^- 
 
 THE LIBRARY 
 
 OF 
 
 THE UNIVERSITY 
 
 OF CALIFORNIA 
 
 LOS ANGELES 
 
 GIFT OF 
 
 Dr. Emil Bogen
 
 VACCINE AND SERUM THERAPY 
 
 Including also a Study of Infections, Theories 
 OF Immunity, Opsonins and the 
 Opsonic Index 
 
 EDWIN HENRY SCHORER, B. S., M. D., 
 
 Assistant Professor of Parasitology and Hygiene, University of Missouri; 
 
 FORMERLY ASSISTANT ROCKEFELLER INSTITUTE FOR MeDICAL 
 
 Research, New York City. 
 
 ILLUSTRATED. 
 
 St. Louis 
 
 C. V. MOSBY CO. 
 
 1909.
 
 Copyrighted by C. V. Mosby Co., 1909.
 
 iiiomedicaj , 
 
 Libtaxj 
 
 I^Of CONTENTS. 
 
 I. INFECTIONS. Page 
 
 Course of infection 7 
 
 II. IMMUNITY. 
 
 Natural and acquired. Active and passive 12 
 
 Theories of immunity: Klebs and Pasteur, Chauveau, Metchnikoff, Salmon 
 and Smith, Fodor, Behring and Knorr, Ehrlich's side chain theory; opsonin 
 theory, Wright and Douglas; Neufeld and Rimpau 14 
 
 III. OPSONIC INDEX. 
 
 Leishman's method of estimating phagocytic power 23 
 
 Wright and Douglas' opsonic index 23 
 
 Serum; leucocytes; bacterial emulsion; strength of bacterial emulsion; mix- 
 ture of bacteria, leucocytes and serum; smears, preparation, fixing and 
 staining; examination of smears; calculation of the opsonic index 24 
 
 IV. CRITICISMS AND MODIFICATIONS OF WRIGHT'S OPSONIC INDEX 
 DETERMINATIONS. 
 
 Mechanical technique; serum; bacterial emulsions; leucocytes; calculation 
 and determination of the opsonic index, Simon, Strong; leucocj^tes ex- 
 amined 37 
 
 V. OPSONIC INDEX IN HEALTH AND DISEASE. 
 
 Importance of opsonins in immunity and accuracy of the method of deter- 
 mining the opsonic index; changes in opsonic index in disease; changes in 
 opsonic index due to vaccines 49 
 
 VI. NATURE OF OPSONINS. 
 
 Specificity; effect of heat; structure of opsonins; relation to leucocytosis; rela- 
 tion to stimulins; opsonins and aggressins; influence of chemicals and 
 reaction; non-bacterial opsonins 58 
 
 VII. VACCINE THERAPY. 
 
 Bacterial Vaccines: dosage; organisms used in vaccination; preparation of 
 
 vaccines; control of injections, opsonic index and clinical 64 
 
 Staphylococcus, streptococcus, gonococcus, pneumococcus, tuberculosis, colon 
 Bacillus, Bacillus typhosus. Bacillus dysenteriae, cholera, and less com- 
 mon infections 73 
 
 Specific vaccines for diseases of unknown etiologj'': hydrophobia, small-pox. ... 81 
 
 Future of opsonins and vaccines 85 
 
 VIII. SERUM THERAPY. 
 
 Historical: Untoward effects of serum injections, serum disease and In'per- 
 susceptibility to serum; indications for immune serum injections; con- 
 centration and purification of serum; dried immune serum; oral adminis- 
 tration of serum 87 
 
 Anti-toxic serum: Diphtheria antitoxin; tetanus anti-toxin 99 
 
 Anti-bacterial serum: Anti-streptococcic syrum; anti-meningococcic serum; 
 anti-gonococcic serum; anti-pneumococcic serum; anti-typhoid serum; 
 anti-dysenteric serum; anti-staphylococcic serum 109 
 
 fi^fif).Q5
 
 PREFACE 
 
 Treatment of individual diseases with medicines or b}^ methods having 
 a selective curative action has until recently been limited. With the establish- 
 ment of the germ theory of certain diseases and the development of informa- 
 tion concerning immunity, new methods of specific treatment were made pos- 
 sible and are now practiced under the name of serum and vaccine therapy. 
 As might be expected, the medical profession has been much interested in these 
 methods of treatment, and applied them whenever possible. The develop- 
 ment of vaccine and serum therapy has been slow, the methods have had to 
 be revised and in some cases the results obtained have been found to be other- 
 wise than was at first expected. Because of this, much confusion has arisen. 
 The practitioner has not been able to keep pace with the developments and 
 literature on these subjects, and finally has been forced to depend on the state- 
 ments and recommendations coming from serum and vaccine laboratories, 
 enthusiasts and ever exploiters. 
 
 In this work an attempt has been made to state concisely and accurately 
 the present knowledge concerning vaccines and immune sera. An effort has 
 been made to establish theoretical and experimental evidence as well as clin- 
 ical application of. the specific treatment of bacterial diseases. To accomplish 
 this, some space is given to infections in general, the theories of immunity, 
 with especial emphasis on the opsonic theory of immunity, as well as the par- 
 ticular methods of vaccine and serum therapy. 
 
 Considerable space has been given to opsonins, the opsonic index and the 
 importance of opsonins in health and disease. This has been done because 
 since 1904 no subject lias appeared more prominently or frequently in medical 
 literature than that concerning opsonins, opsonic immunity, and bacterial 
 vaccines. In the first presentations of discoveries on this form of immunity 
 and specific treatment of bacterial diseases, great possibilities were promised. 
 Methods, which would, according to Wright, give uniform success in treat- 
 ment of the large class of bacterial infections and diseases, naturally received 
 immediate and general attention by the medical Avorld and were at once quite 
 generally applied. This has been followed b}- much indiscriminate and un- 
 scientific use of tliese methods of specific treatment so that in the minds of
 
 mail}-, opsonins and vaccine therapy, have gone into disrepute as did tuber- 
 culin neaily twenty years ago. We now know tuberculin has many appli- 
 cations of importance in the diagnosis and treatment of tuberculosis, though 
 this knowledge lias been gained some years later than it would have been had 
 it not been for improper exploitation. In the hope of avoiding a repetition 
 of such an effect as far as opsonin and bacterial vaccines are concerned, this 
 subject is given considerable attention. The opsonic index technique which 
 is given here is the one taught the writer by Dr. W. G. Ross, who was for two 
 years a pupil of Sir A. E. Wright. 
 
 An attempt has been made to bring the su])jects taken up as nearly up 
 to date as is possible. It is hoped that this work may furnish to the medical 
 student and practitioner information which may lead him to a better under- 
 standing of the nature of infections and the subjects of immunit}^, and active 
 and passive immunization. 
 
 Columbia, Missouri, April, 1909.
 
 CHAPTER I. 
 
 INFECTIONS 
 
 By the term infection we understand the entrance of micro- 
 parasitic living agents into the body tissue or substance, and 
 the occurence of definite symptoms of disease as the restdt of 
 the multiplication and action of the invading organisms. Infec- 
 tions do not always occur when pathogenic microorganisms are 
 present in the body tissues, certain conditions being necessary 
 for the establishment of a bacterial disease. These conditions 
 may be broadly divided into two classes; those dependent upon 
 the biological properties of the infecting organism, and those 
 dependent upon the conditions in the host and tissues invaded. 
 
 Of the biological characters of importance in the infecting 
 organism in the production of a bacterial disease, the most im- 
 portant are, that the microorganisms must be able to multiply 
 rapidly and greatly in the tissues of the body, and that they must 
 be able to produce poisons or substances harmful, either to some 
 or to all, of the tissues of the bod}'. The number of organisms 
 necessarv' to cause disease varies with different species. Many 
 more staphylococci are necessary to cause the formation of a 
 furuncle than of anthrax bacilli to cause anthrax. The property 
 to produce substances poisonous to the body tissues generally 
 determines virulence. That virulence is of great importance is 
 evident from the fact that innumerable so-called saprophytic 
 bacteria which are present in the different parts of the body, grow 
 and multiply there but because in their growth sufficient poison 
 is not produced, cannot cause an infection. The virulence of an 
 organism, according to numerous investigators, depends upon 
 the presence of certain substances in the parasite which reduce 
 the resistance of the body and its tissues. These substances are 
 various!}' designated as lysins and aggressins.
 
 8 VACCINE AND SERUM THERAPY. 
 
 Besides conditions in the causal organism, certain conditions 
 must exist in the host or body so that infection can occur. Cer- 
 tain animals are insusceptible to the action of certain species of 
 microorganism, thus for example, the horse and other domestic 
 animals are naturally immune to venereal diseases. Again cer- 
 tain organisms can only produce disease when they are present 
 in certain parts of the body and have entered the body by certain 
 portals of entry. The spirillum cholerae can only produce its 
 typical form of disease when it has gained entrance through the 
 small intestine. Invasion of the dermis or epidermis by this or- 
 ganism produces no disease. The bacilli producing tuberculosis as 
 well as certain other organisms, can cause infection when the}- 
 have entered through the skin or through the mucous membrane. 
 Certain organisms as Bact. diphtheriae, B. tuberculosis, and B. 
 typhosus, show predilections for certain tissues but will also pro- 
 duce diseased conditions in other parts of the body. 
 
 In addition to the non-susceptibility of the host, the body 
 possesses certain natural barriers to disease which must be over- 
 come before infections can be produced. The unbroken skin 
 usually offers a barrier to infection. Age, sex, race, occupation, 
 etc. , at times account for certam resistance to invasions of organ- 
 isms. The resistance furthermore varies for the different organ- 
 isms, as is evidenced by the fact that for B. tetanus a wound 
 is necessaiy to produce lock-jaw, while for the glanders bacillus 
 the slightest abrasion of the muccus membrane will furnish a 
 focus for an infection. The body fluids, lymph glands, phagocytes, 
 all offer resistance to infection. These barriers are overcome in 
 various ways and under different conditions, so that while indi- 
 viduals may be immune at one time they may, at another time, 
 be susceptible to the same infectious agent. 
 
 COURSE OF INFECTIONS. 
 
 From the foregoing it is evident that not only must the in- 
 fectious organism come into contact with the body tissues to 
 produce disease, but the organism must be able to grow, multi- 
 ply, produce its poison, and overcome the resistance of a body 
 susceptible to its action. The symptoms and signs of infections 
 do not appear until a certain time after the invasion b\' the micro-
 
 INFECTIONS. 9 
 
 organism. This period is known as the "incubation period," and 
 varies according to the biological characters of the infecting or- 
 ganism, but is also influenced by the number and virulence of 
 the organisms and the individual susceptibility of the host. 
 
 The course of a disease is determined by conditions produced 
 by the specific organism and partly by the distribution of these 
 organisms. But even here it is to be noted that the course of 
 the disease varies. Many of these variations we are by no means 
 able to explain. 
 
 The symptoms and signs produced by an organism will vaiy 
 as the organism acts locally or generally. In local infections 
 the most marked disturbance occurs at the portal of entiy of the 
 microorganism, while in general infections the reaction manifests 
 itself in all, or a large part, of the body. The infecting microor- 
 ganism may produce disease either in a mechanical wa}^ because 
 of large numbers, or, as is the case of most all infections, because 
 of toxins which may act locally or be distributed over a large part 
 or the whole, of the body. Some organisms, as the bacilli of diph- 
 theria and of tetanus, produce extracellular toxins, while other 
 organisms, as those causing typhoid fever and cholera, have 
 intracellular toxins which are liberated supposed^ when the 
 organisms disintegrate. 
 
 After organisms have entered the body the distribution varies 
 with the species. Thus we see that staphylococci form furuncles, 
 carbuncles and pustules in localized areas, typhoid bacilli though 
 causing lesions in the intestine are usually present in the blood 
 stream, while the tetanus bacilli form a toxin which is distributed 
 through the entire body. At times microorganisms do not remain 
 localized at the portal of entry but pass on to the lymphatic 
 glands and other parts of the body, here producing pathological 
 conditions. In some infections the microorganisms gradually 
 involve more and more of the tissues of the body. Microorgan- 
 isms may first lodge in one part of the body and produce disease 
 in that part and from these primary lesions, other parts of the 
 body may become infected. When the blood stream is not onh^ 
 the canier of microorganisms but becomes the place for gro\^i:h 
 and reproduction of the same, a septicaemia arises. Wliile almost 
 any of these conditions may be produced by any of the pathogenic 
 bacteria, still certain organisms are more Hkely to produce one
 
 10 VACCINE AND SKRUM THERAPY. 
 
 or another of these conditions. Resultant of this tendency of 
 microorganisms to produce particular kinds of conditions, the 
 prognosis for infections varies with the species of microorganism 
 ])roducing the lesions or condition. 
 
 The same species of microorganism will not always be distri- 
 buted in an identical manner; the location of the lesion and the 
 condition produced var\dng with the properties of the particular 
 strain in question and the particular part of the body infected. 
 The appearance of general symptoms will vary greatly with the 
 rapidity of the absolution of the toxin. Experimentally, intra- 
 venous and intraperitoneal injections of microorganisms are fol- 
 lowed by symptoms earlier and more consistently than are sub- 
 cutaneous injections. 
 
 The number of bacteria invading the tissues is of some im- 
 portance. If few bacteria are present they mav die without 
 producing infections, while if larger numbers are present some 
 of them will be likely to grow^ and produce disease. 
 
 At the point of infection, or portal of entry, the effects pro- 
 duced will var\^ with the infecting organism, some producing 
 specific forms of inflammation along with those common to many 
 bacteria. Even with the same species of microorganism the 
 reaction will vary with the prevalence and virulence of the invad- 
 ing organism and with the anatomical structure of the part of 
 the body involved. The local reaction, however, is not only pro- 
 duced when the bacteria enter the tissues of the body but also 
 when the toxins produced by the microorganism are present in 
 the tissues. The most commonly seen reaction at the portal of 
 entry is that which results in pus formation, in which, due to 
 chemotaxis, leucocytes accumulate. While a local reaction is 
 usually produced at the portal of entr}^ of bacteria, the reaction 
 at times may be slight or absent entirely. A local reaction when 
 produced is generally regarded as of importance in the protection 
 of the body against the invasion of bacteria. 
 
 Besides the local reaction, general reactions usually follow 
 in all severe infections. General reactions occur as a result of 
 the action of toxins produced by the microorganisms when they 
 are absorbed by body tissues. The general symptoms produced 
 vary according to the location of the primary lesion, the extent 
 of the process, the peculiarities of the organism, and the resistance
 
 INFECTIONS. 11 
 
 of body tissues. The more common general reactions are fever, 
 digestive disturbances, effects on the nervous system, leucocvtosis, 
 anaemia, and enlargement of the spleen and other glands. 
 
 Infections and infectious diseases may be divided into two 
 classes ; one in which the disease runs a self -limited course, recovery 
 coming after a definite period of time has elapsed, while in the 
 other class the course ma}" be extended over a very indefinite 
 period of time. 
 
 The elimination of the casual or etiological factor of infections 
 and infectious diseases occurs in various ways. When the seat 
 of the disease is on the surface of the body, the organisms are elim- 
 inated with the diseased tissues, but when the organisms are 
 found in ]:arts of the body where this is impossible, the microor- 
 ganisms must enter the general circulation, be allowed to pass 
 through the glandular tissues and be eliminated with the secretions 
 and excretions. In the blood and the body tissues, bacteria 
 are in many cases destroyed by bacteriolytic and bactericidal sub- 
 stances. Sometimes after an apparent recovery from the disease, 
 viable organisms may remain and at a later time again give rise 
 to disease.
 
 CHAPTER II. 
 
 IMMUNITY 
 
 Immunity may be defined as, non-susceptibility to a disease, 
 or as the ability to resist the action of the causes of the disease. 
 The body may be immune because of inherited properties or 
 because it has become so during life. Immunity because of in- 
 herited properties is called "natural" immunity, while the mi- 
 munity acquired during life is called "acquired" immunity. 
 
 Natural immunity is demonstrated by the non-susceptibility 
 of the hen to the action of the tetanus bacillus. It is an im- 
 munity of race or species. This immunity at times may be re- 
 duced or removed by hunger, exhaustion, exposure to cold, etc. 
 Certain closely related races or species of animals sometimes show 
 a natural immunity and at times a natural susceptibility to the 
 same infecting agent. This non-susceptibility is frequently called 
 a natural resistance and at times is only an apparent immunity, 
 depending in these cases on the common natural barrier to the 
 entrance and development of disease producing organisms. xVgain, 
 what may be regarded as a natural immunity is, in part at least, 
 only a resistence to infection due to the inability of organisms to 
 reach viable tissues. This is the case when the acidity of the 
 stomach is sufficient to kill cholera organisms before they reach 
 the epithelium of the intestine. 
 
 Acc[uired immunity only results after a pathological condi- 
 tion exists or has existed. The individual becomes immune, 
 because he has survived a natural course of the disease, as is the 
 case following an attack of scarlet fever; because he has gone 
 through a modified form of the disease, as is the case in vaccination 
 against small-pox; or, because he receives substances prepared by 
 some other individual or animal tliat has gone through a natural
 
 IMMUNITY. 13 
 
 or modified course of the disease. Acquired immunity may be 
 either active or passive. 
 
 Experimental active immunization is usually called vacci- 
 nation and generally produces in the individual a modified form 
 of the disease. All, or nearly all, of the symptoms are less severe 
 than in the natural course of the disease. The individual in this 
 case produces his own immunity. In artificial or intentional in- 
 oculation, the etiological factors, or more particularly, the causal 
 organisms injected must be so modified that the natural course 
 of the disease will not follow the inoculation or injection. Ex- 
 perimentally acquired active immunity is produced by the in- 
 jection of living or killed microorganisms, or of toxins produced 
 by these organisms. When living organisms are injected their 
 virulence is usually reduced by passage through animals, growth 
 at high temperatures, on artifical media, in the presence of chem- 
 icals, or growth in the presence or absence of oxygen, etc. Active 
 immunity may also be acquired as a result of injection of living 
 virulent organisms into such parts of the body where the disease 
 will not be produced. The first amounts injected are usually 
 smaller than those in subsequent injections. When bacteria are 
 injected the immunity produced is a bacterial immunity and 
 when toxins are injected a toxin immunity results. 
 
 In passively acquired immunity, the individual that becomes 
 immune does little or nothing toward obtaining this immunity. 
 It results from the injection of immunizing substances which 
 have been prepared by an actively immunized individual or 
 animal. 
 
 There are two classes of immunizing substances; those 
 that act on bacteria, said to be anti-bacterial; and those that 
 act on toxins, called anti-toxic. Of these two classes of immune 
 substances, the anti -toxic has been more efficient in passive im- 
 munization. 
 
 After the formation of immunizing substances they do not 
 remain indefinitely within the body, but are lost through the ex- 
 cretions either as immune bodies or as immtme bodies broken 
 down by the body cells. On this the difference in persistance of 
 natural and acquired immunity is partly based, for in acquired 
 immunity the supply of immtme bodies is exhausted, while in 
 natural immunity new substancss are constantly formed.
 
 14 VACCIXE AND SERUM THERAPY. 
 
 THEORIES OF IMMUNITY. 
 
 Acquired immunity, as has been stated, results because some 
 individual or animal has gone through a natural or modified 
 course of the disease. While acquiring immunity, the body and 
 its tissues have in some way been modified. Various theories 
 have been advanced to explain this phenomenon. 
 
 Klebs and Pasteur tried to explain the changes that occur 
 in the acquisition of immunity by assuming that in going through 
 a natural or modified course of disease, certain substances, nec- 
 essarv as food for the parasites, are used up. As the result of 
 immunization, the food necessarv^ for the microorganism is con- 
 sumed and the individual is immune to a certain organism be- 
 cause this organism cannot obtain the food it needs for its ex- 
 istence and production of the disease. 
 
 Chauveau assumes that in immunization certain products 
 of bacterial metabolism are retained in the body of the immun- 
 ized animal or individual, which products protect the bod\' tissues 
 from further invasions by that particular parasite. 
 
 Metchnikoft', in 1883, formulated a theory according to which, 
 during immunization, certain white blood cells and cells of certain 
 organs acquire the ability to engulf and destroy the attenuated 
 bacteria. This results in acquiring the abihty to engulf and de- 
 stroy more virulent forms of this same species of microorganisms. 
 
 EHRLICH'S SIDE CHAIN THEORY OF IMMUNITY. 
 
 In 1887 Salmon and Smith, Foa and Bonome, Roux and 
 Chamberland, and others found that immunity could be produced, 
 not only bythe injection of bacteria, but also, as a result of the injec- 
 tion of the products of bacterial metabolism. As a result, a chemi- 
 cal theory of immunity was advanced. According to this theory 
 the tissues of the body are chemically changed by immunization. 
 
 Fodor, in 1887, was the first to observe that the normal body 
 fluids, especially the blood, contain certain substances able to 
 destroy bacteria. Buchner, Behring, and Nuttall, soon after 
 Fodor's observation also recognized the bactericidal powers of 
 certain sera. In 1889 Buchner reported that the cell-free blood 
 serum contains certain substances which he called alexines. 
 Alexines, he found, have the property of destroying bacteria.
 
 EHRLICH S THEORY OF IMMUNITY. 15 
 
 In 1888 Hericourt and Richet, and in 1889 Babes and Lepp re- 
 ported investigations which showed that by injections of blood 
 serum from animals having acquired immunity to certain diseases, 
 immunity to these same diseases can be conferred to other ani- 
 mals. Soon after this Behring and Kitasato reported successful 
 immunization of rats to tetanus by means of injections of blood 
 serum from ral^bits immunized to tetanus. From the work of these 
 investigators there developed the humeral theory of immunity. 
 
 Behring and Knorr found that the toxic product of the meta- 
 bolism of the tetanus bacillus, could, without the presence of the 
 bacillus, be used in immunization. The serum thus produced, they 
 found would protect against the disease and the injection of the 
 by-products of the tetanus bacillus. They assumed from this 
 that toxin is neutralized by the immunizing substance in the .'m- 
 mune serum, as a base is neutralized by an acid. 
 
 Ntunerous theories have been advanced to explain the facts 
 observed and reported, but of all these theories one stands out 
 prominently — Ehrlich's side chain or receptor' theory, advanced 
 by Ehrhch in 1897. 
 
 i\ccording to this theory, every living cell consists of a domi- 
 nating nucleous (Leistungskem) and of side chains or receptors. 
 The paradigm of this picture is to be found in the benzol ring 
 with its side chains. The side chains or receptors of a cell are of 
 many different kinds and serve usually to anchor and assimilate 
 the food stuffs. At times, however, by means of the receptors 
 the cells are combined with substances, not foods but cell poisons. 
 The combination of the receptors of the cell with the receptors of 
 foods differs from the coinbination of receptors of the cell and re- 
 ceptors of toxins. The combination of the receptors of the cell 
 with the receptors of foods is so loose that after undergoing certain 
 changes which are of value in assimilation of the food and the 
 nourishment of the cell the food stuff is again eliminated without 
 having injured the receptor. Toxins on the other hand, combine 
 so firmly with the receptor of the cell that they cannot again 
 be separated. As a result of this, receptors anchoring toxins 
 are lost to the cell. 
 
 When a certain limit of anchoring of receptors by toxins has 
 taken place the cell peri hes, and if a sufficient number of cells 
 which have vital functions to perform are destroyed, death of
 
 16 VACCINE AND SERUM THERAPY. 
 
 the individual follows When, however, the toxins do not anchor 
 enough receptors or are not potent enough to destroy the cell 
 and the organism, immunity results or follows. 
 
 In 1896, Weigert advanced the hypothesis that when fssue 
 is lost the regeneration that follows is not only sufficient to restore 
 the amount of tissue lost, but actually results in overproduction 
 of this tissue. This process is observed in the over-production 
 of segments in reptiles and of cell proliferation in granulating 
 wounds in man and the animals. Ehrlich assumes that when 
 the toxins combine with the receptors of the cell, the receptors 
 are lost to the cell and as a result the cell is stimulated to repro- 
 duce the destroyed receptors. The process does not stop, how- 
 ever, when all lost receptors have been reproduced, for more re- 
 ceptors will be produced than were anchored by the toxins. The 
 excessive production of receptors as a result of the stimulus fol- 
 lowing the anchorage of the receptors by toxins, Ehrlich assumes, 
 leads to a disturbance of the equilibrium of the cell. As a result 
 of 'this, the surplus receptors are thrown off and constitute what 
 is known as the anti-body. This process can be actually ob- 
 served in the lower animals in which extra parts, produced 
 as a result of destruction of some parts, are cast off or lost. 
 
 The principal function of all receptors and side chains is to 
 provide for the nutrition and metabolism of the cells. Receptors, 
 and hence immune bodies, however, are not all of the same com- 
 position or even of the same general structure. In order to ex- 
 plain the different functions and actions of different immune 
 bodies, Ehrlich has assumed that the receptors may be of simple 
 constitution and structure or they may be complex. Any cell 
 of the body may have large numbers of the same and different 
 kinds of receptors. Ehrlich divides this large number of receptors 
 into three orders. 
 
 RECEPTORS OF THE FIRST ORDER. 
 
 These are receptors that are of relatively simple constitution 
 and structure and combine with substances that can be easily 
 and readily anchored. Bacterial toxins anchor receptors of 
 this order. The receptor here consists of only one haptophore, 
 or combining group, which combines directly with the hapto- 
 phore group of the bacterial toxin. This order of receptors and
 
 RECEPTORS FIRST AND SECOND ORDER. 
 
 17 
 
 immune bodies is demonstrated in Figure 1, and represents the 
 type of receptor on which is based the action of bacterial toxin 
 and the formation of anti-toxin. 
 
 /oxo-i'Hotfe. Q-ifo</f 
 
 X OrfoetT 
 
 ~Fr^E.e. "TfecsfTOT^, 
 
 ^ — ^o%iM Tfaoor To 
 
 Fig. 1. 
 
 RECEPTORS OF THE SECOND ORDER. 
 
 The receptors of the second order are distinguished from 
 those of the first order in that the receptors here have in addition 
 to the haptophore group, a zymophore group. This zymophore 
 group acts on the larger food particles, making them more readily 
 assimilable. In a similar manner it acts on the bacterial cell. 
 Receptors of this kind, possessing haptophore and zymophore 
 groups, are broken off from the cells and circulate in the blood as 
 agglutinins and precipitins after immunization by the injection 
 of certain bacteria. The haptophore group of the immune body 
 in an agglutinating or precipitating serum combines with the 
 bacterial cells. The zymophore group, however, does not com- 
 bine with anything but exerts its influences entirely through the 
 haptophore group. The zymophore group is destroyed by age, 
 acids, heating to 65° C, etc. Receptors and immune bodies of 
 the second order are represented in Figure 2. 
 
 jB^crs 
 
 •21 y/«^07'«<J7f e (^ffOUP 
 
 
 Fig. 2.
 
 18 
 
 VACCINE AND SERUM THERAPY 
 
 RECEPTORS OF THE THIRD ORDER 
 The receptors of the third order are Hkewise adapted to the 
 anchorage of bacteria. These receptors, however, have two com- 
 bining groups: one for the anchorage of cells or food substances 
 and the other for the anchorage of substances which, acting through 
 the receptors, can produce a ferment-like action. This latter 
 substance is called an activating substance and is present in nor- 
 mal sera. It is known either as complement or alexin. 
 
 Complement is easily destroyed by ageing, acids, heat, 55° C, 
 being sufficient to destroy it. When the receptors of this order 
 are anchored by bacteria in numbers not sufficient to kill the cell 
 but to stimulate it to over-production of receptors, the extra 
 receptors are thrown off. The receptors thrown off constitute 
 the immune body, amboceptor or substance sensibilitrice of Bor- 
 det. They have two combining or haptophore groups, one for 
 the combination with bacterial or other cells and substances, 
 called the "cytophyUc" group; the other for combination with 
 alexin or complement, called the " complement ophore" group. 
 It is by means of complement that the amboceptor is able to 
 dissolve bacteria, red blood cells and other substances. Immune 
 sera containing receptors of the third order are bacteri- 
 olytic, haemolytic, or cytol3rtic, depending upon wdiether they, 
 together with complement, can dissolve bacteria, red blood cells, 
 or other cells. The receptors of the third order are graphically 
 represented in Figure 3. 
 
 
 Tfec-p-rntf J2L OffDETf 
 
 (^yrOTHf UlC (f-rfOi/P, 
 
 Fig. 3. 
 
 It will be observed that receptors of the first, second or tliird 
 order can be produced in excess to form immune body or anti-
 
 ANTI-BODIES. U) 
 
 body, specific immune bodies are produced for many substances, 
 toxins, bacterial cells, red blood cells, ferments, etc. As a result 
 of this multiplicity of stibstances that can cause the cells to 
 produce anti -bodies, anti- bodies ma)^ be anti- toxic, aggluti- 
 nating, precipitating, lytic, etc. To produce agglutination, 
 precipitation or lysis it is necessary that a ferment-like sub- 
 stance be a part of, or able to combine with, the immune body. 
 This ferment -like substance, as stated before, is labile and lost 
 on ageing, heating, etc. When this is lost the serum containing 
 the immune body is said to be inactive. In normal serum there 
 is present a substance called complement which can again reacti- 
 vate a lytic serum. The agglutinating power, however, cannot 
 be restored by the addition of fresh complement. Complement 
 can be preserved for long periods of time if the serum is dried 
 without heat soon after the blood is drawn. Whether there is 
 only one complement or a multiplicity of the same has not been 
 definitely decided, Ehrlich assuming that there is a multiplicity 
 of the same, while Bordet and Buchner claim there is only one. 
 
 It has been demonstrated that anti-toxic substances are im- 
 portant in preventing the action of toxins on the body cells, while 
 lytic substances have been found to protect the body by the 
 solution and destruction of bacteria. Whether the agglutinat- 
 ing or precipitating substances are of importance in destroying 
 bacteria, is by no means certain. Numerous investigators as- 
 sume that they injure and change the bacterial cells or substances 
 used in immunization, while others claim that they exert no 
 such action. It has been quite definitely observed that it is pos- 
 sible to cultivate and grow bacteria that have undergone ag- 
 glutination. 
 
 As has been stated earlier, immune bodies are formed only 
 for such substances as are able to combine firmly w^ith the recep- 
 tors of the cells, and it is on this assumption, that Ehrlich explains 
 the impossibility of producing immunity to certain poisons, as 
 the alkaloids, strychnine and morphine. 
 
 The cells whose receptors anchor the substances and cells for 
 which immune substances are formed, are probably widely dis- 
 tributed throughout the body. The particular tissues in which 
 anti-bodies are formed has not been determined and probably 
 varies for the difi'erent substances to which immunization can be
 
 20 VACCIXI-: AXD SKRl'M THI-:RAPy. 
 
 obtained. It seems quite definite that in certain cases, internal 
 organs may contain more immune substances than the blood se- 
 rum. After immune bodies have heen formed they do not remain 
 permanently in the body, but are gradually lost through destruc- 
 tion in the body or lost with the excretions. 
 
 Ehrlich's views on the formation of immune bodies are cjuite 
 generally accepted. His views, however, concerning the com- 
 bination of toxin and anti -toxin, agglutinable substances and 
 agglutinins, and substances capable of solution and lysins are 
 not as universally accepted. Ehrlich assumes that the union of 
 immune bodies and the specific substances is a chemical combina- 
 tion. Bordet assumes that in lytic sera the immune body acts 
 as a mordant which sensitizes the substance capable of solution 
 to the action of the complement. Field has explained the inter- 
 action of immune bodies and specific substance according to the 
 principles of colloid chemistry, so that for example in a toxin 
 and anti-toxin combination one is adsorbed (physical rather than 
 chemical) by the other. 
 
 OPSONIN THEORY OF IMMUNITY. 
 
 It has been stated earlier that in 1883, Metchnikoff advanced 
 his phagocytic theory of immunity. According to this theory 
 the presence or absence of immunity depends upon the ability of 
 phagocytes to engvilf and destroy bacteria. Metchnikoff had 
 given serum little consideration in immunity although he believed 
 that some sera contain substances which stimulate the leucocytes 
 to engulf bacteria. These substances he called "stimulins." The 
 phagocytic theory, however, was practically replaced by the 
 humeral theory of immunity because Fudor, Buchner, Behring, 
 Nuttall and others had found that serum containing no cells 
 whatever can destroy bacteria by solution. It was also found 
 that antitoxin, which combines with toxin, is carried in serum. 
 
 From 1887 on the humeral theory of immunity gained a 
 strong foothold and it was not until 1895 that attention was again 
 called to tlie function of the leucocytes in immunity. In this 
 year Denys and LeClef reported experiments from which they 
 concluded that in immunization certian changes are produced 
 in the serum, which make it possible for the leucoc}'i:es to engulf 
 bacteria. In their conclusions they state that leucocytes from a
 
 OPSONIN THEORY OF IMMUNITY. 21 
 
 normal rabbit on the addition of normal rabbit serum, have only 
 slight phagocytic action for the streptococcus. If, however, 
 leucocytes from a normal rabbit, or from a streptococcus-immune 
 rabbit are added to the serum from an animal vaccinated 
 with streptococci, the leucocytes actively engulf and destroy 
 the streptococci. When streptococci, able to produce erysipelas 
 in a normal rabbit, are injected under the skin of a rabbit vacci- 
 nated against streptococci, disease is not produced. This pro- 
 tection is due, according to these investigators, especially to the 
 ability of the leucocytes to destroy streptococci. They attribute 
 the increased phagocytosis to an action of the immune serum on 
 the leucocytes. 
 
 The results obtained and conclusions arrived at by Denys 
 and LeClef were repeated by Bordet, who was not able to verify 
 the results obtained by these investigators. Mennes found that 
 immunity to Mic. pneumoniae depends on the action of serum 
 which produces active phagocytosis but was not certain that 
 this action is due to stimulation of the leucocytes. 
 
 In 1903 Wright and Douglas pointed out that there are cer- 
 tain substances in serum that so affect bacteria that they are 
 more easily taken up and disposed of by the leucocytes. This 
 substance they called "opsonin." They found opsonin present in 
 normal and immune serum and from their investigations decided 
 that the amount present in serum is variable, and can be increased 
 or decreased by the injection of killed cultures of bacteria. To 
 determine the amount of opsonin present in blood they modified 
 the method of estimating phagocytic power introduced by Leish- 
 man in 1902. According to their method the average number 
 of bacteria taken up by leucocytes, when bacteria, leucocytes and 
 serum are mixed together and allowed to remain together for a 
 certain time, is determined. The ratio of the average number 
 of bacteria per leucocyte when patient's seruin and serum from 
 a healthy individual determine the phagocytosis, they called the 
 opsonic index. The methods employed in the determination of 
 this opsonic index will be taken up later. 
 
 In 1904 Neufeld and Rimpau, entirely independently of 
 Wright and Douglas referred to the two well-known elements, 
 antitoxin and bactericidal substances, in immune serum and stated 
 that in anti -streptococcic and anti-pneumococcic serum they had
 
 22 VACCINE AND SERUM THERAPY. 
 
 found a third factor of importance in immunity. This element, 
 accordmg to these investigators, sensitizes the corresponding 
 bacteria and so modifies theva that they are more readily engulfed 
 by the leucocytes. This third factor in immunity they called 
 "bacteriotropin." It is to be noticed that Neufeld and Rimpau 
 worked with virulent streptococci and pneumococci. These or- 
 ganisms are not ingested by the leucocytes in a mixture of leuco- 
 cytes, virulent streptococci or pneumococci and normal serum. 
 However, when, instead of normal serum, a specific anti-serum 
 takes part in the mixture there is ingestion even of cultures of 
 virulent strains of these organisms. They furthermore showed 
 that the serum does not act on the leucocytes but exerts its in- 
 fluence on the bacteria, sensitizing them to the action of leuco- 
 cytes. 
 
 There seems now to be but little doubt that opsonin and 
 bacteriotropin are the same substances, the greatest difterence 
 that can be assigned, being that bacteriotropins are probably 
 what Wright and Douglas have called "immune opsonins." 
 
 Most investigators now take an intermediate position on the 
 phagocytic and humeral theories of antibacterial immunity. It is 
 quite generally accepted that both serum and leucoc\^es contain 
 substances, which, acting after the manner of ferments, are able 
 to dissolve bacteria. Just as all important manifestations of life 
 are found in the normal and pathological cellular elements, so also 
 the means of defense against harmful agents is probably closely 
 related to the condition and functions of cells which prepare and 
 secrete the protective substances by means of which bacteria and 
 other harmful agents are destroyed or neutralized.
 
 CHAPTER III. 
 
 THE OPSONIC INDEX 
 
 From the foregoing chapter it is seen that since the work 
 of Denys and LeClef in 1895, it has been known that serum is of 
 importance in phagoc3rtosis. Little attention, however, was 
 given to the various observations on this subject until the work 
 of Wright and Douglas was presented. Although Neufeld and 
 Rimpau discovered probably the same substances as Wright and 
 Douglas, these investigators are seldom referred to in the discusion 
 of the substances that change bacteria so as to prepare them tor 
 ingestion by leucocytes. There are two definite scientific reasons 
 for the great prominence given to the work of Wright and Doug- 
 las: one is that they advanced and improved a technique for the 
 determination of the amount of phagocytosis, by means of which 
 the opsonic index could be determined; the other is to be found 
 in the widespread interest in the methods they advanced for 
 active immunization of patients by the injection of killed cultures. 
 
 In 1902 Leishman presented a method for the quantitative 
 determination of phagocytosis. He mixed equal quantities of 
 patient's blood and bacterial emulsion, which he then incubated 
 for a time in a moist chamber. After incubation he made cover 
 glass spreads which he dried, fixed and stained. On these slides 
 he counted the number of bacteria ingested by the leucocytes, 
 from which the average number per leucocyte was determined. 
 This average he compared with the average per leucocyte obtained 
 when normal blood instead of patient's was added to the bacterial 
 emulsion. Leishman did not take into consideration the action 
 of serum on bacteria or leucocytes but devised merely a "method 
 of estimating phagocytic power." 
 
 Wright and Douglas, in their work on the determination of 
 the opsonic index modified Leishman 's method to meet their 
 theory on phagocytosis. According to their observation opsonin
 
 24 VACCINE AND SERL'M THERAPY. 
 
 is a substance present in all serum. The amount varies in the 
 different sera and as the amount varies so also will the amount of 
 phagocytosis vary. To determine the phagocytic power it is 
 necessary to have the three factors of phagocytosis in the mix- 
 ture. These three factors are found in the blood serum, leuco- 
 cytes and bacteria. A mixture of these three must be allowed 
 to remain together for a definite period of time, after which the 
 average number of bacteria taken up by the polymorphonuclear 
 neutrophiles must be determined. This average number of bac- 
 teria per leucocyte is called the "phagocytic index." The 
 phagocytic index obtained when serum from the patient is mixed 
 and incubated with leucocytes and bacteria, is compared with 
 the phagocytic index obtained when serum from a healthy in- 
 dividual is added to and incubated with a similar amount of the 
 same emulsions of bacteria and leucocytes. The result obtained 
 is called the "opsonic index." 
 
 The technique here given is essentially the one developed 
 by Wright and Douglas and demonstrated in New York City in 
 1906 by Wright and taught by his associate, Dr. Ross. It is 
 given somewhat in detail inasmuch as it is the one most generally 
 used, even though many modifications have been suggested and 
 followed by different investigators. 
 
 SERUM. . 
 Blood from the individual whose serum is to be used in the 
 determinations of the opsonic index, is collected as follows: A 
 small glass capsule with one curved capillary limb is made as is 
 indicated in Fig. 4. This tube is brought to a needle's point at 
 "b" by heating over the pilot flame of a Bunsen burner. This 
 
 Fig. 4. 
 
 point is later used to puncture the finger or lobe of the ear. The 
 end "a" of the capsule is left open. One of the fingers of the
 
 COLLECTION OF SERUM. 
 
 jLO 
 
 hand is cleaned with alcohol and water, after which a bandage, 
 handkerchief or rubber band is quite firmly tied around this fin- 
 ger. The windings of the bandage, handkerchief or rubber band 
 are started at the base of the finger and run gradually toward 
 the tip of the finger as more turns are put on. In this way there 
 is produced an accummulation of blood in the veins and capil- 
 laries. Now, with the pointed end of the capillary bulb, the fin- 
 ger is pricked at a point about a quarter of an inch back of the 
 nail, the needle end is broken off enough to open the capillary 
 end. The open end "a" is now held close to the drop of blood 
 which gradually fills the bulb; this is indicated in Fig. 5. 
 
 Fig. 5. 
 
 When enough blood has been drawn and collected in the bulb, the 
 bulb is held between the fingers at "a" and "c" and the end"b" is 
 heated and sealed off. As the end "b" cools the blood will draw 
 away from the end "a" and finally all the blood will collect in 
 the bulb proper. After this the bulb is again taken hold of at 
 the curve "c" and by a rapid swing of the arm, as is practiced 
 in shaking down a clinical thermometer, the blood is thrown down 
 into the sealed end "b" as is indicated in Fig. 6. The blood is 
 now allowed to clot in order to expel the serum. The serum 
 may also be separated by hanging the bulb at "c" over the arm 
 of the centrifuge, and centrifuging until the clot and serum have
 
 26 VACCINE AND SERUM THERAPY. 
 
 well separated. The tube may now be opened by filing and break - 
 incr at "c." 
 
 Fig. 6. 
 
 According to the original method of Wright and Douglas, 
 blood was obtained from the patient and from a number of 
 healthy individuals. Equal parts of the normal serum were taken 
 and mixed together or "pooled." This was done so as to get a 
 better normal serum. After it has once been established that 
 serum from a certain healthy individual has an opsonic index 
 of 1 . for a particular organism when compared with pooled 
 serum, this individual's serum replaces the pooled serum. After 
 this has been established, the opsonic index of any patient for 
 this organism is determined by dividing his phagocytic index 
 by the phagocytic index of the "normal individual," as he may 
 now be called. 
 
 LEUCOCYTES. 
 
 The leucocytes used in the determination of the opsonic 
 index are usually obtained from the blood of supposedly healthy 
 individuals, most frequently from the blood of the investigator 
 himself. No particular stress, however, is laid upon the individ- 
 ual from whom the blood is obtained. 
 
 To obtain blood, preferably, the middle finger is cleaned, con- 
 gested and punctured in the same manner as was followed in the 
 collection of serum. Ten drops of blood are collected in about 
 lOc.c. of a normal salt solution containing one per cent of sodium 
 citrate. The sodium citrate is added to keep the blood from 
 clotting. The tube containing the mixture is then centrifuged 
 in a centrifuge of not too great speed. When the speed exceeds 
 1,200 to 1,500 revolutions per minute, the cells are too closely
 
 LEUCOCYTES EMULSION. 27 
 
 packed together and form clumps. The mixture is centrifuged 
 until the corpuscles are thrown to the bottom of the tube and 
 the fluid above is clear, although it may be slightly straw 
 colored. The supernatent fluid is then drawn off with a 
 pipette. The corpuscles are washed free from serum and sodium 
 citrate by again filling the tube with normal salt solution, mix- 
 ing thoroughly and centrifuging until there again is a clear super- 
 natent fluid. If the material in the centrifuge tube is now ex- 
 amined, one sees a clear fluid above and a red fluid in the lower 
 part of the tube. The red fluid below, however, is not of the 
 same shade throughout. The uppermost part consists of a gray- 
 ish red layer, the leucocytes. These are layered above the red 
 blood cells because of the difference in specific gravity of the 
 leucoc5rtes and red blood cells. It is from the gray layer that 
 the leucocytes, to be used in the determination of the opsonic 
 index, are obtained. 
 
 To obtain the leucocytes the clear fluid above is drawn oft' 
 with a pipette and then with a clean pipette of about 1 . 6m. m. 
 inside diameter, the leucocytes are removed. To do this the 
 pipette firmly held in the hand, all of the air in the nipple 
 is expelled, and then as the pressure on the nipple is gradually 
 released, the open end of the pipette is held on the surface of the 
 uppermost layer of grayish red color. This fluid will contain 
 many red blood cells and also a relatively large number of leuco- 
 cytes. After this fluid has been drawn into the larger part of 
 the pipette the capillary end is sealed off. This fluid is called 
 "leucoc}'i:ic cream." Wright and Douglas state that in their 
 experience there is no variation of the ability to engulf bacteria 
 within the space of a few hours but that after three days the 
 phagocytic power decreases to one-half or one-third of what it 
 was when freshly drawn. 
 
 BACTERIAL EMULSION. 
 
 Inasmuch as Wright had early decided that opsonins are 
 specific, such species or varities of bacteria are used in the deter- 
 mination of the opsonic index as may be of importance in the 
 bacterial infection. In the selection of the particular culture to 
 be used, there are two sources— either the different cultures iso- 
 lated from the lesion are used or else the same species and varieties
 
 28 
 
 VACCINE AXD SERUM THERAPY. 
 
 of microorganisms are taken from a stock culture. Usually liv- 
 ing freshly grown bacteria are used to make emulsions. The 
 principle, or practically only exception to this has been in the 
 determination of the opsonic index for the bacillus of tubercu- 
 losis, for which often killed and old cultures of the organism have 
 been- used to make the bacterial emulsion. 
 
 In all cases it is intended that the bacteria shall be well sep- 
 arated and suspended uniformly in salt solution. The tech- 
 nique for making the bacterial suspension varies with the differ- 
 ent organisms, some of which it will be necessary to consider 
 separately. The organisms for which the opsonic index is de- 
 termined may be divided into three classes. 
 
 A. Many of the organisms belonging to this 
 class grow on the ordinary media, while for 
 others, media containing blood or other body 
 fluids are necessary. The organisms occur 
 singly or in pairs and when present in larger 
 groups the cell aggregates can be easily 
 broken up. 
 
 (a). Mic. pyogenes aureus, albus and citreus, 
 B. coli, B. typhosus, B. pyocyaneus, etc., are 
 grown on slant agar for twenty-four hours 
 at 37° C. Various investigators, including 
 Wright, have used iour to five hour cultures 
 of these organisms. 
 
 (b). Mic. gonorrhoeas has usually been 
 grown on hydrocele or human blood agar. 
 Cole and Meakins obtained their cultures from 
 growth on agar in which 0.5 c.c. of fresh 
 blood had been added to 10 c.c. of ordinary 
 agar. The age of the cultures used has varied 
 from four to twenty-four hours incubation 
 at 37° C. 
 
 (c). Mic. meninigitidis, Mic. pneumoniae, 
 etc., produce good growth on sheep serum 
 agar. The cultures have usually been incubated at 37° C. from 
 twelve to twenty-four hours. 
 
 To each culture of the proper age abotit ten drops of sterile 
 normal salt solution are added. With a platinum loop the cul- 
 
 FiG. 7.
 
 BACTERIAL EMULSION. 29 
 
 ture is washed off the media and with a thick walled capillary 
 pipette, with the end broken off squarely, the suspension is drawn 
 into the pipette. The open end of the pipette is firmly pressed 
 against a watch glass, as indicated in Fig. 7. When this is done 
 only a small crevice will remain between the end of the pipette 
 and the watch glass. The bulb is now compressed and the bac- 
 teria suspended in the salt solution are forced out through the 
 small crevices. This is done to break up the small clumps of bac- 
 teria. 
 
 B. The organisms belonging to this class grow on the same 
 kind of media as do those belonging to the first group. The or- 
 ganisms of this group form cell aggregates that are not easily 
 broken up. The streptococci are the most important organisms 
 in this class. Streptococci frequently grow in long chains, 
 varying from two to thirty cocci. It is evident that a leuco- 
 cyte may be able to engulf one or more cocci without being able 
 to engulf a chain consisting of twenty or thirty cocci. The method 
 for breaking up ordinary clumps will not suffice for breaking up 
 chains of streptococci. 
 
 While observing the opsonic index for streptococci in ery- 
 sipelas, the writer found it necessary to adopt some method to 
 break up the long chains of streptococci frequently isolated from 
 the lesions in erysipelas. This method consisted in the addition 
 of 2 to 3 c.c. of sterile salt solution to each twenty-four hour cul- 
 ture on glycerine, glucose agar. After washing off the growth, 
 the emulsion was put into a small test tube containing sterile 
 sea sand; the tube was sealed in a flame and shaken for one and 
 one-half hours in the shaking machine. The sand and emulsion 
 in the tube were centrifuged for one minute and the supernatent 
 fluid drawn off. In this way short chains, from two to four 
 cocci, were obtained. 
 
 Another method was devised by Wright of Harvard University. 
 This method differs only from the one of Wright and Douglas in the 
 substitution of a paraffine block for the watch glass. When the 
 open end of the pipette is held against the paraffine the crevices 
 very small so that the chains are more completely broken up. 
 This method gives very satisfactory results. . 
 
 C. This class includes practically only one species of mi- 
 croorganisms, the bacillus of tuberculosis. The preparation of
 
 30 VACCINE AND SERUM THERAPY. 
 
 suspensions of tubercle bacilli is attended by numerous difficul- 
 ties, because this organism when grown on artificial media forms 
 conglomerated masses. According to Wright and Douglas the 
 living tubercle bacilli are heated to 100° C. before breaking up 
 the clumps. Later Wright modified this techinque by heating 
 the bacilli to 100° C. on three successive days. The clumps after 
 this are ground up in an agate mortar or in a watch glass, two or 
 three drops of 0. 1 per cent salt solution being added at a time until 
 two or three c. c. have been added. One-tenth per cent salt 
 solution is used in making this suspension because with greater 
 concentration the bacilli are again clumped. Later Wright added 
 1 . 5 per cent salt solution in maknig up the emulsion of tubercle 
 bacilli because he found that this concentration is necessary to 
 prevent spontaneous phagocytosis. When the heated bacilli are 
 thoroughly rubbed and suspended in 1 . 5 per cent salt solution 
 a homogeneous mixture, containing but few clumps and many 
 isloated bacteria, results. This mixture is then centrifuged at 
 high speed for about ten minutes. After this the supernatent 
 fluid is drawn off, and enough salt sokition added to get the right 
 concentration of the emulsion. 
 
 While this method gives a fairly homogenous suspension of 
 tubercle bacilli, still in an emulsion made in this way many of 
 the tubercle bacilli are broken up. In determining the opsonic 
 index it is necessary to count the number of bacilli taken up by 
 the leucocytes, and when there is fragmentation of bacilli it is 
 necessary either to count each fragment as one bacillus, or else to 
 determine the fractional part of a bacillus. Either of these 
 methods is most unsatisfactory. 
 
 Sellard and Jeans have emulsified the living tubercle bacilli 
 in the same manner that has been used for the emulsification of 
 other bacteria. After this they have killed the bacilli by exposing 
 the emulsion to sunlight for a number of hours — ten hours being 
 sufficient to kill all tubercle bacilli present. In such emulsions 
 they have gotten no spontaneous clumping, no fragmentation, nor 
 spontaneous phagocytosis. 
 
 Walker has recommended a method, according to which Dor- 
 sett's egg medium is heavily inoculated with an actively growing 
 culture of the bacillus of tuberculosis. After fourteen to eighteen 
 hours of incubation at 37° C, salt solution is squirted over the
 
 MIXTURE OF BACTERIA, LEUCOCYTES AND SERUM. 31 
 
 culture, which is now rubbed off into the salt solution, the clumps 
 being broken up with a platinum needle. The suspension is then 
 filtered first through a loosely packed cotton filter and later through 
 a filter made with scraped filter paper. Filtration is repeated 
 until all clumps are removed, after which the clump-free suspen- 
 sion is heated to 75° C. for twenty to thirty minutes. 
 
 STRENGTH OF BACTERIAL EMULSION. 
 
 The strength of bacterial emulsion most commonly used has 
 a slightly opalescent appearance. It has been found that cloudi- 
 ness may be absent or be only very slight and still the emulsion 
 ma}^ contain too many bacteria. According to Wright's instruc- 
 tions the strength of the emulsion to be used is one which gives 
 an average count of from five to eight bacteria for leucocytes 
 when normal serum, leucocytes and the suspension of bacteria 
 are incubated for the proper length of time. Walker has obtained 
 better results with heavier suspensions of bacteria and diluted 
 serum. It is not very difficult to make an emulsion of staphy- 
 lococci, for after a bit of experience it can be determined by the 
 naked eye whether the suspension of this species is heavy enough 
 or not. The tubercle bacillus, however, presents greater diffi- 
 culties, it frequently being necessary to actually make a trial 
 test for the bacterial emulsion. Simon and others have proposed 
 the numerical determination of the number of bacteria per c. c. 
 in the bacterial emulsion. 
 
 MIXTURE OF BACTERIA, LEUCOCYTES AND SERUM. 
 
 From the definition of the opsonic index, it is evident that 
 similar quantities of the same factors must be taken to make com- 
 parative mixtures. Furthermore, according to the methods de- 
 vised by Wright, equal amounts of each factor are mixed to- 
 gether. When it is desired to use diluted serum equal volumes 
 may still be used if the serum be diluted properly before taking 
 the volume. 
 
 In order to get equal volumes Wright has made a capillary 
 pipette, the walls of the capillary part of which are thick. The 
 end of the pipette is broken oft' squarely. About a quarter or 
 one-half inch from the open capillary end a mark is made with a 
 soft wax pencil. This pipette is shown in Fig. 8. By means of
 
 32 VACCINE AND SERUM THERAPY. 
 
 a rubber teat which is attached to the large end of the pipette, 
 leiicocytic cream is drawn into the capillary end up to the mark. 
 Then the capillary end is withdrawn from the leucocytic emulsion 
 and the emulsion in the tube is drawn about one quarter inch 
 further up into the tube. After this the capillary end of the 
 pipette is immersed in the bacterial emulsion, which is taken into 
 the tube up to the mark. The tube is withdrawn and again a 
 small amount of air drawn into the capillary tube. After this the 
 serum is drawn in, again taking the amount necessary to fill the 
 capillary pipette to the mark. Walker has suggested that small 
 quantities of serum and leucocytic cream be put into small tubes 
 and that serum and leucocytes be taken only once from each of 
 these small tubes. This is done in order to avoid carrying ma- 
 terials from one ttibe into another. 
 
 The capillary pipette now contains equal volumes of leuco- 
 cytic cream, bacterial emulsion, and serum, as is show^n in Fig. 9. 
 
 Fig. S. 
 
 Fig. 9. 
 
 All three volumes are now forced out onto a glass slide, being drawn 
 in and out with the pipette in order to mix the three parts thor- 
 oughly. When this has been done the mixture is drawn into the 
 tube and allowed to come about half way up the capillary part 
 when the open end is sealed off in a pilot flame of the Bunsen 
 burner. Walker mixes the three factors in a small test tube 
 taking precautions against air bubbles. Slides, however, have 
 seemed better adapted especially because air bubbles are more 
 easily avoided. 
 
 After sealing the capillary pipette, containing the mixture of 
 leucocytes, bacteria and serum, it is incubated at 37° C. Realiz- 
 ing that the pipette containing the mixture will not assume the 
 temperature of the thermostat if it is merely exposed to the air 
 in the thermostat, Wright originated a so-called opsonizer. 
 
 The length of time for which the mixture is incubated varies 
 for the different organisms. It is to be noted that up to a certain
 
 wSMEARS. 33 
 
 limit the longer the period of incubation the more marked will 
 be the phagocytosis. The incubation time must in some cases 
 be limited because of solution and agglutination of bacteria. 
 In all instances the mixture containing patient's serum and 
 that containing normal serum must be incubated at the same 
 temperature and for the same length of time. The time of in- 
 cubation varies from five to thirty minutes, depending upon the 
 species of microorganism and the properties of the blood serum 
 tested. 
 
 After incubation the nipple is removed and the capillary 
 end of the pipette is broken off, the nipple is then replaced and 
 the leucocytes, bacteria and serum are again thoroughly mixed 
 on a glass slide, after which smears are made. 
 
 SMEARS. 
 
 If reference again is made to the principle involved in the 
 determination of the opsonic index, it will be noted that the index 
 shows the ratio of the number of bacteria taken up by the poly- 
 morphonuclear neutrophiles w^hen patient's serum is a part of 
 the mixture, to the number of bacteria taken up by the same 
 class of leucocytes when normal serum is a part of the mixture. 
 It is therefore necessary to count the number of bacteria in the 
 polynuclear leucocytes. In order to have an abundance of leuco- 
 cytes the grayish red layer is taken off the centrifuged blood. 
 This contains a relatively larger number of white blood cells than 
 does blood freshly drawn. If a drop of this leucocytic cream 
 is spread out by dropping a cover glass onto it, fixed, and stained, 
 many red blood cells and some w^hite blood cells wall be found in 
 the field of the microscope. It w^ould, how^ever, be a tedious 
 task to find fifty or one hundred polynuclear neutrophiles. Wright 
 and Douglas have devised an ingenious method to gather the leuco- 
 cytes together in certain parts of the slide. The method of doing 
 this is based on the difference in size of red blood cells, mononu- 
 clear and polynuclear leucocytes; the polynuclear and large mon- 
 onuclear cells being largest in size. 
 
 The method of Wright and Douglas is as follows: A slide is 
 cleaned and slightly scratched by rubbing with jeweler's emory 
 paper to slightly roughen the surface of the glass slide. On the 
 left end of this slide a drop of the incubated mixture, which has
 
 34 
 
 VACCINE AND SERUM THERAPY. 
 
 been thoroughly mixed after incubation, is placed. Then a 
 slide with a smooth edge is made into a spreader by breaking 
 off a corner. This is done so that the two margins of the spread 
 shall be on the slide instead of running over the edges of the same. 
 
 Fig. 10. 
 
 The narrowed end of the spreader is now touched to the slide 
 and the drop of blood allowed to run to the under edges of the 
 spreader. The spreader, held at an angle of about 35° to 45° 
 is drawn over the slide as is indicated in Fig. 10. The spread 
 on the slide will have an outline as is indicated in Fig. 11. 
 
 The different steps taken in making the spread are essential 
 to secure good smears. When the spreader is pressed firmly 
 enough against the slide while it is being drawn over the same, 
 
 Fig. 11. 
 
 the polynuclear leucocytes, being larger than the red blood cells, 
 will slide out at the edge of the spreader or be drawn to the end 
 of the spread. Care must be taken not to make the smear too 
 thin. 
 
 FIXING AND STAINING OF THE SMEARS. 
 
 After the smear has been made it must be fixed and stained 
 for examination. Fixing and staining is usually accomplished 
 by the ordinary blood stains, Leishman's, Wright's, Jenner's all 
 giving good results, except when the index for the tubercle bacillus 
 is to be determined. The writer, however, has gotten more sat-
 
 CALCULATION OF OPSONIC INDEX. OO 
 
 isfactory results for organisms, other than the bacillus of tuber- 
 culosis, by fixing the spread with methyl alcohol for one minute, 
 washing off with water and then staining with Loeffler's methy- 
 lene blue for three to five minutes. In staining slides on which 
 the index for the tubercle bacillus is to be obtained, a modification 
 of Wright's method has given most satisfactory results. The smears 
 are first fixed in a saturated solution of bichloride of mercury which 
 is then washed oft' with water. After this the slide is immersed 
 in a jar containing Ziehl's carbol-fuchsin . The stain is heated 
 by placing the jar in a heated water bath. After five minutes 
 of staining in. hot carbol-ftichsin, the stain is washed off and the 
 smear is decolorized in a mixture of 97 parts of alcohol and 3 
 parts of concentrated hydrochloric acid. The smear is counter 
 stained in a solution of one-half grams each of sodium carbon- 
 ate and methylene blue in 100 c. c. of water. This counter-stain 
 acts rapidly and has the advantage that if the slide be over 
 stained in the process it can be easily decolorized with warm water. 
 
 EXAMINATION OF SMEARS. 
 
 The spreads are examined by first going over the slide with 
 the low power of the microscope to determine whether the slide 
 has been properly stamed and also to find the part of the slide 
 where the polynuclear leucocytes are most numerous. Usually 
 it is found that these are most abundant near the margins and 
 at the end of the smear. Wright has recommended that those 
 at the end of the smear be examined. 
 
 Under the high power of the microscope the number of bac- 
 teria engulfed by a definite number of polynuclear leucocytes is 
 determined. The number of leucocytes examined varies. Wright 
 based many of his determinations on examinations of twenty 
 leucocytes. Most investigators, however, have counted the num- 
 ber of bacteria in fifty or more polynuclear leucocytes. 
 
 The average number of bacteria per leucocytes is spoken 
 of as the phagocytic index. 
 
 CALCULATION OF THE OPSONIC INDEX. 
 
 The opsonic index of Wright is determined by dividing the 
 phagocytic index obtained when patient's serum is used with
 
 36 VACCINE AND SERUM THERAPY. 
 
 a certain leucocytic cream and bacterial suspension, by the pha- 
 gocytic index obtained when serum from the healthy individual 
 is used with the same leucocytic cream and bacterial emulsion. 
 This may be illustrated in a concrete case as follows : If the aver- 
 age number of staphylococci phagocytosed as determined by 
 counting the cocci in fifty leucocytes is eight when the patient 
 serum is used, and ten when normal serum is used, then eight- 
 tenths or . 8 is the opsonic index.
 
 CHAPTER IV. 
 
 CRITICISMS AND MODIFICATIONS 
 
 OF WRIGHT'S OPSONIC INDEX 
 
 DETERMINATIONS 
 
 Wright's publications on opsonins, the part they play in im- 
 munity and the method of determination of the opsonic index 
 lead to much investigation on these subjects. Many mvestiga- 
 tors followed Wright's methods closely while some have followed 
 what they supposed were Wright's methods. The first reports 
 of work done by investigators other than Wright and his pupils, 
 agree strikingly with the results obtained by Wright. Later 
 results, however, were not as favorable either before or after 
 Wright himself and some of his pupils had demonstrated and 
 taught his technique. For a time want of personal skill and 
 ability, improper methods of work, and lack of ability to manipu- 
 late were supposed to account for results which did not agree 
 with those obtained by Wright. Numerous investigators, how- 
 ever, after having received instructions from Wright and his 
 pupils on the methods of determining the opsonic index, seriously 
 questioned the methods of Wright. This list of investigators 
 includes Park, Simon, Baldwin, Cole, Moss, Potter, Bolduan, 
 Walker, and many others. On the other hand there are num- 
 erous investigators who have not questioned the technique and the 
 results obtained by following the methods of Wright. These 
 investigators followed principally the opsonic index in the various 
 bacterial diseases and made efforts to determine the nature of 
 opsonins, their importance and behavior in the various diseases. 
 
 The opsonic index, according to Wright, is obtained when 
 the average number of bacteria ingested per leucocyte in a mix- 
 ture of patient's serum, bacterial emuslion and leucocytes in- 
 cubated for a certain period of time, is divided by the average
 
 38 VACCINE AND SERUM THERAPY. 
 
 number of bacteria ingested !)y the leucocytes in a mixture of 
 the same quantity of serum from a normal individual, the same 
 bacterial emulsion and leucocytic cream, incubated for the same 
 length of time at the same temperature. In the process of the 
 determination of the opsonic index mechanical technique, serum, 
 bacterial emulsion, leucocytic cream, and calculation of the aver- 
 age number of bacteria ingested are of importance. Inasmuch 
 as the results obtained by the different investigators are so 
 discordant and inconsistent, all of these factors have been 
 considered, emphasized and, in the minds of some at least, 
 improved. 
 
 MECHANICAL TECHNIQUE. 
 
 The mechanical part of the technique of Wright has prob- 
 ably been less criticised than any other part of his method. Var- 
 ious investigators have reported their technique in full and some 
 at least have shown that they have not interpreted Wright's ex- 
 planations of the same correctly, while others have modified the 
 technique in order to give greater accuracy to the method of the 
 determination of the index. 
 
 Only some of the modifications suggested can be mentioned. 
 Barber, thinking that the mark on the pipette as made by the 
 wax pencil, is too wide and therefore leads to inaccuracies in the 
 amounts used in the mixtures, has suggested that the mark on 
 the capillary pipette be made with a glass hair dipped in Bismarck 
 black. This suggestion, however, is not generally followed as it 
 is possible for one experienced in handling the capillary pipette 
 to draw fluids to the upper or lower edge of the wax pencil mark 
 with as much accuracy as it is to take it to the mark made by the 
 glass hair. 
 
 Walker has suggested the use of a water bath for the incu- 
 bation of the mixture of serum, leucocytes, and bacterial emul- 
 sion. Undoubtedly this has advantages over incubation for 
 short periods of time in the air in the thermostat. Wright's op- 
 sonizer, however, is more convenient and almost immediately 
 brings the pipette to the temperature of the metal of the opsonizer. 
 
 For the collection of serum. Walker has suggested that the 
 capsule be held in position by inserting it into a slit in a slide 
 box. Unless one is taking his own blood, however, it is
 
 NORMAL SERUM. 39 
 
 about as convenient to hold the capsule in the hand as to hold 
 the punctured finger near the table on which the slide box rests. 
 
 Walker has also advocated that small quantities of full 
 strength or diluted sera and leucocytic cream be put into separ- 
 ate small test tubes in order to avoid carrying bacterial emulsion 
 to the lencocytic cream from which other preparations are to be 
 made later. This method is certainly to be recommended. 
 
 Walker has also modified Wright's methods concerning the 
 capillar}^ pipette containing the mixture. In making opsonic 
 index determinations, Walker breaks off the large end of the cap- 
 illary pipette and seals both ends of the capillar}^ tube. When 
 this is done it becomes necessary to again fit the capillary part 
 of the pipette to a larger tube to which a rubber teat can be fas- 
 tened. This Walker accomplishes by fastening a thin rubber 
 sheet over the open end of the large pipette. Thisworks as a dia- 
 phragm and can be easily punctured by the capillary tube. 
 
 SERUM. 
 
 Immunity to and recovery from numerous infections accord- 
 ing to Wright's theory is due to the presence of opsonin. Opsonin 
 prepares bacteria for ingestion by the leucoc5rtes and is a sub- 
 stance contained in the serum. In the determination of the 
 opsonic index the ratio of bacteria ingested by the leucocytes is 
 established by mixing bacteria and leucocytes in one case with 
 patient's serum and in another case with serum from healthy 
 individuals. It is thus evident that the serum is the important 
 and valuable factor. 
 
 Normal Serum. 
 
 In the determination of the opsonic index numerous in- 
 vestigators have been struck by the apparently unexplainable 
 indices found. This has lead to the investigation of the 
 opsonic index obtained with serum from the normal individual, 
 to deterinine whether the index does not vary in the normal, as 
 well as in the infected, individual. 
 
 If reference is made to the technique, as proposed to obtain 
 the normal phagocytic index, it will be observed that Wright 
 recognized that there is a difference in the amount of opsonin 
 present in serum from the normal individual. He states that
 
 40 VACCIXE AND SERUM THERAPY. 
 
 the opsonic index of a normal individual will vary from O.S to 
 1.2. To obtain the true amount of opsonin in normal serum, he 
 pools equal quantities of sera from several normal individuals. 
 However, after it has been established that a healthy individual 
 has an opsonic index of 1.0, as determined by estimation with 
 pooled normal sera, he uses serum from this individual as a normal 
 serum. Wright's own determinations, from which he concedes 
 that there are variations in the amount of opsonin present in 
 the blood of healthy individuals, discredit the accuracy either 
 of the method of determining the opsonic index or the A^alue of 
 determining the same. If the opsonic indices of two normal in- 
 dividuals is . 8 and 1 . 2 respectively on the same day as deter- 
 mined by CO parison with pooled normal serum, then as com- 
 pared to each other their opsonic indices vaiy from 0.66 to 1.5 
 respectively. It is thus evident that from Wright's concession 
 the index of a normal individual may vary from . 66 to 1.5 
 when one serum is used as a control for the other. 
 
 Indices for normal serum as actually determined will not 
 even vary within these limits. The writer sometime ago reported 
 results on a series of ten specimens of blood from ten healthy 
 adults, studied to determine the differences in opsonic power in 
 healthy individuals. The leucoc3rtes were washed twice with 
 0.85 salt solution. The number of staphylococci taken up by 
 100 polynuclear leucocytes was counted in two preparations, sepa- 
 rately incubated. The phagocytic indices as determined by two 
 workers A and B are as follows: 
 
 Source. A B 
 
 N 9.48 7.63 
 
 J 9.16 5.20 
 
 B 8.75 6.80 
 
 McL 7 . 27 4.37 
 
 Z 6.63 5.12 
 
 C 6.60 5.37 
 
 S 5.89 4.82 
 
 5.84 4.57 
 
 H 5.49 6.03 
 
 Ns 4.91 4.37 
 
 The great error introduced by this method becomes evident 
 upon the determination of the opsonic indices when each serum 
 is used for comparison with all of the others. In the series A, 
 the normal opsonic index varies from 0. 52 to 1 . 93, while in series 
 B it varies from 0.57 to 1.75. Moreover, the indices obtained 
 by the two workers A and B vary considerably for the same sera.
 
 DILUTIONS OF SERUM. 41 
 
 Bolduan has reported determinations of the opsonic index 
 of normal individuals and finds that as far as the bacillus of tuber- 
 culosis, staphylococci and streptococci are concerned, there is 
 marked variation not only in different individuals but also from 
 day to day. 
 
 Moss has found opsonic indices for staphylococci to vary 
 from 0.18 to 0.56 in normal rabbits. 
 
 The same serum has also been tested a number of times 
 with the same leucocytes and bacterial emulsion. Bolduan re- 
 ports opsonic index determinations for a single serum which 
 vary from 1 . 05 to 1 . 46. The writer has reported determinations 
 made by taking five specimens of blood from the five fingers 
 of the same hand of a healthy adult. The leucocytes used for 
 the determination were washed twice in 1.5 per cent potassium 
 citrate in normal salt solution. The phagocytic indices for 
 Mic. pyogenes aureus are shown in the table: 
 
 1st 100 leukocytes 2d 100 leukocj'tes Phagocytic 
 
 counted. counted. index. 
 
 Specimen 1 166 196 1.810 
 
 2 176 183 1.795 
 
 3 198 199 1.985 
 
 4 185 175 1.800 
 
 5 230 190 2.100 
 
 If the indices for each specimen of blood be compared with 
 all the others, the opsonic index as determined by counting the 
 number of cocci in two hundred cells will be found to vary from 
 0.85 to 1.22. 
 
 It thus seems evident that if normal serum varies as much 
 as has been found, the variations from the normal, unless marked, 
 can indicate but little. The most that would seem warranted 
 is to determine whether a serum is "high" or "low" in opsonic 
 content, as the case may be. 
 
 Dilutions of Serum. 
 
 Wright has usually used undiluted serum in the determina- 
 tion of the opsonic index. Simon, Lamar and Bispham have 
 shown that by the dilution of some sera there often is a rapid 
 exhaustion of phagocytic power of the leucocytes. They have 
 observed that undiluted pig's serum manifests a most intense 
 opsonizing effect on staphylococci but that this action diminishes 
 markedly upon dilution of the serum. Human serum, on the 
 other hand, though not having as marked an initial opsonizing
 
 42 VACCINE AND SERUM THERAPY. 
 
 power, will retain this power on much greater dilution. They 
 liave fiirther found that in human beings, who are supposedly 
 in good health, the phagocytic power in concentrated serum will, 
 in some cases, diminish in proportion to the degree of dilution, 
 while with the serum of other individuals, more rapid exhaustion 
 takes place. 
 
 Moss has compared the opsonic index in various dilutions 
 of serum and has found that the index obtained in one dilution 
 is not proportional to that obtained in all the other dilutions. 
 
 Walker's reports on the results obtained by the dilution of 
 serum differ from those obtained by Moss. Walker has found 
 that wHth many bacteria, undiluted normal serum will opsonize 
 so many bacteria in a heavy suspension of bacteria that the leu- 
 cocytes will contain so many microorganisms, that counting is 
 impossible. If the bacterial suspension be made less heavy fewer 
 bacteria will be taken up by the leucocytes, but under this 
 condition the serum will not be exhausted of its opsonin and 
 part will be lost in the estimation of the opsonic index. A 
 serum containing much less opsonin may opsonize just as many 
 bacteria in a light bacterial suspension as a serum containing 
 more opsonin. "When thin suspensions of bacteria for which 
 much opsonin exists in the serum are used it generally happens 
 that both the sera sensitize all the bacteria so that the work if 
 accurately done will produce ecivial phagocytic indices for both — 
 in other terms, an opsonic index of unity — regardless of the real 
 relation of the serum. Phagoc^^tic indices proportional to the 
 sera tested may readily be obtained by diluting all the sera equally 
 to a sufficient degree, and using with these diluted sera a thick 
 bacterial suspension." For some bacteria as B. typhosus, cer- 
 tain strains of streptococci and tubercle bacilli, Walker does not 
 recommend the dilution of serum, while for staphylococci, 
 some strains of streptococci and tubercle bacilli and for B. coli 
 he does. 
 
 Walker's views concerning the utilization of all the opsonin 
 capable of sensitizing the particular species of bacteria by the 
 use of heavy bacterial suspension and diluted sera, must be favor- 
 ably received. Before they can be accepted and followed it will, 
 however, be necessary to disprove the results of Moss and others 
 concerning the dilution of opsonin by the dilution of the serum.
 
 BACTERIAL EMULSIONS. 43 
 
 BACTERIAL EMULSIONS. 
 
 Wright, in his technique, attempts in every case to break 
 up all clumps of bacteria and tries to get such strength of suspen- 
 sion that the number of bacteria per leucocyte can be easily 
 determined. With certain organisms trial tests are necessary. 
 After this, the suspension may be diluted or bacteria added as 
 may be indicated. 
 
 Most investigators have experienced difficulty in obtaining 
 suitable suspensions and Wright himself has changed his tech- 
 nique for making bacterial suspensions from time to time. The 
 difficulties that have been experienced are of two kinds: those 
 dealing with the strength of the bacterial suspension, and those 
 concerning the condition of the microorganisms in the suspension. 
 
 Difficulties depending upon the strength of the bacterial 
 suspension. Numerous investigators have given definite figures 
 to be obtained for the phagocytic index with normal serum. In 
 most cases it is desirable that the phagocytic index for normal 
 serum be between six and fifteen. Simon has found that when 
 the emulsion contains between 666,000 and 2,000,000 microor- 
 ganisms per cubic millemeter the best results are obtained. Re- 
 cently Walker has emphasized certain facts that must be taken 
 into consideration in determining the strength of the bacterial 
 emulsion. If the suspension does not contain enough bacteria 
 to exhaust all the opsonin in the strongest serum, a certain amount 
 of opsonin will, according to Walker, be lost in the estimation of 
 the opsonic index. In order to get the right strength of bacterial 
 suspension. Walker tests the same with serum diluted 1 to 30 
 and 1 to 15. If the 1 to 15 dilution of serum shows a phagocytic 
 index twice as great as that obtained in the 1 to 30 dilution then 
 enough bacteria are present in the suspension. Too such a sus- 
 pension he, however, adds more of the culture, because having 
 more bacteria than are absolutely necessary will not change the 
 opsonic index but will certainly furnish enough bacteria to ex- 
 haust the serum of its opsonin. Only with the staphylococci 
 will a too heavy suspension effect the true index. This Walker 
 believes is due to the action of a product of bacterial growth. 
 To overcome the excessive phagocytosis when heavy suspensions 
 of bacteria are used. Walker dilutes serum. However, as has been
 
 44 VACCINE AND SERUM THERAPY. 
 
 mentioned before, there are'conflicting views concerning the effect 
 of dilution of serum for which the opsonic index is to be 
 determined. 
 
 DIFFICULTIES ARISING BECAUSE OF THE PARTICULAR 
 SPECIES OF BACTERIA IN THE SUSPENSION. 
 
 Anyone who has made opsonic index determinations has at 
 times had sHdes to examine in which the bacteria were not 
 well stained and in which the microorganisms were apparently 
 clumped. Frequently only shells of organisms remain and at 
 times the bacteria are completely dissolved. These difficulties 
 are apparently dependent upon the particular culture used but 
 to a greater extent upon the agglutinins and lysins present 
 in different sera. To overcome this difficulty young cultures 
 are preferred because they stain better than do old cultures. 
 To overcome agglutination and lysis the serum is in some cases 
 heated to 55° or 65°. The clumping of bacteria in opsonic 
 index preparations is given little attention by Wright. Other 
 investigators have, how^ever, regarded clumping of bacteria 
 as responsible for the occasional enormous differences of the 
 indices determined. Walker gives tables which indicate the 
 objection of clumps on the uniformity of phagocytosis and has 
 prepared a technique by which he obtains clump-free bacterial 
 suspensions. According to this technique the bacteria are rub- 
 bed gently in a mortar with small, and gradually increasing, 
 quantities of salt solution until a thick suspension is obtained. 
 This suspension is then filtered through moistened scraped filter 
 paper until in a stained specimen no more clumps are found. 
 Even though a technique may be devised to obtain relatively 
 clump-free suspensions of bacteria, this will not do away with the 
 clumping occasionally observed in opsonic index preparations, 
 because the action of agglutinins in undiluted or only slightly 
 diluted sera w^ill again cause clumping of the bacteria. 
 
 LEUCOCYTES. 
 
 There has been but little criticism of the method of collection 
 of leucocytes, as proposed by Wright. Simon has used 0.1 per 
 cent ammonium oxalate instead of 1.0 percent sodium citrate 
 in salt solution. Some investigators have drawn the corpuscles
 
 CRITICISM OF CALCULATION OF OPSONIC INDEX. 45 
 
 off after the first centrifugalization while others have attempted 
 to remove the serum of the blood and the citrate or oxalate 
 solution by several washings with normal salt solution. 
 
 To the source of the leucocytes Wright attaches little impor- 
 tance, while most other investigators have aimed to obtain leuco- 
 cytes froiTL the blood of apparently healthy individuals. The 
 work of Peterson and Hiss and Zinsser indicates that the leuco- 
 cytes contain substances that are of importance in immunity 
 and that these substances vary in the leucocytes of normal and 
 immunized animals. For this and other reasons it seems advis- 
 able in all cases to obtain the leucocytes from the blood of normal 
 individuals. 
 
 Walker draws the blood from which the leucocytes are to 
 be obtained into citrate solution heated to 37° C. The leuco- 
 cytic cream is kept at this temperature up to the time of mix- 
 ture with serum and bacterial emulsion. He believes this to be 
 of great importance. In experiments performed by the writer 
 and others, this has not been found to be of especial value in the 
 production of uniform phagocytosis, nor do Walker's tables 
 show a marked uniformity, for the number of bacteria per 
 leucocyte vary froin 1 to 1 7 in twenty-five leucocytes examined 
 on the same slide. 
 
 CALCULATION AND DETERMINATION OF THE 
 OPSONIC INDEX. 
 
 According to Wright's method the opsonic index is deter- 
 mined and calculated by dividing the degree of phagocytosis ob- 
 tained with patient's serum by that obtained with serum from a 
 normal individual. 
 
 Simon has proposed an entirely different method for the 
 calculation of the opsonic index. He had observed that with 
 many species of bacteria, due to lysis and fragmentation, great 
 difficulty is experienced in determining the phagoc3rtic index and 
 that the strength of emulsion greatly changes the amount of 
 phagocytosis. To remedy this, Simon first proposed that the 
 percentage of phagocytic cells be determined by counting 50 cells 
 taken in sequence. Later Simon compared the percentage of 
 phagocyting leucocytes with patient's serum to the percentage 
 of phagocyting cells with pooled normal serum. The value ob-
 
 46 VACCINE AND SERUM THERAPY. 
 
 tained for pooled normal serum he called 1.0, and the index de- 
 termined in this way he called the percentage index as contrasted 
 to Wright's bacillary index. These two indices Simon finds can 
 be made to agree to the second decimal place. 
 
 Moss and others have found that the absolute numbers of 
 cells phagocytosing depends in normal or slightly diluted sera on 
 the strength of the bacterial suspension, and that in low dilutions 
 practically all the cells can be made to engulf bacteria. It is 
 evident that by Simon's method only sera having low indices can 
 be investigated, unless the bacterial emulsion or serum are di- 
 luted. Simon has recommended that light bacterial suspensions 
 and at times diluted sera be used so as to be able to determine 
 the percentage index. 
 
 Strong, in his early experiments with anti-plague serum, 
 determined the index according to Wright's method. Later, 
 however, he substituted this method by one in which the highest 
 dilution of an immune serum which gives a marked phagocytosis 
 is compared to the same dilution of a normal serum. If, with a 
 certain dilution of an immune serum marked phagocytosis is ob- 
 served while the same dilution of a normal serum gives only slight 
 phagocytosis, the immune serum is regarded as having an increased 
 opsonin content. Even on this method Strong places little re- 
 liance. 
 
 Although Simon prefers to accept his index to Wright's 
 when there is disagreement between the two, most investigators 
 have preferred Wright's opsonic index. 
 
 LEUCOCYTES IN WHICH THE BACTERIA 
 ARE TO BE FOUND. 
 
 According to Wright's method the bacteria are to be 
 counted in the pohmuclear neutrophiles. These cells when 
 Wright's technique is followed will be found along the margins 
 and end of the spread. 
 
 In all work on the determination of opsonic indices it has been 
 found that there are great differences in the number of bacteria 
 taken up by the individual leucocytes. Moreover, great difTer- 
 ences have been found in the phagocytic index as determined by 
 different investigators examining the same vslide. Even the same
 
 LEUCOCYTES EXAMINED. 47 
 
 investigators have obtained markedly different phagocytic indices 
 on the same slide. The writer has reported work showing that 
 there is not always agreement between results obtained by count- 
 ing the bacteria in 100 and 200 polynuclear leucocytes on the 
 same slide. Bolduan, Moss, Cole, Jeans and Sellards, Simon and 
 others have published results showing striking differences in the 
 counts obtained in 50, 100, 200 and more cells. Bolduan supposes 
 that counting the number of bacteria ingested by 100 or 150 cells 
 will give a fairly correct average. Potter, Dittman, and Bradley 
 believe counts in 100 cells suffice in most cases. Moss, in an effort 
 to determine how many leucocytes must be examined, has de- 
 cided that after counting the bacteria in three hundred cells there 
 is still an error of ten per cent ; and when 50 cells only are counted 
 his experiments show a variation of from . 8 per cent to 30 per 
 cent. Most investigators determine the ninnber of bacteria taken 
 up by at least 100 leucocytes. 
 
 Large numbers of leucocytes must be examined because of 
 the variability of the number 'of bacteria taken up by the indi- 
 vidual leucocytes. Cells which are apparently normal as far as 
 can be determined by staining and which may contain an average 
 of from 5 to 8 bacteria per leucocyte, will phagocyte from 20 to 
 oO microorganisms at a inaximum while other leucocytes may 
 take up none. This difference in the number of bacteria taken 
 up by individual leucocytes has been supposed to be due to the 
 leucocytes themselves and to clumps in the bacterial emulsion. 
 Walker has suggested that the leucocytes be kept constantly at a 
 temperature of 37° C, and that they be well mixed after incuba- 
 tion with serum and bacterial emulsion. Undoubtedly all in- 
 vestigators have mixed the leucocytes, bacteria and serum well 
 after incubation and before making the spreads to be examined. 
 This procedure, however, does not overcome the differences m 
 numbers of bacteria per leucocyte, as is shown in Walker's tables, 
 in which the variations in numbers of bacteria ingested range 
 from 1 to 15, 2 to 16, 1 to 17, and 4 to 16 in four sets of 25 leuco- 
 cytes examined. It cannot be concluded from this that there is 
 a uniformity of phagocytosis, although Walker's tables do show 
 a uniformity of phagocytic index in groups as small as 25 leuco- 
 C5rtes. It is evident, however, that if 25 leucocytes only were 
 to be examined, the opsonologist might, after he had examined
 
 48 VACCINE AXD SERUM THERAPY. 
 
 24 leucocytes, have some choice for the 25th one to he examined, 
 which choice would change the result from 1 to 17. Based on 
 this selection the opsonic index could be materially lowered or 
 raised as might be desired. 
 
 In one of the early contributions on the subject of opsonic 
 index, Simon, Lamar, and Bispham recommend that in making 
 the spread of the mixture after incubation, the spreader slide be 
 merely kept in contact with the blood without touching the lower 
 slide, for, "Otherwise it may happen that most of the leucocytes 
 containing organisms, are carried to one end, while only the empty 
 cells are found in the intervening space." In the laboratories 
 of the Johns Hopkins Hospital, Cole, Moss, and Jeans and Sellard 
 have made determinations of pliagocytic indices determined in 
 different parts of the slide. It was found that the leucocytes 
 collected near the edge of the smear contain decidedly more bac- 
 teria than those toward the center of the slide. By dividing a 
 slide into three zones they found that at the end of the smear the 
 leucocytes contain more bacteria, than in the first and middle 
 zones. To explain these differences these men assume that the 
 polymorphonuclear leucocytes containing the largest number of 
 bacteria are so increased m size that they are drawn to the end 
 of the slide while the smaller ones drop out earlier. These results 
 indicate that, contrary to Wright's suggestion, one ought not to 
 determine the number of bacteria at the end of the spread. 
 
 Wright's method of the determination of the opsonic index 
 has yielded such marked differences in results that more work on 
 this subject seems justifiable. It must be remembered that the 
 opsonic index is of no value whatever unless carried out with the 
 greatest care and by some one who has had considerable exper- 
 ience. Modifications of Wright's technique have arisen with too 
 much rapidity to make it possible to lay down any definite rule 
 for the determinattion of the opsonic index. However there are 
 only few modifications that have made it possible to get more 
 reliable determinations of Wright's opsonic index.
 
 CHAPTER V. 
 
 OPSONIC INDEX IN HEALTH AND 
 
 DISEASE. 
 
 The active participation and function of the white blood cells 
 in the protection of the body against infection was first carefully 
 studied by Metchnikoff. He regarded the leucocytes as cells 
 whose object is to remove from the body and its tissues bacteria 
 and foreign material. On this he based his theory of immunity. 
 It is not strange that Leishman, Wright, and others should have 
 suspected differences in phagocytosis in health and disease. In 
 1902 Leishman, and especially Wright, emphasized the difference 
 in piiagocytic ratio observed in individuals with infections run- 
 ning favorable or unfavorable courses. While trying to find a 
 method by which to control the administration of vaccine, Wright 
 discovered opsonins. With the discovery of opsonin and the 
 development of a method for the determination of the same, it 
 was evident that the opsonic index in health and disease should 
 be investigated. Wright found that in individuals with localized 
 or chronic infections there is usually a decrease in the opsonin 
 content of the blood. This decreased opsonin content as deter- 
 mined by the opsonic index, according to Wright's theories, is 
 due to the fact that in local infections but little of the bacterial 
 substance is absorbed, and so gives rise to but a small amount of 
 active immunity. When this is the case the local infection be- 
 comes chronic. 
 
 By the method of the determination of the opsonic index, 
 Wright has demonstrated that following the injection of vaccines 
 made from the cultures of infecting organisms, there is first a 
 drop in the opsonic index and later a rise of the same. The drop 
 in the index he calls the "negative" phase and the rise the "posi- 
 tive" phase. The observation of negative and positive phases 
 was made by Ehrlich in 1892, who found that following in-
 
 50 VACCINE AND SERUM THERAPY. 
 
 jection of tetanus toxin there is at first a slight diminution in the 
 anti-toxin in the milk of the animals injected while later there 
 is an increase in the amount of anti-toxin. Likewise Solomonson 
 and Madsen found that similar results are produced in the blood 
 when diphtheria toxin is injected. Agglutinins, precipitins, bac- 
 teriolysins, after injection of killed cultures of bacteria, are also 
 at first decreased and later increased. 
 
 Wright and Douglas did not, as has been supposed by many, 
 find that all pathogenic bacteria are sensible to opsonins. They 
 divide the pathogenic bacteria into four groups: 
 
 a. Those bacteria that are sensible to bactericidal, bacter- 
 iol3rtic and opsonic action of the normal human blood. B. ty- 
 phosus and Spir. cholerse belong to this group. 
 
 b. Those bacteria that are sensible to opsonic and especially 
 bactericidal action of normal blood serum. Examples of this 
 group are to be found in B. coli and B. dysenteriae. 
 
 c. Those bacteria that are definitely sensible to opsonin 
 but not to the bactericidal action of normal blood. B. pest is, 
 Mic. melitensis and Mic. pneumonias belong to this group. 
 
 d. Those bacteria that are insensible to the opsonic and bac- 
 tericidal action of normal human blood serum. Examples of 
 this group are found in the bacillus of diphtheria. 
 
 Practically all of Wright's observations on opsonic immuni- 
 zation were determined in man. In the human, following the 
 injection of killed cultures, the negative phase is visually of short 
 duration, lasting from 24 to 48 hours. The positive phase may 
 not, but usually follows the negative phase and lasts from 3 to 
 4 days to two weeks. In active immunization Wright reasons 
 the curve of protected substances may be affected in different 
 ways. If the dose of vaccine injected is very small, the negative 
 phase may be omitted, while if a very large dose is injected it will 
 be marked and the highest index in the positive phase may not be 
 as high as it was at the time of injection. Usually in active im- 
 munization a number of injections of vaccine must be made. 
 These may produce one of three effects on the opsonic index: 
 
 a. If injections are made after the index has returned to 
 the normal, the index curve may show a series of indices above 
 and below the normal. This is what happens, according to Wright,
 
 CURVE OF PROTECTIVE SUBSTANCES. 
 
 51 
 
 when the individual is allowed to recover completely between 
 injections. The curve is illustrated in Fig. 12A. 
 
 b. The injections may be given at such times that one nega- 
 tive phase is superimposed upon another negative phase and in 
 this way produce a cummulative effect in the duration and degree 
 of the negative phase. This result is obtained according to Wright 
 when active immunization is pushed rapidly or forced. The curve 
 of protective substance is shown in Fig. 12B. 
 
 c. The injection may be made at such times and in such 
 amounts that there is a summation of positive phases. This is 
 the result sought for in immunization. The curve representing 
 the amount of protective substances in this case is represented 
 in Fig. 12C. 
 
 Fig. 12. 
 
 Wright, in his treatment of infections by the injection of vac- 
 cine, tries to inject such doses at such intervals of time as to give 
 him a continuous "high tide phase." It is to be noted, however, 
 that even with the most favorable summation of positive phases 
 Wright has not obtained an index as high as 4 . except in rare in - 
 stances. This, of course, is entirely out of proportion with the amount 
 of other immune bodies produced by active immunization. 
 
 Before the principle and methods as formulated by Wright 
 can be accepted it is necessary to decide (a) that opsonins are of
 
 52 VACCINE AND SKRUM THKRAPY. 
 
 importance in iminimity and that the method of determjnin!:^ the 
 opsonic index is accurate; (b) that in the natural recovery from 
 an infection the opsonic index changes, as Wright aims to change 
 it, by the injection of bacterial vaccine; and (c) that the injection 
 of vaccine produces the changes described by Wright. 
 
 IMPORTANCE OF OPSONIN IN IMMUNITY AND ACCURACY 
 
 OF THE METHOD OF THE DETERMINATION 
 
 OF THE OPSONIC INDEX. 
 
 Spontaneous phagocytosis has been described from time to time. 
 With the introduction of Wright's opsonin theory and the develop- 
 ment of the technique of opsonic index determination, phagocytosis 
 has been found to occur, in most cases, only when fresh serum is 
 present. Neufeld and many others have found that contrary to 
 Wright's statement phagocytosis is not entirely dependent upon 
 opsonins, for bacteria with little virulence may be taken up by 
 the leucocytes even though no serum is present. The existence 
 of opsonin, however, and its relation to phagocytosis can no longer 
 be doubted; the part it plays in immunity has not yet been 
 satisfactorily demonstrated. Many of the objections made to 
 Metchnikoff's phagocytic theory of immunity present themselves 
 in the opsonic theory of immunity. It has been found that highly 
 virulent bacteria are not nearly as readily ingested by leucocytes 
 as are less virulent ones. Bacteria engulfed by leucocytes are 
 not always dissolved and disposed of for it has been demonstrated 
 by various investigators that certain species of bacteria produce 
 or secret substances that are antogonistic to and actually kill 
 leucocytes. Even if it is accepted that opsonin plays an important 
 part in immunity, the opsonic index determined by Wright's 
 method, need not necessarily be an index of the patient's im- 
 munity to certain infectious microorganisms. Agglutinins, bac- 
 teriolysins, and anti-toxins, all of which are substances better 
 known than are opsonins, are only circumstantial evidence of 
 greater or less importance in the complex phenomenon of immu- 
 nity. The body has various means of defense against bacterial 
 invasion and action so that the assumption that all the protec- 
 tive substances and means against bacterial diseases and infec- 
 tions manifest themselves in sucli a manner that they can 1)e de- 
 termined frotn the opsonic index seems unreasonable.
 
 OPSONINS IN IMMUNITY. ol'-, 
 
 In the previous chapter the accuracy of the method for the 
 determination of the opsonic index has been criticised. It is 
 certain that the method is compHcated, requires a considerable 
 amount of experience, and with all the modifications and improve- 
 ments in the technique is still subject to such great error that 
 even in experienced hands the determinations of the opsonic 
 index must be regarded as of doubtful value. 
 
 CHANGES IN THE OPSONIC INDEX IN NATURAL 
 RECOVERY FROM AN INFECTION. 
 
 Wright, at various times, has reported observations on 
 the phagocytic reaction and opsonic index in infections running 
 favorable and unfavorable courses. He has found that recovery 
 and increased opsonic index accompany each other. Hektoen 
 has reported that in pneumonia the opsonic index is at first low 
 and rises as the patient's condition improves so that at the crisis 
 the index is above normal. In patients that have a persistently 
 low opsonic index in this disease death usually follows. Tunni- 
 cliff has found that in scarlet fever the opsonic index for strepto- 
 cocci is below normal early in the disease and, as the acute symp- 
 toms subside, it rises above normal. Later on it again becomes 
 normal. Ruediger has found in erysipelas a sharp rise in the 
 index for streptococci as the temperature begins to fall. Hamilton 
 isolated pseudo-diphtheria bacilli in 75 per cent of cases of acute 
 otitis media and found wide variations in the index for this organ- 
 ism in these cases. Clark has found that the typhoid opsonic 
 index drops before a relapse in typhoid fever. 
 
 The writer in a study on the opsonic index in erysipelas made 
 observations on the changes in the opsonic index in unvaccinated 
 patients to determine the relation between the opsonic index 
 for Streptococcus erysipelatos and recovery from, migration, re- 
 currence and desquamation in erysipelas. The most instructive 
 of these cases are the two which follow: 
 
 Case I. — S., a man, aged thirty-eight years, who had a migra- 
 tory, recurrent erysipelas of the face, ears, scalp, and neck, was 
 admitted to the hospital on the fourth day of his disease. When 
 his face was involved, his index w^as 0.7; w^hen it was desquamat- 
 ing, the index had risen to 1.3; when his face was again involved
 
 54 
 
 VACCINE AND SERUM THERAPY. 
 
 seven days later, the index was 1.4; and when it was desquamat- 
 ing again, the index had dropped to O.S. When his neck became 
 involved, one day after his face began to desquamate for the 
 second time his index was 1.0; two days later, with an index of 
 1.1, his back became involved, and three days later, with an 
 index of 3 . 2, his shoulders and neck w^ere again involved, and two 
 days later, with index of 1.1, tlie back and shoulders began to 
 desquamate. (Chart I.) 
 
 DISEASE i ■' '' ; S y 1« 11 12 13 U Ij 10 17 18 19 20 21 22 23 21 23 26 27 28 21) 30 31 
 
 Chart I. — Temperatrue (unbroken line) and opsonic index (broken line) in a case of 
 
 erysipelas (Case I). 
 
 Case II. — A., a man, aged twenty-four years, suffering w^ith 
 erysipelas of the face and ears, was admitted on the eighth day of 
 the disease with an index of 0.9. The next day his temperature 
 dropped and desquamation began; his index was found to be 1.1. 
 Subsequently the index, though the patient was perfectly well, 
 remained below^ unity. (Chart II.) 
 
 8-0 10 
 
 TEMPCRATURE 
 
 11 12 13 14, 
 
 UPSONIC INDEX 
 
 Chart II. — Temperature (unbroken line) and opsonic index (broken line) in a case of 
 
 erysipelas (Case II). 
 
 In these two cases there has been no constant change of index 
 corresponding with desquamation and recovery.
 
 OPSONIC INDEX IN RECOVERY FOR INFECTION 
 
 55 
 
 To further study the relation of the opsonic index to the 
 course of the disease, the indices of aU patients who had received 
 no vaccine were tabulated with regard to the day of the disease, 
 and an average opsonic index for each day was determined. 
 
 Table Showing the Opsonic Index at the Time of Admission, before Killed 
 Cultures were Injected, and the Day of the Disease. 
 
 
 
 
 
 Day 
 
 of Disease. 
 
 
 
 
 
 ^ 
 
 1 
 
 2 
 
 3 
 
 4 
 
 5 
 
 6 
 
 7 
 
 8 
 
 9 
 
 10 
 
 11 
 
 0.6 
 
 1.2 
 
 0.6 
 
 0.7 
 
 0.7 
 
 0.4 
 
 0.9 
 
 0.8 
 
 1.1 
 
 0.6 
 
 1.0 
 
 0.3 
 
 1.1 
 
 1.1 
 
 1.1 
 
 1.0 
 
 
 1.1 
 
 0.8 
 
 0.9 
 
 
 0.7 
 
 
 1.0 
 
 2.4 
 1.7 
 
 1.4 
 1.5 
 0.7 
 1.0 
 1.2 
 
 0.9 
 0.6 
 1.1 
 
 
 0.6 
 1.6 
 
 1.3 
 
 1.7 
 
 
 1.0 
 
 0.45 
 
 1.1 
 
 1.45 
 
 1.1 
 
 0.86 
 
 0.4 
 
 1.05 
 
 0.96 
 
 1.23 
 
 0.6 
 
 0.9 
 
 Average. 
 
 The composite chart indicates that erysipelas causes an in- 
 crease of the opsonic index which reaches its maximum about the 
 third day of the disease, and is followed by a gradual fall. The 
 subsequent course of the chart represents, in very large part, ob- 
 servations made upon recurrent, migratory, and complicated 
 cases. Ruediger found that the index was high during an attack 
 of erysipelas. 
 
 
 
 
 
 <! ^V 
 
 
 ,' 5.^ 
 
 
 - -• ^« - 
 
 - . : -^--*^ _ -4- 
 
 1 .- -is 
 
 JJ iJri N 
 
 
 j = = -- = ;- 5 --- 
 
 'I : i-^i^ 
 
 y ^ ,' 
 
 ^ - s 
 
 _,'^ - - A ,«^'- 
 
 
 ._ _ _ -* ^^_- 
 
 
 
 
 
 
 
 
 
 
 
 _ i 
 
 X_.X -- 
 
 9 10 ' n 
 
 Chart III. — To show the average opsonic index and the daj^ of disease before killed 
 cultures of streptococci were injected. 
 
 It is to be noted that the determinations made and repre- 
 sented in the lastcurve do not represent the index in any one case.
 
 56 VACCINE AND SERUM THERAPY. 
 
 The indices in individual cases are so variable and show such great 
 irregularity that determinations of the opsonic index in any case 
 give little indication of the severity of the disease and is of no 
 value in prognosis. 
 
 Most investigators have found that in normal individuals 
 the curve of opsonic index is very irregular. Moss in thirteen 
 observations on one individual determined indices which varied 
 from 0.52 to 1.95 and in another from 0.42 to 1.66. Bolduan 
 reports observations in w^hich one patient's serum varied in opsonin 
 content as determined by Wright's opsonic index from 0.82 to 
 2 . 50 in seventeen days. Inasmuch as the opsonic index during 
 infections shows no greater variation than during health it can 
 hardly be accepted that determinations of opsonic indices are of 
 much assistance in diagnosis and prognosis in disease. 
 
 Up to the present, most investigators have found that in the 
 natural course of infection there is no regular curve of the opsonic 
 index and that in the natural recovery from an infection the 
 changes in opsonic index are not those which Wright attempts 
 to obtain by injection of killed cultures of bacteria. 
 
 CHANGES IN OPSONIC INDEX PRODUCED BY INJEC- 
 TIONS OF KILLED CULTURES. 
 
 Wright has observed that the injection of killed cultures is 
 usually followed by a change in opsonic index for the organ- 
 isms injected. If the amount of killed cultures injected is large, 
 he states there is a marked negative phase, after which there 
 may be either a marked or slight increase in the opsonic index. 
 If the amount of killed cultures injected is small, the negative 
 phase may be small or absent entirely and following this there 
 may or may not be a positive phase. Wright has attempted to 
 regulate injection of killed cultures so as to get as small a nega- 
 tive phase and as large a positive phase as is possible. To accom- 
 plish this, he has determined the number of killed cultures in emul- 
 sions and has injected certain numbers of bacteria at a dose, the 
 number varying with the species of the organism and the opsonic 
 index changes obtained by their injection. In some cases Wright 
 has resorted to autoinoculation, which is accomplished by mas- 
 sage or manipulation of the infected part. This partly removes
 
 OPSONIC INDEX FOLLOWING VACCINE INJECTIONS. .")7 
 
 the toxins from the diseased tissues to healthy tissues where im- 
 mune substances are actively produced. The amount of mas- 
 sage and manipulation Wright regulates by the changes produced 
 in the opsonic index. 
 
 While numerous investigators have reported results of vac- 
 cination based on opsonic index determinations and have ob- 
 tained, after injections, almost always a uniform rise in the opsonic 
 index, most investigators have not found this to be the case. 
 Even though Wright claims to have obtained changes in opsonic 
 index showing considerable regularity, yet on examination of 
 his charts one sees curves of most marked irregularity; the index 
 is not constant in normal individuals ; shows no consistent increase 
 or decrease in different stages of infection, and is influenced by 
 many non-specific processes as menstruation, exercise, food, etc. 
 Numerous investigators have found that even though there may 
 be first an increase in opsonic index following vaccination, yet 
 this is soon followed by a fall of the index and still improvement 
 of the condition will be observed. 
 
 The results obtained on determinations of opsonic index in 
 health and disease are so variable that its importance as a means 
 of diagnosis, prognosis and indication of actual immunity 
 must be doubted. It is definitely certain that the index is of 
 no value whatever unless carried out carefully by one having had 
 considerable experience m its determination.
 
 CHAPTER VI. 
 
 THE NATURE OF OPSONINS 
 
 The theories of Wright concerning opsonins have received 
 attention chiefly because of the apparent value of their quanti- 
 tative determination in health and disease and in the regulation of 
 dosage and interspacing of injections of bacterial vaccines. Nu- 
 merotis investigators, however, have attempted to determine their 
 importance in immunity, their structure, and the conditions 
 which influence their action. 
 
 Wright has found opsonins, the substances which prepare bac- 
 teria for phagocytosis, present in the serum of normal, diseased 
 and vaccinated individuals. Neufeld and Rimpau, on the other 
 hand discovered substances which prepare bacteria for ingestion 
 by leucocytes, present in certain immune serum. Inasmuch as 
 both the opsonin of Wright and the bacteriotropin of Neufeld and 
 Rimpau perpare bacteria for phagocytosis, they are by many 
 considered as identical substances. Various investigators, while 
 admitting that these substances may be identical in some sera, 
 do not regard them as the same substances in all sera. Wright 
 has regarded opsonin as an important factor in immunity, but 
 has made no distinction between the opsonin present in normal 
 and immune sera. Numerous investigators, however, have dem- 
 onstrated and emphasized certain differences in the bacteriotropic 
 substances in normal and immune sera. 
 
 SPECIFICITY. 
 
 While Wright and Douglas did not discuss the question of 
 specificity of opsonins, still it is evident that they consider opso- 
 nins as specific. The opsonic index in disease, according to Wright, 
 shows certain deviations from the normal for certain microor- 
 ganisms, while for other organisms it deviates but little from the 
 opsonic index obtained with normal serum. On this_]observation
 
 EFFECT OF HEAT ON OPSONIN. 59 
 
 Wright has based a method for the diagnosis of certain microor- 
 ganisms in infections. This can, however, only be regarded as 
 logical if opsonins are specific. Bulloch and Western found that 
 in normal serum, by absorption tests, the specificity of opsonin 
 for B. pyocyaneus, B. tuberculosis and staphylococci could be 
 deterinined. Muir and Martin, Simon, Russell, Potter, Dittman 
 and Bradley, and others, have found that normal opsonins are 
 not specific. Muir and Martin, and Russell, however, by absorp- 
 tion tests have determined that immune opsonins are quite spe- 
 cific. vSimon, however, has not been able by investigation to 
 prove the existence of specific immune opsonin. 
 
 EFFECT OF HEAT ON OPSONIN. 
 
 Wright has consistently assumed that normal and immune 
 opsonins are identical and has preferred to regard all opsonins 
 as thermolabile. There are, however, on the other hand, a large 
 group of observations which show that while opsonins in immune 
 serum resist a temperature of 55° C. for one hour, opsonins in 
 normal serum will no longer be able to prepare bacteria for phag- 
 ocytosis by the leucocytes after such an exposure to heat. These 
 observations show plainly that immune opsonm is thermostable, 
 while normal opsonin is thermolabile. The basis for Wright's 
 assumption that they are all thermolabile is not evident, inas- 
 much as Wright and Reid have shown that in the serum of cer- 
 tain patients suffering from tuberculosis there is a thermostable 
 opsonin. On this observation Wright has based a method for 
 the diagnosis of tuberculosis, which can only have for a founda- 
 tion the assumption that in tuberculosis specific immune bodies 
 which are heat resisting, are produced. From the various inves- 
 tigations reported, normal and immune opsonins manifest a marked 
 dift'erence in ability to withstand heat. 
 
 STRUCTURE OF OPSONINS. 
 
 Opsonins have, by numerous investigators, Savtchenko, 
 Besredka, Loehlein and Dean, been regarded as identical with 
 amboceptors (fixateur). Muir and Martin have shown that not 
 every immune body produces an opsonizing effect. Hektoen has, 
 from a series of experiments, decided that opsonins are distinct 
 substances or anti-bodies. Neufeld and Rimpau, Neufeld and
 
 60 VACCINE AND SERUM THERAPY. 
 
 Toepfer, Keith, BuUocli and Atkin, agree with Hektoen and Wright 
 and Douglas that opsonic action is due to the presence of hith.erto 
 unknown distinct bodies. 
 
 Before this can be accepted, however, it will be necessary to 
 repeat many of the experiments that have been made, inasmuch 
 as in England and America especially, these investigations were 
 made at a time when no distinction was made between normal 
 and immune opsonins. 
 
 The existence of normal and immune opsonins is now quite 
 generally accepted. Neufeld in a consideration of the causes of 
 phagocytosis, states that he believes bacteria and foreign bodies 
 are only tkken up by the leucocytes when the latter are stimu- 
 lated. He bases this assumption on the phagocytosis of red blood 
 cells by leucocytes, which occurs only when a special haemotropic 
 serum is present. In the haemotropic serum, according to Neu- 
 feld, the physico-chemical condition is so changed that a part of 
 the body is modified so as to serve as a stimulus or appetizer for 
 the phagocytes. Virulent organisms dissolve with greater diffi- 
 culty and give off less appetizer, and becatise of this there is less 
 phagocytosis of virulent organisms than of organisms with de- 
 creased virulence. Spontaneous phagocytosis, according to Neu- 
 feld, is due to changes in the cell, one of these changes accident- 
 ally stimulating the leucocytes to phagocytosis. In immuniza- 
 tion Neufeld believes a specific immune substance is produced, 
 which substance modifies bacterial or other cells so that they will 
 serve to stimulate the leucocytes to phagocytosis. Immune opso- 
 nin or bacteriotropin, as he prefers to call it, is a thermostable 
 substances which does not require complement. Normal opsonin, 
 on the other hand, is believed by Neufeld to produce its action 
 because of normal amboceptor and complement, which gently 
 dissolves bacteria and in this way stimulates the leucocytes to 
 phagocytosis. The assumptions of Neufeld are borne out by 
 numerous investigators. 
 
 Immune opsonins resist temperatures up to 55° for one hour, 
 65° C. at times not being sufficient to destroy their action. If 
 the opsonizing action of immune serum is once lost it cannot be 
 regained by the addition of fresh complement. Muir and Martin 
 have found that inactivated immune opsonin absorbs little or no 
 complement. Because of the properties of immune opsonins, they
 
 STRUCTURE OF OPSONINS. 01 
 
 are generally regarded as belonging to the anti- bodies of 
 the second order of Ehrlich. They apparently possess two groups, 
 the opsonophore and the haptophore. Of these the opsonophore 
 group is destroyed by heat, age, acids, etc. It is thus seen that 
 immune opsonins resemble the agglutinins and precipitins in 
 structure and by some investigators bave been thought to be 
 identical with agglutinins. 
 
 Normal opsonins, though not acknowledged by Wright to 
 be different from immune opsonins, have characteristics by which 
 they differ from immune opsonins. Normal opsonins resemble 
 complement in that they are absorbed or fixed by sensitized and 
 non-sensitized bacteria, blood corpuscles, specific precipitates and 
 indifferent bodies, and exhibit thermolability and susceptibility 
 to deterioration by age. Noguchi has found that normal opsonins 
 resemble complement in that they are highly labile bodies, loose 
 their action on standing several days, are preserved for a long 
 time when present in blood m a dry state, and in this condition 
 can be heated to 135° C. without destruction of their functions. 
 Recently Muir and Martin, Levaditi and Inman, and Huhne and 
 Neufeld have ascribed the action of normal opsonins to comple- 
 ment. Cowie and Chapin have found that normal guinea pig 
 serum restores the opsonic power to normal serum which has been 
 heated to 55° C. They believe from their experiments that opso- 
 nins (normal ?) exert their action because of an amboceptor-com- 
 plement group. Hektoen has recently published results of experi- 
 ments from which he concludes that the activating element is free 
 from the opsonin and therefore he believes that opsonins belong to 
 the third order of anti-bodies of Ehrlich. 
 
 It now seems quite definitely established that the action of 
 normal and immune opsonins is due to entirely different factors 
 in immunity, and that immune opsonins are distinct anti -bodies 
 probably belonging to those of the second order of Ehrlich. 
 
 RELATIONS OF OPSONINS TO LEUCOCYTOSIS. 
 
 No consistent relation has been found to exist between changes 
 in opsonic index and general or localized increase in the number 
 of leucocytes. Simonds and Baldauf have recently found that fol- 
 lowing an injection of heated bacteria there is a decrease in leuco- 
 cytes after which there is a marked increase in leucocytes with an
 
 62 VACCINE AND SERUM THERAPY. 
 
 ultimate return to the normal. These changes in the number of 
 leococytes they find precede the changes in the opsonic index. 
 When the index is at its height the number of leucocytes has re- 
 turned to the normal. Various methods designed to increase the 
 number of leucocytes have been resorted to but have all been of little 
 avail in increasing the opsonin content in the blood as determined 
 by Wright's opsonic index. If, however, the leucocytes can be 
 stimulated to phagocytosis an increase in the number of leucocytes 
 in the infected region must certainly be of value. Opsonins are 
 by some supposed to originate in the leucocytes. The importance 
 and possibility of this becomes more evident from the work of 
 Peterson and Hiss and Zinsser. Potter believes that the opsonic 
 index varies with the source of leucocytes. 
 
 RELATION OF OPSONINS TO STIMULINS. 
 
 Metchnikoff has described a series of experiments in which 
 the introduction of serum, either from normal or immunized ani- 
 mals, greatly increased phagocytosis. This action, he supposes, 
 is exerted on the leucocytes and is of great importance in phago- 
 cytic immunity. The substance m the serum which he regards as 
 stimulating leucocytes he calls "stimulin." Stimulins are prob- 
 ably the same as opsonins, even though up to the present time, 
 in the observations reported, their action is on the leucocytes and 
 not on the bacteria. 
 
 OPSONINS AND AGGRESSINS. 
 
 Endotoxins or the aggressins of Bail, have by some been sup- 
 posed to account for decreased phagocytosis or low opsonic in- 
 dices obtained with some sera. Aggressins are supposed to injure 
 the leucocytes and inhibit their action. The part the endotoxins 
 play in preventing phagocytosis has not yet been determined. 
 Dorr, Sauerbek and others have shown that aggressins are not 
 difinitely specific. Neufeld from experiments has decided that 
 the lack of phagocytosis with virulent organisms does not depend 
 upon the injury to leucocytes and for this reason does not believe 
 that decreased phagocytosis is due to the action of aggressins.
 
 OPSONINS, STIMULINS, AGGRESSINS. G3 
 
 INFLUENCE OF CHEMICALS AND REACTION ON 
 • OPSONINS. 
 
 Various salts, even in small amounts, influence opsonic action 
 markedly. Noguchi has shown that in normal serum, reaction 
 exerts considerable influence on phagocytosis, it being most marked 
 when the serum to be tested is neutral in reaction. 
 
 NON-BACTERIAL OPSONINS. 
 
 Opsonins have been found to be produced for other cells than 
 bacteria. Hektoen has reported opsonins for blastomycetes and 
 red blood cells. Likewise it is possible to get marked phagoc5rtosis 
 of inert particles of charcoal, stains, etc., upon the addition of 
 certain sera.
 
 CHAPTER VII. 
 
 VACCINE THERAPY 
 
 For centuries it has been known that following an attack of 
 certain acute infectious diseases there remains a certain loss of 
 susceptibility to the contraction of a second attack of the same 
 disease. Early in the eighteenth century this experience was 
 utilized by vaccination for small-pox. When it was found, about 
 the middle of the nineteenth century, that many acute and in- 
 fectious diseases have for their etiological factor certain micro- 
 organisms known as bacteria, attempts were made to induce im- 
 munity by inoculation of killed or attenuated cultures of these 
 organisms. Pasteur in this way successfully immunized against 
 chicken cholera, but while a certain amount of success by the 
 injection of killed cultures was obtained, still it was found imprac- 
 ticable for reasons that are not understood nor can be considered 
 here. Recently, however, Haffkine has successfully immunized or 
 vaccinated against cholera and Pfeiffer and Wright against ty- 
 phoid fever. It would prove impracticable to employ active im- 
 munization against all organisms with which man may be infected, 
 for this would require frequent injections of all kinds of organisms. 
 Active immunization to be applicable generally must be such that 
 it can be instituted after infection has occured. In this Pasteur 
 was successful in the disease of hydrophobia. It was found, how- 
 ever, that when the method of active immunization is begun after 
 infection has occured, there is a superimposition of a mild form 
 of the disease upon a severer form, thus accentuating the disease. 
 This observation lead to a relatively long period of cessation of 
 this method of treatment. 
 
 In recent times active immunization after contraction of the 
 disease, has again become important. This has l^een due espec-
 
 BACTERIAL VACCINES. 65 
 
 ially to the efforts of Wright. It is important to note that the 
 method of vaccination was used by Wright before he had dis- 
 covered opsonins. From his later observations Wright has decid- 
 ed that opsonins are of the utmost importance in immunity to 
 some organisms, and are decreased in amount during most infect- 
 ions but that they can be increased by the injection of bacterial 
 vaccine. 
 
 BACTERIAL VACCINES. 
 
 It was stated in a former chapter that in bacterial infections 
 the infected body absorbs bacterial substances and products. 
 According to the amount of substance and products absorbed 
 immune substances are produced. When much is absorbed suf- 
 ficient immune substance is usually produced to cure the disease, 
 but when little is absorbed not enough immune body is produced 
 to cure the infection which then becomes chronic. To produce 
 enough immune substances to affect a cure from an infection 
 Wright and Douglas have employed the well known principle of 
 active immunization. The method they have suggested is to 
 actively immunize by injection at the proper time, of proper doses 
 of killed cultures of the organisms causing the infection. The 
 effects of injection of killed cultures or vaccines according to these 
 investigators is to produce certain changes in the opsonin content. 
 Wright and Douglas attempt to immunize by giving injections 
 of such numbers of bacteria as will produce only a slight negative 
 phase which lasts only a short time and is followed by a rise in 
 opsonic index which lasts for some days. When the index begins 
 to fall after the subsidence of the positive phase, another injec- 
 tion is made and the effect on the opsonic index is determined. 
 An attempt is made to inject such doses at the second and sub- 
 sequent injection that there shall again be only a slight negative 
 phase and that during the positive phase the opsonic index shall 
 be higher than following the first injection. An effort is made to 
 keep the opsonic index of the individual above normal. If the 
 index shows a marked drop after any injection, the dose has been 
 too large. By means of vaccines Wright claims that the anti- 
 bacterial power of the blood can be increased for any microbe 
 invading the body. In 1906 Wright was able to say that he had 
 by this method achieved uniform success.
 
 66 VACCINE AND SERUM THERAPY. 
 
 DOSAGE. 
 
 Wright has, by means of the opsonic index determined the 
 proper dosage for the different bacterial vaccines and while he as 
 not stated that definite amounts must be used, still he has sug- 
 gested certain doses. His principle in dosage is to use the smallest 
 dose that will give a good rise in the opsonic index and never to 
 increase the amount injected until it has been ascertained that 
 the doses which have been injected have been too small to produce 
 a good rise in the opsonic index. 
 
 The doses of vaccine recommended by Wright are as follows : 
 
 Bacillus roli 5 to 50 million bacteria 
 
 Micrococcus gonorrhoeae 5 to 50 million bacteria 
 
 Micrococcus pneumoniae 10 to 50 million bacteria 
 
 Bacillus pyocyaneus 5 to 5 thousand million bacteria 
 
 Micrococcus pj^ogenes 50 to 1 thousand million bacteria 
 
 Streptococcus pyogenes 10 to 25 milHon bacteria 
 
 Bacillus tj'hosus 5 to 50 million bacteria 
 
 Koch's New Tubercuhn 1-1,000 to 1-400 milligrams 
 
 These doses are by some considered purely arbitrary, because 
 the personal characteristics of the individual to be immunized, 
 the duration, extent and severity of the infection, must all be con- 
 sidered in active immunization. While these doses may be arbi- 
 trary they at least offer some guide as to the number of organisms 
 to be injected at a time. At times in active immunization the in- 
 jection of vaccine is accompanied by injection of specific immune 
 serum. This will pave the way for larger doses and more powerftil 
 vaccine injections. 
 
 Injection of the doses recommended by Wright usually pro- 
 duces no constitutional symptoms. Wright has stated that at 
 times malaise, slight general muscular pain and headache are 
 observed on the day following the injection but as the positive 
 phase comes on there is a marked buoyancy of spirits and stimu- 
 lation. Most investigators on this stibject have never observed 
 untoward symptoms when the small doses recommended by Wright 
 are injected, while others have at times observed a rise of tem- 
 perature, nausea, vomiting, etc. According to the principle of 
 active immunization probably the greatest amount of immunity 
 results when there is some reaction for in active immunization the 
 individual or animal undergoes a modified or less severe form of 
 the disease.
 
 BACTERIAL A^ACCINE. 67 
 
 The point of injection of bacterial vaccines is considered by 
 Wright to be of importance in the success of vaccination. Those 
 regions of the body are selected where there is rapid absorption 
 by the lymph and blood. It has been found that on subcutan- 
 eous injection greater amounts of immune body are formed than 
 upon intravenous injection. This is especially illustrated in 
 the production of certain haemolytic sera. 
 
 If Wright's method of vaccine treatment of bacterial diseases 
 is to be judged it must be remembered that the injection of killed 
 cultures did not originate with Wright, but that Wright has added 
 opsonic index determinations to control the time and doses of 
 these injections. Opsonic index determinations have, however, 
 been found to be open to so many sources of error, of such great 
 variability in health, disease and after injections of vaccines, that 
 many clinicians have decided upon the dosage and inter-spacing 
 of injections by clinical symptoms rather than based upon opsonic 
 index determinations. Most clinicians have, in the first injection,^ 
 used the dosage recommended by Wright but in subsequent in- 
 jections have used such amounts as seemed warranted and indi- 
 cated. Hektoen, adhering to the reliability of opsonic index de- 
 terminations, holds that the index is of diagnostic and prognostic 
 importance, while Park, Cole, Bolduan and others regard the index 
 as unreliable for such purposes. 
 
 ORGANISMS USED IN VACCINATION. 
 
 Many of the species of pathogenic bacteria have been used in 
 active immunization. Two kinds of vaccine treatment have been 
 employed, one consists of the injection of killed cultures of the 
 causal organisms and the other of the autoinoculation from the 
 infected focus by means of massage and manipulation. 
 
 In the injection of vaccines, so called "stock culture" vac- 
 cines and "personal" or "autogeneous" vaccines have been used. 
 It has been found that for some organisms, vaccines made from 
 stock cultures will give as good results as will personal or auto- 
 geneous vaccines. Stock vaccines have the advantage that no 
 time need be lost in the preparation of the vaccine, and for nearly 
 all of the species of bacteria causing infections amenable to treat- 
 ment by active immunization, stock vaccines are equally as good 
 as autogeneous vaccines. The principle exceptions are the vac-
 
 68 VACCINE AND SKRUM THERAPY. 
 
 cines for Streptococcus pyogenes, Mic. pneumoniae and, accord- 
 ing to some, for the Mic. gonorrhoeae. 
 
 Some investigators have used vaccines containing several 
 species of bacteria. Serum and vaccine manufacturers have pre- 
 pared vaccines suitable for active immunization against one or 
 many different sepcies. Wright at one time made no particular 
 effort to identify the species of the causal organism, simply mak- 
 ing a vaccine from the cultures obtained by inoculation with 
 material from the lesion. This is open to man 3^ objections, for 
 at times the causal organisms may be such that it will not grow 
 on ordinary media or else the causal organisms may not grow as 
 well as secondary invaders and saprophytic species. Because of 
 the mixed and undetermined vaccines used in immunization it 
 may frequently happen that those organisms, for which immuni- 
 zation is necessary or beneficial, are not injected. 
 
 In all cases it ought clearly to be remembered that if bene- 
 fits are to be derived from active immunization the vaccine must 
 consist of a suspension of the causal organism. To determine 
 these, microscopic examinations of the material from the lesion, 
 and isolation of the organisms when possible ought to be made. 
 This cannot be too strongly emphasized, especially to the practi- 
 tioner who can buy from the druggist vaccines said to be good 
 for boils, acne or whatever the condition may be, while as a mat- 
 ter of fact the vaccine may not contain the organism causing the 
 particular lesion which is to be recovered from as a result of active 
 immunization. This has undoubtedly been the cause of some of 
 the failures observed by some clinicians and has, as was the case 
 with tuberculin, decided the practitioner against the use of bac- 
 terial vaccines in certain cases of infection in which vaccine might 
 have been of value. 
 
 PREPARATION OF BACTERIAL VACCINES. 
 
 While killed cultures of bacteria have for some time been 
 used in active immunization, the amounts injected had been only 
 indefinitely determined. Wright and Douglas originated and de- 
 scribed a method for the preparation and standardization of bac- 
 terial vaccines to be used in immunization. The causal organisms 
 according to this method are grown on artifical culture media 
 after which tliey are suspended in sterile salt solution. The num-
 
 CONTROL OF INJECTION OF VACCINE. 69 
 
 ber of bacteria per cubic centimeter of the suspension is deter- 
 mined by drawing into a capillary pipette one volume of the 
 bacterial suspension, and one or more volumes of fresh blood 
 from a puncture in the finger. These volumes are then mixed 
 well and a drop of the mixture placed on a clean slide. With a 
 spreader which is placed in front of the drop, a smear is made. 
 This is then allowed to dry, is fixed and stained with a suitable 
 stain, usually Loeffler's methylene blue. The slide is then counted 
 by determining the number of red blood cells and bacteria in 
 from ten to twenty-five fields of the high power of the microscope. 
 Normal human blood contains five million red blood cells per 
 cubic millimeter. After having established the ratio of red blood 
 cells to the bacteria in the suspension, it is easy to determine the 
 number of bacteria per cubic millimeter or cubic centimeter. 
 The tube containing the bacterial suspension is now sealed and 
 heated in a water bath, to 60 or 65° C. for one hour. After kill- 
 ing the bacteria in the suspension, a dilution, suitable for injection, 
 is made in a bottle containing 50 c. c. of sterile salt solution. 
 The bottle is closed with a rubber cap and sufficient carbolic acid 
 or lysol is added to make an ultimate dilution of 0.5 per cent. 
 After this the vaccine is tested for sterility on suitable culture 
 media. To obtain vaccine from the bottle a drop of pure lysol 
 is placed on the rubber cap. This is done to sterilize the outer 
 surface of the rubber cap. Then the sterile hypodermic needle 
 is inserted through the cap, the bottle turned up and the piston 
 of the syringe is withdrawn until the desired amount of vaccine 
 has been taken into the barrel of the syringe. 
 
 CONTROL OF INJECTION. 
 
 While Wright and his pupils have claimed to regulate the 
 dosage and inter-spacing of injections entirely by the opsonic 
 index, others have found the opsonic index too unreliable to serve 
 as a guide in the administration of bacterial vaccines. Wright 
 and his pupils have not always based their decisions on dosage 
 and time for injection, on determinations of the opsonic index. 
 It frequently happens that at the time of the first injection of 
 vaccine their patients have an opsonic index well above normal. 
 In most cases the treatment is based on determinations made by 
 serum drawn twenty-four hours earlier. The time for the second
 
 70 VACCINE AND SERUM THERAPY. 
 
 and subsequent injection is not always controlled, even by Wright 
 and his pupils, by the opsonic index. 
 
 In accepting opsonins as important factors in immunity other 
 immune substances must not be lost sio^ht of. Various investi- 
 gators have designed methods by which the action of agglutinins 
 and bacteriolysins is to be inhibited, so that opsonic indices may 
 be determined and used as a guide for the dosage and inter-spac- 
 ing of injections. The justifications for such regulation of dosage 
 and determination of the proper time for injection is not evident. 
 In a patient suffering with cerebro-spinal meningitis, from the 
 spinal fluid of which Mic. meningitidis was cultivated, the writer 
 tried to govern the dosage and inter-spacing of injections of 
 meningococcus vaccine by the opsonic index. After three to 
 four minutes of incubation of the mixture of leucocytes, men- 
 ingococcus suspension and patient's serum, all of the organisms 
 w^ere found to be dissolved. It is evident that lysins in this case 
 were probably of more importance in immunity than were opso- 
 nins. Clark has proposed that in determining the opsonic index 
 of serum of typhoid patients, the serum be heated to 56° C. in 
 order to destroy the lysins for the typhoid bacillus. By follow- 
 ing out this method Clark finds that relapses follow upon a drop 
 in the opsonic index. How much more important would it prob- 
 ably be to determine the curve for lysins in a case where lysins 
 are present in such amount that it is necessary to destroy their 
 action before opsonic index determ'nations can oe made? This 
 would seem more rational, especially because our knowledge con- 
 cerning lysins in immunity is greater than it is concerning opsonins, 
 which may or may not play any part in immunity. 
 
 According to the determinations of the opsonin content of 
 the blood, Wright and his pupils have found that the opsonic 
 index begins to rise two to five days after injection, and usually 
 reaches its height after about five to eight days. In most cases 
 adherents of the opsonin theory make injections every four to 
 eight days. The appearance in the blood, however, of the better 
 known anti-bodies comes somewhat later. Bacteriolysins are 
 found to be present at times in the spleen twenty-four hours after 
 injection of culture. They do not, however, appear in quantities 
 in the blood until five to nine or fourteen days later. Agglutinins 
 are found to be present in the blood from eight to twelve days
 
 ANTI-BODIES AFTER VACCINE INJECTIONvS. 71 
 
 after injection of cultures. Typhoid agglutinins ""usually do not 
 appear in a typhoid patient's blood until after ten'^days of the 
 disease have elapsed. Furthermore, it is noted that anti-toxins, 
 bactericidins and agglutinins can be enormously increased by 
 correct inter-spacing of injections and proper dosage. Wright 
 himself has called attention to the value of determining these 
 different substances in immunity before injections are repeated. 
 If injections are made entirely according to the opsonic index, they 
 may be made before the bactericidal and other protective sub- 
 stances have increased in amount, several negative phases may 
 thus be superimposed on each other, and the protective substances 
 be mtich decreased. With the role of opsonins in immunity in 
 doubt, the difference in sensibility of different species of microor- 
 ganisms to opsonins established, and the presence of marked 
 bacteriolytic action of certian sera for certain species of infecting 
 organisms observed, the control of the process of active immuni- 
 zation by the opsonic index alone does not seem justifiable. The 
 immune bodies of importance in immunity to the particular species 
 of bacteria ought to be established so that the dosage and inter- 
 spacing of injections could be regulated by determinations of the 
 presence of these substances. Meakins has, on this basis, studied 
 immunity to dysentery bacilli. Streptococcus pyogenes, Micro- 
 coccus pyogenes and Bacillus tuberculosis. Such methods would 
 undoubtedly result in a better accomplishment of a higher grade 
 of active immunity. 
 
 It is necessary, however, to refer to the great success obtained 
 by Wright, and others following Wright's teachings, in the treat- 
 ment of bacterial infections and diseases according to the deter- 
 minations of the opsonic index. Wright has stated that he has 
 not achieved success only in a certain percentage of cases, but that 
 he has had uniform success by following his method of active im- 
 munization. Ross has given a list of diseases successfully treated 
 based on the determinations of the opsonic index. This list in- 
 cludes furunculosis, pustular acne, sycosis, cystitis, otitis media, 
 empyema, gleet, gonorrhoeal rheumatism, acute gonorrhoea, local- 
 ized tuberculosis, lupus, tuberculosis of the joints, bladder, kid- 
 ney, epididymis, glands, peritoneum, iris and lungs. Surely a 
 method that will eive uniform success for as large a list of dis-
 
 72 VACCINE AND SERUM THERAPY. 
 
 eases as this, is one tliat must be of tlie utmost importance in 
 clinical medicine. 
 
 Because the methods for the determination of immune bodies 
 are still so inaccurate and in many cases unreliable, and because 
 the defensive agencies of the animal body against bacterial in- 
 vasion and multiplication are so manifold and complex, numer- 
 ous investigators have relied upon the clinical control of dosage 
 and inter-spacing of injections of bacterial vaccine. At all events, 
 the value of active immunization by injection of bacterial vaccines 
 will ultimately rest upon clinical results. While it is relatively 
 easy in most cases of infection to tell when complete recovery 
 has taken place, still it is difficult to tell what clinical symptoms 
 and signs are associated with the best method of immunization. 
 The conditions ultimately desired are definitely known, but the 
 conditions in the diseased body which will produce these ulti- 
 mately desired effects, are little understood, Probably the best 
 illustration of this is seen in the use of tuberculin in the treatment of 
 tuberculosis. Tuberculin has been known and used in treatment 
 for nearly twenty years, and still today it is an open question 
 whether the dosage shall be regulated to give an anti-toxic or an 
 anti -bacterial immunity. Clinical evidence and experience is 
 deceiving as is well demonstrated in the literature on tuberculin. 
 
 With some organisms, and in certain cases treated by the 
 vaccine method, excellent results have been obtained when the 
 dosage and interval between injections has been determined by 
 clinical signs and symptoms. The number of cases in which 
 the treatment is controlled clinically is probably larger than the 
 number of those treated according to Wright's method. Many 
 investigators, although determining the opsonic index during the 
 process of immunization, have found that it is quite as safe to 
 administer vaccine when controlled by clinical results as by op- 
 sonic index determinations. In clinical control of vaccine treat- 
 ment, it is aimed to have the dose so small and the injections so 
 far apart that immunization is accomplished without the pro- 
 duction of clinical symptoms. The doses that are usually given 
 are those recommended by Wright, though some have used larger 
 doses without the production of any reaction. 
 
 In most clinics the doses injected are gradually increased. 
 This is also the procedure recommended by Wright. If this
 
 STAPHYLOCOCCUS VACCINE. 73 
 
 method is followed, it frequently happens that there may be an 
 exacerbation in the pathological condition. This is especially 
 observed in furunculosis produced by the staphylococci. Fre- 
 quently following the second injection of a vaccine of this organ- 
 ism a new furuncle will appear. The writer has obtained better 
 results by first injecting the largest dose and then at subsequent 
 injections to give constantly decreasing doses. The reasons for 
 following this method are based entirely upon clinical experience 
 and may be due to the fact that if the second and subsequent 
 injections are larger than the preceding one, too much of the im- 
 mune substance is used up in combining with the bacterial vac- 
 cine injected or there may be an actual hypersusceptibility to 
 these organisms. 
 
 The interval between injections when the opsonic index is 
 not determined, lies usually between seven and ten days, which 
 is about the time required for the production of known immune 
 substances. Whenever a reaction follows an injection the in- 
 terval between injections is lengthened so as to allow sufficient 
 time for the disappearance of all symptoms. 
 
 Staphylococcus Infections. The various infections caused by 
 the Micrococcus pyogenes group have been considered most satis- 
 factory for vaccine treatment. In the list of staphylococcus infec- 
 tions treated by bacterial vaccines, Ross includes boils, carbuncles, 
 acne, sycosis, felons, styes, and septic wounds. The results ob- 
 tained in the treatment of furuncles and carbuncles by killed 
 cultures of the infecting organisms have varied. Probably the 
 best results have been obtained in localized surface infections. 
 In the treatment of these cases it has been found that autogen- 
 ous vaccines are not necessary. It is, however, important in all 
 cases to determine the species and variety of organisms causing 
 the infection and to use this variety in the treatment of the path- 
 logical conditions. The staphylococcus vaccines with which the 
 writer has obtained the best results are those made from different 
 isolation of the same variety of organisms. Thus a Mic. pyogenes 
 aureus vaccine which has given good results is one made from
 
 74 VACCIXK AND SERUM THERAPY. 
 
 three isolations, the organisms being isolated from the lesions of 
 chronic marked furunculosis, from an acute boil on the face, and 
 from a patient having multiple pustules on the back of the neck. 
 Most patients suffering with fumucles and carbuncles show a 
 marked improvement within twenty-four hours after injection 
 and some of these patients remain well after that. Everyone 
 who has treated a number of patients suffering with furuncles or 
 carbuncles must have observed that there occasionally is recur- 
 rence of these conditions after apparent recovery following upon 
 the injection of suitable vaccines. If Wright's method of vacci- 
 nation according to the opsonic index is followed out, there^ are 
 apparently more recurrences than w^hen the injections are given 
 at intervals not less than seven days nor greater than ten days. 
 In all cases the best results will be obtained if a small incision is 
 made in the carbuncle or furuncle twenty-four hours after vacci- 
 nation. By this method the collection of leucoc3rtes or pus is 
 drawm off, the pressure is relieved and the bacteria ingested by 
 the leucocytes are removed. The advantage of this becomes 
 evident when it is realized that staphylococci secrete a leucocidin, 
 which destroys leucocytes. If the leucocytes containing bacteria 
 can be removed before they are killed the value of phagocytosis 
 in immunity is evident. After this the pus is evacuated daily 
 from the furuncle or carbuncle and the wound is not allowed to 
 heal until it has been found that for several days no more pus is 
 being formed. In addition to draining the furuncle or carbuncle 
 through a slight incision, thorough cleansing of the skin with 
 benzine is of great advantage in preventing the formation of new 
 foci. The doses used in most clinics for these conditions are gradu- 
 ally increased, the first injections usually consisting of three 
 hundred million cocci. As was mentioned before, apparently 
 better results are obtained when at the first injection six hundred 
 million cocci constitute the dose, while the later doses contain 
 fewer cocci, at times only one hundred million. While this method 
 of vaccination does not depend for its results only on the effects 
 of vaccines, still it is a method which gives good results without 
 the formation of large scars. 
 
 Acne vulgaris has been successfully treated by vaccination 
 only in certain cases. The value of bacterial injections in this 
 condition was so emphasized by Wright that for a time it was
 
 STREPTOCOCCUS VACCINE. 75 
 
 the common belief among clinicians that vaccinations with killed 
 cultures of Micrococcus pyogenes albus were applicable in all cases 
 of acne. This, however, is not the case. In the lesions of many 
 patients suffering with acne vulgaris, the white vskin coccus cannot 
 be found. In such cases either the species of organisms causing 
 the pustules must be injected or else vaccine treatment should 
 not be applied. A number of the cases of acne which have been 
 treated by the writer have failed to show microorganisms that 
 could be cultivated. When the vaccine treatment is used other 
 means of treatinent must not be discontinued. Incisions and 
 evacuation of the pustules, cleansing of the skin with benzine, 
 expression of comedones or black heads, the bathing of the infected 
 part in hot water, application of sulphur or other suitable lotions 
 and the regulation of the diet are all of assistance to overcome 
 this disease. Even under these measures the treatment is not 
 always successful. 
 
 The treatment of sycosis barbae or barber's itch with staphy- 
 lococcus vaccines has not always been as successful as has been 
 reported. Two patients with this disease in the City Hospital 
 in New York City, were treated by Ross with injections of staphy- 
 lococcus vaccine. No satisfactory results, however, were obtained. 
 The unsatisfactory results obtained by the injection of staphy- 
 lococcus vaccine is easily explained when it is realized that sta- 
 phylococci cause secondary infections in only some of the cases 
 of sycosis barbas. In these cases only can staphylococcus vaccine 
 be of benefit. 
 
 Streptococcus Infections. Various kinds of streptococcus in- 
 fections have been treated by vaccines. Usually these infections 
 are acute and severe and it often is a question whether sufficient 
 time for the production of immune bodies as a result of vaccina- 
 tion will elapse before death or spontaneous recovery terminates 
 the disease. Arthrites, pneumonia, pericarditis, erysipelas, local 
 infections and endocarditis, etc., have been treated by the use of 
 vaccine. At times striking results have been obtained, while 
 in other cases not treated by vaccination there has been equally 
 striking recovery. One needs only to remember the cases of 
 streptococcus infections of the uterus, of septicaemia and ery- 
 sipelas that recover in a few days without any specific 
 treatment, to realize that it is easy to attribute beneficial results
 
 76 VACCINE AND SERUM THERAPY. 
 
 to the use of streptococcus vaccines in cases in which the same 
 results might have been obtained without them. In 1907, the 
 author treated 37 patients suffering with erysipelas by injections of 
 killed cultures of streptococci isolated from the lesions of ery- 
 sipelas. From the results obtained it was impossible to determine 
 the value of the injection of the killed cultures of streptococci. 
 While the injection of from twenty-five million to one hundred 
 million streptococci did not prevent migration and recurrence, 
 the apparent shortening of the duration of the disease suggests 
 that streptococcus vaccines are of some value. The effect upon 
 the duration of the disease was found to be uncertain because, 
 during the time of the investigation, no suitable cases remained 
 untreated with which comparison could be made. It is known that 
 the duration of the disease varies in different j^ears. Ross and 
 Johnson have recently reported on the treatment of erysipelas 
 with a vaccine made from streptococcus erysipelatos, and conclude 
 that such vaccine exercises a specific and controlling influence on 
 the course of the disease. These investigators make initial injec- 
 tions of from ten to twenty million killed streptococci, and repeat 
 these injections every day or two according to the clinical results 
 obtained. Criticism of the results obtained is not possible because 
 of the meagre reports of the cases treated by these investigators. 
 
 Wright and others have reported successful treatment of 
 streptococcus septicaemia by the injection of streptococcus vac- 
 cine. It must, however, be remembered that streptococcus septi- 
 casmia is at times recovered from even when no specific treatment 
 is given. 
 
 In streptococcus immunization autogenous vaccines have 
 usually been preferred, though Gabritschewsky and others have 
 gotten good results with stock vaccines. 
 
 Gonococcus Infections. Probably the best results of the use 
 of gonococcus vaccines have been obtained in cases of gonorrhoeal 
 arthritis. Cole and Meakins obtained encouraging results in 
 about twenty cases of acute and chronic gonorrhoeal arthritides 
 when treated by injections of stock vaccines of Mi c. gonorrhoeae. 
 Tliese investigators treated their cases according to the opsonin 
 content of the blood as determined by the opsonic index, but 
 feel that probably as good results might have been obtained had 
 the injection been given every ten days, which was about the
 
 GONOCOCCUS AND PNEUMOCOCCUS VACCINE. 77 
 
 time determined upon by the opsonic index. The doses injected 
 in these cases varied from five hundred million to one thousand 
 million cocci. 
 
 Treatment of acute and chronic urethritis by vaccines has 
 given some, but relatively few, beneficial results, and is by no 
 means to be regarded as a sufficient measure to obtain a cure from 
 t his condition. 
 
 Micrococci pneumoniae Infections. Apparently some good re- 
 sults have been obtained in the treatment of chronic infections 
 with this organism. The dose injected varies from ten million to 
 one hundred million. In acute pneumonia the course of the 
 disease is usually too short to derive beneficial results by means 
 of active immunization. 
 
 Tuberculosis. The bacillus of tuberculosis may produce its 
 lesions in almost any part of the body. Bacteriological examina- 
 tion of the lesions produced will vary. In some lesions, especially 
 those localized in the interior of the body such as the glands, 
 joints and bones, the tubercle bacillus alone is responsible for the 
 condition found. In other lesions, as in pulmonary and intestinal 
 tuberculosis, there are present in addition to tubercle bacilli other 
 bacterial species. x\ctive immunization, by the injection of killed 
 tubercle bacilli theroetically will be of benefit in lesions due to 
 the tubercle bacillus alone, while in lesions produced by tubercle 
 bacilli and other species it will not be very efficacious. 
 
 In the specific treatment of tuberculous conditions, various 
 modifications of so-called tuberculin have been used. Old tuber- 
 culin is the concentrated germ-free glycerine bouillon on which 
 tubercle bacilli have been grown for several weeks. Before fil- 
 tration the tubercle bacilli are killed by heat. Deny's bouillon 
 filtrate, "B. F." differ from old tuberculin in that filtration is done 
 before killing the bacteria by heat. Tuberculin R,"T.R." con- 
 tains the powdered tubercle bacilli suspended in salt solution. 
 Usually 1 c. c. contains 2 m. g. of powdered bacilli. Tuberculin 
 0, "T. 0.*' is the supematent opalescent liquid obtained after 
 emulsifications of ground up tubercle bacilli in distilled water, 
 are centrifuged. Bacillen Emulsion, *'B. E." is a suspension of 
 ground up tubercle bacilli in fifty per cent glycerine. It contains 
 5 m. g. in 1 c. c. of suspension.
 
 78 VACCINE AND SERUM THERAPY. 
 
 Our knowledge concerning the effect of tuberculin on the 
 bacillus of tuberciilosis and the tissues of the body in which the 
 bacillus is found, is still indefinite. Before tuberculin can be scien- 
 tifically applied as a therapeutic measure, it is necessary to deter- 
 mine the part played by the moderate reaction in the tissues and 
 what is to be accomplished by immunization with tuberculin. 
 
 The reaction in diseased tissues following the injection of 
 tuberculin may be either mild, in which case, there is redness due 
 to hyperaemia and congestion, or it may be marked, producing 
 necrosis and sloughing of the diseased tissues. In all attempts 
 at immunization with tuberculin the mild reaction is preferred 
 because it does not lead to the liberation of tubercle bacilli as a 
 result of the necrosis of tissues, but does furnish to the lesions 
 an increased blood supply. 
 
 The conception of the immunization accomplished by tuber- 
 culin injections differs, there being two important theories. Koch, 
 Wright and others believe that anti-body, specific to the action 
 of the tubercle bacillus, is produced. This, according to their 
 views, is accomplished through stimulation of all the defenses 
 of the body. In conformity to their views on the objects of 
 tuberculin immunization, tubercle bacilli are injected in the form 
 of "T. R." or "B. E." 
 
 Maragliano, Sahli, Denys, Trudeau, and others adhere to the 
 toxin immunization theory, according to which tuberculin injec- 
 tions lead only to tolerance or immunization to the toxin liberated 
 by the tubercle bacillus. In accordance to this theory, old tuber- 
 culin as well as other tuberculins are injected, the doses being 
 gradually increased to the highest point of tolerance. The lesion 
 according to this theory is only secondarily effected, there being 
 no acquisition of immunity to the tubercle bacillus. 
 
 Tuberculin treatment is usually only undertalvcn in tuber- 
 culosis of the glands, joints and bones, though in some clinics it 
 has also been used in pulmonary and other forms of tuberculosis. 
 Koch would limit its application to such cases as are still 
 curable — -that is, have not advanced too far nor are complicated 
 by other bacteria. In deciding upon cases amenable to tuber- 
 culin treatment Koch relies upon the body temperature as the indi- 
 cator. In uncomplicated and curable cases the temperature does not 
 rise mtich above normal. Various measures liave been taken as
 
 TUBERCULINE, COLON AND TYPHOID BACILLUS VACCINE. 7'.) 
 
 guides in tuberculin treatment of tuberculosis. Wright injects 
 only amounts large enough to produce a positive phase in the 
 opsonic index and in some cases of tuberculous glands and joints 
 has accomplished good results by autoinoculation. Trudeau, 
 Denys and others have regulated the dosage and inter-spacing of 
 injections by clinical signs and symptoms. They usually inject 
 1-1000 m. g. of old or 1-10,000 m. g. of "Bacillen Emulsion." 
 After this, by a long continued treatment they gradually increase 
 the dosage. The doses injected are not increased nor the interval 
 between injections shortened if there are any general or local re- 
 actions, as evidenced by fever, local redness and edema. In this 
 method no stress is laid on the absolute amount of tuberculin 
 injected. When the patient has no tolerance to tuberculin, Tru- 
 dean has usually observed chronic toxaemia and progression of 
 the disease. 
 
 Tuberculin treatment has proven especially beneficial in 
 lesions in the joints, bones, glands, skin and urinary bladder, 
 although good results have been obtained in pulmonary and other 
 formis of tuberculosis. 
 
 Bacillus coli Infections. Different infections produced by 
 Bacillus coli have been treated by injections of vaccines made with 
 this organism. The vaccine method of treatment has been found 
 to be most successful in cases of chronic cystitis due to B. coli com- 
 munis, although cholecystitis, appendix abscesses, endometritis 
 and other local infections with this organism, have at times re- 
 sponded favorably to this method of treatment. The number of 
 bacteria injected is seldom more than fifty million, usually smaller 
 doses than this being used. Vaccines made from cultures isolated 
 from the patient usually give the best results. The injections are 
 repeated about every ten days. 
 
 Bacillus typhosus and Bacillus paratyphosus Infections. The 
 typhoid bacillus group consist of several important members. 
 Recognition of this fact has led to the use of vaccine made from 
 cultures isolated from the patient to be treated. Vaccines made 
 from killed cultures of typhoid bacilli have been used for immuniz- 
 ation as well as for treatment of pathological conditions due to 
 members of this group. 
 
 Pfeiffer and Kolle have made vaccines for immunization 
 against typhoid fever from agar cultures, while in England the
 
 80 VACCINE AXD SERUM THERAPY. 
 
 vaccine usually is made from bouillon cultures. The organisms 
 are killed by one hour's exposure to 53° C. to 60° C. Pfeiffer and 
 Kolle inject sufficient amounts of vaccines to produce a local reac- 
 tion lasting several days, and a general reaction, as is demon- 
 strated by fever and malaise, lasting one to two days. Following 
 the first injection, two to three injections of increasing amounts 
 are made at intervals of eight days. The English method also 
 produces marked disturbances at times. The duration of immunity 
 confen-ed by this method of treatment varies, and is usually re- 
 garded as lasting from one to three years. The results of such im- 
 munization are still doubtful but statistics indicate that immunized 
 individuals are less likely to contract the disease, and that when 
 the disease is contracted it is less severe and less often fatal. 
 
 Typhoid bacillus vaccine has been tried in the treatment of 
 typhoid fever. Here it has met with but little success. The best 
 results with the vaccine have been obtained in cystitis, cholecysti- 
 tis, and the local abscesses following attacks of typhoid fever. The 
 doses injected range usually from five million to fifty million bacilli, 
 the injections being repeated at intervals from eight to ten days. 
 Autogenous A^accines usually give the best results. 
 
 Bacillus dysenteriae Infections. Vaccines made from dysen- 
 tery bacilli have been used in prophylaxis as well as treatment of 
 bacillary dysentery. Inasmuch as there are different strains of 
 these bacilli, it is advisable to use vaccines made from the cultures 
 isolated from the patient or with all of the different strains of B. 
 dysenteriae. 
 
 Shiga has tried various methods of active immunization and 
 has foimd that the best results are obtained either, by injecting 
 subcutaneously anti-dysenteric serum and killed cultures of dysen- 
 tery bacilli, or, by taking by mouth the killed cultures of this organ- 
 ism. By Shiga's m.ethod of simultaneous injection of anti-dysen- 
 teric serum and dysentery vaccine the death rate in Japan due to 
 bacillary dysentery has been much reduced. The duration of im- 
 munity, according to Shiga, is about two months. 
 
 Vaccine treatment of d37-sentery can only be applied in cer- 
 tain cases. By the method of active immunization, immunity can- 
 not be established in much less than eight days, and for this reason 
 only subacute or chronic cases are amenable to treatment by vac- 
 cines. The numiber of cases .treated by this method is still too
 
 IMMUXIZATIOX AGAINST HYDROPHOBIA. 81 
 
 small to warrant a conclusion. It seems quite certain, however, 
 that vaccine therap}^ may prove of value in this disease. It must 
 be borne in mind that there are different strains which can produce 
 the disease and that an autogenous vaccine will most likely give 
 the best results. Usually fifty million bacilli are injected, the inter- 
 val between injections being from eight to ten days. 
 
 Cholera Vaccines. Vaccines made from cholera vibrions have 
 been used in protective immunization. Statistics on this method 
 are still too meagre to warrant definite conclusions on its value. 
 
 Less common Infections. Vaccine treatment has been ap- 
 plied in most all infections produced by bacteria. The results 
 obtained have varied, though Wright believes that the method 
 is applicable in the treatment of all infections caused by bacteria. 
 
 SPECIFIC VACCINES FOR DISEASES OF 
 UNKNOWN ETIOLOGY. 
 
 Rabies, Hydrophobia or Lyssa. This is a disease whose eti- 
 ology is only indefinitely established. The organism responsible 
 for the condition has never been crrown. Bv some investigators, 
 certain peculiar bodies, found in 1903 by Negri in the large nerve 
 cells in the central nervous system, are regarded as the causal factors 
 in the disease. Although these bodies are not iniiversally accepted 
 as the etiological factors, they are quite generally accepted as spe- 
 cific to this disease. 
 
 The inability to establish the etiolog}^ has not prevented the 
 establishment of methods of immunization to this disease. The 
 principle of the method of immunization most generally practised 
 is based on the establishment of an active immunity during the 
 period of incubation of the disease. In man the period of incuba- 
 tion usually lasts from four to six weeks, though it has been found 
 to be as short as fifteen days and as long as one year. The rela- 
 tively long period of incubation makes it possible to establish an 
 active immunity before the symptoms of the disease develop. 
 
 Im^munization is accomplished by repeated injections of so- 
 called 'Tixed virus." Pasteur found that tissues and fluids from 
 rabid animal vary considerabl}^ in virulence, and that the viru- 
 lence of virus can be changed considerably. If successive re-inocu- 
 lations into rabbits be made from the virus of a rabid dog the virus
 
 82 VACCINE AND SERUM THERAPY. 
 
 is increased in virulence for rabbits. After a numljer of passages 
 through rabbits the virus finall}^ can no longer be exalted in viru- 
 lence. This is then called the fixed virus, is usually obtained from 
 the cord and kills a rabbit in six to seven days. This fixed virus 
 can, by drying, be gradually decreased in virulence so that after 
 fourteen days of drying it has lost all virulence for rabbits. To 
 immunize the individual, virus of low virulence is first injected, 
 after which injections are made with virus of greater virulence until 
 at the end of the treatment injections of cord which has dried only 
 from two to three days, are made. 
 
 At the present time there are in the United States m^any so- 
 called "Pasteur Departments" for imimunizat ion against rabies. The 
 particular technique employed in such departments varies in some 
 details, still the fundamental and essential principles are the same. 
 
 In every case treated, an attempt is made to establish definitely 
 the diagnosis of rabies. Two methods are employed to accomplish 
 this : one is to examine for Negri bodies and the other is to make 
 inoculations into rabbits. For both of these methods it is es- 
 sential that the head of the rabid animal be preserved intact and 
 sent to the laboratory for diagnosis. It is therefore recommended 
 that rabid animals be killed by other methods than those which 
 mutilate the head. 
 
 As soon as the diagnosis is established, or if this is impossible 
 because the animal cannot be found, the patient should submit to 
 treatment. This requires from two to four weeks and consists 
 usually in daily injections of cord of hydrophobia rabbits, which 
 has been aseptically dried over caustic potash at a temperature of 
 about 23° C. for varying periods of time. After drying, a part of 
 the cord is suspended in bouillon to which either 0.25 per cent car- 
 bolic acid or glycerine has been added as a preservative. The in- 
 jections are made into the subcutaneous tissue and are usually not 
 followed by much disturbance. If there is an inflammatory reac- 
 tion, it istobe treated as any other cellulitis. During the treatment, 
 the patient may go about his work, his bowels should be kept open 
 and alcoholic excess avoided. Immunization is not fully attained 
 until about two weeks after the last injection of virus. 
 
 Immunization is usually and preferably practiced at the hos- 
 pital of Pasteur departments. The New York City Department of 
 Health has also practised immunization by sending virus to physi-
 
 SMALL-POX VACCIXATION. 83 
 
 cians outside of the City of New York. In such cases a stronger 
 course of treatment is followed and an effort is made to limit this 
 practice to places not more than twenty-four to thirty-six hours' 
 distance from the laboratory. 
 
 The results of Pasteur treatment or active ixmmunization 
 against hydrophobia are most satisfactory. Without specific treat- 
 ment the mortality in patients bitten by rabid dogs varies from 50 
 to 80 per cent, while in patients who have undergone a course of 
 active immunization the death rate is about 0.5 per cent. To get 
 good results, early treatment is essential. Bites on the face and 
 head, and bites by mad ^^olves are not immunized against as suc- 
 cessfully as bites by dogs in other parts of the body. After the 
 symptoms of the disease have developed, active immunization can 
 no longer be expected to be of value. 
 
 Immunization against rabies has also been attempted by the 
 injection of anti-rabic serum. Babes and Lepp in 1889 found that 
 they could protect dogs against rabies by injection of serum from 
 dogs actively immunized to rabies. Tizzoni and Centanni have 
 reported favorable results with anti-rabic senmi. Marie has re- 
 cently advocated the simultaneous injection of virus and anti-rabic 
 serum. By this method it is possible to immunize against hydro- 
 phobia more rapidly. The value of these newer methods has not 
 been proven so that at the present time conclusions as to their 
 value are impossible. 
 
 Small-pox. Smiall-pox is a disease for which the etiological 
 factor has not been determined, although Guanieri, Councilman, 
 Calkins and others have found protozoan parasites in the lesions 
 of the disease. 
 
 For a long time it has been known that one attack of small- 
 pox will protect against subsequent contraction of the disease. 
 This has led to the practice of acquiring immunity against severe 
 forms of the disease by intentional inoculation and contraction of 
 mild forms of the disease. 
 
 In 1796, Jenner inoculated a boy with virus from cow-pox on 
 a dairy-maid's hand, and found that in this w^ay cow-pox can be 
 transmitted and immunity to small -pox be produced. Since then 
 vaccination against small-pox by inoculation of cow-pox has been 
 quite universally adopted. It is now accepted that cow-pox is a 
 modified form of small-pox and that small-pox vaccination is a
 
 84 VACCINE AND SERl^M THERAPY. 
 
 process of active immunization V)y means of living organisms at- 
 tenuated by passage through cows. 
 
 The methods of obtaining virus for vaccination vary. Up to 
 1870, virus was usually obtained from the pustule of the vacci- 
 nated individual. Now, however, vaccine is usually obtained from 
 young calves which have been inoculated on the abdomen and 
 thighs with vaccine from the pustules of healthy children or calves. 
 The pustules on inoculated calves are cleaned and opened on about 
 the third or fourth day and from the material contained in them 
 either so-called "vaccine points" or "glycerinated virus" is made. 
 The animals from which the vaccine is obtained are kept as clean 
 as possible, asepsis and anti-sepsis being employed as much as is 
 possible. When all precautions are taken the vaccine still con- 
 tains many organisms. 
 
 Vaccine points are made by dipping sterile bone slips into the 
 material from the pustule. The vaccine is then dried, kept under 
 aseptic conditions and is ready for use. Glycerinated virus is made 
 by softening the pustular material with glycerine. Glycerine has 
 the additional advantage of killing many bacteria in the virus and 
 makes it possible to put the virus up in tubes. vSmall-pox vaccine 
 is seldom entirely sterile, the organisms present, however, in a well- 
 prepared vaccine, consist of only few species and are usually harm- 
 less. After two to three weeks action of glycerine, vaccine is 
 frequentily free from bacteria. 
 
 The durability of potency and efficiency of small-pox vaccine 
 varies. Some vaccines lose their value in one month while others 
 will remain efficient for three to four months. This emphasizes 
 the importance of using fresh virus for vaccination against sniall- 
 pox. 
 
 The method of vaccination varies. It is the custom now to 
 produce scarifications of one-eighth to one-fourth of an inch in 
 diamxeter on the well-cleaned arm or thigh. When vaccination is 
 made on the arm, the area selected is about the point of insertion 
 of the deltoid muscle. In cleaning the part where vaccination is 
 to be made it is to be remembered that all antiseptics must be re- 
 moved with sterile water before inoculation is made. Scarifica- 
 tion is only made severe enough to produce an exudation of serum, 
 bleeding being avoided as much as possible. After scarification.
 
 FUTURE OF OPSONINS AND VACCINES. 85 
 
 vaccine on the point or from the tube is well rubbed in, the serum 
 is allowed to dry and a dry sterile dressing is put on. 
 
 The reaction produced by successful vaccination usually ap- 
 pears after about three days. Locally, there is at first a papule, 
 which becomes a pustule on the eighth or tenth day. About the 
 end of the second week the vesicle changes to a scab, which comes 
 off and leaves a scar. Constitutional symptoms usually appear 
 about the third day and last until the end of the finst week after 
 vaccination. When there is infection, cellulitis and sloughing fol- 
 low; these conditions are treated as any other infection, but usually 
 ought not occur. 
 
 The duration of immunity varies and is imcertain. The 
 longest time that immunity can be certainly relied upon is two 
 years. The rule for vaccination which is generally adopted and 
 advised consists in vaccination within the first or second year, 
 certainly before entering school, re-vaccination within the tenth to 
 fifteenth year and after that whenever there is an epidemic of 
 sniall-pox or possibility of exposure to the disease. 
 
 The efficiency of vaccination against sriiall-pox cannot be 
 doubted. The mortality in vaccinated individuals is between five 
 and eight per cent, while in the un vaccinated thirty-five to forty 
 per cent of the cases terminate fatally. The disease thus is milder 
 in vaccinated than in unvaccinated individuals. Moreover fewer 
 cases of small-pox occur in the vaccinated than in those not vac- 
 cinated against small-pox. 
 
 FUTURE OF OPSONINS AND VACCINES. 
 
 As the method of determining the opsonic index stands today 
 it is of questionable value in diagnosis and treatment of bacterial 
 diseases. Opsonins may play no part in immunity; on the other 
 hand, they may be quite important, and we be lacking in compre- 
 hension of their value and their determination. Further eft'orts 
 should be made to study them. 
 
 The treatment of the bacterial diseases by killed cultures 
 ought not be cast aside because of the unreliability of the present 
 method of determining the opsonic index, inasmuch as opsonins 
 are not the only immunizing substances produced by the injection
 
 86 VACCINE AND SERUM THERAPY. 
 
 of bacterial vaccines. It seems that the injections of killed cul- 
 tures have been both of prophylactic and curative value. The 
 curative value is probably limited to the treatment of chronic 
 diseases, because time is required to actively immunize. More- 
 over this method may be of especial value in the treatment of 
 infections due to species that have many varieties, for here the 
 individual may be immunized to the special organisms causing 
 the disease. Injections of killed cultures will not cure all infec- 
 tions and bacterial diseases and in some cases many injections 
 may be required to obtain the desired result. 
 
 Attention is again called to the fact there is no vaccine for 
 boils, acne or any other condition, but there are vaccines for im- 
 munization against infections by the Mic. pyogenes aureus, albus 
 or citreus, streptococci, typhoid bacilli, pneumococci, gonococci, 
 etc. Vaccine therapy, to give good results, requires that in all 
 infections treated the causal microorganisms or virus must be 
 determined, before vaccines are resorted to. 
 
 Credit is to be given to Wright, not for the discovery of the 
 method of immunization by injections of killed cultures, but for 
 its revival. The employment of this method of inducing immunity 
 and treating bacterial infections and diseases need not necessarily 
 be associated with opsonins and opsonic immunity.
 
 CHAPTER VIII. 
 
 SERUM THERAPY 
 
 In the discussion of theories of immunity, mention was made 
 of the fact that in 1887 Fodor showed that the juices of the nor- 
 mal living body, especially the blood, possess bacteria-destroying 
 property. Buchner, Behring, and Nuttall were among the first 
 to coroborate the results of Fodor. It was later recognized that 
 the cell-free blood serum of normal animals possesses properties 
 of destroying bacteria. It is to be noted, however, that the blood 
 of every species of animals will not destroy all species of bacteria. 
 Furthermore, while in certain cases the degree of natural immunity 
 corresponds to the amount of the bacteria-destroying substance 
 present, still these two conditions are not always found in the 
 same case. The substance which destroys bacteria, Buchner 
 called "alexine." 
 
 In 1888 Hericourt and Richet made the observation that 
 blood serum from an animal immunized to Staphylococcus pyo- 
 septicus, confers when injected intraperitoneally into rabbits, 
 an immunity to this organism. Babes and Lepp in 1889 reported 
 the possibility of protecting animals against rabies by the injection 
 of body fluid from animals immunized to rabies. However, these 
 results were given but little attention until Behring and his 
 pupils systematically investigated the subject. 
 
 Behring and Kitasato, in 1890, reported that mice can be im- 
 munized to tetanus by the injection of blood serum obtained 
 from rabbits artificially immunized to tetanus. These investi- 
 gators further reported that serum from tetanus-immune animals 
 protects against the primary intoxication produced by the tetanus 
 bacillus. 
 
 In 1888 Roux and Yersin discovered diphtheria toxin. Beh- 
 ring and Wernicke, in 1891, showed that blood serum from diph-
 
 88 VACCINE AND SERUM THERAPY. 
 
 theria immune guinea pigs or rabbits possesses the ability, when 
 injected intraperitoneally, to immunize normal guinea pigs against 
 diphtheria and a diphtheria infection already in progress. 
 
 In 1892 these investigators further found that it is possible 
 to immunize larger animals to diphtheria, and demonstrated in 
 the blood of these animals protective and curative substances 
 which can be transferred by the serum to other animals. The 
 doses necessary to cure the disease were found to be larger than 
 those required to immunize against the same. The amount of 
 protective and curative substance produced was found to vary, 
 to some extent at least, with the degree of active immunization 
 of the animal from which the serum was obtained. It was fur- 
 ther found by Behring that serum from animals immunized to 
 diphtheria will protect against the toxin of Roux and Yersin. 
 
 Based on these observations, Behring has formulated a law 
 which is generally knowm as "Behring's Law." According to 
 this dictum, the blood or blood serum from an animal possessing 
 acquired immunity is able to transmit this immunity to a sus- 
 ceptible individual or animal when this blood or blood serum is 
 injected in the right amounts into the susceptible animal or in- 
 dividual. Individuals naturally immune to a particular infec- 
 tious organism, do not possess in their blood or blood serum im- 
 munizing substances which can be transmitted to another in- 
 dividual. Immune substances which can be transmitted thus 
 are not present naturally but must be produced by a process 
 of immunization which consists either of undergoing the 
 natural course of the disease or submitting to some method 
 of artifical immunization. As a result of these investigations, 
 there has arisen the practice of passive immunization, or, as 
 it is more frequently called, serum therapy. The development 
 of methods and success obtained by passive immunization 
 against the action of tetanus and diphtheria bacilli, led to 
 the belief that it would be a relatively easy matter to make 
 specific sera against all disease-producing bacteria. It was 
 soon found, however, that this is not possible. Acquired 
 anti -toxic immunity has only a relatively limited application. 
 Most pathogenic bacteria hold their special poison so firmly 
 within the cell that the poison is only freed when the cell 
 disintegrates, or when certain stimulating conditions as are found
 
 ANTI-TOXIC AND ANTI-BACTERIAL IMMUNITY. <S9 
 
 in the lDody exist. Conditions favorable to the liberation of 
 toxins have up to the present time been produced for a few 
 bacterial species, so that most bacteria only liberate their 
 toxin in the tissues of the body invaded. As a result of 
 immunization to two widely different substances, toxin and 
 bacterial cells, two different classes of immune sera have been 
 manufactured. Of these two classes, the anti-toxic sera neutralize 
 certain morbific bacterial products, while the anti-bacterial sera 
 destroy the bacterial cells. It is not to be inferred from this 
 that the same serum may not be both anti -toxic and anti- 
 bacterial, for diphtheria toxin and cells, for example, may be 
 used in immunization. Under such conditions the blood serum 
 obtained will have both anti-toxic and anti-bacterial value. 
 
 Acquired anti-toxic iinmunity is an immunity due to a neu- 
 tralization of toxin by a specific anti-toxin. The substance that 
 is formed as a result of the combination of toxin and anti-toxin 
 acts like a harmless and stable compound. Ehrlich, basing his 
 observations on studies of the union of toxin and anti-toxin, 
 classes the anti -toxic bodies formed with the anti-bodies of the 
 first order, that is, the labile susbtance or complement takes no 
 part in the reaction. In natural iinmunity to certain toxins the 
 presence of specific anti-bodies cannot be demonstrated and un- 
 der these conditions immunity is rather a non-susceptibility of 
 the cells to the toxin. Metchnikoff has shown that tetanus toxin 
 circulates freely in the blood of naturally immune animals. In 
 certain other cases of natural immunity toxin combines with cells 
 that are of little importance to the body or which are but little 
 affected by the particular toxin. Acquired anti-toxic immunity 
 thus differs from natural immunity in the formation of immune 
 bodies. These immune bodies are found in the body fluids, 
 especially the blood serum and can be injected into other animals 
 or individuals and in this way confer upon them a passive 
 immunity. 
 
 Acquired anti-bacterial immunity is an immunity depend- 
 ent upon the rapid destruction of bacteria. The process 
 is one of solution or lysis. The anti-bodies which produce lysis 
 belong to Ehrlich's third order of immune bodies, that is, com- 
 plement, which is easily destroyed by age, heat, etc., is necessary 
 to brincr about the solution of bacteria. Natural immunity to
 
 90 VACCINE AND SERUM THERAPY. 
 
 certain bacteria is associated with the presence of lytic bodies. 
 Behring found that blood serum from the white rat which is nat- 
 urally immune to the action of anthrax bacilli, exerts a marked 
 destructive effect on anthrax bacilli, while blood from animals 
 not immune to anthrax bacilli exert no such destructive effects 
 on these organisms. 
 
 On the other hand blood serum from the dog and the 
 pigeon, both of which species are practically immune to the 
 action of anthrax bacilli, posses no demonstrable bactericidal 
 power. It is evident that natural immunity is not in all cases 
 due to bactericidal action of blood serum of non-susceptible 
 animals. Acquired anti-bacterial immunity differs from natural 
 immunity to the action of bacteria, in that definite specific 
 immune bodies are present. These immune bodies are found 
 in the body fluids, especially the blood serum, and dissolve 
 bacteria. 
 
 UNTOWARD EFFECTS OF SERUM INJECTIONS. 
 
 In the conference of passive immunity, blood and blood serum 
 injections are made almost exclusively. Blood transfusions were 
 made by Denis in 1667. While some good results were obtained, 
 fever, emboUi, bleeding, heamoglobinuria, and urticaria were 
 sometimes produced by these blood transfusions. Up to 1894, 
 before diphtheria anti-toxin was first generally used, injections 
 of blood and blood serum were rather rare. Following the wide- 
 spread practice of injection of diphtheria anti- toxin in 
 blood, exanthemata followed in approximately twenty -two 
 per cent of the cases. With the injection of diphtheria anti- 
 toxin in blood serum, exanthemata were produced in only six or 
 seven per cent of the cases. From .time to time after 1894 cases 
 have been reported in which injections of diphtheria anti-toxin were 
 followed by skin manifestations. While the sequellas of serum 
 injections are usually not serious, still at times serious symptoms 
 and even death have been reported as following injections of 
 serum. 
 
 In 1905, V. Pirquet and Schick originated the term "Serum 
 Krankheit," or serum disease. These investigators found that 
 serum disease varies, two rather definite types of the disease 
 being recognized; one type which results from the first injection of
 
 SERUM DISEASE. 91 
 
 serum and another type which follows the second and subsequent 
 injections of serum. 
 
 Serum disease following the first injection of serum mani- 
 fests itself after an incubation period of eight to twelve days, 
 and is largely independent of the amount of serum injected. Of 
 the symptoms of this disease, fever is the most constant, lasting 
 usually during the entire course of the disease. The height of 
 fever, however, is not an indicator of the disease, but from the 
 curve of the temperature a prognosis can be made, because the 
 temperature drops by lysis. Together with fever there is usually 
 an exanthema, appearing most frequently as an urticaria. This 
 appears first about the point of injection, later it is distributed 
 symmetrically and usually itches severely. At times there is swell- 
 ing of the glands, especially of the glands found in the region in- 
 jected. The symptoms of pain in the glands are of prognostic 
 value, in as much as the pain usually disappears or abates before 
 the size of the gland decreases. During the period of incuba- 
 tion the leucocytes are increased in number, but during the height 
 of the disease the mmiber of leucocytes is markedly diminished. 
 Joint pains are found in a small percentage of the cases of serum 
 disease. The metacarpal, hand and knee joints, are most fre- 
 quently effected. Usually these pains do not last long. In cer- 
 tain cases there is edema, but albuminuria is seldom or never 
 present. The mucous membrane is seldom effected, which dis- 
 tinguishes the condition from scarlet fever and measles. The 
 disease is further distinguished from measles by the absence of 
 Koplick spots, coryza, conjunctivitis and increased efflores- 
 cence about the point of injection. From scarlet fever the con- 
 dition is distinguished by its non-communicability by contact, and 
 absence of scaling, nephritis and angina. 
 
 The disease following the second and subsequent injections 
 of serum varies somewhat with the interval between injections. 
 If an injection of serum is made from twelve to forty days 
 after a preceding one, the incubation period is very short, the 
 disease appearing at times in less than one hour after injec- 
 tion. If the interval between injections is from forty da^'s to 
 six months, there may be an immediate reaction, or else an im- 
 mediate reaction with another reaction six to eight days later. 
 If the interval between injections is over six months there is usu-
 
 92 VACCIXE AXD SEKU.M THERAPY. 
 
 ally no immediate reaction, the symptoms appearing six to twelve 
 days after injection. When the interval between injections is 
 six days or less, the disease is not produced. Serum disease 
 follows re-injections more frequently than it does in the first in- 
 jection and is usually produced by smaller amounts of serum. 
 
 The "inmiediate reaction" occuring when re-injection is made 
 within an interval of twelve to forty days, manifests itself in one 
 to six hours after injection and usually reaches its maximum 
 within twenty-four hours after injection. The "second reaction" 
 is not seen after the first injection of serum but occurs most fre- 
 quently when the interval between injections is between forty 
 days and six months. The incubation period for the second 
 reaction is somewhat shorter than it is for serum disease produced 
 by first injections. The symptoms of serum disease produced by 
 re-injection are usually more acute and general but of shorter 
 duration. In many cases there is vomiting. 
 
 When serum disease was first recognized it was supposed to 
 be caused by toxin in the immune serum. However, as early as 
 1894, Heubner expressed doubt as to the importance of specific 
 immune sera in the production of the disease. Later the mani- 
 festations of serum disease were produced by the injection of nor- 
 mal serum. In 1906 Rosenau and Anderson of the Hygienic 
 Laboratory of the Public Health and Marine Hospital vService 
 of the United States, published a work in which experiments were 
 reported on "sudden death" of guinea pigs following serum in- 
 jections. These investigators found that re-injections of normal 
 horse serum into guinea pigs are very poisonous if the interval 
 between injections is more than ten days. The length of time for 
 which this h3^persusceptibility persists has not been definitel}^ 
 determined but lasts at least as long as two years and two days. 
 On the other hand when the interval between injections is less 
 than ten days, normal horse serum will produce no such effect. 
 
 The nature and causes of the reaction have been matters of 
 considerable investigation and contention. The reaction was at 
 first regarded as one resulting from the injection of toxin or poison. 
 The long period of incubation, however, is explained with diffi- 
 culty by such a conception of the phenomenon. While it is true 
 that to produce disease by certain toxins, a period of incubation 
 is necessary, this period is seldom as long as eight to twelve days.
 
 ANAPHYLOXIS. DM 
 
 It can hardly be conceived that horse serum has the abiUty to 
 increase its amount of poison as can bacteria and other organisms 
 capable of self reproduction. Rosenau and Anderson have de- 
 termined quite definitely that the reaction is due to a difference 
 in the susceptibility of individuals and not the toxicity of the 
 serum. 
 
 V. Pirquet and Schick conclude that the phenomenon is due 
 to the presence of specific anti-bodies. According to these in- 
 vestigators the anti-bodies are not precipitins, for man does not 
 produce the precipitins readily with horse serum — in children 
 three weeks are required before the precipitins can be detected 
 and are found in the blood only from four to nine weeks. The 
 "immediate reaction" which occurs when the second injection of 
 horse serum is made twelve to forty days after the first, Pirquet 
 and Schick explain as being due to the already present serum 
 anti-bodies which combine with the immunizing substance in serum 
 and produce poisonous substances, which in turn produce the 
 disease. 
 
 Gay and Southard, in 1907, reported experiments which in- 
 dicate that the theory of Pirquet and Schick is untenable. These 
 investigators beheve that "sudden death" in guinea pigs "sensi- 
 tized to horse serum" is due to a substance they call "anaphylac- 
 tine." This substance is found in normal horse serum, is not 
 absorbed by the tissues of the guinea pig and is ehminated slowly. 
 The anaphylactine in the guinea pig increases the avidity in the 
 cells of the guinea pig, so that when more serum is injected the 
 cells are "overwhelmed in the exercise of their eliminating func- 
 tions, and functional equilibrium is so disturbed that local or 
 general death may follow." Rosenau and Anderson had shown 
 that the hypersusceptibility of the guinea pig for a certain protein 
 is manifested upon a second injection of the corresponding protein. 
 Later they showed that the substance anaphylactine is specific 
 in the same sense. These investigators could not demonstrate 
 this substance in the blood of the sensitized guinea pig during the 
 incubation period, nor at any time in the blood serum of man, 
 monkeys and cats. 
 
 The investigations on the phenomenon of hypersusceptibility 
 of guinea pigs to horse serum in connection with serum disease 
 in man, has lead to a considerable advancement in the m.ethods
 
 94 VACCINE AND SERUM THERAPY. 
 
 of administration of specific immune sera. While anaphylactine 
 has not been found in the blood serum of man, later investigation 
 may show that the phenomenon of serum disease in man corresponds 
 to sudden death and anaphylaxis in guinea pigs. 
 
 The markedly beneficial results of injections of specific im- 
 mune sera in the prevention and cure of certain diseases, have 
 so far outweighed the undesirable and serious results which have 
 followed serum therapy, that most physicians have given little 
 attention to the contra-indications of serum injection. As the 
 indications and contra-indications for injection of immune serum 
 are better understood by physicians fewer objections to serum 
 therapy, scientifically applied, will be made. That serious ob- 
 jections to the injection of immune sera have some foundation, 
 is evidenced by the fact that Rosenau and Anderson have been 
 able to collect a considerable number of cases of sudden deaths 
 in man after the injection of immune serum. It has been noticed 
 from the first that in the cases of sudden death following the in- 
 jection of diphtheria anti-toxin there is marked respiratory em- 
 barrassment. Rosenau and Anderson believe the essential lesion 
 of serum anaphylaxis is localized in the respiratory centers. 
 In collecting statistics on this subject these investigators have 
 found two cases, "and also others have come to our notice," in 
 which sudden death followed the injection of anti-toxin into 
 asthmatics. From these observations Rosenau and Anderson 
 conclude that "the knowledge of the fact that injection of horse 
 serum into asthmatics may be attended with danger, should be 
 considered in the use of anti-toxins." 
 
 While there are some cases in which the injection of horse 
 serum has been followed by sudden death due to a hypersuscepti- 
 bility to horse serum, most of the deaths have undoubtedly been 
 due to other causes. Frequently physicians and laymen wrongly 
 attribute undesirable conditions to the use of diphtheria anti- 
 toxin and other immune sera. In the use of some immune sera, 
 especially diphtheria anti-toxin, recovery from the disease is so 
 rapid and early, that the physician and patient overestimate the 
 physical condition of the patient, and as a result the patient is 
 frequently allowed a certain amount of exercise. This at times is 
 followed by unfavorable symptoms. The condition in these cases is 
 probably due to the action of diphtheria toxin rather than
 
 INDICATIONS OF SERUM INJECTIONS. 95 
 
 of diphtheria anti-toxin. The heart paralysis which develops 
 in some cases is probably only observed because by the use of 
 diphtheria anti-toxin the patient remains alive long enough so 
 that the heart paralysis may be observed. The action of diph- 
 theria toxin on the heart and other organs has been known for 
 some time and in serum laboratories it is not an unusual occurence 
 to have an apparently well horse, which is undergoing iiTimuni- 
 zation to the diphtheria bacillus and its toxin, die very suddenly 
 after exercise. Post diphtheritic paralyses have been observed 
 long before the introduction of the use of diphtheria anti-toxin, 
 and to attribute this condition to anti-toxic serum is to attrib- 
 ute a symptom to a remedy, where the symptom is really one 
 found in the disease treated by the remedy, diphtheria anti-toxin. 
 In all cases treated with immune serum the patient ought to be 
 kept quiet for some time after injection of serum, even though 
 he has apparently recovered from the disease. 
 
 Following the good results obtained in the treatment of 
 diphtheria by diphtheria anti-toxin and the protection against 
 the disease conferred by the so-called immunizing dose of 
 diphtheria anti -toxin, the serum has been used very extensively. 
 In many cases, undoubtedly, serum has been used w^ithout proper 
 indications. Many physicians have used diphtheria anti-toxin 
 even though the diagnosis of diphtheria was not definitely made, 
 because it was assumed that no harm could come from the use of 
 the serum while great benefits might result if the condition were 
 one of diphtheria. Some physicians have injected the immuniz- 
 ing dose fully as indiscriminately ; whole families, attending physi- 
 cian and nurses have received immunizing doses of diphtheria 
 anti-toxin. The indications for protective and curative injec- 
 tions of immune serum ought to rest on a better foundation. 
 This is especially true because hypersusceptibility to serum, or 
 serum disease, is more frequent following re-injections of serum. 
 It is evident that serum ought only to be injected into the indi- 
 vidual when certain indications are present. 
 
 Specific treatment with immune serum is only indicated when 
 the diagnosis, and, in certain cases, the etiological factor has been 
 determined. It frequently happens that a bacterialogical diag- 
 nosis cannot be made immediately. Under such conditions the 
 clinician is warranted in using specific serum if there is such
 
 96 VACCINE AXD SERUM THERAPY. 
 
 clinical evidence as is usually found in infections caused by the 
 corresponding organisms, or if the patient has been exposed to 
 infection from a previously diagnosed case of the communicable 
 disease. 
 
 When injections of immune sera are to be made frequently, 
 and over long periods of time, as is the case with most anti-bac- 
 terial sera, the injections should be made at intervals of less than 
 ten days, because, if the interval is longer than this, serum disease 
 is more likely to develop. 
 
 The indications for injection of immunizing doses are not 
 clear nor definite. Nurses and doctors, however, now rarely re- 
 ceive immunizing doses of immune serum. This is especially 
 true of diphtheria anti-toxin. The reasons for the abandonment 
 of the practice of immunizing doctors and nurses, are based on 
 the fact that doctors and nurses are better able to avoid infec- 
 tion and so usually do not contract the diseases of their patients, 
 and because the protection conferred by passive immunization 
 is only of short duration. Doctors and nurses, because so 
 frequently exposed to infectious diseases, would have to be 
 injected frequently and at different intervals of time to be 
 immune to the various infections of their patients. Serum 
 injections at intervals of more than ten days, however, are more 
 likely to give rise to serum disease. 
 
 The custom concerning immunization of individuals against 
 possible infection because of exposure, varies. It is the rule of 
 some physicians, upon the diagnosis of diphtheria in one member 
 of the family, to give all members of the family an immunizing 
 dose of diphtheria anti-toxin. In some of the large children's 
 hospitals and clinics diphtheria anti-toxin is given to all patients 
 on admission, this being done to avoid the outbreak of diph- 
 theria epidemics. Such preventative measures hardly seem 
 warranted. The custom of most physicians now is to give im- 
 munizing doses of some specific anti-sera to such members of a 
 family or household as are most likely to contract the disease; 
 thus for example, diphtheria anti-toxin is given only to the 
 children in a home in which there is diphtheria. On this basis 
 administration of anti-sera is justifiable, while indiscriminate 
 immunization by the injection of serum is costly and may 
 also sensitize individuals to serum, which might later make the
 
 CONCENTRATION OF IMMUNE SERA. 97 
 
 use of serum difficult or impossible even though injection of 
 immune serum were definitely indicated. While it is true that 
 some sera are ordinarily injected at intervals of two or three 
 weeks without the production of serious results, still it frequently 
 happens that such a degree of hypersensitization to serum de- 
 velops that passive immunization must be given up entirely. 
 
 Since the introduction of serum therapy, large numbers of 
 persons have been injected with immune serum and great bene- 
 fits have been derived from the use of different sera. The cases 
 of serum sickness, especially the rashes, have been relatively fre- 
 quent, yet are so far outweighed by the beneficial results that 
 they must not prevent the physician from the use of immune 
 sera when they are indicated. The cases of sudden death fol- 
 lowing injection of serum fortunately have been very rare. As 
 further knowledge concerning the untoward effects of the injec- 
 tion of horse serum is gained, serum disease and sudden death 
 will become less frequent. The essential points, which have so 
 far been developed, indicate, that specific sera are to be used only 
 after a definite diagnosis of the disease, that there is some danger 
 attending the injection of horse serum into asthmatics, and that 
 untoward effects of serum injections are more frequent when the 
 interval between injections ranges from ten days to several years. 
 
 CONCENTRATION AND PURIFICATION OF SERUM. 
 
 Frequently large amounts of specific immune substances are 
 to be injected in the treatment of infections. Hypodermic in- 
 jection of large amounts of serum, how-sver, is painful and more 
 likely to produce serum disease, especially the rashes. These 
 objectionable features of serum therapy have resulted in an 
 attempt to produce serum of greater concentration and purity. 
 It has been found that bacteria and toxins from certain cultures 
 are more potent and cause animals to produce immtme sera of 
 higher protective value. As a result virulent cultures, or toxins 
 produced by virulent cultures, have been used in active immuni- 
 zation of the animal that is to furnish the immune serum. 
 
 Attempts have been made to obtain an increased potency by 
 separating the immune substance from the non-immune sub- 
 stance of specific serum. Dieudonne, in 1897, showed that the 
 protieds precipitated from diphtheria anti-toxin by acetic and
 
 98 VACCINE AND SERUM THERAPY. 
 
 carbonic acid, contain none of the anti-toxins. In this same 
 year B elf ant i and Carbone found that with the globulins precipi- 
 tated by magnesium sulphate, diphtheria anti-toxin is thrown 
 out. Atkinson, in 1901, and others since then, have shown that 
 during immunization the serum-globulin content of serum in- 
 creases. Gibson and Banzhaf, however, have found that the 
 increase in serum-globulin is not necessarily associated with the 
 accumulation of anti-toxin in the blood. Early in 1906 Gibson, 
 working in the Research Laboratories of the Department of 
 Health of New York City, reported practical methods for con- 
 centrating and purifying diphtheria anti-toxins. According to 
 this method, by half saturation of anti-toxic serum with ammon- 
 nium sulphate, the globulins, nucleo-proteids, and similar sub- 
 stances are thrown out. These contain the immune bodies. The 
 precipitate is again dissolved in a saturated solution of sodium 
 chloride. Now only the anti-toxin fraction and some of ''"he 
 globulins are in solution. After filtration, acetic acid is added 
 to the filtrate to precipitate the anti-toxic globulins which are 
 then filtered off, dried with paper, dialyzed, neutralized and again 
 dialized for several days. After dialysis the solution is made 
 isotonic by the addition of sodium chloride. The sulution is then 
 filtered through a Berkefeld filter to remove any bacteria that 
 may be present and chloroform added as an antiseptic. This 
 method has been attempted for the various immune sera, but up 
 to the present time it is profitable only for the concentration of 
 diphtheria and tetanus anti-toxins. Recently Banzhaf has found 
 that more concentrated anti-toxin may be obtained by heating 
 the serum to 57° for some hours before separating the anti-toxic 
 globulins. By Gibson's method the concentration is increased 
 from three to five times, while by Banzhaf 's from eight to ten times. 
 By the use of concentrated and refined serum the consti- 
 tutional disturbances and rashes are somewhat less frequent and 
 not as severe. The principle advantage of the product obtained 
 by these methods, however, lies in the fact that a large amount 
 of immune substance can be injected in small amounts of material. 
 
 DRIED IMMUNE SERUM. 
 
 Relatively recently, immune sera, especially anti-diphtheritic 
 and anti-tetanic sera,^ have been dried after concentration and 
 refinement. It was mentioned earlier that when serum is dried
 
 ORAL ADMINISTRATION OF IMMUNE SERA. 90 
 
 soon after being drawn, the immune body and especially the 
 complement are preserved longer. Among the advantages of 
 dried immune sera may be mentioned: the lack of deterioration, 
 the cheapness of manufacture; the convenience of carrying about 
 in the medicine case; and the possible administration of immune 
 substances by the mouth. Undoubtedly these products will 
 prove to be of considerable value and are not to be lost sight of. 
 
 ORAL ADMINISTRATION OF IMMUNE SERUM. 
 
 In 1892 Ehrlich discovered that ricin anti-toxins can be ab- 
 sorbed by the intestinal canal. In 1893 Wernicke, working with 
 Behring, established the fact that anti -toxic substances in the 
 body fluids of diphtheria-immune animals are absorbed by the 
 digestive tract; thus, dogs fed on the meat of diphtheria-immune 
 sheep obtain some immunity to diphtheria. The possibility of 
 oral administration of anti-toxic sera has been recently advanced 
 by McClintock and King. These investigators have found that 
 the best results by oral administration of diphtheria anti-toxin 
 are obtained by the following method: "One half -hour before 
 administering the serum the child is given one glass of one per 
 cent sodium bicarbonate solution. When the anti-toxin is given 
 there is added one minim Fl. Ext. Opii and from four to ten 
 minims of saturated solution of salol in chloroform. When pos- 
 sible no food should be given for at least four hours before ad- 
 ministering the serum." This method is adopted to inhibit the 
 digestion of the anti-toxin so as to make the absorption of the 
 unchanged anti-toxin possible. Dried diphtheria anti-toxic globu- 
 lins were found to give satisfactory results. By oral adminis- 
 tration of diphtheria anti-toxin neither serum sickness nor 
 anaphylaxis was observed. At present this method is still of 
 questionable value and requires further investigation. 
 
 ANTI-TOXIC SERA. 
 
 It was stated earlier that sera containing bodies which neu- 
 tralized toxin are called anti-toxic sera. The immune bodies 
 are receptors of the first order of Ehrlich (see p. 16), which have 
 been thrown off as a result of overproduction of certain receptors.
 
 100 VACCINE AND SERUM THERAPY. 
 
 The overproduction of these receptors occurs as a result of the, 
 presence of specific toxins, found in infections with specific or- 
 ganisms, or after experimental injection of certain toxins. As is the 
 case with other anti-bodies, anti-toxins are present to some de- 
 gree in many normal individuals, thus diphtheria anti-toxin was 
 found by Bolton to be present in the blood serum of 30 per cent 
 of the horses examined, and in 50 per cent of the children and 81 
 per cent of the adults examined by Wasserman. Various sub- 
 stances have been used to produce an increase of specific anti- 
 toxins and as a result a considerable number of anti-toxins have 
 been made. Among these may be mentioned the anti-toxins 
 which combine with the toxins of the organisms of diphtheria, 
 tetanus, pyocyaneaus, symptomatic anthrax, and botulism, with 
 snake and spider venoms, with ricin, aprin and robin, and 
 with the glucosids of toad stools and poison ivy. Of the anti- 
 toxic sera, the anti-diphtheritic and anti-tetanic have been of 
 most importance. 
 
 In the treatment of diseases for which anti-toxic sera have 
 been made, it must always be remembered that anti-toxin neu- 
 tralizes prepared toxin and combination can take place only when 
 the receptors of the toxins and anti-toxins are free. As soon as 
 the cells and toxins have combined and the body cells have been 
 injured as a consec^uence, no- amount of anti-toxin can protect 
 the cell from the action of the poison molecule. Anti-toxic 
 bodies can only anchor and render inert or harmless such toxins 
 as are free or have uncombined receptors. This emphasizes the 
 importance of the early administration of anti-toxic serum be- 
 cause at this time the poison molecules can be anchored by the 
 anti-toxic substances instead of combining with the cells of the 
 body. 
 
 DIPHTHERIA ANTI-TOXIN. 
 
 Ferran, early in 1890, and Fraenckel and Behring later in the 
 same year, published methods by which experimental animals 
 can be immunized to diphtheria. Behring and Kitashima, in 
 1891, published methods for the immunization of guinea pigs to 
 diphtheria toxin. In 1892 Behring and Wernicke emphasized 
 the presence of protective substances in serum of diphtheria-im- 
 mune animals. Serum therapy as applied to the treatment of
 
 DIPHTHERIA ANTI-TOXIN. 101 
 
 diphtheria in man began in 1891 and 1892. The first diphtheria 
 anti-toxin was put on the market in August, 1894. Since then 
 diphtheria anti-toxin has been used extensively. 
 
 The method for making diphtheria anti-toxin has gone 
 through various stages of development. All diphtheria anti- 
 toxin used in the United States is made practically after the same 
 method, which is as follows: Young, vigorous and absolutely 
 healthy horses are immunized, although other species of animals 
 have also been used. During immunization the animal receives 
 repeated injections of diphtheria toxin. This toxin is obtained 
 by growing a virulent culture of B. diphtheria on the surface of 
 beef bouillon in an Ehrlenmeyer flask. After one week's growth 
 at a temperature of 35° to 36° C, the culture is rendered sterile 
 by adding carbolic acid in amounts to make a . 5 per cent car- 
 bolic acid solution. After 48 hours the diphtheria bacilli have 
 settled to the bottom, when the bouillon is filtered through a 
 Berkefeld filter. The germ-free bouillon contains the diphtheria 
 toxin and ought to be potent enough so that 0.01 c. c. will kill 
 a 250 gram guinea pig on or before the fourth day after 
 subcutaneous injection. The horse or animal to be immunized 
 receives enough of this toxin in bouillon to kill five thousand 
 guinea pigs of 250 grams each. At the time this amount 
 of toxin is injected the horse also receives an injection of ten 
 thousand units of diphtheria anti-toxin. After three to five days, 
 when the fever has subsided, a somewhat larger dose of toxin and 
 the same amount of anti-toxin are injected. A third injection is 
 made after another interval of from three to five days. After 
 this usually no more anti-toxin is injected with the toxin, the 
 doses of which are constantly increased and injected at intervals 
 of from five to eight days. After about two months of treatment, 
 if the immunization has been successful, the horse will usually 
 tolerate enough toxin at one injection to kill one hundred thousand 
 guinea pigs of 250 grams each. 
 
 At the end of six weeks to two months, samples of blood are 
 drawn and tested as to protective value. If the anti-toxic value 
 is high, the horse is usually bled to death by tapping the jugular 
 vein. The withdrawal of serum is made under aseptic conditions. 
 The bottles containing the blood are slanted and after four to 
 five days the serum is drawn off.
 
 102 VACCINE AND SERUM THERAPY. 
 
 By means of Gibson's or Banzhaf's method the serum is now 
 concentrated and refined. After this it is standardized, i. e., its 
 strength is determined. The United States government deter- 
 mines the unit of diphtheria anti -toxin in the United States, and 
 the Government requires that all diphtheria anti-toxin made by 
 manufacturers, having a United States government license, must 
 conform to this standard. This emphasizes the importance of 
 using only diphtheria anti-toxin made by manufacturers having a 
 United States Government license. Because the toxic value of 
 diphtheria toxin changes with ageing and other conditions, the 
 H3^gienic Laboratory of the U. S. Public Health and Marine Hos- 
 pital Service, issues from time to time standard anti-toxic serum. 
 With this serum the strength of the toxin used in determining 
 the anti-toxic value of serum is gauged. The "immunity unit," 
 or unit of anti-toxin, is the amount of diphtheria anti-toxic serum 
 which will just neutralize one htmdred times the smallest amount 
 of toxin necessary to kill a 250 gram guinea pig in four days. 
 The anti-toxic value of serum is determined by mixing one hun- 
 dred times the smallest fatal dose of fresh diphtheria toxin with 
 varying amounts of the diphtheria anti-toxin, allowing the mix- 
 tures to stand for fifteen minutes and then injecting them into 
 250 gram guinea pigs. The smallest amount of serum which will 
 protect a 250 gram guinea pig for more than four days against one 
 hundred times the smallest amount of diphtheria toxin necessary 
 to kill a 250 gram guinea pig, contains one unit of diphtheria anti- 
 toxin. 
 
 At the time of bleeding inmiunized horses, that is after six 
 to eight weeks of immunization, the serum may contain from 
 one hundred to one thousand units of diphtheria anti-toxin per c. c. 
 The concentrated and refined diphtheria anti-toxin contains usually 
 from three hundred to two thousand units of anti-toxin per c. c. 
 The various manufacturers of diphtheria anti-toxin now furnish 
 their product in syringes containing a determined number of 
 units of anti-toxin. These packages are stamped so as to indicate 
 the date after which they can be exchanged, free of charge, for 
 more recently tested serum. Because of the degeneration in 
 potency of diphtheria anti-toxin in the fluid condition, it is desir- 
 able that no sera be useti after the date when they are to be ex- 
 changed for new sera.
 
 DIPHTHERIA ANTI-TOXIN. 103 
 
 The therapeutic application of diphtheria anti-toxin has given 
 most satisfactory results, and is now recognized and regarded as 
 the specific for diphtheria. The statistics on the death rate from 
 diphtheria have been collected at many different times, and all 
 indicate that, while before diphtheria anti-toxin was used over 
 fifty per cent of the cases resulted fatally, since the • application 
 of this method of treatment, the death rate from diphtheria has 
 fallen below twenty per cent, and according to some statistics to 
 five per cent. In Boston the death rate of diphtheria from 1888 
 to 1894 was 43.2 per cent, while from 1895, after which diphtheria 
 anti-toxin was used extensively, to 1904, inclusive, the death 
 rate has fallen to 11.84 per cent. Still better results will prob- 
 ably be obtained as this method of treatment becomes more uni- 
 versal and is better understood. 
 
 The method of treatment of diphtheria with diphtheria anti- 
 toxin varies. However, some definite principles of treatment 
 have been established. It is very essential that all cases be treated 
 as early as possible. The reasons for this are clear when it is 
 understood that as toxin is liberated by diphtheria bacilli, it tends 
 to combine with the cells of the body. After this union has once 
 taken place the cell is rapidly injured. In order that diphtheria 
 anti-toxin combine with these toxins it is essential that the immune 
 substances be present at the time of the liberation of the toxin. 
 Most statistics show that in ordinary cases of diptheria the death 
 rate is nil when diphtheria anti-toxin is injected on the first day 
 of the disease. Even when treated with diphtheria anti-toxin on 
 the second day, the death rate is below five per cent; however, 
 when specific treatment is not instituted until after the fourth 
 day, twenty per cent of the patients do not recover. 
 
 The dose to be given in the different conditions varies, and 
 no hard and firm rules can be laid down. There are, however, 
 some quite generally accepted rules as to doses. 
 
 The ordinary case of diphtheria, treated on the first day of 
 the disease, receives injections of from two to three thousand 
 units of anti-toxin. When the serum is not given until the second 
 day, usually three to four thousand units are injected. 
 
 In the pharyngeal and laryngeal types of diphtheria larger 
 doses are usually given — seldom less than five thousand units.
 
 104 VACCIXE AND SERUM THERAPY. 
 
 When the disease conies under ol)servation hite or is very 
 severe twenty thousand to one hundred tliousand units may be 
 necessary. Evidence has been obtained that toxin may be taken 
 from the cells with which it has combined when very large doses 
 of anti-toxin are injected. No (^ase of diphtheria should be re- 
 garded as being too severe or too far advanced to be treated by 
 diphtheria anti-toxin, but in such cases large doses should be given, 
 as small doses are of no avail, because they do not neutralize all 
 the toxin present. The age of the patient, unless very young, 
 should have no influence on the amount of anti-toxin injected. 
 
 Diphtheria anti-toxin, as any other therapeutic agent, should 
 be given in sufficient quantities to accomplish the full therapeutic 
 action. To determine this the effects of diphtheria anti-toxin 
 must be recognized. The results of injections of sufficient amounts 
 of anti-toxin are: general improvement of the patient's condi- 
 tion, reduction of fever, improvement of the pulse, but most 
 noticeable of all is the shriveling of the membrane, decrease of 
 discharge, and less fetid odor of the breath. When these effects 
 do not appear from six to eight hours after injection, more diph- 
 theria anti-toxin should be injected. Re-injections usually are 
 larger than first injections, and are indicated at any time when 
 the patient's condition becomes more grave, or when improve- 
 ment does not come in six to eight hours after injection. At 
 times diphtheria anti-toxin does not give satisfactory results in 
 the treatment of diphtheria. In most of these cases, however, the 
 treatment is begun too late or there is infection with other organ- 
 isms. Of these organisms streptococci are most important and 
 are responsible for many of tlie fatal cases of diphtheria. Such 
 cases are at times treated with both diphtheria anti-toxin and 
 anti-streptococci serum. 
 
 Various attempts have been made to administer diphtheria 
 anti-toxin by the mouth. The work of McClintock and King on 
 this method has already been referred to. Before oral adminis- 
 tration of diphtheria anti-toxin can be generally employed, more 
 experimentation will be necessary. 
 
 The use of dried diphtheria anti-toxic globulins, which are 
 dissolved in salt solution before injection, is relatively recent. 
 If the results obtained by their use are as favorable as has been 
 reported, it may be expected that they will be readily taken up
 
 IMMUNIZATION AGAINST DIPHTHERIA. 105 
 
 uy the medical profession. Serum, however, has the advantage 
 of always being ready for use and not requiring sterilisation 
 before administration. 
 
 In some of the severer cases of nasal diphtheria, anti-toxin 
 has been sprayed on the membrane with some beneficial results. 
 Frequently too, it is observed that diphtheria bacilli remains viable 
 in the nose and throat after an apparent recovery from the dis- 
 ease. Such persons frequently are the cause of the spread of 
 diphtheria. Various attempts have been made to remove the 
 diphtheria bacilli from the nose and throat of these apparently 
 well individuals. In addition to the antiseptic methods em- 
 ployed in the treatment of this condition, diphtheria anti-toxin 
 has been applied locally. Recently it has been proposed to ac- 
 tively immunize such individuals by the injection of bacterial 
 vaccines made from the cultures of diphtheria bacilli isolated 
 from the patient. 
 
 Immunization of w^ell individuals against possible diphtheria 
 infection has been practiced for some time. It has been quite 
 definitely proven that injection of a relatively small number of 
 anti-toxic units will' protect from four to six weeks against in- 
 fection w^ith the diphtheria bacillus. The doses generally recom- 
 mended are from three hundred to five hundred units in small 
 children and one thousand units for older children and adults. 
 While the custom concerning the immunization of well persons 
 varies, it is quite generally accepted that children who have been 
 exposed to diphtheria should receive at once immunizing doses of 
 diphtheria, while adults seldom are treated in this way. 
 
 The definitely beneficial results which have been obtained 
 by the use of diphtheria anti-toxin in the treatment of diphtheria, 
 ought to be sufficient to convince the practitioner that diphtheria 
 anti-toxin should be used in practically all cases of diphtheria. 
 Moreover the practioner ought to know enough concerning the 
 symptoms and complications of diphtheria, and the untoward 
 effects of serum injection to distinguish the conditions dependent 
 upon the disease and those dependent upon the serum injected. 
 It is not unusual for the practioner to diagnose the transient serum 
 rashes as erysiplas, and the edema, following serum injections,
 
 106 VACCINE AND SKRl'M THERAPY. 
 
 as those due to Bright's disease. Practioners are undoubtedly 
 largely responsible for the misconceptions of laymen concerning 
 the effects of diphtheria anti-toxin injections. As a result of these 
 misconceptions frequently consent to use diphtheria anti -toxin, 
 when it is definitely indicated, cannot be obtained, and yearly 
 numbers of children whose lives could undoubtedly have been 
 saved are carried to the grave. 
 
 TETANUS ANTI-TOXIN. 
 
 In 1890, Behring and Kitasato immunized mice and rabbits 
 to tetanus by injecting cultures of the bacillus of tetanus. These 
 investigators found that blood from rabbits immunized to tetanus 
 bacilli, is able to protect mice against tetanus. The first reliable 
 anti-tetanic serum to be used in man, was put onto the market 
 in 1896. 
 
 Theprocessof producing anti-tetanic serum, is similar to that 
 employed to produce anti-diphtheria serum — blood is drawn from 
 horses that have received injection of increasing amounts of tetanus 
 toxin. The toxin injected is produced by growing the tetanus bacillus 
 on bouillon. After ten to fifteen days of incubation under anaerobic 
 conditions, the bouillon culture is filtered through a Berkef eld filter. 
 The germ free filtrate contains the toxin, which is present usually in 
 large amounts. At the first injection into the horse usually one-half 
 c. c. of toxic bouillon, together with anti-tetanic serum are in- 
 jected. The amount of toxin is increased at each injection, until 
 finally seven hundred to eight hundred c. c. of toxin are tolerated 
 when given in one injection. After the third injection the anti- 
 tetanic serum is usually omitted. After several months of treat- 
 ment, and complete recovery from the last injection, the horses 
 are bled and the serum is collected. The serum is then concen- 
 trated and refined after the same methods that are used for diph- 
 theria anti-toxin. 
 
 The standardization of tetanus anti-toxin until very recently 
 has been indefinite and unsatisfactory. At the present time 
 different standards exist in the various countries. The unit of 
 anti-toxin for tetanus for the United States has been fixed as that 
 amount of tetanus anti-toxin that will protect a 350 gram guinea 
 pig for ninety-six hours against one thousand times the smallest 
 fatal dose of tetanus toxin. In order that the standard may be
 
 TETANUS ANTI-TOXIN. 107 
 
 the same throughout the United States the Hygienic Laboratory 
 of the United States Pubhc Health and Marine Hospital Service 
 sends out at regular intervals a stable precipitated tetanus toxin 
 which is called the "test dose" and contains one hundred times 
 the smallest fatal dose. It will be observed that a unit of 
 tetanus anti-toxin contains more than ten times as much neu- 
 tralizing value as does a unit of diphtheria anti-toxin. The var- 
 ious anti-tetanic serum producers furnish their product in suitable 
 syringes, and, as is the case with diphtheria anti-toxin, state the 
 date after which it is desirable that the serum be returned if not 
 used. 
 
 The value of tetanus ant i -toxin and the methods for adminis- 
 tration can only be judged after study of the action of the tetanus 
 bacillus and its toxin. Tetanus in man is usually the result of 
 the subcutaneous introduction of tetanus bacilli, some foreign 
 matter and usually saprophytic organisms. Thus the disease 
 follows most frequently upon puncture and contused wounds 
 as the result of injuries made by machinery, pistol shots, trauma- 
 tism especially in stablemen, infection of the naval during or 
 after birth, and sometimes as a result of the injection of con- 
 taminated vaccines and sera. The distribution of the organism 
 is very wide, being found practically everywhere where there is 
 animal habitation. 
 
 After the entrance of the tetanus bacillus, usually there is 
 little change produced in the tissues infected. Suppuration occurs 
 practically only as a result of other organisms. Usually the or- 
 ganisms remain localized. The disease and its symptoms are 
 entirely dependent on the absorption of the toxins produced by 
 the tetanus bacilli. Tetanus toxin circulates in the blood and 
 lymph, but shows no symptoms until it unites with and is ab- 
 sorbed by the end organs of the motor nerves and the central 
 nervous system. To reach the end organs the toxin is carried 
 through the axis cylinders. It is thus seen that when the symp- 
 toms of the disease appear, the toxins have already combined with 
 the cells, are exerting their toxic effects on them and can no longer 
 combine with the anti-toxin which is not taken up by the central 
 nervous system. In cases not treated with tetanus anti-toxin. Rose 
 has found that ninety-one per cent of the cases in which the incuba- 
 tion period is short, terminate fatally; 81.3 per cent of those
 
 108 VACCINE AND SERUM THERAPY. 
 
 having a medium incubation period die, while of the cases with a 
 long incubation period, 52.9 per cent of the patients die. 
 
 Tetanus anti -toxin can only bind tetanus toxin before it is 
 taken up by the nerve cells and axis cylinder. Frequently, how- 
 ever, the disease is not suspected until after the symptoms appear. 
 Because of this the value of tetanus anti-toxin lies mainly in its pro- 
 phylactic application. As a preventative of tetanus, tetanus anti- 
 toxin has proven to be of great value. However to obtain bene- 
 ficial results mjections should be made early, as soon after in- 
 fection as IS possible, and inasmuch as immunity lasts only two 
 to three weeks, the injections should be repeated as long as danger 
 of infection exists. The immunizing dose which is generally 
 used consists of fifteen hundred immunity units. The wounds 
 after w^hich immunizing doses of tetanus anti-toxin are to be 
 used are those made by bums with toy pistols, fire-works, pistol 
 shot wounds, injuries occuring while working about the stable 
 or infected with fertilized garden earth or manure. The value of 
 this method cannot be definitely determined, for if tetanus anti- 
 toxin is used successfully, it is difficult to decide whether or not 
 there has been infection with the tetanus bacillus. It is the be- 
 lief of some, however, that tetanus can be prevented entirely if 
 after all injuries of the kind likely to be infected with tetanus 
 bacilli immunizing doses of tetanus anti-toxin are injected early. 
 Jordan states that in the United States in 1903, there were 4,449 
 Fourth of July injuries, of which 406 cases died of tetanus ; while 
 in 1907, when tetanus anti-toxin was used quite universally for 
 such wounds, there were only sixty-two deaths caused by tetanus, 
 following 4,413 Fourth of July injuries. 
 
 The use of tetanus anti-toxin as a curative measure has not 
 given universal success. This is undoubtedly due to the fact 
 that after the symptoms appear the tetanus toxin has already 
 combined with and injured the nerve cells, and is no longer amen- 
 able to neutralization with tetanus anti -toxin. It is usually 
 conceded that if tetanus anti-toxin is not given within thirty hours 
 after the appearance of symptoms, subcutaneous injections of 
 anti-toxins is of little avail. When the disease has advanced, 
 the large nerves in the vicinity of the infection and the spinal 
 canal have been injected. The therapeutic dose of tetanus anti- 
 toxin administered varies. If it is a case of short incubation, that
 
 TETANUvS ANTI-TOXIN. 109 
 
 is eight days or less, injections of from 15,000 to 20,000 units are 
 made every two to eight hours until there is abatement of the 
 symptoms. These cases usually terminate fatally, even when 
 tetanus anti-toxin is given. The cases where the incubation period 
 is longer are more frequently recovered from. In all cases early 
 injections in large doses are indicated when the symptoms have 
 appeared, and radical methods of injection are justifiable, because 
 most cases without anti-toxin terminate fatally. As a result of 
 tetanus anti-toxin, the death rate in animals as determined by 
 Behring has been found to be reduced from 88 per cent to 40 or 
 45 per cent. The curative value of tetanus anti -toxin in man 
 cannot be well demonstrated by statistics. While the results are 
 not nearly as favorable as those obtained by treating diphtheria 
 with diphtheria anti-toxin, still tetanus anti-toxin ought to be 
 used in all cases of tetanus. 
 
 Relatively recently a dried tetanus anti -toxin has been made. 
 This powder is used principally as a dressing for wounds which 
 are likely to be injected, but it may also be used for injection 
 after dissolving in salt solution. For the latter use it has the ob- 
 jection that it dissolves with difficulty. While relatively little 
 is known concerning it, it has advantages similar to those of dried 
 diphtheria anti-toxin and may prove of considerable value to the 
 medical profession. 
 
 As a result of the decreased number of cases of tetanus fol- 
 lowing Fourth of July wounds, when immunizing doses of tetanus 
 anti-toxin are given, the physician should in all cases of such injury 
 administer to the patient an immunizing dose of tetanus anti- 
 toxin. Moreover all wounds in which tetanus infection might 
 occur, should be thoroughly cleansed and the patient immvmized 
 with tetanus anti -toxin. When s^^mptoms of tetanus have de- 
 veloped the patient ought to have every advantage of any bene- 
 fits that may be derived from the use of large and repeated in- 
 jections of tetanus anti-toxin. 
 
 ANTI-BACTERIAL SERA. 
 
 Sera containing substances which result in the death and 
 destruction of bacteria are called anti-bacterial sera. Acquired 
 anti-bacterial immunity depends on destruction of bacteria be-
 
 110 VACCINE AND SERUM THERAPY. 
 
 fore they have had sufficient chance for multiplication and pro- 
 duction of poisons which kill the cells in the body. For this reason 
 anti-bacterial sera are frequently called bactericidal sera. In 
 the serum of individuals and animals that have acquired anti- 
 bacterial immunity, various kinds of anti-bodies are found. The 
 best known of these bodies are bacteriolysins, opsonins, precipi- 
 tins and agglutinins. These prepared anti-bodies are introduced 
 into the body in passive immunization. The importance and 
 value in immunity of all of these anti-bodies is not known, but it 
 is generally accepted that agglutinins and precipitins are of little 
 or no value, while opsonins and bacteriolysins are considered of 
 importance in anti -bacterial immunity. Bacteriolytic immune sera 
 depend for their action on specific substances called ' 'bacteriolysins, ' ' 
 which dissolve bacteria. The value of opsonins in bactericidal 
 sera to be used in injections for the purposes of conferring passive 
 immunity, is still indefinitely understood, although some investi- 
 gators have attached considerable importance to their presence 
 in sera. 
 
 The anti-bodies in anti-sera called agglutinins and precipi- 
 tins belong to the second order of receptors, the bacteriolysins 
 to the third order, while in regard to opsonins, no statement can 
 be made at the present time — some regarding them as belonging 
 to the second and others to the tliird order of receptors. 
 
 Anti-sera containing receptors of either the second or third 
 order are easily inactivated by heat, age, acids, etc. After inacti- 
 vation they are no longer able to produce agglutination, precipi- 
 tation, lysis, or opsonification. Tlie loss of powder to produce tliese 
 effects depends on the destruction of ferment-like substances, which 
 are a part of the agglutinin and precipitin receptors and are fur- 
 nished to the receptors of the third order by the fresh blood serum, 
 (See page 18.) Inactivated bacteriolytic serum differs from in- 
 activated or aged agglutinating or precipitating serum in that 
 on the addition of fresh serum the anti-bodies producing lysis 
 can be re-activated, while the agglutinating and precipitating 
 properties cannot be restored in this way. Because the different 
 specific bacteriolytic sera used in passive immunization are usually 
 not freshly drawn and therefore have been inactivated by age, 
 the individual or animal immunized must furnish the complement 
 or ferment-like substance so as to make the destruction of bacteria
 
 ANTI-BACTERIAL SERA. HI 
 
 possible. The normal individual in order to utilize all the lytic 
 and possibly the opsonic anti-body in a specific serum, therefore 
 must possess large amounts of complement. Numerous investi- 
 gators, among which may be mentioned Ehrlich, Morgenroth and 
 Metchnikoff, have found that in certain diseased conditions there 
 is an actual decrease in complement. Moreover, as far as can be 
 determined, complement is not increased during disease and im- 
 munization. Richardson has shown that the serum of typhoid 
 patients is not usually able to destroy typhoid bacilli, but that 
 upon addition of fresh serum from a normal individual the typhoid 
 immune serum is able to bring about complete lysis of typhoid 
 bacilli. Numerous similar observations have been made in natural 
 infections in man. In the efficiency of anti-bacterial sera the 
 presence of sufficient amounts of complement undoubtedly plays 
 a large part and in certain cases the lack of complement probably 
 is the cause of failure of beneficial action of certain specific anti- 
 bacterial serum. Various attempts have been made to increase 
 the amount of complement. Ant i -complement has been used 
 to immunize in order to excite the over-production of complement. 
 Injection of fresh complement with the immune serum has been 
 tried by some investigators. Efforts have been made to preserve 
 the complement in immune sera by freezing the serum and keeping 
 it cold until it is injected. Recently dried immune sera have 
 been advocated because it is known that complement is preserved 
 for a long time in serum dried down soon after it is drawn. It is 
 questionable, however, whether by any of these methods enough 
 complement will be present to activate the amount of immune 
 bodies injected. Hiss recently has published results on the cura- 
 tive action of subcutaneous and intraperitoneal injection of ex- 
 tracts of leucocytes from normal rabbits. Of this leucotic extract 
 Hiss says, "The action of the leucocytic extract may be due to 
 the enhancement of the bacteriolytic action of the animal's plasma 
 by the introduction of complement — ^but is most likely chiefly due 
 to the poison neutralizing or destroying bodies." 
 
 The practical application of specific anti -bacterial sera in 
 the treatment of the various infections in man has been tried on 
 a large scale. It has been found that by the early injection of 
 serum from animals actively immunized to various organisms, 
 animals can be rendered non-susceptible to infections with pneu-
 
 112 VACCINE AND vSERUM THERAPY. 
 
 mococci, gonococci, streptococci, typhoid and dysentery bacilli, 
 cholera spirillae and many other microorganisms. Based on these 
 results, many different anti-bacterial sera have been used in passive 
 immunization in man. The results of ant i -bacterial sera in the 
 treatment of diseases of man have, however, not been as satis- 
 factory as those obtained with specific anti -toxic sera. The im- 
 portance of complement has already been emphasized. There are, 
 however, other reasons why anti -bacterial sera have not yielded 
 as good results as anti-toxic sera. 
 
 Anti -bacterial substances, with the aid of complement, have 
 the powder of destroying bacteria but not of neutralizing bacterial 
 toxins. For this reason it is essential that anti-bacterial sera be 
 used early enough in the disease, before the bacteria have had a 
 chance to multiply sufficiently and to set free enough poison to 
 injure and kill the cells. Early injection of specific sera unfortu- 
 nately is not possible in many of the diseases for which specific 
 anti -bacterial sera have been used, but as our methods of early 
 diagnosis are improved earlier administration of these sera will 
 be made. It is to be remembered that it is of the utmost impor- 
 tance that these sera be vised before the infecting organisms 
 have multiplied greatly. 
 
 Another reason of the failure of anti-bacterial sera to cure 
 disease is dependent upon the fact that as the bacteria are dis- 
 solved and destroyed by the action of immune substance and com- 
 plement, the intracellular toxins are liberated. If the body cells 
 are not able to cope with the increased amount of toxin liberated 
 upon the solution of the bacteria, they will die and the gravity of 
 the disease be increased. Hiss has produced evidence to show 
 that the leucocytes contain substances neutralizing these poisons. 
 To have liberated the minimum amount of toxin, anti-bacteriai 
 sera should be administered when the number of invading bacteria 
 is relatively small, which will be early in the disease. 
 
 Anti-bacterial sera are standardized with more difficulty and 
 less reliably than are the anti -toxic sera. Attempts have been 
 made to determine the number of smallest fatal doses of bacteria 
 a given amount of serum will protect against. It is to be remem- 
 bered, however, that with living microorganisms, a multiple of 
 the fatal dose is not as much more severe than a single dose as
 
 ANTI-STREPTOCOCCIC SERUM. llo 
 
 the multiple would indicate. Attempts have also been made 
 to determine the protective value of specific anti-bacterial serum 
 by injecting varying amounts of sera, and bacteria, which have 
 been mixed in vitrio and allowed to act for some time before in- 
 jection. The value of some of the lytic serum, as determined by 
 this method, is high. Frequently it is not possible to test the bac- 
 tericidal power when, as a result of the difficulties of determining 
 the amounts of specific bactericidal substances in the serum, the 
 agglutinating and precipitating values are determined. This 
 method of standardization is not followed because agglutinins 
 and precipitins are regarded as bactericidal substances but because 
 these anti-bodies, to some extent at least, accompany bactericidal 
 power. 
 
 As was stated earlier, specific anti-bacterial sera have been 
 made by injections of practically all the disease producing bacteria. 
 The most important of these are the sera which contain specific 
 substances by which they destroy streptococci, meningococci, 
 pneunococci, typhoid and dysentary bacilli, and staphlycocci. 
 
 ANTI-STREPTOCOCCIC SERUM. 
 
 Anti-streptococcic serum was first made bv Marmorek, in 
 1895. This investigator immunized horses by injections of in- 
 creasing doses of living virulent streptococci. With serum from 
 these animals he was able to confer passive immunity to rabbits 
 and also made attempts to treat erysipelas and pueperal fever in 
 the human. Denys and LeClef have immunized animals with 
 bouillon cultures of streptococci and claim to get favorable results 
 upon injection of serum from the immunized animals. Van der 
 Velde, realizing that there are different strains of streptococci, and 
 that the anti-sera produced are specific for the especial strain 
 used in immunization, immunized animals with the different 
 strains of streptococcic, thus making what is called a "polyvalent" 
 anti-streptococcic serum. 
 
 The sera which are now on the market are almost universally 
 made by immunizing horses with repeated injections of increasing
 
 114 VACCINE AND SERUM THERAPY. 
 
 doses of killed and later living bouillon cultures of numerous 
 strains of streptococci recently isolated from patients. Several 
 months are required to immunize horses from which the immune 
 serum is to be obtained. After testing the serum for sterility it 
 is usually tubed in suitable syringes. 
 
 The method of action of anti -streptococcic serum is little 
 understood. The serum contains agglutinins but these have not 
 been regarded of value in immunization. With the discovery of 
 bacteriotropins by Neufeld and Rimpau and opsonins by Wright 
 and Douglas new light has been thrown on the action of these 
 sera, for it has been found that in anti -streptococcic serum there 
 is present a specific substance which makes streptococci vulnerable 
 to ingestion by leucocytes. 
 
 Standardization and determination of the amount of pro- 
 tective substances in anti-streptococcic serum has not been suc- 
 cessful, because the susceptibility of animals to streptococci varies 
 and because the method of protective action of the serum is little 
 known. Usually the dose injected is regulated according to cubic 
 centimeters of polyvalent anti-streptococcic sera. The potency 
 of this serum decreases relatively rapidly so that it ought not to 
 be used later than six months after the bleeding of the immunized 
 horse. 
 
 Anti-streptococcic serum has been used especially in septi- 
 caemia, local infections following traumatisin, streptococcic pneu- 
 monia, meningitis, rheumatism, erysipelas, pueperal sepsis, scarlet 
 fever, secondary infection complicating tuberculosis, and in fact, 
 in all streptococcus infections. 
 
 The therapeutic and prophylactic results obtained by the use 
 of anti-streptococcic serum vary considerably and are apparently 
 dependent on various conditions, such as grade and kind of im- 
 munizing serum, day of disease on which injections are made, 
 amount of serum injected, virulence of infecting organism, and 
 determination of the invading organism. From the various re- 
 ports which have been received from clinicians who have used 
 these sera, there can be no doubt as to the beneficial results which 
 may at times be obtained from its use. In order that good results 
 may be obtained, however, it is essential that it be definitely 
 determined that the streptococcus is responsible for the disease^! 
 condition, that a high grade of polyvalent serum be given early
 
 ANTI-MENINGOCOCCIC SERUM. 115 
 
 in doses varying from 10 to 40 c. c, and that these injections be 
 repeated at intervals of from four to eight hours. 
 
 After injection of anti -streptococcic serum the patient usually 
 rests more quietly for several hours. This, however, need not 
 indicate that recovery from the disease will follow. The thera- 
 peutic effects of the serum are manifested by the relief of symp- 
 toms, decline ni fever, improvement of the pulse, and subsidence 
 of the nervous symptoms. These effects usually appear within 
 twenty-four hours after injection if the serum is to be of value, 
 and if no relief comes within twenty-four hours after two injec- 
 tions of from 20 to 40 c. c. of serum, no beneficial results from the 
 use of the serum are to be hoped for. No untoward effects of the 
 serum are met with except the occasional skin rashes which have 
 already been discussed elsewhere. 
 
 Anti-streptococcic serum, even though its curative results are 
 uncertain, ought to be used in every case of acute streptococcus 
 infection. The use of the serum in scarlet fever is not universal 
 but probably should be resorted to in the severer cases. In the 
 chronic cases of discharging sinuses, etc., streptococcuc vaccines 
 are at times used to greater advantage than anti-streptococcic 
 sera. 
 
 ANTI-MENINGOCOCCIC SERUM. 
 
 Agglutinating substances were discovered in 1903 by Jaeger, 
 in rabbits experimentally immunized to meningococci. Since 
 then numerous attempts have been made to produce specific 
 anti-sera to be used in passive immunization of the human. It 
 was not until 1906, however, that injection of such anti-meningo- 
 coccic serum was followed with any degree of success. In 1906 
 Kolle and Wasserman reported results on the use of specific 
 meningococcus serum which they had been able to produce in 
 animals either by intravenous or subcutaneous injections of 
 cultures of meningococci killed by heating to 60° C, or by in- 
 jections of extracts of meningococci, obtained by shaking these 
 organisms for four or five days in suspension in distilled water. 
 Jochman in this same year reported results on a specific anti- 
 meningococcic serum prepared after similar methods. The effects 
 obtained by the use of these sera have, however, not been as
 
 IIG VACCINE AND SERUM THERAPY. 
 
 favorable as those obtained by the injection of a serum prepared 
 by Flexner and Jobling. 
 
 The method of preparation of the curative meningococcic 
 serum of Flexner and Jobling, is to inject into the horse first in- 
 creasing doses of killed meningococci, later increasing doses of 
 living meningococci and finally increasing doses of an extract of 
 meningococci. The extract injected contains the endotoxin liber- 
 ated by the action of a meningococcus autolytic enzyme which as 
 Flexner has found, is able to destroy the cell substance of the 
 meningococcus . 
 
 The action of Flexner and Jobling's serum, according to the 
 originators, is three fold. It exerts an anti-toxic action, is bac- 
 tericidal and is bacteriotropic or opsonifying. Of these effects 
 clinicians who have used the serum emphasize especially the ap- 
 parent anti -toxic action. 
 
 Flexner and Jobling have summarized the results of the treat- 
 ment of 400 cases of epidemic cerebrospinal meningitis. Accord- 
 ing to this summary it has been found by the various clinicians 
 using this serum, that the period of illness can be shortened, the 
 chronic lesions largely prevented and the percentage of mortality 
 markedly reduced. Probably the principle reason for the differ- 
 ence in efficiency of Flexner and Jobling's serum as compared to 
 KoUe and Wasserman's and Jochman's specific sera lies in the 
 method of application. Flexner and Jobling have emphasized the 
 importance of reaching the causal organisms with the serum. For 
 this reason lumbar puncture and withdrawal of a certain amount 
 of cerebrospinal fluid is first made, after which, without removing 
 the needle, injection of the serum is made directly into the sub- 
 arachnoid space of the spinal cord. Recently the persistance of 
 Diplococcus intracellularis in the lateral ventricles, after apparent 
 death of the organism in the spinal sub-arachnoid space, has been 
 emphasized by Knox and Sladen. For the treatment of this con- 
 dition. Gushing and Sladen have suggested the advisability of ven- 
 tricular puncture and injection of anti-meningococcus serum. 
 
 The results which have been obtained by the use of anti- 
 meningococcus serum as made by Flexner and Jobling, have been 
 so beneficial in acute and chronic cases of meningitis due to the 
 Diplococcus intracellularis that its use is indicated as a specific 
 therapeutic measure.
 
 ANTI-GONOCOCCIC SERUM. 11 
 
 ANTI-GONOCOCCIC SERUM. 
 
 Various attempts have been made to produce an anti-gono- 
 coccic serum for passive immunization of man. It is a well known 
 fact that in the human no immunity results from an attack of 
 gonorrhea, and for this reason, together with the lack of favorable 
 results from the use of anti-gonococcic serum, most investigators 
 have concluded that a specific immune serum for Mic. gonorrhoeae 
 cannot be produced. In most of the attempts at the production 
 of anti-gonococcic serum old cultures of the organism have been 
 used. 
 
 In 1906, Torrey described an anti-gonococcic serum with which 
 he had attained beneficial results in the treatment of some cases 
 of gonorrheal arthritis. In an attempt to make anti-gonococcic 
 serum, Torrey made the observation that the toxin of the Mic. 
 gonorrhoeae, which is present in old fluid cultures, is toxic for 
 the small laboratory animals. The toxin for the gonococcus has 
 been studied at various times, and has been generally regarded 
 as being derived from the dead and disintigrated bodies of the 
 organism, though by some it has been claimed to be a true ex- 
 tracellular toxin. Torrey found that it is not possible to im- 
 munize the small laboratory animals to this toxin, but that in 
 fact at times a true hypersusceptibility may develop as a result of 
 injections of this toxin. This investigator observed, however, that 
 animals can be immunized to the living and dead organisms of 
 gonorrhea, and that in. the blood of animals so immunized agglu- 
 tinins and lysins are present. Based on these observations, Tor- 
 rey holds that the efficiency of the serum depends upon specific 
 bactericidal substances which act because of lytic rather than of 
 opsonifying power. Phagocytosis, according to this investigator, 
 is of little importance in the destruction of gonococci. 
 
 Torrey and Rogers have prepared an anti-gonococcic serum 
 for use in treatment of gonorrheal infections in man. The method 
 of preparation of this serum, as perfected and recommended by 
 these investigators, and used by the different serum producers is 
 as follows : Strong healthy rams receive intraperitoneal injections 
 of increasing amounts of twenty-four hour old ascitic agar cultures 
 of various, recently isolated, virulent strains of Mic. gonorrhoeae. 
 The culture is suspended in salt solution, and for the first two
 
 118 VACCINE AND SERUM THERAPY. 
 
 injections, this suspension is heated to 65° C. for one-hcdf hour 
 before the injection is made. Nine or ten injections are usually 
 necessary to produce serum of high value. After immunization 
 is completed, the animal is bled from the carotid arteries, and the 
 serum allowed to separate out. After this the serum is collected, 
 filtered and tested for sterility. Immunization is made in rams 
 because blood from these animals is apparently little toxic for the 
 human. Polyvalent serum is used because the imnume bodies for 
 one strain are specific for that strain, and inasmuch as the strains 
 of gonococci vary in the different infections, immune bodies for 
 all strains must be present in the serum. 
 
 Standardization of this serum has not been effectual. It 
 seems quite definite that this serum possesses no anti-toxic nor 
 opsonic value, but is dependent for its efficiency on its lytic action. 
 Because of a lack of any better method of determining the immun- 
 izing value of this serum, determinations have usually been made 
 of its agglutinating value. The amount of serum injected is based 
 on cubic centimeters rather than units of immune substance. 
 
 Torrey and Rogers' anti-gonococcic serum has been tried 
 in the treatment of the various gonorrheal infections. From time 
 to time favorable and unfavorable results have been reported. 
 The greatest value of the serum apparently is manifested in the 
 treatment of the complications of gonorrheal urethritis, such as 
 prostatitis, epididymitis, orchitis, cystitis, salpingitis, endocarditis, 
 pleuritis, meningitis and especially arthritis. The acute condi- 
 tions, urethritis, vaginitis, and conjunctivitis, are little eff'ected 
 by this treatment. 
 
 The method of treatment with anti-gonococcic serum consists 
 of injections of 2 c. c. of serum at intervals of from one to four 
 days, as may be indicated clinically. The injections of serum are 
 made in the loose subcutaneous tissue in convenient parts of the 
 body. In every case treated with anti-gonococcic serum all the 
 other methods known to be of value in the treatment of the par- 
 ticular condition should be employed. Anti-gonococcic serum seems 
 to cause serum disease more frequently than most of the other 
 immune sera. In all cases it is to be remembered that, because 
 this method of treatment frequentty must be carried over a long 
 period of time, serum injections must be made at intervals of not 
 more than seven to eight days.
 
 ANTI-PNEUMOCOCCIC SERUM. 119 
 
 While anti-gonococcic serum, as prepared by Torrey and Rogers' 
 method, has produced beneficial results in many conditions, 
 it is to be remembered that improvement and recovery have not 
 constantly followed its injection. Moreover injection of this 
 serum ought not to be the only method of treatment instituted. 
 
 ANTI-PNEUMOCOCCIC SERUM. 
 
 Man after an attack of pneimionia possesses a certain degree 
 of immunity. Immunity, however, does nnt always follow pneu- 
 monia, nor is it lasting. Early in the history of the diplococcus of 
 pneumonia, attempts were made to artificially immunize animals, 
 with the result that it was found that a certain degree of immunity 
 can be produced. In as much as the pneumonococcus does not 
 produce an extracellular toxin, anti-toxic immunity cannot be 
 produced in animals as a result of artificial immunization with this 
 organism. 
 
 The anti-pneumococcic serum now used is usually taken from 
 horses that have received increasing injections of dead and later 
 of living cultures of recently isolated pneumococci. Most of the 
 sera are polyvalent, the best known being those made by Roemer 
 .and by Pane. The method of action of anti-pneumococcic sera 
 is little understood. It is quite definitely established that it 
 possesses no anti-toxic value. The curative action of the serum 
 then, must depend on its anti -bacterial power. Lysins, until 
 recent times, were supposed to account for the immunizing value 
 of the serum. Neufeld and Rimpau and Wright and others have 
 shown that anti-pneumoccic serum contains bacteriotropic or 
 opsonifying substances which prepare the cocci for ingestion by 
 the leucocytes. 
 
 In the treatment of pneumonia with anti-pneumococcic serum 
 usually relatively large amounts of serum are injected. The 
 treatment as recommended by clinicians who have had most ex- 
 perience, is to inject 20 c. c. into the subcutaneous tissue twice 
 a day until the symptoms are relieved. None of the other known 
 methods of treatment are suspended during serum treatment. 
 
 The results]which have been attained by the use of the serum 
 have varied a great deal. Some observers have lauded the use
 
 120 VACCINE AND SKRUM THERAPY. 
 
 of this serum, but when all cases are considered the results are not 
 convincing as far as the curative effects of the serum are con- 
 cerned. The reasons for the failure of a specific anti-pneumococcic 
 serum are probably dependent on the absence of an anti-toxin 
 in the serum, injection after the disease is far advanced, and the 
 difficulty in determining the species of organism causing the pneu- 
 monia. The latter reason must be evident to the clinician, because 
 the disease of lobar pneumonia may be caused by various differ- 
 ent species of organisms. Although in general, the therapeutic 
 action of anti-pneumococcic sera has not been marked, still 
 physicians who have used it in pneumonia have been impressed 
 with the general improvement of the patient, the lowering of the 
 temperature and the absence of complications, following its use. 
 Important untoward symptoms seldom have developed from its use. 
 While anti-pneumococcic serum is not definitely a curative 
 measure, the physician is warranted in using it whenever the 
 patient's condition becomes serious. In all cases it is to be 
 remembered that if beneficial results are to be obtained wdth 
 this serum it must be injected early. 
 
 ANTI-TYPHOID SERUM. 
 
 Before the discovery of the organism causing typhoid fever, 
 it was knowm that an attack of typhoid fever, usually gives some 
 protection against a second attack of the disease. After the 
 discovery of the typhoid bacillus it was found that animals repeat- 
 edly injected with non -fatal doses of this organism will ultimately 
 be protected against otherwise fatal doses. It was also found 
 that by injecting blood serum from immunized animals, a passive 
 immunity against typhoid bacilli can be conferred to other animals. 
 Based on these results various anti-typhoid sera have been made 
 for the specific treatment of the disease of typhoid fever in man. 
 
 Anti-typhoid sera are usually obtained from horses which 
 have been immunized either by repeated increasing injections of 
 dead and living cultures of typhoid bacilli or typhoid bacillus 
 toxins. Most of the sera prepared have, however, not been anti- 
 toxic but ant i -bacterial. Chantemesse has prepared a so-called 
 anti-toxic serum by injecting horses with increasing doses of pure 
 cultures of typhoid bacilli grown for some time on a macerated
 
 ANTI-TYPHOID SERUM. 121 
 
 Splenic pulp and defibrinated human blood medium. Tavel by 
 repeated injections of two weeks old bouillon cultures, sterilized by 
 the addition of 0.5 per cent of carbolic acid has made a serum 
 which is supposed to possess anti-toxic substances. Most of the 
 sera are, however, made by injecting into horses and other animals, 
 increasing doses of dead and living typhoid bacilli of various 
 strains. For these sera little or no anti-toxic value is claimed, 
 the immunizing properties being dependent on the lytic power of 
 the serum. 
 
 In the treatment of typhoid fever witli these sera, 10 c. c. and 
 frequently larger quantities are injected daily until improvement 
 occurs. 
 
 The results following the use of anti-typhoid serum have been 
 disappointmg. In most cases no effects on the course of the 
 disease have been observed followmg the use of this serum. 
 Chantemesse in 1902, reported, that by the use of his serum the 
 death rate in children due to typhoid fever was 3 per cent, whereas 
 the death rate in all the children of Paris w^ho received no such serum 
 injections, was 19 per cent. Occasionally observers have obtained 
 and reported cases in which improvement is rapid after the in- 
 jection of anti-typhoid serum. As is the case with many of the 
 ant i -bacterial sera, there is in general no influence on the course 
 of the disease. In some cases, however, a drop in the fever and 
 improvement in the pulse and general conditions have been re- 
 ported as resulting from the use of this serum. Various reasons 
 have been assigned for the failure of the curative action of the serum, 
 the most important of which are, the lack of anti-toxic substances 
 which can combine with the toxin liberated after solution and 
 disintegration of the bacilli due to the specific lytic substances in 
 the serum, and, the failure of the body to supply a sufficient 
 amount of complement so as to get the destruction of the invading 
 typhoid bacilli. 
 
 Jez has prepared an extract from the spleen, bone marrow 
 and lymph glands of animals immunized to typhoid bacilli. This 
 extract is usually administered by the mouth, and is supposed to 
 possess anti-toxic properties. The value of this extract is doubtful. 
 
 Anti-typhoid sera are still in the experimental stage. With 
 the sera now produced no marked beneficial results can be hoped 
 for, and are only used in certain severe cases of the disease.
 
 122 VACCINE AND SERUM THERAPY. 
 
 ANTI-DYSENTERIC SERUM. 
 
 Soon after the discovery of the bacillus of dysentery by 
 Shiga in 1892, the treatment of bacillary dysentery by the use of 
 immune sera was undertaken. It was found by Shiga, that the 
 organism which is now regarded as the etiological factor in bacillary 
 dysentery is readily agglutinated with blood from patients suffer- 
 ing with this disease. Following the discovery of the etiological 
 importance of this organism in the dysenteries in Japan this or- 
 ganism was found by Flexner in the dysenteries in the Philippine 
 Islands, and in an epidemic of dysentery in Germany by Kruse. 
 In 1903, Duval and Vedder found this organism in the discharges 
 of dysentery patients in United States, and in 1903, Duval and 
 Bassett, working at the Thomas Wilson Sanitarium near Balti- 
 more, Maryland, isolated this organism from the stools of children 
 suffering with summer diarrhoea. Since Shiga's discovery of the 
 organism, B. dysenteriae has been found in association with dysen- 
 tery in almost all parts of the world, and the etiology of bacillary 
 dysentery now seems well established. While at first it was sup- 
 posed that the different organisms isolated from stools in these 
 cases were the same, later investigation has shown that there are 
 two principal varieties of the species of Bacillus dysenteriae. Of 
 these the original Shiga isolation represents a strain which does 
 not ferment mannite, while the organism isolated by Flexner 
 represents a type which produces acid on mannite. The mannite 
 fermenting type has now been found to include at least two and 
 probably three different strains. The original Shiga type has 
 been found especially in the cases of epidemic dysentery in adults, 
 while the mannite fermenting types are most frequently found, 
 althotigh not exclusively or as the only type, in the stools of chil- 
 dren suffering with summer diarrhoeas. The latter fact empha- 
 sizes the especial importance of the mannite fermenting types of 
 dysentery bacilli to the dysenteries in the United States. 
 
 A further difference has been found between the two main 
 types; the mannite fermenting types do not produce an extra- 
 cellular toxin, while the type, not producing acid on mannite, 
 produces an extracellular toxin. Because of these differences 
 in the ability to prodvice extracellular toxin, antitoxic immunity 
 can be produced by experimental immunization for the Shiga 
 type, while for the mannite fermenting types the production of
 
 ANTI-DYSENTERIC SERUM. 123 
 
 anti-toxic immunity is not possible experimentally. These differ- 
 ences have been recognized recently and only after much work 
 had been done on the treatment of bacillary dysentery with specific 
 immune sera. Because of this the value of serum therapy in 
 dysentery can be determined only from the relatively recent 
 literature on this subject. 
 
 The results obtained by the use of anti-dysenteric serum in 
 the treatment of dysentery have differed markedly. In Japan, 
 by the use of Shiga's anti -dysenteric serum, the mortality of 
 dysentery has been reduced from 22-26 per cent to 9-12 per cent. 
 Vaillard and Dopter have collected statistics on two hundred 
 cases treated with anti-dysenteric serum, in which cases there was 
 a mortality of only 2 per cent. In the United States on the 
 other hand, no great beneficial results have been attained by the 
 use of the serum as is evidenced from the extensive investigations 
 made in 1903 under the direction of Dr. Flexner. 
 
 There are various reasons for the differences in results which 
 have been obtained by the use of anti-dysenteric serum. The 
 patients treated in the United States were largely children under 
 three years of age, while a large percentage of the cases for which 
 favorable results are obtained with serum treatment, occurred 
 in adults. The day of the disease on which serum injections are 
 made is probably earlier in such locations where epidemic dysen- 
 tery is regarded as a serious disease. In the United States many 
 cases of frequency of stool are only simple diarrhoeas, because of 
 this the patient usually receives no medical attention until the 
 disease is well advanced. Probably the most important reason 
 for the difference of the curative value of the different sera is 
 dependent on the differences in the specific substances in the 
 serum. Evidently those sera which have produced the most 
 beneficial results possess anti-toxic properties, while those used 
 in the United States are principally or wholly anti -bacterial. 
 Shiga says of his serum that it is "bactericidal as well as anti- 
 toxic and — therefore is more effective than anti-typhoid serum." 
 Practically all of the anti -dysenteric sera have been polyvalent, 
 i . e. , the animals furnishing the serum have been immunized to the 
 various strains and toxins of dysentery bacilli, as the case may be. 
 
 The method of treatment with anti-dysenteric serum varies 
 with the severity of the disease. Shiga suggests that in mild
 
 124 VACCINE AND SERUM THERAPY. 
 
 cases one dose of 10 c. c. of the serum be injected. In cases of 
 medium severity two injections of ten c. c. each, the interval be- 
 tween injections ranging from six to ten hours, are recommended. 
 In the severer cases, 40 to GO c. c. in all are to be injected, but 
 never more than 20 c. c. daily. The serum used in the United 
 States has been injected in larger amounts, sometimes as much 
 as 100 c. c. being injected in one day. 
 
 The effect of treatment with Shiga's serum has been to 
 decrease the number of stools, cause the blood and pus to disap- 
 pear from the stools, restore the temperature to normal, and to 
 lessen the pain and tenesmus. When this serum is used late in 
 the disease the beneficial effects manifest themselves much later. 
 The serum used in the United States has not influenced the course 
 of the disease to any extent, nor is it possible to determine any 
 particular improvement in the condition of patients so treated. 
 
 Coyne and Auche have reported eleven cases of dysentery 
 produced by the Flexner, or mannite fermenting, type of dysentery 
 bacillus, which were treated very successfully with a polyvalent 
 serum. The curative effects of this serum were probably due to the 
 action of the anti-toxin to the Shiga type of bacillus dysenteriae. 
 
 It is to be hoped that specific anti-toxic sera can be made 
 for the mannite fermenting group of dysentery bacilli, or that 
 Shiga type anti-toxin will be found to be efficient in combatting 
 infections with the mannite fermenting group of dysentery bacilli . 
 This is especially desirable because the mannite fermenting 
 organisms are largely responsible for the childrens' summer 
 diarrhoeas in the United States. 
 
 ANTI -STAPHYLOCOCCIC SERUM. 
 
 Various anti-staphyloccic sera have been made. They are 
 usually obtained from horses and other animals that have received 
 repeated injections of dead and living cultures of these organisms. 
 The method of its action is not clearly understood although any 
 protective value it may have is probably due to lytic and opsoni- 
 fying power. The value of the serum, as it can now be obtained, 
 is inconsiderable, and its injection in the treatment of staphy- 
 lococcus infections is seldom or never warranted.
 
 INDEX OF NAMES. 
 
 Page 
 
 Atkinson 98 
 
 Babes and Lepp 83, 87 
 
 Baldwin 37 
 
 Banzhaf 98, 102, 106 
 
 Barber 38 
 
 Behring 100 
 
 Behring and Kitasato 15, 87, 106 
 
 Behring and Kitashima 100 
 
 Behring and Knorr 15 
 
 Behring and Wernicke 87, 100 
 
 Belfanti and Carbonne 98 
 
 Besredka 60 
 
 Bolduan 37, 41, 47, 67 
 
 Bolton 100 
 
 Bordet 20 
 
 Buchner 14, 87 
 
 Bulloch and Atkin 61 
 
 Calkins 84 
 
 Chamberland 14 
 
 Chantemesse 120, 121 
 
 Chauveau 14 
 
 Clark 54, 71 
 
 Cole 38, 47, 48. 67 
 
 Cole and Meakins 28, 76 
 
 Covne and Auche 124 
 
 D(^n 60 
 
 Denis 90 
 
 Deny s 79 
 
 Denys and LeClef 20, 113 
 
 Dieudonne -'7 
 
 Dorr 63 
 
 Duval and Bassett 122 
 
 Duval and Vedder 122 
 
 Ehrlich 15, 50, 111 
 
 Ferran 100 
 
 Field 20 
 
 Flexner 122, 123 
 
 Flexner and Jobling 116 
 
 Foa and Bonome I"* 
 
 Fodor 14, 87 
 
 Fraenckel 100 
 
 Gabritschewsky 77 
 
 Gay and Southard '*3 
 
 Gibson 98, 102, 106 
 
 Guerini 84 
 
 Hafifkine 65 
 
 Hamilton ^4 
 
 Hektoen 60, 64, 67 
 
 Herricourt and Richet 87 
 
 Heubner 9- 
 
 Hiss and Zinsser 45, 63, 111, 112 
 
 Huhne and Neufeld r^t 
 
 Jaeger H^ 
 
 Jeans and Sellards 30, 4/ , 48
 
 126 INDEX OF NAMES CONTINUED. 
 
 Jenner 84 
 
 Jez 121 
 
 Jochman 115 
 
 Keith (31 
 
 Klebs 14 
 
 Kolle and Wa.sserman 115 
 
 Koch 79 
 
 Kruse 122 
 
 Leishman 23, 49 
 
 Levaditi and Inmaii 62 
 
 Loehleiii 60 
 
 Madsen 50 
 
 Maragliano 79 
 
 Marie S3 
 
 Marmorek •. 113 
 
 McClintock and King 99, 104 
 
 Meakins 72 
 
 Metchnikoff 14, 20, 53, 63, 110 
 
 Moss 38, 41, 42, 46, 47, 48, 57 
 
 Morgenroth Ill 
 
 Muir and Martin 60 
 
 Neufeld 61, 63 
 
 Neufeld and Rimpau 21, 59, 60, 1 14, 1 19 
 
 Neufeld and Toepfer 61 
 
 Noguchi 62 
 
 Nuttall 14, 89 
 
 Pane 119 
 
 Park 37, 67 
 
 Pasteur 14, 65, 82 
 
 Petersen 45, 63 
 
 Pfeitter 65 
 
 Pfeiffer and Kolle 80 
 
 Pirquet and Schick 90, 93 
 
 Potter ; 37 
 
 Potter, Dittman and Bradley 47, 60 
 
 Richardson Ill 
 
 Roemer 119 
 
 Rosenau and Anderson 92, 94 
 
 Ross 34, 72, 76 
 
 Ross and Johnson 77 
 
 Roux 14 
 
 Roux and Yersin 87 
 
 Ruediger 54 
 
 Russell 60 
 
 Sahli 63 
 
 Salmon and Smith 14 
 
 Sauerbek 63 
 
 Savtchenko 60 
 
 Shiga 81, 122, 123 
 
 Simon 37, 41, 43, 44, 45, 46, 47, 48, 60 
 
 Simonds and Baldauf 62 
 
 Solomonson 50 
 
 Strong 46 
 
 Tavels 121 
 
 Tizzoni and Centanni .83 
 
 Torrey and Rogers 117 
 
 Trudeau 79 
 
 Vaillard and Dopter 123 
 
 V. der Velde 113 
 
 AValker 31, 32, 37, 38, 39, 42, 43, 44, 45, 47 
 
 Wasserman 100 
 
 Wright 27, 30, 31, 35, 37, 38, 40, 44, 49, 51, 57, 59, 60, 65, 67, 72, 72, 75, 79, 119 
 
 Wright and Douglas 21, 23, 27, 30, 33, 50, 59, 60, 66, 69, 70, 71, 114 
 
 Wright and Ried 60 
 
 Wright, T. H 29
 
 INDEX. 
 
 Page 
 
 Acne vulgaris 7o 
 
 Agglutinins 17, 19, 50, 53, 71, 110 
 
 Aggressins 7, 63 
 
 Alexines 14, 87 
 
 Amboceptor 18, 60 
 
 Anaphylactine 93 
 
 Anaphylaxis 93 
 
 Anti-bacterial sera 106 
 
 Anti-bodies 16, 19, 89 
 
 Anti-dysenteric serum 122 
 
 historical 122 
 
 differences due to strains of bacilli 122 
 
 preparation of 123 
 
 method of treatment with 123 
 
 results obtained with 124 
 
 Anti-gonococcic serum 117 
 
 preparation of 117 
 
 method of treatment with 118 
 
 results obtained with 118 
 
 Anti-meningococcic serum 115 
 
 Flexner and Jobling serum 116 
 
 administration of > 116 
 
 results of treatment with 116 
 
 Anti-pneumococcic serum 119 
 
 preparation of 119 
 
 method of treatment with 119 
 
 results obtained with 120 
 
 Anti-staphylococcic serum 124 
 
 Anti-streptococcic serum 113 
 
 preparation of 1 1 "^ 
 
 actien of ^ H"! 
 
 method of treatment with Hf 
 
 results obtained with and value of 1 1"^ 
 
 Anti-typhoid serum 12^ 
 
 preparation of 1^0 
 
 method of treatment with 120 
 
 results obtained with 121 
 
 Jez's immune extract ^-^ 
 
 Anti-toxic sera -'9 
 
 diphtheria anti-toxin ^^^ 
 
 tetanus anti-toxin ^0" 
 
 Anti-toxic globulins • • • • 97, 102, 106 
 
 Anti-toxin 17, 19, 53, 89, 99 
 
 Appendix abscesses ^J? 
 
 Arthritis, streptococcus i!^ 
 
 gonococcus ' ' 
 
 Bacillus coli, sensibility of ', , 
 
 suspensions "^ 
 
 vaccine ' ' ' qq inn 
 
 Bacillus diphtheriae, anti-toxni • • ^^' .y 
 
 sensibility of. 
 toxin . . . 
 
 vaccine . .,.-, 
 
 Bacillus dysenteriae anti-serum, sensibilitj' of ■''^- '-- 
 
 vaccine. 
 
 50 
 101 
 105 
 
 SI
 
 128 INDEX. 
 
 Bacillus, pestis sensibility of 50 
 
 Bacillus pyocyaneus, suspension of 28 
 
 vaccine 66 
 
 Bacillus tuberculosis, immunization to Avith tuberculin 78 
 
 suspension of i 29 
 
 slainiufr of 35 
 
 vaccine (tuberculin) 78 
 
 Bacillus typhosus, anti-serum 120 
 
 opsonic inclex determination for 71 
 
 sensibility of 50 
 
 vaccine 80 
 
 Bacterial agglutinins 17, 19, 50, 53, 71. 110 
 
 Bactei'ial emulsions 27, 31 
 
 clumping in 30, 40 
 
 Bacterial precipitins 17, 19, 50, 110 
 
 Bacterial vaccines 64 
 
 autogenous , 68 
 
 colon bacillus 80 
 
 cholera spirillum 81 
 
 control of injections of 70, 73 
 
 dosage of 66 
 
 dj'sentery bacillus 81 
 
 gonococcus 77 
 
 paratyphoid bacillus 80 
 
 preparation of 69 
 
 point of injection of 67 
 
 staphylococcus 74 
 
 streptococcus 76 
 
 stock culture 68 
 
 tuberculosis (tuberculin) 78 
 
 typhoid bacillus 80 
 
 Bacteriotropins 21, 59 
 
 Bacteriolysins 17, 19, 50, 53, 71, 89, 110, 112, 117, 119, 120, 123 
 
 Barber's itch 75 
 
 Behring's law 88 
 
 Calculation of opsonic index 35, 45 
 
 Carbuncles 74 
 
 Chemicals, effect on opsonic index 64 
 
 Chicken cholera 35 
 
 Cholecystitis 80, 81 
 
 Cholera, immunization against 65, 81 
 
 sensibility of spirillum of 50 
 
 vaccine against 81 
 
 Clumping of bacteria in bacterial suspensions 30, 44 
 
 Complement 18, 98, 110, 112 
 
 Complementophore group 18 
 
 Concentration of serum 97, 102, 106 
 
 Cystitis 79, 80, 81, 118 
 
 Cytophylic group 8 
 
 Diphtheria anti-toxin 89, 100 
 
 dried 104 
 
 dosage 103 
 
 oral administration of 104 
 
 preparation of 101 
 
 standardization of 102 
 
 therapeutic action of 104 
 
 treatment with 103 
 
 Diphtheria immunization, active 105 
 
 with anti-toxin 105 
 
 Diphtheria toxin 87, 89, 101 
 
 Dysentery, bacillary, immunization against 81 
 
 vaccine treatment in 81 
 
 serum treatment in 1-- 
 
 Ehrlich's side chain theory 14 
 
 Emulsions, bacterial 27, 31 
 
 Endocarditis, gonococcus 118 
 
 streptococcus 76, 115
 
 INDEX. 129 
 
 Endometritis, colon bacillus infections gO 
 
 gonococcus infections Ug 
 
 Erysipelas ......'....'.. 54, 76 
 
 Fixing of smears for opsonic index det(M-minalions 34 
 
 Fixed virus, hydrophobia g2 
 
 Furunoulosis 74 gg 
 
 Globulins in immune seru gg 
 
 precipitation of gg^ 102, 106 
 
 diphtheria 102 
 
 tetanus 106 
 
 Gonococcus infection, serum therapy II7 
 
 vaccine therapy 77 
 
 HaptophoFe group 16 
 
 Hydrophobia 65, 81 
 
 diagnosis of g2 
 
 Pasteur treatment 81, 83 
 
 Hypersusceptibility to serum gO, 105 
 
 to vaccine 73 
 
 Immune sera 87 
 
 anti-bacterial sera 109 
 
 anti-dysenteric serum 122 
 
 anti-gonococcic serum 117 
 
 anti-meningococcic serum 115 
 
 anti-pneumococcic serum 119 
 
 anti-staphylococcic serum 124 
 
 anti-streptococcic serum 113 
 
 anti-typhoid serum 120 
 
 anti-toxic sera 99 
 
 diphtheria anti-toxin 100 
 
 tetanus anti-toxin 106 
 
 dried 104, 109, 1 1 1 
 
 polyvalent 113 
 
 Immunity, acquired 12, 65, 89 
 
 actice 13, 65 
 
 definition and classification of 12 
 
 passive 13, 89 
 
 theories of 14, 15, 20 
 
 Immunizing substances 13, 88, 89 
 
 elimination and loss of 20 
 
 Infections 7 
 
 course of 8, 11 
 
 elimination of causal factors 11 
 
 general reaction in 10 
 
 incubation period in 9 
 
 local reaction in 10 
 
 Leistungskern 15 
 
 Leucocytes, accumulation of 10 
 
 in immunity 15, 20, 21, 45, 63, 111, 112 
 
 in opsonic index determinations, examination of 35, 46 
 
 source of 26, 44 
 
 Leucocvtosis, relation to opsonic index 62, 111, 112 
 
 Lysis. .' 17, 19, 50, 52, 71, 89, 110, 112, 117, 119, 120, 123 
 
 Meningitis, opsonic index in 70 
 
 serum treatment of 115 
 
 vaccine treatment of 70 
 
 Micrococcus melitensis, sensibility of 50 
 
 Micrococcus meningitidis, anti-serum 115 
 
 suspension of 28 
 
 Micrococcus pneumoniae, anti-serum 119 
 
 sensibility of 50 
 
 suspension of 28 
 
 vaccine 66, 77 
 
 Micrococcus pyogenes, anti-serum 124 
 
 infection, treatment with anti-serum 124 
 
 treatment with vaccine 74 
 
 suspension of, for opsonic index determination 28 
 
 vaccine 74 
 
 Negative i)hase 50, 51
 
 130 INDEX. 
 
 Opsonic index 21, 23, 24 
 
 after injection of bacterial vaccines o7 
 
 bacterial eniulsions for 27, 31, 43, 44 
 
 calculation of 3o, 45 
 
 dilution of serum for 41 
 
 in acute otitis media 34 
 
 in erysipelas 54 
 
 in health and disease 50, 53, 54 
 
 in tuberculosis 60, 79 
 
 hi typhoid fever 54, 70 
 
 leucocytic emulsion for 26, 44, 46 
 
 mixture of bacteria, leucocj^tes and serum for 31, 39 
 
 normal serum for 26, 39 
 
 smears for 33 
 
 staining of smears for 35 
 
 sei'um for 24, 39 
 
 technique for determination of 24, 38 
 
 Opsonin 20, 49, 59, 110 
 
 effect of chemicals on 64 
 
 effect of heat on , 60, 61 , 62 
 
 effect of reaction on 64 
 
 immune and normal 20, 21, 59, 60, 61, 114, 119, 124 
 
 nature of 58 
 
 non-bacterial 64 
 
 specificity of 20, 59, 61, 62 
 
 structure of 60, 61 
 
 importance in immunity 20, 50, 53 
 
 Oral administration of anti-sera 99, 104 
 
 Pericarditis, gonococcus 118 
 
 streptococcus 76, 114 
 
 Phagocytic index 24, 39, 45 
 
 Phagocytosis 20, 21, 45 
 
 causes of 14, 20, 61 
 
 spontaneous 30, 61 
 
 Pneumonia 76, 119 
 
 Pneumococcus infections, serum treatment 119 
 
 vaccine treatment 77 
 
 Pooled serum 26 
 
 Polyvalent immune serum 113, 123 
 
 Positive phase 50, 51, 71 
 
 Precipitins 17, 19, 50, 93, 110 
 
 Pus 10 
 
 Rabies 81 
 
 Reaction, influence on opsonic index 64 
 
 Receptors 15 
 
 first order 16 
 
 second order 17 
 
 third order 18 
 
 Septicaemia 9 
 
 anti-streptococcic serum 114 
 
 streptococcus vaccine 70 
 
 Serum, anti-bacterial 106 
 
 anti-toxic 99 
 
 concentration 97, 102, 106 
 
 disease 90, 105 
 
 dried immune 98 
 
 dried diphtheria anti-toxin 104 
 
 dried tetanus anti-toxin 109 
 
 oral administration of 99, 104 
 
 pooled normal 26 
 
 purification of 97, 102, 106 
 
 therap3^ 87 
 
 Small-pox vaccination 84 
 
 duration of immunity from". 85 
 
 preparation of vaccine for 85 
 
 Smears for determination of opsonic index 33, 48 
 
 examination of 35, 46 
 
 staining of 35
 
 INDEX. ];U 
 
 yi)irilliini eholerae, iinmunization against 81 
 
 sensibility of .^1 
 
 Staphylococci, suspension for opsonic index determination. . 28 
 
 Staphylococcus infections, serum treatment of 124 
 
 vaccine treatment of 74 
 
 Stimulins 20, 03 
 
 Streptococci, .suspension for opsonic index determination 2!) 
 
 Streptococcus infections, serum treatment of 113 
 
 vaccine treatment of 76 
 
 Substance sensibilitrice 18 
 
 Sycosis barbae 75 
 
 Tetanus antitoxin 106 
 
 dried lOlt 
 
 preparation of 106 
 
 standardization of lOG 
 
 treatment with 107 
 
 Toxin 13, 17, 87, 89, 101, 106 
 
 produced by diphtheria bacillus. 87, 101 
 
 produced by tetanus bacillus 106 
 
 Tuberculin 78 
 
 Tuberculosis, treatment with tuberculin 79 
 
 Typhoid bacillus infections, immunization against 65, 80 
 
 serum treatment of 120 
 
 vaccine treatment of 80 
 
 Typhoid bacillus, sensibility of 'A) 
 
 suspensions of for opsonic index determination 71 
 
 Urethritis, gonococcus; serum treatment of 118 
 
 vaccine treatment of 77 
 
 Vaccine therapy 65 
 
 bacterial vaccines 65 
 
 Bacillus coli 79 
 
 Bacillus dysenteriae 81 
 
 Bacillus paratyphosus 80 
 
 Bacillus typhosus 80 
 
 control of injections 70 
 
 dosage of 66 
 
 gonococcus 76 
 
 pneumococcus 77 
 
 preparation of 69 
 
 Sprillum cholera 81 
 
 staphjdococcus • 73 
 
 streptococcus 75 
 
 tuberculin 77 
 
 of unknown etiology, attenuated virus 81 
 
 rabies, virus 81 
 
 immunization with 83 
 
 preparation of 82 
 
 results obtained with 83 
 
 small-pox virus 84 
 
 immunization with 85 
 
 preparation of 84 
 
 results obtained with. 85
 
 Diagnosis and Treatment of 
 Diseases of Women 
 
 By 
 
 H. S. Crossen, M. D. 
 
 Clinical Professor of Gynecology, Medical Department Washington University; 
 
 Gynecologist to the Washington University Hospital, and Chief of the 
 
 Gynecological Clinic; Consulting Gynecologist to the Bethesda 
 
 Hospital, St. Louis Female Hospital, and 
 
 St. Louis City Hospital. 
 
 816 Pages. 700 Illustrations. Price : Cloth $6.00. One-half Morocco, $7.50. 
 Sent anywhere prepaid, upon receipt of Price. 
 
 Chapter 
 
 I. 
 
 Chapter 
 
 IL 
 
 Chapter 
 
 in. 
 
 Chapter 
 
 IV. 
 
 Chapter 
 
 V 
 
 Chapter 
 
 VI 
 
 Chapter 
 
 VII 
 
 Chapter 
 
 VIII 
 
 Chapter 
 
 IX 
 
 Chapter 
 
 X 
 
 Chapter 
 
 XI 
 
 Chapter 
 
 XII 
 
 Chapter 
 
 XIII 
 
 Chapter 
 
 XIV 
 
 Chapter 
 
 XV 
 
 Chapter 
 
 XVI 
 
 Chapter 
 
 XVII 
 
 CONTENTS. 
 
 Gynecologic Examination Methods. 
 
 Gynecologic Diagonsis. 
 
 Gynecologic Treatment. 
 
 Diseases of External Genitals and Vagina. 
 
 Lacerations and Fistula of Pelvic Floor, Perineum, External Geni- 
 tals and Vagina. 
 
 Inflammatorj' and Nutritive Diseases of the Uterus. 
 
 Displacements of the Uterus. 
 
 Fibromyoma of the Uterus. 
 
 Malignant Diseases of the Uterus. 
 
 Pelvic Inflammation. 
 
 Other Affections of Fallopian Tubes, Peritoneum and Connective 
 Tissue. 
 
 Diseases of the Ovary and Parovarium. 
 
 Malformations. 
 
 Disturbance of Functions. 
 
 Invasion of the Peritoneal Cavitj' for the Treatment of Gyneco- 
 logical Diseases. 
 
 After-Treatment of Operative Cases. 
 
 Medico-Legal Points in Gjmecology. 
 
 Appendix — Formulae — Index. 
 
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 (jrolden rvules of Surgery 
 
 Aphorisms Observations Reflections 
 
 On the 
 
 Science and Art of Surgery 
 
 A Guide for Surgeons and Those Who Would Become Surgeons 
 
 By 
 
 Augustus Charles Bernays, A. M., M. D. 
 F. R. C. S., England 
 
 Late Chief Surgeon Lutheran Hospital and for Twenty Years Professor of Surgery 
 and Anatomy, St. Louis, Mo., U. S. A. 
 
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 The Education of a Surgeon. 
 On Scientific Communications to the Litera- 
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 Science and Surger3^ 
 
 About Fees. 
 
 Off with the Cloak of Superstition. 
 Inflammation and the Confusion It Has 
 Caused. 
 
 
 GOLDEN 
 
 RULES OF SURGERY: 
 
 
 Asepsis. 
 
 
 Genito-Urinary. 
 
 Nose. 
 
 Anesthesia. 
 
 
 Operations. 
 
 Goitre. 
 
 Abscesses. 
 
 
 Joints. 
 
 Shock. 
 
 Abdomen. 
 
 
 Ear. 
 
 Oesophagus, 
 
 Appendicitis. 
 
 
 Erysipelas. 
 
 Pelvis. 
 
 Aneurysm. 
 
 
 Gangrene. 
 
 Rectum. 
 
 Arterj^ Bleeding. 
 
 
 Hand and Foot. 
 
 Spine. 
 
 Burns. 
 
 
 Moist Dressing. 
 
 Throat. 
 
 Breast. 
 
 
 Mouth. 
 
 Veins. 
 
 Can Minor Surgical Operations Be Done in OflBce? 
 
 Death Following Miner Surgical Operations. 
 
 Fractiures and Dislocations. Therapeutic ffints. 
 
 Irrigation Drainage of Abdominal Cavity. 
 
 Minor Surgical Cperaticns. Stomach and Intestines. 
 
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 ing, and the Principles of Scientific Treatment. 
 
 By 
 JOHN ZAHORSKY, A. B., M. D. 
 
 Clincal Profos.^or of Pediatrics, ^\'a^hinKt(Jll I'nivcrsity Medical Department, St. Louis; 
 Ex- President Bethesda Societj^; Attending Phy.sician to the Bethe.sda Found- 
 lings' Home; Member of the American Medical Association and of the 
 St. Louis Academy of Science; Editor of the St. Louis Courier 
 of Medicine; Author of "Baby Incubators," etc. 
 
 WITH AN INTRODUCTION 
 
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 E. W. SAUNDERS, M. D. 
 
 Professor of Diseases of Children and Clinical Midwiferv. Washington L'niver.sity, 
 
 St. Louis, Mo. 
 
 370 Pages, Silk Cloth Binding, Price $3, sent Post Paid anywhere en receipt of price. 
 
 CONTENTS. 
 
 Ascites. The Adenoid Face. 
 Acute Pneumonic Consolidation. 
 Intestinal Obstruction. 
 Nervous State. Scrofula. 
 Tuberculosis. 
 
 Introduction. 
 Part I. 
 General Rules of Diagnosis. 
 General Rules. 
 Loss in "\^'eight. Appetite. 
 Convulsions. 
 Physical Examination. 
 Head and Neck. 
 Some Deformities. 
 Teeth and Gums. Dentition. 
 The Enanthemata. 
 Vomiting. Hematemesis. 
 Diarrhea. 
 
 Distended Abdomen. 
 Abdominal Pain. 
 Abdominal Swellings. 
 The Nose and Nasopharynx. 
 The Larynx. 
 
 Anomalies of Breathing. Cough. 
 The Lungs. 
 
 The Heart and Circulation. 
 The Urine. The Eruptions. 
 The Nervous System. 
 Paralysis. 
 
 Tremor, Choreiform Movements. Head- 
 ache, etc. 
 Changes Abo;it the Eyes. 
 Changes About the Ear. 
 Clinical Syndromes. 
 FeA'er. Chronic Fever. 
 Status Gastricus. 
 The Typhoid State. 
 Infantile Atrophy. 
 Gastroenteric Infection, Diarrhea. 
 Chronic Indigestion in Older Children. 
 Chronic Constipation. 
 Peritoneal Irritation. 
 Severe Anemia. Edena. 
 
 Impending Heart Failure. 
 
 The Syndrome of Cerebral Irritations. 
 
 Golden Rules cf Prognosis. 
 
 Part II. 
 
 Golden Rules cf Hygiene and Infant 
 
 Feeding. 
 
 The Nursing Mother. 
 The Wet Nurse. 
 Artificial Feeding. 
 Feeding the Sick. 
 
 Golden Rules cf Treatment. 
 General Therapeutics. 
 The Newly Born. 
 Diseases of the Mouth. 
 The Neck and Scalp. 
 The Throat. 
 
 The Respiratory Organs. 
 G-astroenteric Diseases. 
 Rickets and Scurvy. 
 Lleart and Circulation. 
 The Blood. 
 
 The Genito-L'rinary Organs. 
 The Nervous Sj^steni. 
 Specific Infectious Diseases. 
 Malaria. Cerebro-Spinal Fever. 
 Diphtheiia. Intubation. 
 Tuberculosis. Pertussis. 
 Mumps. Sei:ticemia. 
 Rheumatism and Endocarditis. 
 Syphilis. The Exanthemata. 
 The Severe Infectious Fevers. 
 The Skin. 
 Formularv. 
 
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 Golden Rules of Dietetics 
 
 By A. L. BENEDICT, A. M., M. D. 
 
 Consultant in Digestive Diseases, City and Riverside Hospitals and Attendant in 
 
 same; Mercy Hospital, Buffalo; Member of the Academy of Medicine and 
 
 of American Gastro-Entrological Association, etc.; 
 
 Author of Practical Dietetics. 
 
 CONTENTS. 
 
 Part I. 
 
 Phy.siologic Chemistry. 
 
 Daily Recjuirements of the Human Body. 
 
 Standard Diet in Health. 
 
 Quantative Estimate of Diet. 
 
 Approximate Methods of Checking Diet Weight and the Excretions. 
 
 Transmutability and Reservation of Food. 
 
 Waste of Food. 
 
 Predigestion of Food. 
 
 Emergency Methods of Introducing Nourishment. 
 
 Preserved Foods. 
 
 Methods of Cooking. 
 
 Compositions of Natural and Commercial Foodstuffs. 
 
 Food Adjuncts. 
 
 Purine Bodies. 
 
 Important Constituents of Foodstuffs. 
 
 Distinctly Deleterious Foodstuffs. 
 
 General Hygiene of Eating. 
 
 Diet Tests. 
 
 Condensation of Atwatcr & Bryant's Analysis of Foodstuffs. 
 
 Part II. 
 
 Principles of Dietetics According to General Pathologic Conditions. 
 
 Infant Feeding. 
 
 Diet in Critical Physiologic Periods. 
 
 Diabetis, Glycosuria. 
 
 Obesity and Leanness. 
 
 Chronic Diseases of Nitrogeneous Metabolism. 
 
 Diseases of the Urinary Organs. 
 
 Diseases of the Ductless Glands. 
 
 Diseases of the Liver. 
 
 Diseases of the Pancreas. 
 
 Diseases of the Digestive Organs. 
 
 General Preversion of Digestive Functions. 
 
 Functional Intestinal Diseases. 
 
 Organic Intestinal Diseases. 
 
 Diseases of the Heart and Blood Vessels. 
 
 Blood Diseases. 
 
 Hemorrhagic Diseases. 
 
 Bone Diseases. 
 
 General Principles of Feeding in Fevers. 
 
 Infectious and Parasite Diseases. 
 
 Respiratory Diseases. 
 
 Skin Diseases. 
 
 Diseases of the Nervous System. 
 
 Surgical Emergencies and Operations. 
 
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 A VERY 
 YOUNG OVUM IN SITU 
 
 By 
 
 G. Leopold, 
 
 Dresden 
 Authorized Translation By 
 
 W. H. VOGT, A. M., M. D. 
 
 Obstetrician and Gynecologist, Lutheran Hospital, St. Louis, Mo. 
 
 65 Pages Text. 35 Pages Lithographic Illustrations in Colors 
 
 Price, Cloth $3.50 
 
 Publisher's Announcement 
 
 The importance of an understanding of embryolog}' is becoming more and more 
 apparent. The student now realizes that anatomy is much better understood — where 
 it is worked out from the standpoint of embryonic development — than where it is 
 learned in its crude state in the dissecting-room. The surgeon realizes that he can bet- 
 ter grasp the relationship of structures when he is familiar ^^^th their formation from 
 the embrj'o. The scheme of development as Avorked out by Leopold represents the latest 
 work along this line. The work is most scientific and cannot fail to interest all who 
 are seeking the fundamental truths of embryonic development. 
 
 THE C. V. MOSBY MEDICAL 
 BOOK AND PUBLISHING CO. 
 
 Grand Avenue and Olive Sreet, :: St. Louis, Missouri.
 
 rLxamination of 1 he lL 
 
 ar 
 
 By 
 Selden Spencer, A. B., M. D. 
 
 Instructor of Otology in the Washington University Medical Department, 
 
 St. Louis, Mo. 
 
 With an Introduction 
 
 By 
 
 H. N. Spencer, M. D., LL. D. 
 
 Professor of Otology, Medical Department Washington University, 
 
 St. Louis, Mo. 
 
 67 PAGES OF TEXT 
 5 FULL PAGE PLATES 
 12 OTHER ILLUSTRATIONS 
 PRICE $1.00 
 
 CONTENTS. 
 
 Methods of Procedure (General Consideration). 
 
 The External Ear. 
 
 Diseases of the Canal. 
 
 The Middle Ear. 
 
 The Middle Ear (Continued), Non-Suppurative Conditions. 
 
 The Middle Ear (Continued), Post-Suppurative Conditions. 
 
 The Middle Ear (Continued), Suppurative Conditions. 
 
 The Middle Ear (Continued), Purulent Otitis Media. 
 
 The Middle Ear (Continued), Purulent Otitis Media. 
 
 The Middle Ear (Continued), Operations in Chronic Purulent Otitis 
 
 Media. 
 The Internal Ear. 
 Hearing Tests. 
 Intra-Cranial Complications. 
 Exercises in the Surgical Anatomy of the Temporal Bone. 
 
 THE C. V. MOSBY MEDICAL 
 BOOK AND PUBLISHING CO. 
 
 Grand Avenue and Olive Street, St. Louis, Missouri. 
 
 Chapter 
 
 I. 
 
 Chapter 
 
 II. 
 
 Chapter 
 
 III. 
 
 Chapter 
 
 IV. 
 
 Chapter 
 
 V. 
 
 Chapter 
 
 VI. 
 
 Chapter 
 
 VII. 
 
 Chapter 
 
 VIII. 
 
 Chapter 
 
 XI. 
 
 Chapter 
 
 X. 
 
 Chapter 
 
 XI. 
 
 Chapter 
 
 XII. 
 
 Chapter 
 
 XIII. 
 
 Chapter 
 
 XIV.
 
 Suggestive Therapeutics, 
 Applied Hypnotism 
 
 and Psychic Science 
 
 By H. S. MUNRO, A. M., M. D. 
 
 Americus, Ga. 
 376 Pages, Octavo. Price, S3. 00. Sent anywhere upon receipt of price. 
 
 Publisher's Announcement. 
 
 That suggestion is an important factor in the treatment of diseases is no longer 
 denied bj' those that keep abreast of medical progress. The medical journals are replete 
 with articles from the leading alienists and internists of this country. The profession 
 in Europe has been alive to the importance of this subject for many years and much 
 has been written on it by svich men as Schofield, Bernheim, Forell and Duboise. These 
 writings have been in the nature of research work and have not been devoted to the 
 practical application of this branch of therapeutics in everyday practice. The book 
 herein described is designed to give a practical working guide to the general practitioner 
 in the application of suggestive therapeutics. Its purpose is to aid the profession in 
 reaching a correct understanding of a subject that has been shrouded in mj'stery and 
 used by the cjuack and charlatan in many cases to discredit scientific medicine. The 
 indorsements that have been given the author by prominent phj-sicians and surgeons 
 are the best recommendations the book can have. 
 
 CONTENTS. 
 
 Introduction. 
 
 Suggestion: Its Uses and Abuses. 
 
 Hypnotism: A Demonstration of the EfBciencj^ of Suggestion. 
 
 Technicjue of Inducing the Hj-pnotic State. 
 Theorj' and Practice of Suggestive Therapeutics. 
 Simple Suggestions, or Suggestion ^\'ithout Hj-pnotism. 
 Hj-pnotic Suggestive Therapeutics Applied in 3Iedicine, Surgery. 
 The Psychological Factor in Ob.stetrics. 
 Training the Subconscious Self for Health and Strength. 
 Correct Diagonsis a Safeguard Against Blunders. 
 Philosoph}^ and Religion and Their Relation to Health. 
 Conservation of Energj', Education and Control of Emotions. 
 
 Breathing, Relaxation, Dietetics, Exercise, etc. 
 Roughing It as a Means of Health. 
 Are All Specialists Egotists? 
 Personality as a Factor in Therapeutics. 
 Environment: Its Influence in Therapeutics. 
 Brutality of Frankne-ss: Hone.stj' Imperative. 
 
 Physical and Mental Hygiene; Character as a Resource of Health. 
 Suggestion in Education, Character Building, etc. 
 Moral Stamina a Therapeutic Power; The Higher Art in Therapeutics, 
 
 and the True Physician. 
 Chapter XX. Self-Mastery as a Fine Art. 
 
 Chapter 
 
 I. 
 
 Chapter 
 
 II. 
 
 Chapter 
 
 III. 
 
 Chapter 
 
 IV. 
 
 Chapter 
 
 V. 
 
 Cahpter 
 
 VI. 
 
 Chapter 
 
 VII. 
 
 Chapter 
 
 VIII. 
 
 Chapter 
 
 IX. 
 
 Chapter 
 
 X. 
 
 Chapter 
 
 XI. 
 
 Chapter 
 
 XII. 
 
 Chapter 
 
 XIII. 
 
 Chapter 
 
 XIV. 
 
 Chapter 
 
 XV. 
 
 Chapter 
 
 XVI. 
 
 Chapter 
 
 XVII. 
 
 Chapter 
 
 XVIII. 
 
 Chapter 
 
 XIX. 
 
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 Arteriosclerosis 
 
 ETIOLOGY, DIAGNOSIS, PROGNOSIS, PROPHYLAXIS AND 
 
 TREATMENT 
 
 By 
 
 L. M. Warfield, A. M., M. D. 
 
 With an Intrcducticn by 
 
 H. S. Thayer, Baltimore, Md. 
 
 8 ORIGINAL ILLUSTRATIONS. 
 
 150 PAGES. 
 
 PRICE $2.00 
 
 PUBLISHER'S ANNOUNCEMENT 
 
 This book is particularly opportune. Tlie rapid pace which American.s are living, 
 the worrj^ and mental strain under which the majority of their time is spent, has made 
 this a nation of arterio-sclerotics. The author has laid stress upon prophylaxis as well 
 as given the most rational treatment known to modern times. The text is embellished 
 with instructive original illustrations. Sent anywhere on receipt of price. 
 
 Diseases of The Skin 
 
 By 
 
 A. H. Ohmann-Dumesnil, A. M., M. E., M. D., Ph. D., etc. 
 
 Formerly Professor of Dermatology and Syphilology in the St. Louis College for .Medical 
 Practioners; the St. Louis College of Physicians and Surgeons; the Marion- 
 Sims College of Medicine; Member of the St. Louis Medical Society, 
 of the Missouri State Medical Association, of the American 
 Medical Association, of the 1st, 2d, 3d, 4th, 5th 
 and 6th International Dermatological 
 Congress, etc. 
 
 THIRD EDITION 
 
 THOROUGHLY REVISED AND ENLARGED 
 
 150 ORIGINAL ILLUSTRATIONS 
 
 600 PAGES. PRICE: CLOTH $4.00. MOROCCO, $5.50 
 
 PREFACE 
 
 This book is not a treatise. The intention has been to make of it a practical guide 
 to the easy recognition of skin diseases, as well as to their successful treatment. The 
 remedies which have been recommended are such as may be found in every practitian s 
 armamentarium nwdicinorum. ISo attempt has been made to write an elaborate work, 
 but rather to furnish, in a clear, concise manner, just that information most desired by 
 medical students and general practitioners. 
 
 TABLE OF CONTENTS. 
 
 Prognosis. 
 
 Symptomatology. 
 
 Classifications. 
 
 Diet in Skin Di.seascs. 
 
 Food Eruptions. 
 
 Aj^pendix. 
 
 C. V. MOSBY MEDICAL BOOK AND PUBLISHING CO. 
 
 Grand Avenue and OUve Street, :: :: St. Louis, Missouri. 
 
 Chapter 
 
 L 
 
 The Skin. 
 
 Chapter 
 
 VIII. 
 
 Chapter 
 
 IL 
 
 Anatomy. 
 
 Chapter 
 
 IX. 
 
 Chapter 
 
 in. 
 
 Physiology. 
 
 Chapter 
 
 X. 
 
 Chapter 
 
 IV. 
 
 Diagnosis. 
 
 Chapter 
 
 XL 
 
 Chapter 
 
 V. 
 
 Etiolog3^ 
 
 Chapter 
 
 XII. 
 
 Chapter 
 
 VI. 
 
 Pathology. 
 
 Chapter 
 
 XIII. 
 
 Chapter 
 
 VII. 
 
 Therapeutics. 
 

 
 Office Treatment of Rectal Diseases 
 
 By 
 
 R. D. Mason, M. D. 
 
 Professor of Rectal Diseases in the Creighton University, 
 Omaha, Nebraska. 
 
 New 4th Edition. 367 Pages. 87 Illustrations. Price $2.50 
 
 Tuberculosis of the Nose and Throat 
 
 By 
 
 Lorenzo B. Lockord, A. B., M. D. 
 Denver, Colorado 
 
 Consulting Laryngologist to the Agnes Memorial Hospital, Denver. 
 
 504 Pages. 85 Illustrations. 64 of which are colored. Price $5.00 
 
 THE C. V. MOSBY MEDICAL BOOK AND PUBLISHING CO. 
 
 St. Louis, Mo.
 
 Hand Book of Rectal Diseases 
 
 By 
 
 L. J. Hirschman, M. D. 
 
 Professor of Clinical Proctology Detroit College of Medicine and Surger3^ 
 
 150 Illustrations 
 400 Pages. Including 2 colored Plates. Price $4.00 
 
 Gonorrhea in Women 
 
 By 
 Palmer Findley, M. D. 
 
 Professor of Gynecology in the Medical Department of the University of Nebraska, 
 
 Omaha, Nebraska. 
 
 128 Pages. Royal Octavo. Price $2.00 
 
 Chronic Constipation 
 
 By 
 J. A. McMillian, M. D. 
 
 Professor of Therapeutics in the Detroit College of Medicine and Surgery, 
 
 Detroit, Michigan. 
 
 257 Pages. Price $2.00. 
 
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 St. Louis, Mo.
 
 UNIVERSITY OF CALIFORNIA LIBRARY 
 
 Los Angeles 
 This book is DUE on the last date stamped below. 
 
 TH^ IJBRARY 
 UNIVERSITY OF CALIFORNIA 
 
 LOS AWGELES
 
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