iii., p. 214. Isaeff, Ann. Inst. Past., vol. vii., p. 259. Casagrandi, 
 quoted by Oppenheimer. Mennes, Zeit. f. Hyg., vol. xxv., p. 413. Carnot 
 and Fournier, Arch. Med. Exp., 1900, p. 357. 
 
 Serum Treatment. Washbourn, B. M. J., February 27, 1897, p. 510 ; 
 and with Eyre, ibid., 1899, p. 1247 ; and Journ. Path, and Bact., vol. v., 
 p. 13. Eyre, vide infra. Pane, Cent. f. Bakt. I. O., vol. xxi., p. 664. 
 Knauth, Deut. Med. Woch., 1905, p. 452. Castresana, Rev. de Ther., 
 1905, No. 1 8. Tyler, Journ. Amer. Med. Assoc., 1901, p. 1540. Mennes, 
 vide supra. 
 
 Vaccine Therapy, Opsonins, etc. MacDonald, Path. Studies, Univer. 
 Aberdeen. Eyre, Lancet, February 22, 1908. Neufeld and Rimpau, Zeit. 
 f. Hyg., vol. li., p. 283. Graham, Journ. Inf. Dis., vol. v., p. 273. Butler 
 Harris, Practitioner, May, 1908. Briscoe and Williams, ibid. 
 
 Gonococcic Infections; Toxin. Wassermann, Berlin. Klin. Woch., 
 1897, P- 685 ; and Zeit. f. Hyg., vol. xxvii., p. 298. Christmas, Ann. 
 Inst. Past., vol. xi., p. 609. Nicolaysen, Cent. f. Bakt. I. O., vol. xxii., 
 
 P- 305- 
 
 Serum Diagnosis, Immunity, etc. Torrey, Journ. Med. Res., vol.- xvii., 
 p. 347, and vol. xix., p. 471. Teague and Torrey, ibid., vol. xvii., p. 223. 
 Meakins, Johns Hopkins Hosp. Bull., 1907, p. 255. Ricketts, Infection 
 and Immunity. Bruckner and Christeanu, Comptes Rendus Soc. Biol., 
 vol. Ix., May, June. Miiller and Oppenheim, Wien. Klin. Woch., vol. xix., 
 p. 894. Bruck, Deut. Med. Woch., 1906, p. 1368. Vannod, ibid., 1906, 
 p. 1984. Rogers, Cent. f. Bakt. I. O., vol. xxxix., p. 279. 
 
 Vaccine Treatment, Opsonins, etc. Wright, Lancet, August 17 and 24, 
 1907. Allen, Vaccine Therapy. Rons, Arch. Int. Med., vol. i., p. 433. 
 Cole and Meakins, Bull. Johns Hopkins Hosp., 1907, p. 223. Butler and 
 Long, Journ. Amer. Med. Assoc., 1908, p. 744. 
 
 Meningococcic Infections : Toxins, Immunity. Lepierre, Journ. Phys. 
 et Path. Gen., vol. v., p. 547. Houston and Rankin, B. M. J., Novem- 
 ber 16, 1907. Davis, Journ. Inf. Dis., vol. ii. 
 
 2 8 
 
434 BIBLIOGRAPHY 
 
 Agglutination. Kutscher, Deut. Med. Woch., 1906, p. 1849. Alice 
 Taylor, Lancet, July 6, 1907. 
 
 Serum Treatment. Kolle and Wassermann, Deut. Med. Woch., 1906, 
 p. 609. Ruppel, ib'id., 1906, p. 1366. Markl, Cent. f. Bakt., vol. xliii., 
 p. 95. Levy, Deut. Med. Woch., 1908, p. 139. Emmett Holt, B. M. J., 
 October 31, 1908. Flexner and Jobling, Journ. Exp. Med., 1908, pp. 141, 
 690. Jochman, Deut. Med. Woch, 1906, p. 788. Meyer and Ruppel, 
 Mediz. Klin., 1907, No. 4, and Cent. f. Bakt. I. O., 1907. Wassermann, 
 Deut. Med. Woch., 1907, p. 1585. 
 
 Vaccine Therapy, Opsonins, etc. McKenzie and Martin, ibid., October 31, 
 1908, and Journ. Bact., 1908, vol. xii., p. 539. Davis, Journ. Inf. Dis., 
 vol. ii., and vol. iv., p. 538. Houston, B. M. J., November 16, 1907. 
 Mackenzie, ibid., June 15, 1907. 
 
 Malta Fever. Wright and Smith, Lancet, March 6, 1897. Birt and 
 Lamb, Lancet, September 9, 1899. Eyre, J. W. H., and Shaw, H. E. A., 
 Report of Royal Society's Comm. on Med. Fever, part v. Bassett-Smith, 
 Journ. Trop. Med. and Hyg., 1907, and Journ. Hyg., vol. vii., p. 115. 
 Eyre, Lancet, 1908, June 13, 20, and 27. 
 
 Tubercle ; Tuberculin Reaction. Koch, Deut. Med. Woch., 1890, p. 1028, 
 and 1891, pp. 101, 1188. (See also 1890, p. 1053 et seq.) Babes, Zeit. f, 
 Hyg., vol. xxxii. Marmorek, Comptes Rendus Soc. Biol., 1903, p. 1650. 
 Ehrlich, Inter. Kong. f. Hyg., 1900. Trudeau, Baldwin, and Kinghorn, 
 Journ. Med. Res., vol. xii., p. 169. Weil and Nakajama, Munch. Med. 
 Woch., 1906, p. 1001. Cohn, Berlin. Klin. Woch., 1908, p. 1309. Richet, 
 Comptes Rendus Soc. Biol., 1905, p. 109. Citron, Berlin. Klin. Woch., 
 I 97> P- TI 35- Marmorek, Lancet, December 12, 1903 (in diagnosis 
 especially). Arloing, Journ. de Phys. et Path. General, 1903, p. 677. 
 V. Bergmann, Deut. Med. Woch., 1890, p. 1073, and Munch. Med. Woch., 
 p. 824. Beck, Arch. f. Kinderheilkunde, 1903, p. i. Lowenstein, Kraus, 
 and Levaditi, vol. i., p. 1019 (with full bibliography). Lingelsheim, 
 Deut. Med. Woch., 1898, p. 583. Armand-Delille, These de Paris, 1903, 
 abs. in Bull. Inst. Past., vol. ii., p. 73. For an account of the main forms 
 of the tuberculins, see Allen's Vaccine Therapy, and Gamble, Pharma- 
 ceutical Journal, February 16, 1909. 
 
 Tuberculinin Treatment. Koch, Deut. Med. Woch., 1891, No. 3. Denys, 
 Comptes Rendus Congr. Tuberc., 1898, p. 497. Petruschky, Berlin. Klin. 
 Woch., 1899, pp. 1 1 20, 1141. Moller and Kayserling, Zeit. f. Tuberkulose, 
 
 1902, p. 4. Bandelier, Deut. Med. Woch., 1898, p. 798 ; ibid., Zeit. f. Hyg., 
 vol. xliii., p. 335. Pardoe, Lancet, December 16, 1905. Spengler, Deut. 
 Med. Woch., 1897, No. 36. Lowenstein and Rappoport, Zeit. f. Tuber- 
 kulose, vol. v., p. 6. Stone and Miller, Medical Record, March 28, 1908. 
 Hamburger, Munch. Med. Woch., vol. Iv., p. 1741. 
 
 Serum Treatment. Maragliano, Berlin. Klin. Woch., 1903, pp. 563, 593. 
 Marmorek, Berlin. Klin. Woch, 1903, p. 1108. 
 
 Tuberculin in Immunization of Animals. Macfadyen, Journ. Comp. Path, 
 and Therap., 1901, p. 136; 1902, p. 60. Behring, Berlin. Klin. Woch., 
 
 1903, p. 233, and Deut. Med. Woch., 1903, p. 689. Behring, Romer, and 
 Ruppel, Beitr. zur Exp. Therap., vol. v. Pearson and Gilliland, Univ. 
 Penns. Med. Bull., vol. xviii., No. 2. Neufeld, Deut. Med. Woch., 1904. 
 Baumgarten, Berlin. Klin. Woch., 1905, No. 3. Vallee and Rossignol, 
 Bull. Soc. Med. Vet. Pratique, 1906, p. 39. 
 
 Cuti-Re action. V. Pirquet, Klin. Studien iiber Vaccination and Vac- 
 cinale Allergic (Deut. Wien., 1907) ; Berlin. Klin. Woch., 1907. Vallee, 
 Comptes Rendus Acad. des Science, 1907, No. 22. Ferrand and Lemaire, 
 La Presse Medicale, 1907, p. 617. Dufour, Bull. Soc. Med. Hop. de Paris, 
 1907. Engel and Bauer, Berlin. Klin. Woch., 1907, p. 1169. Lignieres, 
 Bull. Soc. Cent. Med. Vet., 1907, p. 517. Wolff-Eisner and Teichman, 
 Berlin. Klin. Woch., 1908, p. 65. 
 
 Ophthalmo-Reaction. Wolff-Eisner, Berlin. Klin. Woch., 1907. Cal- 
 mette, Comptes Rendus Acad. des Sciences, 1907. Vallee, ibid. Moro 
 
BIBLIOGRAPHY 435 
 
 and Dagonoff, Wien. Klin. Woch., 1907, August. Calmette, La Clinique, 
 August, 1907. Chantemesse, Comptes Rendus Acad. de Med., July 20, 
 1907. Deut. Med. Woch., September 26, 1907. 
 
 Vaccine Treatment. Vide numerous articles by Wright and his fellow- 
 workers (in his Collected Studies), especially Clinical Journal, Novem- 
 ber 9, 1904. Trans. Med. Chi. Soc., vol. Ixxxix., and the succeeding 
 articles in the discussion, Lancet, August 17 and 24, 1907. Reyn and 
 Peterson, Lancet, April 4, 1908. Latham, Spitta, and Inman, Proc. Roy. 
 Soc. Med., April, 1908. Torton, International Clinics (eighteenth series), 
 vol. ii., p. 23. Riviere, B. M. J., October 26, 1907. Whitfield, Practitioner, 
 May, 1908. Briscoe and Williams, ibid. Allen, Vaccine Therapy. Car- 
 malt Jones, Science Progress, April, 1909. Patterson, Lancet, Janu- 
 ary 25, 1908. Inman, ibid. 
 
 Typhoid Fevsr ; Toxin. Chantemesse, Prog. Med., 1898, p. 245 ; Deut. 
 Med. Woch., 1907, p. 1572. Presse Med., 1904, p. 681. Macfadyen and 
 Rowland, Proc. Roy. Soc., vol. Ixxi., p. 77. Conradi, Deut. Med. Woch., 
 1903, p. 26. Pfeiffer and Kolle, Zeit. f. Hyg., vol. xxi., p. 203. Besredka, 
 Ann. Inst. Past., vol. xx., pp. 149, 304. Neisser and Shiga, Deut. Med. 
 Woch., 1903, p. 6r. 
 
 Immunity, Bactericidal Power of Blood, Opsonins, etc. Leishman, Jour. 
 R. A. M. C., 1907. Evans, Laming, Journ. Path. Bact., 1904, p. 42. 
 Shiga, Berlin. Klin. Woch., 1904, p. 79. Richardson, Journ. Med. Res., 
 vol. xiii. Wright, Lancet, September 14, 1901. Harrison, Journ. R. A. 
 M. C., 1907, p. 472. Stern and Korte, Berlin. Klin. Woch., 1904. Klien, 
 Johns Hopkins Hosp. Bull., 1907, p. 245. Neufeld and Kuhn, Arb. a. d. 
 K. Gesundh., vol. xxv., p. 164. 
 
 Vaccine Treatment (Prophylactic). Wright, Short Treatise on Anti- 
 Typhoid Inoculation (Constable, 1904) ; ibid., Lancet, September 6, 1902 ; 
 ibid., B. M. J., October 10, 1903. Luxmore, Journ. R. A. M. C., 1907. A 
 good account of the subject is by Netter, Bull. Inst. Past., vol. iv., pp. 873, 
 921, 969, and 1024. Shiga, Berlin. Klin. Woch., 1904, p. 78. Friedberger 
 and Moreschi, Deut. Med. Woch., 1906, p. 1986. 
 
 Curative. Richardson, Boston Med. and Surg. Journ., vol. Ivii., p. 449. 
 Cholera : Toxin. Wassermann, Zeit. f. Hyg., vol. xiv., p. 35. Westbrook 
 Ann. Inst. Past., vol. viii., p. 318. Pfeiffer, Zeit. f. Hyg., vol. xi., p. 373, 
 and vol. xvi., p. 268, and vol. xx., p. 198. Metchnikoff, Roux, and Tau- 
 relli Salimbeni, Ann. Inst. Past., vol. x., p. 257. Kraus, Wien. Klin. 
 Woch., vol. xix., p. 655. Brau and Demei, Ann. Inst. Past., vol. xx., 
 p. 578. Macfadyen, Cent. f. Bakt., vol. xlii., p. 365. 
 
 Serum Treatment. Kraus, Wien. Klin. Woch., 1909, No. 2. Macfadyen, 
 Lancet, August 25, 1906. 
 
 Vaccine Prophylaxis. Haffkine, Bull. Inst. Past., vol. iv., pp. 697, 737. 
 Fischera, Cent. f. Bakt. I. O., vol. xli., pp. 576, 671, and 771 (with full 
 bibliography). 
 
 Plague : Prophylaxis. Haffkine, B. M. J., June 12, 1897 '< ibid., B. M. J., 
 September 24, 1898 ; ibid., Proc. Roy. Soc., 1899., vol. Ixv. ; ibid., Gov. 
 Central Press, 1900, 1903, 1904. Burch, N. Y. Med. Journ., September, 
 1902. Forsyth, Lancet, December 12, 1903. Lustig and Galeotti, 
 B. M. J., October 9 and November 27, 1897. Bannerman, Cent. f. Bakt. 
 I. O., vol. xxix., p. 857. Kolle and Otto, Deut. Med. Woch., 1904, p. 493. 
 Lustig and Galeotti, Deut. Med. Woch., 1897, P- 22 7- 
 
 Sero-Therapy. Yersin, Ann. Inst. Past., vol. xi., p. 8-1. Metchnikoff, 
 ibid., vol. xi., p. 737. Zabolotny, ibid., vol. xiii., p. 833. Calmette and 
 Salimbeni, ibid., vol. xiii., p. 865. Dugardin-Beaumetz, Bull. Inst. Past., 
 1906, p. 453. Choksy, Report on Treatment of Plague, Bombay, 1906, 
 and Lancet, 1900, p. 291. Clemow, Lancet, May 6, 1899, p. 1212. Cairns, 
 Lancet, 1903, May 9. Symmers, Cent. f. Bakt. I. O., vol. xxv., p. 460. 
 Markl, Zeit. f. Hyg., vol. xlii., p. 244. 
 
 Glanders : Immunity. Nicolle, Ann. Inst. Past., vol. xx., pp. 625, 698, 
 and 801 (especially p. 828). Kleine, Zeit. f. Hyg., vol. xliv., p. 183. 
 
 282 
 
436 BIBLIOGRAPHY 
 
 Mallein. The directions given at the Royal Veterinary College, London, 
 are given in Hewlett's Serum-Therapy. See also Jowett's Blood -Serum 
 Therapy. 
 
 The only full account of the subject is in Kraus and Levaditi, vol. i., 
 p. 1090 (Wladimoroff). 
 
 Agglutination. Bonome, Cent. f. Bakt. I. O., vol. xxxviii., p. 60 1. 
 Heanly, Lancet, February^, 1904, p. 364. Feodorowsky, Bull. Inst. 
 Past., vol. ii., p. 127. 
 
 Dysentery : Toxin. Rosenthal, Deut. Med. Woch., 1904, p. 235. Todd, 
 ,/ Journ. of Hyg., vol. iv., p. 480. Conradi, Deut. Med. Woch., 1903, p. 26. 
 Ludke, Berlin. Klin. Woch., 1906, pp. 3, 54. Besredka, Ann. Inst. Past., 
 vol. xx., p. 304. Neisser and Shiga, Deut. Med. Woch., 1903, p. 61. 
 
 Serum. Kruse, Deut. Med. Woch., 1903, pp. 6, 49. Shiga, Cent. f. 
 Bakt. I. O., 1903, No. 7 ; ibid., Deut. Med. Woch., 1901, pp. 744, 
 765, and 783 ; ibid., Zeit. f. Hyg., vol. xli., p. 355 (in Ehrlich's Collected 
 Studies), and vol. lx., p. 75. Vallard and Dopter, Ann. Inst. Past., vol. xx., 
 p. 321. Flexner, Bull. Johns Hopkins Hosp., vol. xi., p. 231. Besredka, 
 vide supra. Doerr in Kraus and Levaditi's Hardbuch (with bibliography). 
 Coyne and Auche, Comptes Rendus Soc. Biol., vol. Ixiv., p. 829. Ruffer 
 and Willmore, B. M. J., October 17, 1908, vol. ii., p. 1176. Heller, Cent, 
 f. Bakt. I. O., vol. xlii., p. 30. 
 
 Vaccine Treatment. Shiga, Cent. f. Bakt. I. O., vol. xxxiv., p. 392. 
 Forster, Indian Med. Gaz., 1907, p. 201 (quoted by Allen). Newman, 
 Lancet, May 16, 1908, p. 1410. Kolle and Strong, Deut. Med. Woch., 
 1906, p. 413. 
 
 Anthrax: Toxin. Conradi, Zeit. f. Hyg., vol. xxxi., p. 287 (with full 
 bibliography to date). 
 
 Immunity, Serum Reactions, etc. Sobernheim, Berlin. Klin. Woch., 
 1897, p. 910 ; ibid., Zeit. f. Hyg., 1899, p. 891. Bail, Cent. f. Bakt. I. O., 
 vol. xxvii., p. 10 ; ibid., vol. xxxiii., pp. 343, 610 ; vol. xxxvi., pp. 266, 
 287 ; vol. xxxvii., p. 270. Bail and Petterson, ibid., vol. xxxiii., p. 756, 
 and vol. xxxiv., pp. 450, 540. Gengou, Ann. Inst. Past., vol. xiii., p. 642. 
 Hektoen, Journ. Inf. Dis., vol. iii., p 103. Horton, ibid., vol. iii., p. no. 
 Ascoli, Zeit. f. Hyg., vol. Iv., p. 44. Bandi, Cent. f. Bakt., vol. xxxvii., 
 p. 464. Gruber and Futaki, Deut. Med. Woch., 1906, p. 1589. Cler, 
 Arch. Sc. Med., vol. xxix., 1905. 
 
 Serum Treatment. Legge, Lancet, March 25, 1905, in which a good 
 account of the subject and the more important references are given. 
 
 Diphtheria: Immunization to the Bacilli. Bandi, Cent. f. Bakt. I. O., 
 vol. xxxiii., p. 535. Rist, Comptes Rendus Soc. Biol., 1903, p. 978. 
 Lipstein, Cent. f. Bakt. I. O., vol. xxxiii., p. 305. 
 
 Opsonic Action. Tunnicliffe, Journ. Inf. Dis., vol. v. Reque, ibid., 
 vol. iii., p. 441. 
 
 No literature concerning the use of diphtheria antitoxin need be given. 
 
 Tetanus. A full account of the toxin is given in Oppenheimer, with full 
 bibliography. 
 
 Action on the Nervous System. Gumprecht, Deut. Med. Woch., 1894, 
 p. 546. Meyer and Ransom, Arch. Exp. Path., vol. xlix., p. 369. Marie 
 and Morax, Ann. Inst. Past., vol. xvi., p. 818, and vol. xvii., p. 335. Roux 
 and Borrel, ibid., vol. xii., p. 225. Vaillard and Vincent, ibid., vol. v., 
 p. i. Marie, Bull. Inst. Past., vol. i., p. 633. Fletcher, Brain, 1903, 
 
 P- 383- 
 
 Immunity. Vide Metchnikoff, L'Immunite, especially p. 179 (English 
 edition, p. 169) and p. 412 (p. 392). In the same work much information 
 will be found regarding the action of tetanus toxin on different animals. 
 
 Antitoxin. Vide Hewlett's Serum-Therapy, where the process of manu- 
 facture is given. 
 
 Local Application of Antitoxin. Calmette, Comptes Rendus Acad. Sci., 
 vol. cxxxvi., p. 1150. 
 
 Syphilis. The literature of the serum diagnosis of syphilis has already 
 
BIBLIOGRAPHY 437 
 
 assumed formidable proportions. Wassermann, Neisser, Bruck, and 
 Schucht, Zeit. f. Hyg., vol. lv., p. 451. Wassermann, Berl. Klin. Woch., 
 I9 O 7. P- 1599- Wassermann and Plaut, Deut. Med. Woch., 1906, p. 1769. 
 Wassermann and Meier, ibid., 1907, p. 1287. Neisser, Bruck, and Schucht, 
 Deut. Med. Woch., 1906, p. 1937. Bruck and Stern, ibid., 1908, p. 401. 
 Schutze, Berlin Klin. Woch., 1907, p. 126. Levaditi and Marie, Comptes 
 Rendus Soc. Biol., vol. Ixii., p. 872. Levaditi and Yamanouchi, ibid., 
 vol. Ixiii., p. 740, and vol. Ixiv., pp. 275, 349, and 720. Marie, Levaditi, 
 and Yamanouchi, ibid., p. 169. Citron, Berlin. Klin. Woch., 1907, p. 1370. 
 Michaelis, ibid. Meier, ibid., p. 1636. Weil and Braun, Berlin. Klin. 
 Woch., 1907, p. 1570, and Wien. Klin. Woch., 1908, p. 151. Klausner, 
 Wien. Klin. Woch., 1908, p. 214. Landsteiner, Miller and, Potzl, Wien. 
 Klin. Woch., 1907. Forges and Meier, Berlin. Klin. Woch., 1908, p. 731. 
 Elias, Neubauer, Forges, and Salmon, Wien. Klin. Woch., 1908, p. 748. 
 Simplified forms of technique are given by Noguchi, Journ. Exp. Med., 
 1909, p. 392, and Caulfeild, Journ. Med. Res., 1908, p. 507. 
 
 Rabies. -An excellent account of modern views on the immunity to 
 rabies is given by Marie, Bull. Inst. Past., vol. vi., 1908, pp. 705 and 753. 
 
 See also Schneder, Zeit. f. Hyg., vol. xlii., p. 362. Remlinger, Bull. 
 Inst. Past., vol. ii., pp. 753 and 792. 
 
INDEX OF AUTHORITIES CITED 
 
 ABEL, 1 18, 173 
 Albarran, 193 
 Allen, 367, 370, 384 
 Amos, 193, 209 
 Anderson, 312 
 Andrewes, 293, 359 
 Armand-Delille, 170, 196 
 Arrhenius, 80, 83 et seq., 119 
 Arthus, 311 
 Arzt, 417 
 Ascoli, 193, 233 
 Atkinson, 63, 67 
 Axenfeld, 366 
 Ayer, 188, 190 
 
 Bail, 220, 291, 292, 308, 340, 406 
 
 Bannerman, 404 
 
 Bassenge, 392 
 
 Bassett-Smith, 291, 375 
 
 Baumgarten, 250 
 
 Bechhold, 217 
 
 Beebe, 192 
 
 Bearing, von, 61, 132, 379 
 
 Bergengriin, 247 
 
 Bernard, 193 
 
 Besredka, 28, 1 13, 172, 190, 242, 314, 
 
 393 
 
 Bickel, 284 
 Bier, 31 
 Bierry, 192 
 
 Biltz, 90, 217, 324, 327, 328 
 Blum, no 
 Blumenthal, 106 
 Bolton, -.94 
 Bordet, 88, 153, 168, 180, 209, 212, 
 
 219, 228, 299, 322 
 Bousfield, 374 
 Bram Pusey, 197 
 Bredig, 320 
 Brieger, 54, 61, 411 
 Briscoe, 180, 248, 286, 333, 374 
 Brodie, 70 
 Browning, 286 
 Briick, 169, 285, 306 
 Brunton, Sir L., 224 
 Buchner, 54, 58, 86, 179 
 
 Bulloch, 127, 139, 1 80, 184, 259, 269 
 
 270, 286, 289, 290, 348, 378 
 Buxton, 215, 352 
 
 Cairns, 402 
 
 Calmette, 70, no, 122, 199, 33, 
 
 393- 403. 4 T 3 
 Casagrandi, 365 
 Castellani, 217 
 Cattani, 334 
 Centanni, 196, 418 
 Chantemesse, 304. 391 
 Chapin, 283 
 Charrin, 193, 204 
 Chatenay, 112 
 Chauveau, 18, 35 
 Cherry, 48, 70, 329 
 Choksy, 403 
 Citron, 293 
 Cler, 407 
 Cohn, 54, 411 
 Cole, 352 
 Collins, 220 
 Conradi, 181, 396, 405 
 Corpechot, 193, 197 
 Courmont, 107 
 Cowie, 283 
 
 Crendiropoulo, 209, 211, 214 
 Crofton, 283 
 Currie, 309 
 
 D 
 
 Danysz, 327 
 Davis, 372 
 Dean, 263, 283 
 Delaware, 193 
 Delbriick, 155 
 Delecarde, 122 
 Delezenne, 182, 196, 256 
 Denys, 179, 257 
 Descatello, 224 
 Dineur, 211 
 Dmitrevsky, 108, 127 
 Doerr, 293 
 Douglas, 257, 286 
 Doyon, 107 
 Dreyer, 87, 216, 324 
 
 439 
 
440 
 
 INDEX OF AUTHORITIES CITED 
 
 Duclaux, 54 
 
 Dudgeon, 262 
 
 Dungern, von, 159, 192, 230, 327, 
 
 39i 
 Durham, 204 
 
 Ehrlich, 16, 33, 45, 69 et seq., 87, 94, 
 
 105, 125, 142 et seq., 200 
 Eisenberg, 216, 230, 343 
 Eisenzimmer, 418 
 Emden, von, 214 
 Ewing, 235 
 Eyre, 14, 34, 266, 288, 365, 366, 374 
 
 j.' 
 
 Falloise, 182, 183 
 
 Ferran, 400 
 
 Field, 320, 329 
 
 Figari, 193 
 
 Flexner, 155, 373 
 
 Forner, 418 
 
 Forster, 397 
 
 Frankel, 207 
 
 Friedberger, 162, 171, 281, 392 
 
 Friedemann, 217 
 
 Frouin, 207 
 
 G 
 
 Gay, 171, 172, 188, 190, 223, 312 
 
 Girard-Mangin, 326, 329 
 
 Gengou, 153, 169, 182 
 
 Golovine, 197 
 
 Goodall, 316 
 
 Goodman, 132, 310 
 
 Gordon, 359 
 
 Gruber, 204, 208, 211 
 
 Guedini, 170 
 
 Guldberg, 81 
 
 Guseff, 180 
 
 H 
 
 Haffkine, 401, 403 
 Hahn, 182 
 
 Hamburger, 297, 316 
 Hankin, 54, 179 
 Hardy, 252, 320 
 Harris, 55 
 Harris, Butler, 395 
 Hartoch, 416 
 Hekma, 297 
 Hektoen, 222 
 Henri, 326, 329 
 Herter, 115 
 Hewlett, 198 
 Hime, 285 
 Hoffmeister, 155 
 Hogyes, 419 
 Hoke, 231, 341 
 Houston, 371, 372, 373 
 
 Ignowtowsky, 44 
 Inman, 268 
 Irons, 304 
 Isaeff, 204, 364 
 
 J 
 
 acobi, 55 
 
 agic, 326 
 
 enner, 20 
 
 ex-Blake, 216, 324 
 
 ochmann, 373 
 
 ones, Wharton, 224 
 Joos, 215 
 Jungano, 196 
 
 K 
 
 Kanthack, 70, 184, 244, 252, 334 
 
 King, 132 
 
 Kirschbruch, 219 
 
 Kitashima, 61 
 
 Klein, 188 
 
 Klemperer, 364 
 
 Klien, 263 
 
 Knorr, 73, 133 
 
 Koch, 300, 305 
 
 Kolle, 373, 392 
 
 Korschun, 181 
 
 Kossel, 70 
 
 Kraus, 209, 226 
 
 Kruse, 397 
 
 Kutscher, 372 
 
 Kyes, 155 
 
 Lamb, 232 
 
 Lambotte, 181 
 
 Landsteiner, 190, 220, 222, 326, 417 
 
 Lastschenko, 181, 184 
 
 Laubry, 170 
 
 Lazar, 184 
 
 Leclef, 257 
 
 Ledingham, 128, 259, 286, 295, 416 
 
 Leishman, 261, 284 
 
 Le Play, 193, 197 
 
 Levaditi, 180, 183, 286, 335, 336, 417 
 
 Levy, 373 
 
 Liepmann, 195 
 
 Lignieres, 387 
 
 Lindemann, 193 
 
 Lingelsheim, von, 379 
 
 Lipstein, 409 
 
 Loffler, 173 
 
 Longcope, 180 
 
 Lubarsch, 139, 183 
 
 Lustig, 403 
 
 M 
 
 Macdonald, 265, 282, 365 
 Macfadyen, 58, 181, 390, 396, 399 
 Mackenzie, 373 
 
INDEX OF AUTHORITIES CITED 
 
 44 I 
 
 Madsen, 41, 76, 80, 82, 83 et stq., 86, 
 
 93. H9 
 
 Malvoz, 211, 406 
 
 Manouelian, 335 
 
 Maragliano, 383 
 
 Marenghi, 71 
 
 Marie, 419 
 
 Markl, 257, 259, 402 
 
 Marmorek, 17, 306, 583 
 
 Martin, 282, 373 
 
 Martin, S., 54 
 
 Marx, 184, 214 
 
 Massart, 299 
 
 McClintock. 132 
 
 McFarland, 383 
 
 Meakins, 177, 370 
 
 Mendel, 55 
 
 Mennes. 257 
 
 Metchnikoff, 43, 59, 60, 93, 107, 109, 
 113, 125, 134, 137, 152, 166, 179, 
 184, 190, 204, 238 et seq., 289, 293, 
 
 335, 46 
 Meyer, 411, 415 
 Morax, 107 
 
 Moreschi, 171, 172, 392 
 Morgenroth, 45, 88, 177, 181 
 Moro, 381 
 
 Muir, 88, 163, 164, 282, 286, 341 
 Miiller, 220, 417 
 Myers, 82, 228, 234 
 
 N 
 
 Nefedieff, 193 
 Neisser, 50, 70, 173, 217, 236, 323, 
 
 324. 4 J 5 
 Nernst, 87 
 Netter, 317 
 
 Neufeld, 257, 284, 285, 365 
 Nicolle, 170, 209, 211, 219 
 Nikayama, 292 
 Nocard, 381 
 Noguchi, 155, 232 
 Norris, 227 
 Norton, 395 
 Nuttall, 139, 228, 232, 250 
 
 Obermayer, 232, 234 
 Osborne, 55 
 Otto, 207, 312 
 
 P 
 
 Paget, Sir James, 361 
 Pane, 366 
 Panichi, 209 
 Parascandalo, 360 
 Park, 206, 220 
 Parvu, 170 
 
 Pasteur, 15, -8, 19, 34 
 Pauli, 298, 321, 324, 327 
 
 Pearce, 191 193 
 
 Perrin, 320 
 
 Peskind, 223 
 
 Petrie, 181 
 
 Petterson, 182 
 
 Pfeiffer, 58, 140, 162, 171, 184, 214, 
 
 281, 392 
 Pick, 232, 234 
 Pierallini, 353 
 
 Pirquet, von, 303, 308, 315, 381 
 Ponder, 296 
 Porges, 417 
 Posselt, 206 
 Potzl, 417 
 Pozerski, 170 
 
 R 
 
 Rankin, 372 
 Ransom, 106, 411, 415 
 Reagh, 215 
 Reid, 282, 375 
 Remy, 406 
 Richet, 309 
 Ricketts, 369 
 Rimpau, 257, 365, 392 
 Rist, 409 
 Roger, 204, 221 
 Rdmer, 106, 108, 351, 366 
 Rosenau, 297, 312, 
 Rosenfeld, 418 
 Rosenow, 282, 291, 343 
 Rossignol, 387 
 Rostane, 317 
 Roux, 86, 93, 414 
 Rowland, 58 
 Ruffer, 211, 214 
 Ruppel, 373 
 
 S 
 
 Sachs, 154, 172, 236, 327 
 Sagasser, 206 
 Salmon, 25, 39 
 Salmonsen, 93 
 Sanarelli, 250 
 Satchenko, 407 
 Schattenfroh, 181 
 Schereschewsky, 418 
 Schick, 315 
 Schmidt, 196 
 Schutze, 228, 234 
 Sclavo, 198, 407, 408 
 Sellards, 295, 297 
 Shattock, 262 
 Shiga, 199, 397, 405 
 Siedentopf, 319 
 Simon, 263 
 Smith, 25, 39, 215 
 Smith, Henderson, 286 
 Smith, Theobald, 311 
 Sobernheim, 406, 408 
 Soudakewitch, 336 
 Southard, 312 
 
442 
 
 INDEX OF AUTHORITIES CITED 
 
 Stern, 232, 233 
 Stillmarck, 38, 55 
 Stockman, 29 
 Sturli, 224 
 
 T 
 
 Takaki, 106 
 Tauber, 366 
 
 Tchistovitch, 125, 227, 336 
 Teague, 320, 329 
 Tizzoni, 334 
 Todd, 396 
 
 Torrey, 215, 352, 368 
 Tunnicliffe, 123, 262, 267, 270 
 
 U 
 
 Uhlenhuth, 228, 232, 235, 236 
 Uschinsky, 54 
 
 Vaillard, 86, 93, 113, 411 
 
 Vallee, 387 
 
 Van de Velde, 179 
 
 Vincent, 113 
 
 Volk, 216 
 
 W 
 
 Waage, 81 
 Walker, Ainley, 17, 183, 208, 219 
 
 Walker, R., 262 
 Washbourn, 364 
 
 Wassermann, 44, 57, 71, 106, 184, 228, 
 232, 235, 293, 303, 306, 337, 373, 
 
 392, 4*5 
 
 Wechsberg, 50, 70, 173, 323 
 Weidenreich, 225 
 Weichardt, 195 
 Weigert, 98 
 Weil, 291, 292 
 Weinberg, 170 
 Welch, 59, 219 
 Western, 270, 395 
 Whitfield, 278, 299 
 Widal, 317 
 Wiltshire, 223 
 Woltmann, 194 
 Wood, Cartwright, 62, 65 
 Wright, Sir A., 25, 127, 189, 199 211, 
 
 257 * seq., 333, 350, 360, 375, 42 
 
 Yersin, 403 
 
 Zammit, 375 
 Ziemka, 235 
 Zsigmondy, 319 
 
INDEX 
 
 ABRIN, 54; action on conjunctiva, 108 
 
 Abscess, cure of, 349 
 
 Absorption of complement. See Fixa- 
 tion of complement 
 
 Acne, 358 
 
 Acquired immunity, 19 
 
 Active immunity, 20. See Glossary 
 
 Addiment, 143. See Glossary 
 
 Adsorption, 90, 392 
 
 Age in relation to immunity, 8 
 
 Agglutination : by chemical substances, 
 211 ; mechanism of, 212; salts in, 
 209, 326 
 
 Agglutinins, 204 (see Glossary) ; action 
 of heat on, 205 ; chemical nature of, 
 214 ; to B. diphtheria, 409 ; to B. 
 dysenteria, 398 ; effects of tempera- 
 ture on, 208, 216 ; formation of, 208 ; 
 in normal blood, 99 ; to gonococci, 
 368 ; mechanism of action, 212 ; to 
 meningococci, 372 ; to pneumococci, 
 365 ; relation with cytolysins, 207 ; 
 role in immunity, 208 ; sensitiveness 
 of bacteria to, 219; specificity of, 205, 
 217; tostaphylococci, 359; to strepto- 
 cocci, 360; to tubercle bacilli, 383; to 
 typhoid bacilli, 389 ; to V. cholera, 400 
 
 Agglutinogen, 210. See Glossary 
 
 Agglutinoids, 47, 210, 216, 323. See 
 Glossary 
 
 Aggressins, 291 (see Glossary) ; speci- 
 ficity of, 292 
 
 Air, vitiated, 12 
 
 Albumoses in bacterial cultures, 54 
 
 Alcohol, 13 
 
 Alexins, 139 (see Glossary) ; source of, 
 179 
 
 Allergia, 308 
 
 Amboceptor. 141 (see Glossary) ; action 
 as opsonin, 285 ; formation of, 147 ; 
 methods of investigating, 185; source 
 of, 184 
 
 Amoeba, phagocytosis in, 238 
 
 Anaesthesia. 12 
 
 Anaphylaxis, 309. See Glossary 
 
 Anaphylactin, 313 
 
 Anthrax bacilli : immunity to, 405 ; 
 phagocytosis of, 244, 250, 252 ; 
 prophylaxis, 23, 407 ; toxins of, 405 ; 
 treatment of, 408 ; vaccination 
 against, 18, 23, 407 
 
 Anti-abrin, 108 
 
 Anti-agglutinin, 219 
 
 Anti-aggressin, 292 
 
 Anti-amboceptor, 160 
 
 Anti-antibodies, 104 
 j Anti-autolysin, 150 
 
 Antibodies, site of production of, 105,351 
 
 Anticomplement, 157 
 
 Anticrotin, 327 
 
 And enzymes, 48 
 
 Anti-epithelial serum, 197 
 
 Antigen, 101. See Glossary 
 
 Antihsemolysin, 109 
 
 Anti-intestinal serum, 195 
 
 Antileucolysin, 50 
 
 Antileucotoxin, 190 
 
 Antilysin, 160 
 
 Antispermotoxin, 109, 160 
 
 Antistaphylolysin, 52, 358 
 
 Antistreptocolysin, 360 
 
 Antitoxin : administration by mouth, 
 131 ; formation of, 60, 97, 114; in 
 normal blood, 93, 99 ; production by 
 toxoids, 99 ; reactions with toxin, 69 ; 
 role in immunity, 119; role in re- 
 covery, 119 ; unit of, 72 
 
 Antituberculin, 161, 307 
 
 Arsenic, absorption by leucocytes, 113 
 
 Arthus' phenomenon, 311. See Glossary 
 
 Atoxyl in trypanosomiasis, 6, 16 
 
 Atreptic immunity, 35. See Glossary 
 
 Atropin, absorption by leucocytes, 113 
 
 Aqueous humour, opsonin in, 286 
 
 Auto-agglutinin, 104, 222 
 
 Auto-anticomplement, 158 
 
 Autohsemolysin, 149. See Glossary 
 
 Auto-inoculation, 268, 382 
 
 Autonephrotoxin, 192 
 
 443 
 
444 
 
 INDEX 
 
 B 
 
 B. anthracis. See Anthrax 
 Bacillus of hotulismus, toxin of, 40 
 B. coli : diseases due to, 393 ; im- 
 munity to, 393 ; toxins of, 393 ; 
 
 vaccine treatment, 394 
 B. diphtheria. See Diphtheria 
 Bacillus of dysentery, toxins of, 396 
 B. pyocyaneus : antagonism to anthrax, 
 
 40 ; antitoxin, 121 ; hsemolysin of, 
 
 53 ; leucolysin, 50 
 B. tetani. See Tetanus 
 B. typhosus. See Typhoid 
 Bacteria, immunity to, 331 
 Bacterial hsemolysins, 40, 44 
 Bactericidal serum, therapeutic use of, 
 
 198 
 Bactericidal power of blood, 139 ; of 
 
 serum, measurement of, 188 
 Bacteriolysis, 139. See Glossary 
 Bacterio-precipitin, 226, 231 
 Bacteriotropin. See Glossary 
 Bazillen emulsion, 384 
 Bleeding large animals, 65 ; small, 
 
 185 
 
 Blood, human, test for, 233, 235 
 Blood-relationship, 234 
 Boils, 358 
 Bone-marrow, reaction of, in infections, 
 
 34i 
 Bordet-Gengou phenomenon, 153, 168. 
 
 See Glossary 
 
 Bovine tuberculosis, diagnosis of, 380 
 Brain substance and tetanus toxin, 44, 
 
 106 
 Bright's disease, 13, 193 
 
 Calcium chloride, agglutination by, 
 
 211 
 
 Calcium lactate, use of, 317, 391 
 
 Calmette's test, 303, 382 
 
 Capsules (bacterial), function of, 343 
 
 Carbuncles, 358 
 
 Castellani's absorption reaction, 217 
 
 Cayman, reaction to tetanus toxin, 60 
 
 Cellulo-humoral theory, 249 
 
 Cerebro-spinal fever, 371 
 
 Cerebro-spinal fluid, 373, 374 
 
 Cervical catarrh, 395 
 
 Chemotaxis, 112, 244, 294, 341. See 
 Glossary 
 
 Chicken cholera, aggressin to, 291 
 
 Cholecystitis, 395 
 
 Cholera, 398; bacteriolysis in, 140, 337 ; 
 diagnosis of, 399 ; endotoxin of, 57, 
 59 ; Pfeiffer's test in, 140, 400 ; pro- 
 phylaxis, 400 ; toxins, 398 
 
 Cholesterin, action on toxins, 107 
 
 Coagulation of blood, liberation 
 complement in, 182 
 
 Coagulation of proteids, 321 
 
 Cobra-lecithid, 156 
 
 Cold in causation of disease, 9 
 
 Colchicine, latent period of, 41 
 
 Colitis, mucous, 395 
 
 Colloidal chemistry, 319 
 
 Colloids, 90 ; agglutination of, 217 
 
 Complement, 14.2, 145 (see Glossary) ; 
 as opsonin, 285 ; deviation of, I73> 
 323; (endo-), 156; fixation of, 170; 
 methods of research on, 185 ; ori- 
 gin of, 179, 252 ; specificity of, 
 286 
 
 Complementoid, 158. See Glossary 
 
 Complementophile haptophore group, 
 146 
 
 Complementoids, 47. See Glossary 
 
 Conjunctivitis, 368, 370 
 
 Copula, 141 
 
 Crisis (in pneumonia), 365 
 
 Cuti-reaction, 303, 381 
 
 Cystitis (B. coli}, 394 
 
 Cytase, 142, 167, 254. See Glossary 
 
 Cytolysins, 190 et seq. (see Glossary) ; 
 bacterial, 40 
 
 Cytophile haptophore group, 145 
 
 Cytotoxin, 197 
 
 1) 
 
 Danysz effect, 327. See Glossary 
 
 Daphnia, phagocytosis in, 238 
 
 Dead bacteria as vaccines, 24 
 
 Dendroccelum, digestion in, 241 
 
 Desmon, 141. See Glossary 
 
 Deuterotoxin, 75 
 
 Deviation of complement, 173, 323. 
 See Glossary 
 
 Diabetes, 13 
 
 Digestion, intracellular, 241 
 
 Diphtheria antitoxin : dosage of, 410 ; 
 in normal blood, 93 ; standardiza- 
 tion of, 45 
 
 Diphtheria bacillus, antiserum against, 
 409 
 
 Diphtheria : diagnosis of, 409 ; latency 
 of, 33 ; local immunity to, 30 ; pro- 
 phylaxis, 410 ; toxin of, 40 ; action 
 of, 49 ; neutralization of, 72, 85 ; 
 standardization of, 45 
 
 Diphtheritic paralysis, 72, 80, 87 
 
 Dissociation, 28, 85 
 
 Dominant complement, 154. See 
 Glossary 
 
 Dosage of vaccines, 24 
 
 Dysentery, 396 ; bacillus, agglutination 
 of, 220 ; prophylaxis of, 397 ; treat- 
 ment of, 397 
 
INDEX 
 
 445 
 
 Eclampsia, cytolytic theory of, 195 
 
 Eel serum: immunity to, 125, 130, 135 ; 
 precipitin for, 228 
 
 Ehrlich's phenomenon, 328. See Glos- 
 sary 
 
 Electrolysis of toxins, 90, 92 
 
 Endocomplement, 156. See Glossary 
 
 Endothelial cells as phagocytes, 246 
 
 Endotoxin, 56, 339. See Glossary 
 
 Enterokinase, 256 
 
 Enzymes : analogies with toxins, 42 ; 
 proteolytic, in pus, 337 
 
 Epitoxoid, 76 
 
 Epitoxonoid, 327 
 
 Ergophore group. See Glossary 
 
 Erysipelas, treatment of, 362 
 
 Evolution, 130, 165 ; of bacteria, 221, 
 
 344 
 
 Exhaustion, Pasteur's theory of, 34 
 Exotoxins, 48 (see Glossary) ; chemical 
 
 nature of, 53 
 
 False rise, 274 
 
 Fatigue, 10 
 
 Fixation of complement, 153, 168, 236. 
 
 See Glossary 
 
 Fixator, 141, 167. See Glossary 
 Flagella, agglutination of, 227 
 Food, insufficient, II 
 Fowl cholera, 18, 22 
 Frog, action of tetanus toxin on, 45 
 Frontal sinus suppuration, 367 
 
 Gastrotoxin, 194. See Glossary 
 
 Gengou's reaction. See Glossary 
 
 Giant cells, 378 
 
 Gleet, opsonic index in, 368 
 
 Gonococci : immunity to, 369 ; local 
 immunity to, 30, 369 ; opsonic index 
 to, 368 ; vaccines in disease due to, 
 370 
 
 Group reactions, 217. See Glossary 
 
 H 
 
 Hsemagglutinin, 221. See Glossary 
 Hsemolysins : bacterial, 40, 44, 50 ; 
 
 serum, 141 et seq. 
 Haemolysis, 40, 141 (see Glossary) ; 
 
 by silicic acid, 326 ; methods of 
 
 research, 185 
 Hsemolysoids, 46, 51 
 Hsemopsonin, 245, 273, 285 
 Haptines, 95. See Glossary 
 Haptophore group, 46. See Glossary 
 Hepatotoxin, 192 
 Heterolysins, 149 
 Hog cholera, vaccination against, 39 
 
 Horse-flesh, test for, 237 
 Horse-sickness, vaccination against, 28 
 Hydatids, diagnosis of, 170 
 Hypersensitiveness to toxins, 61, 121. 
 See Anaphylaxis 
 
 Ichthyotoxin, 101, 125 
 
 Immune body, 141. See Glossary 
 
 Immunisin, 141 
 
 Immunitas non sterilisans, 16, 33, 331 
 
 Immunity : acquired, 19 ; active, 20 ; 
 atreptic, 35 ; bacterial, 331 ; defini- 
 tion of, I ; due to loss of receptors, 
 125 ; local, 29 ; mixed, 28 ; natural, 
 7 ; of leucocytes, 128 ; passive, 26 ; 
 to toxins, 115; to toxins, natural, 
 
 134 
 
 Incitor element. See Glossary 
 Indol, 115 
 Infection, definition of, 5 ; predisposing 
 
 causes of, 9 
 Interbody, 141 
 Intermediary body, 141 
 Ions, 80 
 
 Iritis, gonococcal, 370 
 Isoagglutinin, 221. See Glossary 
 Isolysin, 149 
 Isoprecipitin, 234 
 
 K 
 
 Koch's phenomenon, see tuberculin. 
 
 See Glossary 
 Kraus's reaction, 209 
 
 Latency of bacteria) 33 ; of tubercle 
 bacilli, 387 
 
 Latent period of toxins, 41 
 
 Lecithin : action on toxins, 107 ; role 
 in haemolysis, 156, 180 
 
 Lens, crystalline, precipitin to, 233 
 
 Lethal dose, minimal, 42 
 
 Leucocytes : absorption of toxins by, 
 44, 113 ; as source of complement, 
 179 ; chemotactic attraction of, 112 ; 
 degeneration of, 122 ; during starva- 
 tion, etc., 12 ; immunity of, 128 ; 
 in combating toxins, 120, 137 ; in 
 Metchnikoff s theory, 242 ; prepara- 
 tion of emulsions of, 257 
 
 Leucocytosis in prognosis, 112, 341 
 
 Leucolysins, 49, 358 
 
 Leucopsenia, 341 
 
 Leucotoxic serum, 190 
 
 Leucotoxins, 49, 358 
 
 Liver, phagocytosis in, 336 
 
 Local lesion, 33 ; cure of, 346 
 
 Local immunity, 29, 125 
 
 Lungs, phagocytosis in, 248, 336 
 
446 
 
 INDEX 
 
 M 
 
 Macrocytase, 152, 254. See Glossary 
 
 Macrophage, 247. See Glossary 
 
 Malaria, immunity to, 33 
 
 Mallein, 303 
 
 Malta fever, 374 ; treatment of, 375 
 
 Meats, recognition of, 237 
 
 Meningococcus, toxins of, 371 ; phago- 
 cytosis of, 371 
 
 Meningitis : serum treatment, 373 ; 
 vaccine treatment, 374 
 
 Micrococcus melitensis, agglutination of, 
 
 375 
 
 Microcytase, 152, 254. See Glossary 
 Microphage, 247. See Glossary 
 Minimal lethal dose, 42 
 Monospora, phagocytosis of, 239, 240 
 Mytilo-congestine, 309 
 
 N 
 
 Nasik vibrio, toxin of, 41 
 Natural immunity, 7 
 Negative phase, 62 (see Glossary) ; in 
 
 opsonic index, 274 ; summation of, 
 
 276 
 Neisser-Wechsberg phenomenon, 173, 
 
 323. See Glossary. 
 Nephrotoxin, 192. See Glossary 
 Nerves, peripheral, cytolytic serum for, 
 
 196 
 
 Neutralization of poison?, 116 
 Neurotoxin, 196 
 New tuberculin, 384 
 Nicotin, absorption by liver, 116 
 Nitrites, production of, in cholera, 37 
 Nucleo-proteids as antigens, 192 
 
 Ophthalmo-reaction, 303, 304, 382 
 
 Ophthalmotoxic serum, 197 
 
 Opsonic index, 261 ; in acute diseases, 
 265; in chronic diseases, 268; in 
 diphtheria, 267 ; effect of dilution of 
 serum, 264 ; effect of vaccines, 274 ; 
 in erysipelas, 270 ; false rise in, 274 ; 
 to gonococci, 270 ; to meningococci, 
 371 ; pre-agonal rise in, 280 ; to 
 pneumococci, 265, 361, 365 ; in 
 staphylococcic diseases, 266 ; tubercle 
 bacilli, 268, 382 
 
 " Opsonins-therapy," 277 ; in tubercle, 
 
 385 
 
 Opsonins: etTect of temperature on their 
 action, 295 ; fundamental experi- 
 ments on, 257 ; Metchnikoffs views 
 on, 289 ; nature of, 273 ; origin of, 
 288 ; presence in plasma, 286, 333 : 
 specificity of, 270 ; thermostability, 
 259, 282 ; technique of researches on, 
 259-264; thermolabile, 285 ; thermo- 
 stable, 259, 282 
 
 Orthophosphoric acid, agglutination by, 
 
 216 
 Osteomyelitis, 358 
 
 Passage, 15, 219 
 
 Passive immunity, 26. See Glossary 
 
 Peritoneum, phagocytosis in, 248, 250, 
 
 35 2 
 
 Perlsucht tuberculin, 384 
 
 Pfeiffer's phenomenon, 141. See Glos- 
 sary 
 
 Phagocytic index, 260 
 
 Phagocytosis, 238 et seq.; action of 
 salts in, 297; in circulating blood, 
 333 ; in peritoneum, 248, 250, 352 ; 
 influence of source of leucocytes, 290 ; 
 influence of temperature, 295 ; influ- 
 ence of virulence, 343 ; nature of, 295 
 
 Phagolysis, 181, 353 
 
 Philocytase, 141 
 
 Phthisis, 12 
 
 Pigmentolysin, 197 
 
 Piroplasma bigeminum, 21 
 
 Pirquet's (von) reaction, 303, 381 
 
 Placentolysin, 195 
 
 Plague, 402 ; prophylaxis of, 403 ; 
 serum treatment of, 403 
 
 Plasma, complement in, 182; opsonin 
 in, 286, 333 
 
 Pleuralistic conception, 151 
 
 Pleuropneumonia of cattle, 22 
 
 Pneumonia. See Pneumococci 
 
 Pneumococci : agglutinins to, 365 ; 
 immunity to, 365 ; in childhood, 8 ; 
 opsonic index to, 266 ; serum treat- 
 ment, 366 ; vaccine treatment, 366 ; 
 virulence, 366 
 
 Poisons : difference from toxins, 37 ; 
 neutralization of, 116 
 
 Polyceptor. See Glossary 
 
 Polyvalent serum, 363 (see Glossary) ; 
 
 vaccine (see Glossary) 
 ! Positive phase, 62. See Glossary 
 : Potato bacillus, phagocytosis of, 248 
 j Precipitins, 226 (see Glossary) ; speci- 
 ficity of, 232, 235 
 
 Precipitoid, 228, 323. See Glossary 
 
 Precipitogenoid, 231. See Glossary 
 
 Predisposing causes, 9 
 
 Preparator, 141. See Glossary 
 
 Pro-agglutinin, 210 
 
 Prostatotoxin, 196 
 
 Proteids, coagulation of, 321 
 
 Prototoxin, 75 
 
 Prototoxoid, 73 
 
 Pro-zones, 216, 323. See Glossary 
 
 Pus, enzymes of, 337 
 
 Pyocyaneus (B. ) : antagonism to anthrax, 
 40 ; toxin of, 57 
 
 Pyocyanolysin, 53 
 
INDEX 
 
 447 
 
 R 
 
 Rabies, vaccination against, 23, 419 ; 
 
 virus of, 15 
 Reactions : curative effects of, 281 ; 
 
 tubercle, etc., 300 
 Receptors, 95 (see Glossary) ; loss of, 
 
 I2 5. 343 ; sessile, 126, 160 / 
 
 Relapsing fever, recovery from, 335 r 
 Retention theory. Chauveau's, 35 
 Reversible reactions, 81 
 Ricin, 38, 54, 55 
 
 Rinderpest, vaccination against, 21 
 Ringworm, local immunity to, 30 
 
 Salts, role of, in agglutination, 209, 
 
 211 
 
 Septicsemia, 332 
 
 Serum : anti-anthrax, 408 ; anticholera, 
 400 ; antidiphtheritic, 409 ; anti- 
 meningococcic, 373 ; antipneumo- 
 coccic, 366 ; antiplague, 403 ; anti- 
 streptococcic, 363 ; antitetanic, 413 ; 
 antityphoid, 390-392 ; bacteriolytic, 
 use of, 198; disease, 315; toxin, 
 62 
 
 Side-chains, 95 
 
 Side-chain theory, 94. See Glossary 
 
 Smith's (Theobald) phenomenon, 311 
 
 Specific inhibition, 230 
 
 Specificity, 19, 105 (see Glossary) ; 
 of agglutinins, 205, 217; of cyto- 
 lysins, 191; of precipitins, 232 
 
 Spectrum of toxin, 73 
 
 Spermotoxin, 109, 190 
 
 Spleen, phagocytosis in, 334 
 
 Staphylococcus pyogenes : bacteriolysis, 
 337; leucolysin of, 50, 358; im- 
 munity against, 359 ; recovery, 347 ; 
 toxins of, 388 ; vaccine treatment of, 
 
 359 
 
 Staphylolysin, 51, 52, 358 
 Starvation, II 
 Sdmulins, 122, 295 
 Stomach, immunity of, 29 
 Streptococci : immunity to, 360 ; serum 
 
 treatment of disease due to, 362 ; 
 
 toxins of, 359 ; vaccine treatment of 
 
 disease due to, 362 
 Streptococcus pyogenes : hcemolysin of, 
 
 5) 359 I leucolysin of, 50 
 Streptocolysin, 50 
 Substance sensibilatrice^ 141 
 Surface-tension, 213, 298 
 Swine erysipelas, 19, 22 
 Symbiosis of leucocytes, 248 
 Sympathetic ophthalmia, 197 
 Syncytiolysin, 195 
 Syphilis, 415 ; Wassermann's reaction 
 
 in, 415 
 
 ; Teianolysin, 52, 83, 411 
 
 Tetanospasmin, 52, 411 
 
 Tetanus: diagnosis of, 410; immunity 
 to, 412 ; passive immunity to, 27 ; 
 prophylaxis of, 413; treatment of, 
 414 ; toxin, 40, 47, 411 ; absorption 
 of, by brain, 44, 106 ; leucocytes, 44; 
 tissues, 44; action of, 49, 116; on 
 various animals, 133 ; antitoxin to, 
 413 ; effect of temperature on, 45 
 i Texas fever, vaccination against, 21 
 ! Thyrotoxin, 197. See Glossary 
 
 Tick fever, 335 
 
 Tissue immunity. See Local immunity 
 
 Toxalbumin, 54 
 
 Toxins : action of, 41 ; composition of, 
 47 ; electrolysis of, 90, 92 ; hyper- 
 sensitiveness to, 61, 309; immunity 
 to, 115 ; spectrum, 73 ; standardi- 
 zation of, 45, 71 ; union with tissues, 
 100 
 
 Toxoids, 46, 47, 80 (see Glossary) ; pro- 
 duction of antitoxin by, 99 ; use in 
 immunization, 62 
 
 Toxone, 73, 80. See Glossary 
 
 Toxonoid, 88 
 
 Toxophore group, 46. See Glossary 
 
 Trichina spiralis, 29 
 
 Trichotoxin, 192. See Glossary 
 
 Tritotoxin, 76 
 
 Trypanosomiasis, 6, 1 6 
 
 Tubercle bacillus : antibodies for, 337 ; 
 immunity to, 377 ; opsonic index 
 to, 377 ; phagocytosis of, 251, 377 ; 
 toxins of, 314, 376 ; toxins of, Mar- 
 morek's, 383 
 
 Tuberculin : dilution of, 380 ; immuni- 
 zation to, 303 ; old, 300, 379 ; re- 
 action, 301, 378 ; theories of reaction, 
 305, 306, 308 ; reaction in cattle, 
 380 ; R, 383 ; therapeutic use of, 
 
 384 
 
 Tuberculosis : diagnosis of, 379 ; op- 
 sonin therapy of, 385 ; prophylaxis 
 of, 386 ; prophylaxis in cattle, 387 
 
 Tulase, 387 
 
 Typhoid bacillus : agglutinins to, 206, 
 210, 215 ; agglutinins in normal 
 blood, 99 ; endotoxin of, 53 ; haemo- 
 lysin of, 53 ; immunity, 388 ; in 
 peritoneum, 353 ; latency of, 33 ; 
 phagocytosis of, 263, 389 j virulence 
 of, 17, 343 
 
 Typhoid fever : bacterisemia in, 332 ; 
 bacteriolysis in, 337 ; opthalmo-re- 
 action in, 304 ; prophylaxis of, 28, 
 390 
 
 Typholysin, 53 
 
 Tyrosin : action on toxins, 107 
 
448 
 
 INDEX 
 
 U 
 
 Ulcerative endocarditis, 332 
 Ulcus serpens, treatment. of, 366 
 Unitarian theory of complement, 152 
 Unit of toxin, 72 ; of antitoxin, 72 
 Uraemia, cytolytic theory of, 192 
 
 Vaccine treatment : for gonococcic 
 diseases, 370 ; for Malta fever, 375 ; 
 for meningitis, 374 ; for pneumo- 
 coccic diseases, 366 ; for staphylo- 
 coccic diseases, 359 ; for strepto- 
 coccic diseases. 362 ; theory of, 276 ; 
 tubercle (see Tuberculin) 
 
 Vaccines : chemical, 25, 39 ; of dead 
 bacteria, 24 ; dosage of, 24 ; method 
 of preparation, 1 8 
 
 Vaccinia, 22 
 
 Venom (snake), '155 
 
 Vibrio, Nasik, 41 
 
 Vibrio Metchnikovi, bacteriolysis of, 
 
 173 
 
 Vibrio Nordhafen, bacteriolysis of, 174 
 Virulence, 14 ; capsule formation, 
 effect of, on, 343 ; changes in, 15, 17 ; 
 diminution in, methods of produc- 
 tion, 18 ; increase in, methods of 
 production, 15, 17, 219; influence 
 on phagocytosis, 295 ; mechanism 
 of, 343 
 
 Virus (see Glossary), 22 ; fixed, 15 ; of 
 the streets, 15 
 
 W 
 
 Wassermann's reaction, 416 
 Wet, in causation of disease. 9 
 Widal reaction, 389 
 
 Zones of inhibition, 216 
 
 Zoological affinities : immunity in re- 
 lation to, 8 ; relations to precipitins, 
 234 
 
 K. LEWIS, 136, GOWER STREET, LONDON, W.C. 
 
Yf 8^/35 
 
 fet 
 
 BIOLOGY 
 
 LIBRARY 
 
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 UNIVERSITY OF CALIFORNIA LIBRARY