1 
 -m 
 
BACTERIOLOGY 
 
 MEDICINE AND SURGERY. 
 
 A PRACTICAL MANUAL 
 
 FOR 
 
 PHYSICIANS, HEALTH OFFICERS AND STUDENTS, 
 
 BY 
 
 WM. HALLOCK PARK, M.D., 
 
 ASSOCIATE PROFESSOR OF BACTERIOLOGY AND HYGIENE, UNIVERSITY AND BELLE- 
 VUE HOSPITAL MEDICAL COLLEGE, AND ASSISTANT DIRECTOR OF THE 
 RESEARCH BACTERIOLOGICAL LABORATORIES OF THE DE- 
 PARTMENT OF HEALTH, CITY OF NEW YORK. 
 
 ASSISTED BY 
 
 A. R. GUERARD, M.D., 
 
 ASSISTANT BACTERIOLOGIST, DEPARTMENT OF HEALTH, CITY OF NEW YORK. 
 ILLUSTRATED WITH 87 ENGRAVINGS AND 2 COLORED PLATES. 
 
 LEA BROTHERS & CO., 
 NEW YORK AND PHILADELPHIA. 
 
BIOLOGY 
 
 LIBRARY 
 
 G 
 
 Entered according to the Act of Congress, in the year 1899, by 
 
 LEA BROTHERS & CO., 
 In the office of the Librarian of Congress. All rights reserved. 
 
 DORNAN, PRINTER. 
 
[ 
 
 PREFACE. 
 
 IN the following pages the attempt has been made 
 to group together those facts in Bacteriology which 
 will constitute a sufficient text-book for the student and 
 which are of direct practical value to the physician 
 and health officer. Laboratory technique is given in 
 its essentials and to such an extent as is necessary to 
 make bacteriological methods plain to the physician, 
 to guide him in making the simple examinations pos- 
 sible in his office, and to show him under what 
 conditions he can obtain diagnostic or other help from 
 bacteriological examinations in laboratories. The phy- 
 sician can readily understand and apply the essentials 
 of bacteriology, but the actual carrying out of the 
 more difficult examinations should be left to the 
 trained bacteriologist. 
 
 Such subjects as the chemical changes produced by 
 bacteria, infection, immunity, the nature and use of 
 protective serums, and the diagnostic value of bacte- 
 riological cultures, are particularly emphasized, since 
 knowledge of such subjects is of special importance to 
 
 91547 
 
iv PREFACE. 
 
 the practising physician, in that it enables him- to ob- 
 tain an intelligent grasp of the nature of the infectious 
 diseases. 
 
 The methods used in the laboratory for the isolation 
 and identification of the typhoid, tubercle, and diph- 
 theria bacilli have been given with especial fulness, as 
 bacteriological examinations of the discharges of per- 
 sons suspected to have typhoid fever, tuberculosis, or 
 diphtheria are now generally made for these bacteria 
 in the laboratories of the health departments of even 
 the smaller cities, because of the manifest importance 
 to the public of knowing where such sources of infec- 
 tion exist. 
 
 In preparing this book the best works have been 
 freely consulted. Of these, those of Fliigge and 
 Sternberg, on General Bacteriology, and those of 
 Abbott and Mallory and Wright, on Technique, should 
 perhaps be especially mentioned. 
 
 My sincere thanks are due to Dr. Hermann M. 
 Biggs, the Director of the Bacteriological Laboratory, 
 and to my colleagues in it, who have so freely furnished 
 me with the results of their original investigations. I 
 wish also to especially acknowledge my indebtedness 
 to Dr. A. R. Guerard, who has given me invaluable 
 aid in the preparation of the book. The illustrations, 
 with the exception of those on malaria and cholera, 
 for which I am indebted to Drs. Welch and Dunham, 
 
PEE FA CE. 
 
 are almost entirely from photographs taken from 
 cover-glass preparations and cultures by Dr. Edward 
 R. Learning, Instructor in Photography in the Medical 
 Department of Columbia University. 
 
 NEW YORK, November, 
 
CONTENTS. 
 
 INTRODUCTION. 
 
 PAGE 
 
 Historical Sketch of the Development of Bacteriology . . 17 
 
 CHAPTER I. 
 
 The General Characteristics of Bacteria Their Morphology 
 
 and Structure Vegetative and Spore Forms ... 33 
 
 CHAPTER II. 
 
 The Chemical Composition of Bacteria The Conditions Suit- 
 able for their Growth ....... 50 
 
 CHAPTER III. 
 
 Vital Phenomena of Bacteria Motion, Heat, and Light Pro- 
 ductionChemical Effects ( Ferments, Ptomai'ns, Toxins) . 58 
 
 CHAPTER IV. 
 
 The Relation of Bacteria to Disease ..... 85 
 
 CHAPTER V. 
 Immunity . . . . . . . . . . 102 
 
 CHAPTER VI. 
 
 Theories of Infection, Immunity, and Recovery . . . 118 
 
 CHAPTER VII. 
 Infection 128 
 
viii CONTENTS. 
 
 CHAPTEK VIII. 
 
 PAGE 
 
 The Effect of Light, Electricity, Pressure, Agitation, Drying, 
 and Association with Other Micro-organisms upon Bac- 
 teria 134 
 
 CHAPTEK IX. 
 Effect of Temperature upon Bacteria 144 
 
 CHAPTER X. 
 The Destruction of Bacteria by Chemicals .... 151 
 
 CHAPTEK XL 
 
 Practical Disinfection and Sterilization (House, Person, In- 
 struments, and Food) Sterilization of Milk for Feeding 
 Infants 168 
 
 CHAPTEK XII. 
 
 The Preparation, Staining, and Microscopical Examination 
 
 of Bacteria . 197 
 
 CHAPTEK XIII. 
 
 The Cultivation of Bacteria Sterilization of Media Appa- 
 ratus . .212 
 
 CHAPTEK XIV. 
 
 The Use of Animals for Diagnostic and Test Purposes . . 236 
 
 CHAPTER XV. 
 
 The Procuring of Material for Bacteriological Examination 
 
 from those Suffering from Disease ..... 240 
 
 CHAPTER XVI. 
 
 Bacteriological Examination of Water and Air The Con- 
 tamination and Purification of Drinking Waters . . 245 
 
CONTENTS i x 
 
 CHAPTER XVII. 
 
 PAGE 
 
 The Classification of Bacteria ...... 257 
 
 CHAPTER XVIII. 
 
 Bacillus of Tuberculosis (Koch's Tubercle Bacillus) . . 263 
 
 CHAPTER XIX. 
 
 Bacilli Showing Similar Staining Reactions to those of the 
 Tubercle Bacilli Syphilis Bacillus Smegma Bacillus 
 Leprosy Bacillus Grass Bacilli 311 
 
 CHAPTER XX. 
 Influenza Bacillus ......... 320 
 
 CHAPTER XXI. 
 Diphtheria Bacillus 329 
 
 CHAPTER XXII. 
 Tetanus Bacillus 335 
 
 CHAPTER XXIII. 
 
 Bacillus Typhosus ( Eberth-Gaff ky's Bacillus of Typhoid 
 
 Fever ; Bacillus Typhi Abdominalis) .... 402 
 
 CHAPTER XXIV. 
 
 Bacillus Coli Comnmnis (or Colon Bacillus of Escherich) . 444 
 
 CHAPTER XXV. 
 
 Pneumobacillus ( Friedlander's Bacillus) . . . 455 
 
 CHAPTER XXVI. 
 
 The Producers of Abscess, Cellulitis, Septicaemia, etc. (the 
 
 Staphylococci and Micrococcus Tetragenus) . . . 461 
 
x CONTENTS. 
 
 CHAPTEE XXVII. 
 
 PAGE 
 
 Streptococcus Pyogenes (Streptococcus Erysipelatis ; Strep- 
 tococcus of Pus ; Streptococcus Pathogenes Longus) . 476 
 
 CHAPTER XXVIII. 
 
 Micrococcus Lanceolatus (Pneumococcus ; Micrococcus Pneu- 
 moniae Crouposae of Sternberg ; Micrococcus of Sputum 
 Septicaemia and Diplococcus of Fraenkel ; Diplococcus 
 Pneumonias of Weichselbaum ) . . . - . 498 
 
 CHAPTEE XXIX. 
 
 Diplococcus Intracellularis Meningitidis . . . . 516 
 
 CHAPTER XXX. 
 
 Micrococcus Gonorrhoaae (Gonococcus Neisser) . . . 522 
 
 CHAPTER XXXI. 
 
 Bacillus Pyocyaneus (Bacillus of Green and of Blue Pus) 
 Bacillus Proteus Vulgaris Bacillus of Malignant (Edema 
 Bacillus Aerogenes Capsulatus . .... . 535 
 
 CHAPTER XXXII. 
 
 Bacillus Anthracis (Anthrax Bacillus) Bacillus Anthracis 
 
 Symptomatici . . . 547 
 
 CHAPTER XXXIII. 
 
 Spirillum Choleras Asiaticae (Koch's Comma Bacillus of 
 
 Asiatic Cholera) 568 
 
 CHAPTER XXXIV. 
 
 Spirilla Resembling that of Cholera The Spirillum of Re- 
 lapsing Fever 589 
 
CONTENTS. xi 
 
 CHAPTER XXXV. 
 
 PAGE 
 
 Glanders Bacillus 598 
 
 CHAPTER XXXVI. 
 
 Bubonic Plague Bacillus Yellow Fever Bacillus Whoop- 
 ing-cough Bacillus . . 606 
 
 APPENDIX. 
 
 BRIEF DESCRIPTIONS OF A FEW REPRESENTATIVE 
 
 PATHOGENIC MICRO-ORGANISMS WHICH 
 
 ARE NOT BACTERIA 
 
 CHAPTER XXXVII. 
 
 The Streptothrix Group ( Actinomyces) Favus and Ring- 
 worm Fungi Yeast ....... 615 
 
 CHAPTER XXXVIII. 
 
 Plasmodium Malarise (Malarial Parasites; Laverania) 
 Amoeba Coli (Amoeba Dysenteriae of Councilman and 
 Lafleur ; Dysenteric Amoeba) 626 
 
 CHAPTER XXXIX. 
 
 Vaccine The Micro-organism of Smallpox and Cowpox . 648 
 
 CHAPTER XL. 
 Rabies (Hydrophobia) 659 
 
BACTERIOLOGY IN MEDICINE AND SURGERY. 
 
 
 INTRODUCTION. 
 
 ALTHOUGH most of the more important discoveries 
 in bacteriology which place it on the footing of a 
 science are of comparatively recent date, the founda- 
 tion of the study of vegetable and other micro-organisms 
 was laid over two centuries ago. From the earliest 
 times its history has been intimately associated with 
 that of medicine. Indeed, it is only through the inves- 
 tigations into the life-history of micro-organisms in their 
 relation to disease that our present knowledge of the 
 etiology, course, and prevention of the infectious diseases 
 has been acquired ; and it is only by the practical ap- 
 plication of the principles and methods of bacteriology 
 that many diseases can be positively diagnosed or the 
 problems which present themselves to the sanitarian be 
 certainly solved. The prominent position which bac- 
 teriology already holds toward medicine is, moreover, 
 daily increasing in importance. Original discoveries are 
 constantly adding to the list of known germ diseases, 
 and the outlook is favorable for eventually obtaining 
 through serums or through the toxic substances of the 
 micro-organisms themselves means for immunizing 
 against, if not curing, many of the specific infections. 
 Even at present bacterial products and protective 
 
 2 
 
18 BACTERIOLOGY. 
 
 serums are used successfully as preventives in many of 
 the infectious diseases and as a cure in several. An 
 acquaintance, therefore, with the main facts and results 
 of bacteriology is as necessary to the education of the 
 modern physician as a knowledge of anatomy, pathol- 
 ogy, chemistry, or any of the allied sciences. 
 
 But before entering into a detailed consideration of 
 the subject, it may be interesting and instructive to 
 review briefly the most important steps which led up 
 to the development of the science, and upon which its 
 foundation rests, in which we shall see that the vast 
 results obtained by bacteriology were gained only 
 through long and laborious research, and after many 
 obstacles were met and overcome by indomitable per- 
 severance and accurate observation and experiment. 
 
 The first authentic observations of living microscopi- 
 cal organisms of which there is any record are those of 
 Athanasius Kircher, in 1671. This original investi- 
 gator demonstrated the presence in putrid meat, milk, 
 vinegar, cheese, etc., of <e minute living worms," but 
 did not describe their form or character. 
 
 Not long after this, in 1675, Anthony von Leeuwen- 
 hoek observed in rain-water putrid infusions, and in 
 his own and other saliva and diarrhoeal evacuations 
 living, motile " animalculse " of most minute dimen- 
 sions, which he described and illustrated by drawings. 
 Leeuwenhoek was a linen-draper by trade, living at 
 the time of his discoveries in Amsterdam, but he prac- 
 tised the art of lens-grinding, in which he eventually 
 became so proficient that he perfected a lens superior 
 to any magnifying glass obtainable at that day, and 
 with which he was enabled to see objects very much 
 smaller than had ever been seen before. " With the 
 
INTRODUCTION. 19 
 
 greatest astonishment/' he writes, " I observed distrib- 
 uted everywhere through the material which I was 
 examining animalcules of the most microscopic size, 
 which moved themselves about very energetically." 
 The work of this observer is conspicuous for its purely 
 objective character and absence of speculation ; and his 
 descriptions and illustrations are done with remarka- 
 ble clearness and accuracy, considering the imperfect 
 optical instruments at his command. There is little 
 doubt that Leeuwenhoek really saw some of the larger 
 species of micro-organisms which we now recognize as 
 bacteria, probably spirilla. 
 
 It was not until many years later, however, that any 
 attempt was made to define the characters of these 
 minute organisms and to classify them. The first to 
 make such an effort was Otto Friedrich Miiller, in 
 1786 ; but having no means of obtaining pure cultures 
 all the earlier botanists naturally fell into serious errors 
 in the classification of bacteria. Thus various motile 
 organisms, which are now known to be of vegetable 
 origin, were commonly included under the infusoria, 
 which are unicellular animal organisms. 
 
 Ehrenberg, in 1838, thus describes under the gen- 
 eral name Vibrioniens four genera of filamentous bac- 
 teria : 
 
 1. Bacterium filaments linear and inflexible. 
 
 2. Vibrio filaments linear, sinuous, flexible. 
 
 3. Spirillum filaments spiral, inflexible. y. 
 
 4. Sperochcete filaments spiral, flexible. 
 Dujardin, in 1841, also placed the vibrioniens of 
 
 Ehrenberg among the infusoria, describing them as 
 extremely slender, filiform animals without appreciable 
 organization and without visible locomotive organs. 
 
20 BACTERIOLOGY. 
 
 Perty, in 1852, drew attention to the vegetable 
 origin of these minute organisms ; Robin, in 1853, 
 suggested their relationship to the algae ; Davaine, in 
 1859, emphasized this fact ; and since it has been con- 
 firmed by the investigations of Cohn, Nageli, and 
 others. Bacteria are now generally believed by bacte- 
 riologists to be vegetable organisms, schizomycetes, or 
 fission-fungi, closely allied to the algse. 
 
 From the earliest investigations into the life-history 
 and properties of bacteria these micro-organisms have 
 been thought to play an important part in the causa- 
 tion of infectious diseases. The doctrine of contagium 
 aminatum was based upon the discoveries of Athana- 
 sius Kircherand Leeuwenhoek, and the " animalculse " 
 then observed in organic materials were believed to be 
 the cause of the great epidemics of the day, such as 
 the plague. Shortly after these first investigations, 
 Lange and Hauptmann advanced the opinion that 
 puerperal fever, measles, smallpox, typhus, pleurisy, 
 epilepsy, gout and many other diseases were due to 
 animal contagion. Andry and Linne, in 1701, as- 
 sumed the same cause for syphilis, and Lancisi, in 
 1718, for malaria. In fact, so wide- spread became the 
 belief in a causal relation of these minute organisms to 
 disease that it soon amounted to a veritable craze, and 
 all forms and kinds of diseases were said to be pro- 
 duced in this way, upon no other foundation than that 
 these organisms had been found in the mouth and in- 
 testinal contents of men and animals, and in water. 
 
 Among those who were especially conspicuous at 
 this time for their advanced views on the germ-theory 
 of infectious diseases was Marcus Antonius Plenciz, a 
 physician of Vienna. This acute observer, who pub- 
 
INTRODUCTION. 21 
 
 lished his views in 1762, maintained that not only 
 were all infectious diseases caused by micro-organisms, 
 but that the infective material could be nothing else 
 than a living organism. On these grounds he en- 
 deavored to explain the variations in the period of 
 incubation of the different infectious diseases. He also 
 insisted that there were special germs for each infectious 
 disease by which the specific disease was produced. 
 Plenciz believed, moreover, that these organisms were 
 capable of multiplication in the body, and suggested 
 the possibility of their being conveyed from place to 
 place through the air. He also made original investi- 
 gations into the process of decomposition, and having 
 found " animalcube " in all decomposing matter, he 
 became so thoroughly convinced of the causative rela- 
 tion of these organisms to the process that he formu- 
 lated the law that decomposition takes place by means 
 of living organisms, and is possible only through their 
 increase. 
 
 These views, it is true, were largely speculative, and 
 rested upon insufficient experiment; but they were so 
 plausible, and the arguments put forward in their sup- 
 port were so logical and convincing, that they continued 
 to gain ground, in spite of considerable opposition and 
 ridicule, and in many instances the conclusions reached 
 have since been proved to be correct. The fact that 
 infectious diseases were of sudden occurrence, breaking 
 out often in isolated places, and that they frequently 
 remained clinging for long periods to certain localities, 
 leaving others unaffected, was evidence that they were 
 not produced by a gaseous infective agent. Moreover, 
 the mode of infection, its unlimited development among 
 large numbers of individuals, and gradual spread over 
 
22 BACTERIOLOGY. 
 
 wide areas the incubation, course of, and resulting 
 immunity in recovery from infectious diseases all 
 pointed to the probable cause being a living organism. 
 
 Amoug other distinguished men of the day whose 
 observations exerted a most powerful influence upon 
 the doctrine of infection may be mentioned Henle. 
 His writings (Pathological Investigations, 1840, and 
 Text-book of Rational Pathology, 1853), in which he 
 described the relation of micro-organisms to infectious 
 diseases, and defined the character and action of bac- 
 teria upon certain phases and symptoms of these affec- 
 tions, are remarkable for their clearness and precision. 
 
 But, meanwhile, the question which most interested 
 these investigators into the cause of infectious diseases 
 was, Whence are these micro-organisms derived which 
 were supposed to produce them ? Were they the result 
 of spontaneous generation due to vegetative changes in 
 the substances in which the organisms were found, or 
 were they reproduced from similar pre-existing organ- 
 ismsthe so-called vitalistic theory ? This question is 
 intimately connected with the investigations into the 
 origin and nature of fermentation and putrefaction. 
 
 Among those who advocated the theory of spon- 
 taneous generation was Neidham, who, in 1749, at- 
 tempted to prove by experiment the truth of his opin- 
 ions. He placed a grain of barley in a watch-glass 
 containing water, covered it carefully, and allowed it 
 to germinate. On later examination he found bacteria 
 present, which he maintained were the result of changes 
 in the grain itself due to its germination. 
 
 In 1769, Spallanzani showed by another experiment 
 that the theory of spontaneous generation was incor- 
 rect. He demonstrated that if putrescible infusions 
 
INTR OD UCTION. 23 
 
 of organic matter were placed in symmetrically sealed 
 flasks and then boiled the liquids were sterilized ; 
 neither were living organisms found in the solutions, 
 nor did they decompose ; and the infusions remained 
 unchanged for an indefinite period. 
 
 It was objected to these experiments that the high 
 temperature to which the liquids had been subjected so 
 altered them that spontaneous generation could no 
 longer take place. This objection was met by Spall- 
 anzani by cracking one of the flasks and allowing air 
 to enter, when living organisms and decomposition 
 again appeared in the boiled infusions. 
 
 Another objection raised was that in excluding the 
 oxygen of the air by hermetically sealing the flasks the 
 essential condition for the development of fermentation, 
 which required free admission of this gas, was inter- 
 fered with. This objection was then met by Schulze, 
 in 1836, by causing the air admitted to the boiled 
 decomposable liquids to pass through strong sulphuric 
 acid. Air thus robbed of its living organisms did not 
 produce decomposition ; whereas when no such precau- 
 tions were taken with the air admitted the boiled solu- 
 tions quickly fell into putrefaction, and living organ- 
 isms were found to be present. 
 
 Schwann, in 1839, obtained similar results in another 
 way; he deprived the air admitted to his boiled liquids 
 of micro-organisms by passing it through a tube 
 which was heated to a temperature high enough to 
 destroy them. To this investigator is also due the 
 credit of having discovered the specific cause the yeast 
 plant, or saccharomyces cerevisice of alcoholic fermen- 
 tation, the process by which sugar is decomposed into 
 alcohol and carbonic acid. 
 
24 BACTERIOLOGY. 
 
 Helmholtz, in 1843, repeated and confirmed 
 Schwann's experiments with calcined air. He found 
 that the free admission of air so treated to boiled 
 organic infusions was not capable of producing fer- 
 mentation of any kind. 
 
 Again, it was objected to these experiments that the 
 heating of the air had perhaps brought about some 
 chemical change which hindered the production of fer- 
 mentation. Schroeder and von Dusch, in 1854, then 
 showed that by a simple process of filtration, which 
 has since proved of inestimable value in bacteriological 
 work, the air can be mechanically freed from germs. 
 By placing in the mouth of the flask containing the 
 boiled solutions a loose plug of cotton, through which 
 the air could freely circulate, it was found that all 
 suspended micro-organisms could be excluded, and 
 that air passed through such a filter, whether hot or 
 cold, did not cause fermentation of boiled infusions. 
 
 Similar results were obtained by Hoffmann in 1860, 
 and by Chevreul and Pasteur in 1861, without a cotton 
 filter, by drawing out the neck of the flask to a fine 
 tube and turning it downward, leaving the mouth open. 
 In this case the force of gravity prevents the suspended 
 bacteria from ascending, and there is no current of air 
 to carry them upward through the tube into the flask 
 containing the boiled infusion. 
 
 Tyndall later showed (1876), by his well-known in- 
 vestigations upon the floating matters of the air, that 
 in a closed chamber, in which the air is not disturbed 
 by currents, all suspended particles settle to the bottom, 
 the superincumbent air being optically pure, as is 
 proved by passing a ray of light through it. He dem- 
 onstrated that the presence of living organisms in 
 
INTR OD UCTION. 25 
 
 decomposing fluids was always to be explained either 
 by the pre-existence of similar living forms in the in- 
 fusion or upon the walls of the vessel containing it, or 
 by the infusion having been exposed to air which was 
 contaminated with organisms. 
 
 These facts have since been practically confirmed on 
 an enormous scale in the preservation of food by the 
 process of sterilization. Indeed, there is scarcely any 
 biological problem which has been so satisfactorily 
 solved or in which such uniform results have been ob- 
 tained ; but all through the experiments of the earlier 
 investigators irregularities were constantly appearing. 
 Although in the large majority of cases it was found 
 possible to keep boiled organic liquids sterile in flasks 
 to which the oxygen of the air had free access, the 
 question of spontaneous generation still remained un- 
 settled, inasmuch as occasionally, even under the most 
 careful precautions, decomposition did occur in such 
 boiled liquids. 
 
 This fact was explained by Pasteur in I860 by ex- 
 periments showing that the temperature of boiling 
 water was not sufficient to destroy all living organ- 
 isms, and that, especially in alkaline liquids, a higher 
 temperature was required to insure sterilization. He 
 showed that at a temperature of 110 to 112 C. (230 
 to 233 F.), however, which he obtained by boiling 
 under a pressure of one and one-half atmospheres, all 
 living organisms were invariably killed. 
 
 Pasteur at a later date (1865) demonstrated that the 
 organisms which resist the boiling temperature are, in 
 fact, reproductive bodies, which he described under the 
 name of " corpuscles ovoides " or " corpuscles bril- 
 lants " now known as spores. Perty, in 1852, and 
 
26 BACTERIOLOGY. 
 
 Robin, in 1853, had observed these highly refractile 
 bodies ; but it was not until 1876 that the development 
 of spores was carefully investigated and explained by 
 Cohn and later by Koch. These observers showed that 
 certain rod-shaped organisms possess the power of pass- 
 ing into a resting or spore-stage under peculiar conditions 
 of growth, and when in this stage they are much less 
 susceptible to the injurious action of higher tempera- 
 tures than when in their normal vegelative condition. 
 
 With this discovery the controversy of spontaneous 
 generation was finally settled. If these micro-organ- 
 isms, some of them being capable of producing the 
 more resistant spores, were present in the air, dust, 
 soil, water, etc., it was easy enough to explain the 
 irregularities in the foregoing experiments ; nor was it 
 any longer to be doubted that these bacteria, through 
 their products, were the cause, not the effect, of fer- 
 mentation and putrefaction, and that when organic sub- 
 stances were completely sterlized and protected against 
 the entrance of living germs from without, no develop- 
 ment of micro-organisms occurred in them. 
 
 Stimulated by the establishment of the fact that fer- 
 mentation and putrefaction were due to the action of 
 living organisms reproduced from similar pre-existing 
 forms, the study of the causal relation of these micro- 
 organisms to disease was taken up with renewed vigor. 
 Reference has already been made to the opinions and 
 hypothesis of the earlier observers as to the microbic 
 origin of infectious diseases. The first positive grounds, 
 however, for this doctrine, founded upon actual ex- 
 periment, were the investigations into the cause of cer- 
 tain infectious diseases in insects and plants. Thus 
 Bassi, in 1837, demonstrated that a fatal infectious 
 
INTRODUCTION. 27 
 
 malady of the silkworm muscardine was due to a 
 parasitic micro-organism. Pasteur later devoted several 
 years' study to an exhaustive investigation into the 
 same subject ; and in like manner Tulasse, in 1864, 
 and Kiihne, in 1855, showed that certain specific affec- 
 tions in grains, the potato, etc., were due to the inva- 
 sion of parasites. 
 
 Very soon after this it was demonstrated that micro- 
 organisms were the cause of certain infectious diseases 
 in man and the higher animals. Bacteriological re- 
 search has always been of special interest to physicians. 
 Many of the most distinguished physicians of the day, 
 in the earlier history of the science, concerned them- 
 selves in these investigations, and the progress made 
 during the past fifteen or twenty years has been largely 
 due to their work. Davaine, a famous French physi- 
 cian, has the honor of having first demonstrated the 
 causal relation of a micro-organism to a specific infec- 
 tious disease in man and animals. The anthrax bacil- 
 lus was discovered in the blood of animals dying from 
 this disease by Pollender, in 1849, and by Davaine, 
 in 1850 ; but it was not until 1863 that the last-named 
 observer demonstrated by inoculation experiments that 
 the bacillus was the cause of anthrax. These experi- 
 ments were subsequently confirmed by Pasteur, Koch, 
 and others. 
 
 The next discoveries made were those relating to 
 wounds and the infections to which they are liable. 
 Rindfleisch, in 1866, and Waldeyer and von Reckling- 
 hausen, in 1871, were the first to draw attention to the 
 minute organisms occurring in the pysemic processes 
 resulting from infected wounds, and occasionally fol- 
 lowing typhoid fever. Further operations were made 
 
28 BACTERIOLOGY. 
 
 in erysipelatous inflammations secondary to injury by 
 Wilde, Orth, von Recklinghausen, Luthomsky, Bill- 
 roth, Ehrlich, Fehleisen, and others, agreeing that in 
 these conditions micro-organisms could always be de- 
 tected in the lymph-channels of the subcutaneous 
 tissues ; and through numerous experiments on ani- 
 mals the pathogenic character of the micro-organisms 
 found in erysipelas, suppuration from wounds, diph- 
 theria, puerperal fever, etc., was established by Oertel, 
 Huester, Birsch-Hirschfeld, Narsiloff, Classen, Letz- 
 erich, Leber, Frisch, Eberth, Klebs and others. 
 
 The brilliant results obtained by Lister, in 1863 
 1870, in the antiseptic treatment of wounds, to prevent 
 or inhibit the action of infective organisms, exerted a 
 powerful influence on the doctrine of bacterial infec- 
 tion, causing it to be recognized far and wide and 
 gradually lessening the number of its opponents. 
 
 The next important discovery was that of Ober- 
 meier, a German physician, who, in 1873, announced 
 having found in the blood of patients suffering from 
 relapsing fever a minute spiral, actively motile micro- 
 organism the spirochcete Obermeieri which is now 
 generally recognized as the specific infectious agent in 
 this disease. 
 
 In 1877, Weigert and Ehrlich recommended the use 
 of the aniline dyes as staining agents in the micro- 
 scopical examination of micro-organisms in cover-glass 
 preparations. 
 
 In 1878, Koch published his important work on 
 traumatic infectious diseases. 
 
 Hausen, in 1879, reported the discovery of bacilli 
 in the cells of leprous tubercles, which, from subsequent 
 researches, are believed to be the cause of leprosy. 
 
INTRODUCTION. 29 
 
 Neisser, in the same year (1879), discovered the 
 " gonococcus " in gonorrhoeal discharges. 
 
 In 1880, Eberth and Koch independently observed 
 the typhoid bacillus, but it was not until 1884 that 
 Gaffky published his important researches, and proved 
 the etiological relation of this bacillus to typhoid fever. 
 
 In the same year (1880) several important communi- 
 cations in bacteriological research appeared. Pasteur 
 published his discovery of the bacillus of fowl cholera 
 and his investigations upon the attenuation of the virus 
 of anthrax and of fowl cholera, and upon protective 
 inoculation against these diseases. 
 
 Sternberg and Pasteur independently observed (1880) 
 a pathogenic micrococcus in saliva, which was subse- 
 quently proved by Fraenkel and others (1885) to be 
 the organism most commonly associated with acute 
 croupous pneumonia the " diplococcus pneumoniae" 
 and now recognized as the usual cause of that disease. 
 
 In 1881, Koch made his fundamental researches upon 
 pathogenic bacteria, the result of which was the estab- 
 lishment of a foundation upon which bacteriology of the 
 future was to rest. He introduced solid culture media 
 and the " plate method 7 ' for obtaining pure cultures, 
 and showed how different organisms could be isolated, 
 cultivated independently, and by inoculation of pure 
 cultures into susceptible animals made, in many cases, 
 to reproduce the specific disease of which they were the 
 cause ; and he laid down the laws by which it may be 
 proved that a micro-organism is the specific cause of 
 a disease. It was in the course of this work that the 
 Abbe system of substage condensing apparatus was first 
 used in bacteriology and that Weigert's method of 
 staining was generally employed. 
 
30 BACTEEIOLOG T. 
 
 In 1882, Koch published his discovery of the tubercle 
 bacillus. 
 
 The same year (1882) Pasteur published his in- 
 vestigations upon "rouget" or hog erysipelas. In 
 this year, also, his first communication upon rabies 
 appeared. 
 
 In 1882, also, Loemer and Schiitz discovered the 
 bacillus of glanders. 
 
 The cholera spirillum, or " comma bacillus/' was 
 discovered by Koch in 1884. 
 
 The diphtheria bacillus was discovered during the 
 same year (1884) by Loemer, though it had been ob- 
 served by Klebs the year before (1883). 
 
 Rosenbach, in 1884, by the application of Koch's 
 methods, fixed definitely the characters of the various 
 micro-organisms found in the pus from acute abscesses, 
 etc. 
 
 The tetanus bacillus was also discovered in 1884 by 
 Nicolaier. Carle and Rattone showed that tetanus is 
 an infectious disease communicable to man by inocula- 
 tion. Kitasato, in 1889, obtained the bacillus in pure 
 cultures. 
 
 In 1892, Pfeiffer and Canon independently discov- 
 ered a bacillus which is believed to be the specific 
 cause of influenza. 
 
 In 1894, Kitasato, the Japanese bacteriologist, during 
 a visit to China, discovered the bacillus of the bubonic 
 plague. 
 
 These include all the most important pathogenic 
 bacteria, the discovery of which is of special interest 
 to medical students and physicians. We cannot close 
 this brief historical review, however, of the progress of 
 our knowledge in this department of science, without 
 
INTR OD UCTION. 31 
 
 referring to the recent discovery of the antitoxins of 
 diphtheria and tetanus, the protective inoculations 
 against rabies, the plague, cholera, etc., and the pecu- 
 liar characteristics of the serum of those ill with infec- 
 tious diseases. These discoveries, in which the names 
 of Pasteur, Koch, Behring, Kitasato, Roux, Pfeiffer, 
 and Widal are among the most prominent, mark an 
 epoch in the history of bacteriology and scientific medi- 
 cine. Lately, attention has also been given to the 
 smaller group of the animal parasites, the protozoa, 
 which may prove to be the source of infection in many 
 diseases, such as the exanthemata, in one of which 
 smallpox they have already been apparently found. 
 
CHAPTER I. 
 
 THE GENERAL CHARACTERISTICS OF BACTERIA 
 THEIR MORPHOLOGY AND CHEMICAL COMPOSITION. 
 
 BACTERIA are among the smallest of all known liv- 
 ing organisms, the largest of them having a diameter 
 of only a few micromillimetres, while the smallest do 
 not measure more than a fraction of a micromillimetre. 
 Structurally and morphologically they are extremely 
 simple, though biologically very variable. Through 
 their ability to derive their carbon from tartrates and 
 their nitrogen from ammonia or its salts, they are 
 ranked in the vegetable kingdom. They obtain their 
 food entirely through the surface absorption of soluble 
 nutritious substances. They are reproduced by trans- 
 verse division, and in some respects resemble the fungi ; 
 hence called by Nageli fission-fungi, or schizomycetes. 
 They are also closely allied to certain kinds of algae, 
 though they must receive their nourishment from living 
 or dead organic material, since they are without chloro- 
 phyll, the green coloring matter possessed by the higher 
 plants, by means of which they are enabled, in the 
 presence of sunlight, to decompose CO 2 , NH 3 , and H 2 S 
 into their elementary constituents. A few varieties of 
 unicellular organisms resemble bacteria in all their 
 known characteristics, except that they possess chloro- 
 phyll or substances similar to it. Others, still, which 
 have no chlorophyll, are able in the absence of light to 
 build up organic substances synthetically. Bacteria, 
 
 3 
 
34 BACTERIOLOGY. 
 
 especially the motile forms, are also closely allied to 
 some of the micro-organisms which belong to the ani- 
 mal kingdom. If we exclude the micro-organisms 
 containing chlorophyll, bacteria may be defined as 
 extremely minute vegetable organisms; without chloro- 
 phyll, consisting of single spherical, rod-shaped, or 
 corkscrew-like cells or aggregates of such cells, between 
 whose protoplasm and nucleus it has been as yet im- 
 possible to differentiate with certainty. 
 
 Bacteria occur as saprophytes or refuse-eaters and as 
 parasites. Saprophytic bacteria are such as commonly 
 exist independently of a living host, obtaining their 
 supply of nutriment from soluble food-stuffs in dead 
 organic matter. Parasitic bacteria, on the other hand, 
 live on or in some other organism, from which they 
 derive their nourishment for the whole or a part of 
 their existence. Those bacteria which depend entirely 
 upon a living host for their existence are known as 
 strict parasites ; those which can lead a saprophytic 
 existence, but which can also thrive within the body 
 of a living animal, are called facultative parasites. 
 The strict saprophytes, which represent the large 
 majority of all bacteria, while they destroy refuse, are 
 not only harmless to living organisms but perform 
 many important functions in nature without which 
 existence would be impossible, such as the destruction 
 of dead organic material through decomposition, putre- 
 faction, and fermentation. The parasites, on the con- 
 trary, though some of them may multiply in the secre- 
 tions or on the surface of the body without injury to 
 the animal upon which they depend for their exist- 
 ence, are usually harmful invaders, giving rise through 
 tHe lesions brought about in the body tissues by their 
 
GENERAL CHARACTERISTICS OF BACTERIA. 35 
 
 growth and products to derangements which are known 
 as acute or chronic infectious diseases. 
 
 Numerous attempts have been made by various 
 authors to classify bacteria systematically, but usually 
 with the proviso that the system was only a temporary 
 one. The classification of the older naturalists and 
 botanists was based generally upon purely morphological 
 peculiarities. As this depended, at times, upon slight 
 variations that were seen to occur in the size and shape 
 of one and the same species, it naturally resulted in a 
 more or less complicated arrangement. In this place 
 the morphological character of the bacteria will alone 
 be given, their classification being left until the general 
 characteristics of bacteria have been considered. 
 
 ih 
 MORPHOLOGY. 
 
 The basic forms of the single bacterial cells are 
 threefold the sphere, the rod, and the segment of a 
 spiral. Although under different conditions, the form 
 of any one species may vary considerably, yet these 
 three main divisions under similar conditions are per- 
 manent; and, so far as we know, it is never possible 
 by any means to bring about changes in the organisms 
 that will result in the conversion of the morphology of 
 the members of one group into that of another that 
 is, micrococci always, under suitable conditions, produce 
 micrococci, bacilli produce bacilli, and spirilla produce 
 spirilla. 
 
 The form of the bacterial cells at their stage of com- 
 plete development must be distinguished from that 
 which they possess just after or just before they have 
 divided. As the spherical cell develops preparatory to 
 
36 BACTERIOLOGY. 
 
 its division into two cells it becomes elongated and ap- 
 pears as a short oval rod; at the moment of its division, 
 on the contrary, the transverse diameter of each of its 
 two halves is greater than their long diameter. A 
 short rod becomes in the same way, at the moment 
 of its division, two cells, the long diameter of each of 
 which may be even a trifle less than its short diameter, 
 and thus they appear on superficial examination as 
 spheres. 
 
 As bacteria multiply the cells produced from the 
 parent cell have a greater or less tendency to remain 
 attached. In some varieties this tendency is extremely 
 slight, in others it is marked. This union may appear 
 simply as an aggregation of separate bacteria or so close 
 that the group appears as a single cell. According to 
 the method of the cell division and the tenacity with 
 which the cells hold together, we get different group- 
 ings of bacteria, which aid us in their differentiation and 
 identification. Thus whether the bacterial cell divides 
 in one, two, or three planes, we get forms built in 
 one, two, or three dimensions. If we group bacteria 
 according to the characteristic form of the cells, and 
 then subdivide them according to the manner of their 
 division in reproduction and the tenacity with which 
 the newly developed cells cling to one another, we will 
 have the following varieties : 
 
 1. Spherical Form, or Coccus (Figs. 1 to 4). The size 
 varies from about Q.3/J. as minimum diameter to 3// as 
 maximum. The single elements are at the moment of 
 their complete development, so far as we can deter- 
 mine, absolutely spherical; but when seen in the process 
 of multiplication through division the form is seldom 
 that of a true sphere. Here we have elongated or lancet- 
 
GENERAL CHARACTERISTICS OF BACTERIA. 37 
 
 shaped forms, as frequently seen in the diplococcus of 
 pneumonia, or the opposite, as in the diplococcus of 
 gonorrhoea, where the cocci appear to be flattened 
 against one another. Those cells which divide in one 
 
 FIG. l, 
 
 FIG. 2. 
 
 ****> *"*V. 
 
 Single coccus, grouped irregularly. Diplococcus of pneumonia, with sur- 
 Staphylococcus. rounding capsule. 
 
 FIG. 3. 
 
 FIG. 4. 
 
 TBb 
 
 J^m 
 
 &$&L 
 
 Streptococcus. 
 
 Tetracoccus. 
 
 direction only and remain attached are found in pairs 
 (diplococci) or in shorter or longer chains (streptococci). 
 Those which divide in two directions, the one at right 
 angles to the other, form bunches of four (tetrads). 
 
38 BACTERIOLOGY. 
 
 Those which divide in three directions and cling to- 
 gether form packets in cubes (sarcinse). Those which 
 apparently divide irregularly in any axis form irregu- 
 larly shaped, grape-like bunches (staphylococci). 
 
 There are a considerable number of bacteria which 
 appear to frequently assume spherical forms, or at least 
 forms so like spheres that they cannot be differentiated 
 from them, and yet under other conditions they generate 
 rod-like forms. These apparently spherical bacteria 
 we can properly regard as short forms of bacilli, which, 
 owing to the rapidity of division, are for the time being 
 of the same size in both diameters. Under suitable 
 conditions, however, the true rod- shape is always de- 
 veloped. 
 
 2. Rod Form, or Bacillus. The type of this group is 
 the cylinder. The length of the fully developed cell 
 is always longer than its breadth. The size of the 
 cells of different varieties varies enormously, from a 
 length of 30// and a breadth of 4// to a length of 0.2// 
 and a breadth of 0.1 //. The largest bacilli met with 
 in disease do not, however, average over 3/*. In des- 
 cribing their forms bacilli are roughly classed as slender 
 when the ratio of the long to the transverse diameter is 
 from 1 : 4 to 1 : 10, and as thick when the proportions 
 of the long to the short diameter is approximately 1 : 2. 
 
 The characteristic form of the bacillus is one with a 
 straight axis, uniform thickness throughout, and flat 
 ends (Fig. 13, page 47); but there are many exceptions 
 to this typical form. Thus frequently the motile bac- 
 teria have rounded ends (Fig. 10, page 43); many of 
 the more slender forms have the long axis bent; some 
 few species, such as the diphtheria bacilli (Fig. 5), in- 
 variably produce many cells whose thickness is very 
 
GENERAL CHARACTERISTICS OF BACTERIA. 39 
 
 unequal at different portions. Spore formation also 
 causes an irregularity of the cell outline (Fig. 12). 
 
 The bacilli divide only in the plane perpendicular to 
 their long axis. A classification, therefore, of bacilli 
 
 FIG. 5. 
 
 FIG. 6. 
 
 ^~ ^-^ 
 
 i, single and in threads. 
 
 \& j3fc > * > 7 . \ /*V&~ OF T 
 
 $Sg\ ( UNWEF 
 
 ^f|-/^|^j V.C/Li^^ 
 
 Small bacilli, mostly in pairs. 
 
 according to their manner of grouping is much simpler 
 than in the case of the cocci. We may thus have bacilli 
 as isolated cells, as pairs, or as longer or shorter chains. 
 3. Spiral Form, or Spirillum. The members of the 
 third morphological group are spiral in shape, or rather 
 
40 
 
 BACTERIOLOGY. 
 
 segments of a spiral. Here, too, we have large and 
 small, slender and thick spirals. The twisting of the 
 long axis, which here lies in two planes, is the chief 
 characteristic of this group of bacteria. Under normal 
 
 FIG. 8. 
 
 FIG. 9. 
 
 Medium-sized spirilla. 
 
 conditions the twisting is equal throughout the entire 
 length of the cell. The spirilla, like the bacilli, divide 
 only in one direction. A single cell, a pair, or the 
 union of two or more elements may thus present the 
 appearance of a short segment of a spiral or a comma- 
 shaped form, an S-shaped form, or a complete spiral 
 or corkscrew-like form. 
 
 Among uncommon morphological peculiarities in 
 true bacteria may be mentioned dichotomy, or branch 
 formation that is, a side growth projecting from the 
 bacterial cell. True dichotomous branching has occa- 
 sionally been observed in the bacilli viz., the bacilli 
 of tuberculosis, diphtheria, and glanders. 
 
 Summary of Morphological Forms of Bacteria. 1. 
 Coccus, or micrococctis. Spherical or subspherical 
 forms. 
 
GENERAL CHARACTERISTICS OF BACTERIA. 41 
 
 a. Single coccus, grouped irregularly. 
 
 b. Diplococcus, forming pairs. 
 
 c. Streptococcus, forming chains, often showing 
 
 paired cocci. 
 
 d. Tetracoccus, forming fours by division through 
 
 two planes of space. 
 
 e. Sarcina, forming packets of eight members by 
 
 division through three planes of space. 
 
 2. Bacillus. Oblong or cylindrical forms, having 
 one dimension greater than any other, more or less 
 straight, and never forming spirals. 
 
 a. Single bacillus. 
 
 b. Diplobacillus and streptobacillus, forming twos 
 
 or longer chains, the bacilli attached end to 
 end. 
 
 c. Filaments, or thread-like growths, in which 
 
 divisions into bacilli of the normal length 
 are not apparent, or occur irregularly and 
 transversely, to the long axis of the growth. 
 
 3. Spirillum. Cylindrical and curved forms, con- 
 stituting complete spirals or portions of spirals. 
 
 The determination of morphological characters for 
 the description of bacteria should always be made from 
 fully developed cultures; those which are too young 
 may present, as already noted, immature forms, due to 
 rapid multiplication, while in old cultures altered or 
 degenerated forms may be observed. 
 
 When growth is obtained upon different media, varia- 
 tions, especially in size, may sometimes be observed. 
 These differences should always be described, together 
 with a note of the media upon which they were devel- 
 oped and a statement as to whether such variation is a 
 marked feature of the species under consideration. 
 
42 BACTERIOLOGY. 
 
 The conditions of temperature and of nutrition which 
 favor growth are very various for different species, so 
 that no fixed temperature, medium, or age of growth 
 can be determined upon as applicable to all species. 
 Morphological descriptions should always be accom- 
 panied by a definite statement of the age of the growth, 
 the medium from which it was obtained, and the tem- 
 perature at which it was developed. 
 
 It is further advisable that the appearance observed 
 in growths developed upon a solid and in a liquid 
 medium should be recorded. 
 
 The structure of bacterial cells has recently attracted 
 considerable attention among naturalists. According to 
 Fischer and Migula, the bacterial cells consist of a cell- 
 membrane, a protoplasmic layer, and a central fluid; no 
 nucleus was observed by them. In salt solutions and 
 when dried upon a cover-glass a shrinkage of the pro- 
 toplasmic layer with partial dissolution of the cell-wall 
 occurs, due to the abstraction of water. This process 
 is known as plasmolysis, and it explains the occurrence 
 of the clear, unstained spaces so frequently seen in 
 the stained cover-glass preparations which have erro- 
 neously been taken for spores. In water, or by 
 the continued action of salt solution, this shrink- 
 age does not take place. In many species of bacteria, 
 such as the diphtheria bacilli, there is observed in the 
 interior of the cells, on suitable staining, a peculiar 
 granulation, to which Bab6s has given the name of 
 metachromatic bodies, but which Ernst on more careful 
 study has termed sporagenous granules. 
 
 With regard to the cell membrane, it should be 
 noticed that it is frequently not sharply defined and 
 often difficult to demonstrate. In many species of 
 
GENERAL CHARACTERISTICS OF BACTERIA. 43 
 
 bacteria, however, commonly known as capsule bacteria, 
 as shown in Fig. 2, the cell membrane or the outer layers 
 of the membrane are so much thickened that the bacteria 
 seem to be surrounded by a gelatinous envelope or cap- 
 sule, which is distinguished by a diminished power of 
 staining with the ordinary aniline dyes. The demonstra- 
 tion of this capsule may be of help in differentiating 
 between certain bacteria e. g., some forms of the 
 streptococcus and pneumococcus. A peculiarity of 
 
 FIG. 10. 
 
 Faintly stained flagella attached to heavily stained bacilli. 
 
 the capsule bacteria is that, except very rarely, they 
 exhibit this envelope only when grown in the animal 
 body or in special' culture media, such as liquid blood 
 serum, bronchial mucus, etc. ; grown on nutrient 
 gelatin, agar, or potato the capsule is only visible under 
 very exceptional conditions, and then not distinctly. 
 
 The outer surface of bacteria when occurring in the 
 form of spheres and short rods is almost always smooth 
 
44 BACTERIOLOGY. 
 
 and devoid of appendages ; but the longer rods and 
 spirals are usually provided with fine hair-like append- 
 ages or flagella, which are their organs of motility. 
 These flagella, either singly or in numbers, are some- 
 times distributed over the entire body of the cell, or 
 they may form a tuft at one end of the rod, or only 
 one polar flagellum is found. The polar flagella 
 appear on the bacteria shortly before division. The 
 nature of flagella is little understood; they are believed 
 by some to be formed of protoplasmic material which 
 penetrates the cell membrane, and probably have the 
 property of protrusion and retraction. So far as we 
 know, the flagella are the only means of locomotion 
 possessed by the bacteria. They are not readily 
 stained, special staining agents being required for 
 this purpose. The envelope of the bacteria, which 
 usually remains unstained with the ordinary dyes, 
 then becomes colored and more distinctly visible than 
 is commonly the case. Occasionally, however, some 
 portion of the envelope remains unstained, when the 
 flagella present the appearance of being detached from 
 the body of the bacteria by a narrow zone. Unfor- 
 tunately, many of the methods employed for staining 
 flagella cause them to become degenerated, so that their 
 perfect demonstration is often very difficult. In cul- 
 tures of richly flagellated bacteria peculiar pleated 
 masses sometimes are observed, consisting of flagella 
 which have been detached and then matted together. 
 Bacteria may lose their power of producing flagella for 
 a series of generations. Whether their power be per- 
 manently lost or not we do not know. 
 
 The vegetative reproduction of bacteria takes place by 
 division. When development is in progress a single 
 
GENERAL CHARACTERISTICS OF BACTERIA. 45 
 
 cell will be seen to elongate, in the case of spherical 
 bacteria only slightly, and in the rod-shaped organ- 
 isms considerably in one direction. Over the centre 
 of the long axis thus formed will appear a slight in- 
 dentation in the outer envelope of the cell ; this inden- 
 tation increases in extent until there exists eventually 
 two individuals. As a rule, the cells separate from 
 one another soon after division, but occasionally they 
 remain together for a time, forming pairs and chains. 
 Under certain conditions of nutrition long threads or 
 filaments are formed, whirh, however, when put in 
 contact with new food, break up into fragments. At 
 times, when the culture media are exhausted or nearly 
 so, the bacilli and spirilla will be found to go on divid- 
 ing, with little or no increase in length, and thus coc- 
 cus-like forms result ; but when these are given fresh 
 food under suitable conditions they elongate and repro- 
 duce the usual shaped organisms. According to recent 
 investigations on the subject of cell reproduction, the 
 division of the cell starts from the protoplasmic layer, 
 the central space being passively destroyed, and the 
 outer envelop is only secondarily concerned in the 
 process. This would indicate that the central space 
 is not a true nucleus, otherwise the division of the 
 nucleus should precede the cell division. The com- 
 plete process of cell reproduction in most varieties 
 occupies, under favorable conditions, about twenty to 
 thirty minutes. 
 
 But although elongation in the greater diameter 
 and transverse division is the rule for the majority of 
 bacteria, there are certain groups, as the sarcinse, for 
 example, which divide more or less regularly in three 
 directions. Instead of becoming separated from each 
 
46 BACTERIOLOGY. 
 
 other as single cells, the tendency then is for the seg- 
 mentation to be incomplete, the cells remaining together 
 in masses. The indentations upon these masses or 
 cubes, which indicate the point of incomplete fission, 
 give to these bundles of cells the appearance commonly 
 ascribed to them that of a bale of rags. As already 
 said, division in two opposite directions results in the 
 formation of a group of forms as tetrads. Division 
 irregularly in all directions results in the production 
 of clusters. The rod-shaped bacteria never divide 
 longitudinally. 
 
 Spore-formation must be distinguished from vegeta- 
 tive reproduction. This is the process by which the 
 organisms are enabled to enter a stage in which they 
 resist deleterious influences to a much higher degree 
 than is possible for them in the growing or vegetative 
 condition. There are two kinds of spores which have 
 been described : 1. Endospores, which are strongly 
 refractile and glistening in appearance, oval or round 
 in shape, and developed within the interior of the cell. 
 They are characterized by the power of resisting to a 
 considerable extent the injurious influences of heat, 
 desiccation, and chemical disinfectants. 2. Arthro- 
 sporeSy or jointed spores, developed not within the cell 
 but as a sprout like separation of one of its extremi- 
 ties. These jointed bodies are believed by some to 
 have also greater resisting power to desiccation, etc., 
 than the ordinary cells, though less than the endo- 
 spores, and to serve the purpose of reproductive 
 elements. Recent researches into the formation of 
 arthrospores, however, have resulted in nothing defi- 
 nite, and the question of their existence even in bac- 
 teria still remains open. In describing the biological 
 
GENERAL CHARACTERISTICS OF BACTERIA. 47 
 
 characters, therefore, of the various organisms whenever 
 spores are mentioned, it will be understood that only 
 the endogenous spores are meant. 
 
 FIG. 11. 
 
 Unstained spores in slightly distended bacilli. (The spores are the light 
 spots in heavily stained bacilli.) 
 
 FIG. 12. 
 
 FIG. 13. 
 
 
 Spores in distended ends 01 
 bacilli. 
 
 Unstained spores in centre of bacilli 
 arranged in chains. 
 
 The production of endospores in the different species 
 of bacteria, though not identical, is very similar. To 
 observe the formation of spores in any species it is best 
 to employ a streak culture on nutrient agar or a potato 
 culture, which should be kept at the temperature nearest 
 
48 BACTERIOLOGY. 
 
 the optimum of the organism to be examined. If at the 
 end of twelve, eighteen, twenty-four, thirty, thirty-six 
 hours, etc., specimens of the culture are observed first 
 unstained in the hanging drops, and then, if round or 
 oval, highly refractile bodies are seen, they should be 
 stained for spores. 
 
 According to Fischer motile bacteria always come to 
 a state of rest or immobility previous to spore-forma- 
 tion. Several species first become elongated. The 
 anthrax bacillus does this, and a description of the 
 method of its production of spores may serve as an 
 illustration of the process In the beginning the pro- 
 toplasm of the elongated filaments is homogeneous, 
 but after a time it becomes turbid and finely granular. 
 These fine granules are then replaced by a smaller 
 number of coarser granules, which are finally amalga- 
 mated into a spherical or oval refractile body. This 
 is the spore. As soon as the process is completed there 
 appears between two spores a delicate partition wall. 
 For a time the spores are retained in a linear position 
 by the cell membrane of the bacillus, but this is later 
 dissolved or broken up and the spores are set free. 
 Not all the cells that make the effort to form spores, 
 as shown by the spherical bodies contained in them, 
 bring these to maturity; indeed, many varieties, under 
 certain cultural conditions, lose their property of forming 
 spores. The following are the most important spore 
 types : (a) The spore lying in the interior of a short 
 undistended cell ; (6) the spore lying in the interior of 
 a short undistended cell forming one of the elements of 
 a long filament ; (c) the spore lying at the extremity 
 of an undistended cell much enlarged at that end the 
 so-called " head spore ;" and (d) the spore lying in 
 
GENERAL CHARACTERISTICS OF BACTERIA. 49 
 
 the interior of a cell very much enlarged in its central 
 portion, giving it a spindle shape. 
 
 The germination of spores takes place as follows : 
 By the absorption of water they become swollen and 
 pale in color, losing their shining, refractile appear- 
 ance. Later a little protuberance is seen upon one side 
 or at one extremity of the spore, and this rapidly 
 grows out to form a rod which consists of soft-growing 
 protoplasm enveloped in a membrane which is formed 
 of the endosporium or inner layer of the cellular en- 
 velope of the spore The outer envelope, or exosporium, 
 is cast off, and may be seen in the vicinity of the 
 newly-formed rod. Sometimes the vegetative cell 
 emerges from one extremity of the oval spore, and 
 in other species the exosporium is ruptured and the 
 bacillus emerges from the side. 
 
 In old cultures of bacteria, where the deleterious sub- 
 stances have developed and the food-stuffs have been 
 largely used, there are frequently found very irregular 
 or distorted forms, due to the abnormal development and 
 division of the bacterial cells under the unfavorable 
 conditions present. These are spoken of as involution 
 or degenerated forms. If these deformed cells are placed 
 under suitable conditions they produce again normally 
 fashioned organisms. 
 
CHAPTER II. 
 
 THE CHEMICAL COMPOSITION OF BACTERIA THE 
 CONDITIONS SUITABLE FOR THEIR GROWTH. 
 
 Chemical Composition. Qualitatively considered, the 
 bodies of bacteria consist largely of water, salts, fats, 
 and albuminous substances. There are also present, 
 in smaller quantities, extractive substances soluble in 
 alcohol and in ether. According to Cramer, there is 
 no grape-sugar found in any bacterial species, but many 
 bacteria contain amyloid substances which give a blue 
 reaction with iodine. True cellulose has been found in 
 the bacillus subtilis and an organism closely allied to 
 the bacillus coli; the tubercle bacillus also forms cellu- 
 lose in the animal body, though no cellulose has been 
 found in cultures of the tubercle bacillus. But from 
 these and from cultures of a " capsule bacillus from 
 water/ 7 allied to the pneumococcus of Friedlander, 
 large quantities of a gelatinous carbohydrate similar 
 to hemi-cellulose have been obtained. Nuclein, first 
 demonstrated by Vanderville, is only found with diffi- 
 culty; but the nuclein bases xanthin, guanin, and 
 adenin have been found in considerable amounts. 
 There is a group of bacteria which contain sulphur 
 viz., the begyiatoa and another group, the cladothrix, 
 is capable of separating ferric oxide from water con- 
 taining iron. 
 
 Some light has been thrown upon the chemical com- 
 
CHEMICAL COMPOSITION OF BACTERIA. 51 
 
 position of bacteria, quantitatively, by the studies of 
 Cramer, though so far only a few species have been 
 thoroughly investigated. The percentage of water 
 contained in bacteria grown on solid culture media, as 
 well as the amount of ash, depend largely on the 
 composition of the media. Thus the bacillus prodigio- 
 sus when grown on potato contains 21.5 per cent, of 
 dry residue and 2.7 per cent, of ash ; when cultivated 
 on turnips it contains 12.6 per cent, of dry residue and 
 1.3 per cent, of ash. Beside the concentration of the 
 culture, its temperature and age also influence the 
 amount of residue and ash produced. The residue 
 varies, moreover, in its composition in the same species 
 under the influence of the culture media employed. 
 Thus the Friedlander pneumonia bacillus grown on 
 nutrient agar containing peptone yields of residue : 
 
 With 1 per cent. With 5 per cent. 
 
 peptone. peptone. 
 
 Nitrogenous matter . 71.7 per ct. 79.8 per ct. 
 Extractives . . . 10.3 " 11.3 " 
 
 Ash .... 13.9 " 10.3 " 
 
 With 1 per ct. peptone 
 
 + 5 per ct. glucose. 
 Nitrogenous matter .... 63.6 per ct. 
 
 Extractives . . . . . .22.7 " 
 
 Ash . 7.8 " 
 
 It would thus appear that an additional quantity of 
 peptone in the culture media tends to increase the per- 
 centage of nitrogenous matter in the bacillus, while 
 the addition of glucose decreases it. 
 
 The cholera spirillum shows still greater variations 
 in the residue when grown in soda bouillon containing 
 albumin than in Uschinsky's medium, which is free 
 from albumin. Thus Cramer found as an average 
 
52 BACTERIOLOGY. 
 
 yield of residue from five different varieties of cholera 
 spirilla : Albumin 65 per cent, and ash 31 per cent, 
 when grown in soda bouillon, while in Uschinsky's 
 solution there was only 45 per cent, albumin and 11 
 per cent. ash. The five varieties of spirilla which in 
 soda bouillon yielded almost exactly the same quanti- 
 ties of albumin and ash, in the other medium free from 
 albumin exhibited a very variable composition. This 
 shows how little dependence can be placed upon any 
 single chemical or cultural reaction for the differentia- 
 tion of two species of bacteria. Judging from the per- 
 centage composition of the cholera spirilla when grown 
 in the Uschinsky medium, these five varieties, taken 
 from Paris, Hamburg, Shanghai, etc., might be con- 
 sidered to be different species, whereas they were prob- 
 ably merely varieties of the same species of bacteria. 
 
 CONDITIONS OF GROWTH. 
 
 Culture Media. Although there are among the bac- 
 teria related to disease a number which are met with 
 only in the bodies of living animals or plants, and, 
 therefore, so far as we know, strictly parasites, yet 
 most pathogenic bacteria can be cultivated more or less 
 readily in artificial culture media under suitable con- 
 ditions, as, for example, the tubercle bacillus and the 
 gonococcus. The majority of bacteria which occur 
 usually as saprophytes are easily cultivated artificially; 
 but there are some, such as various 'micro-organisms 
 found in the saliva and in water, which, with our 
 present knowledge, are either difficult or impossible to 
 cultivate. 
 
 All bacterial culture media must contain an abun- 
 
CONDITIONS OF GROWTH. 53 
 
 dance of water ; salts are also indispensable, and there 
 must be organic material as a source of carbon and 
 nitrogen. The greater number of important bacteria 
 and all the pathogenic species thrive best in media 
 containing albuminoid substances and of a slightly 
 alkaline reaction. The demands of bacteria in the 
 composition of the culture media vary very consid- 
 erably. There are some species of water bacteria, 
 for instance, which require so little organic material 
 that they will grow in water that has been twice 
 distilled. In such cases development probably takes 
 place owing to some contamination of the water, or 
 else through the decomposition of the ammonia and 
 carbonic acid in the air. A certain species will 
 grow abundantly in water containing ammonium car- 
 bonate in solution and no other source of carbon and 
 nitrogen. This shows the power of some bacteria of 
 producing cell substance from the simplest materials a 
 power which belongs to the higher plants which obtain 
 their nourishment from the air through their chlorophyl 
 and the assistance of sunlight. Few bacteria, how- 
 ever, of any importance in medicine are so easily satis- 
 fied, though there are many species which are able 
 to develop without the presence of albumin and in 
 comparatively simple culture media, such as the culture 
 liquid proposed by Uschinsky, or the simpler one of 
 Voges and Fraenkel, which consists of: Water, 1000; 
 sodium chloride, 5 ; neutral sodium phosphate, 2 ; 
 ammonium acetate, 6; and asparagin, 4. In these 
 media many bacteria grow well. 
 
 When we consider in detail the source of the more 
 important chemical ingredients of bacteria we find that 
 their nitrogen is most readily obtained from diffusible 
 
54 BACTERIOLOGY. 
 
 abuminoid material and less easily from ammonium 
 compounds. Their carbon they derive from albumin, 
 peptone, sugar and other allied carbohydrates, glycerin, 
 fats, and other organic substances. It is an interest- 
 ing fact, that even compounds which in considerable 
 concentration are extremely poisonous, can, when in 
 sufficient dilution, provide the necessary carbon; thus 
 some derive it from carbolic acid in very dilute solu- 
 tions. Another species of bacteria isolated by Wino- 
 gradsky were shown by him to derive their carbon 
 from CO 2 . 
 
 The value of substances as a source of .nutrition is 
 often influenced by the presence of other materials, as, 
 for instance, the value of asparagin is increased by the 
 presence of sugars. Further, material from which 
 nitrogen and carbon cannot be directly obtained still 
 become assimilable after being subjected to the influ- 
 ence of bacterial ferments. The profound and diverse 
 changes produced by the different ferments make it 
 almost impossible to establish, except in the most gen- 
 eral way, the nutritive value of any mixture for a large 
 number of bacteria through a simple knowledge of its 
 chemical composition. The special culture media, such 
 as bouillon, blood-serum, etc., for the development of 
 bacteria will be dealt with in a later chapter. 
 
 The relation of bacteria to oxygen: The majority abso- 
 lutely require oxygen for their growth, but a consider- 
 able minority fail to grow unless it is excluded. A 
 knowledge of this latter fact we owe to Pasteur, who 
 divided bacteria into aerobic and anaerobic. Between 
 these two groups we have those that can grow either 
 with or without the access of oxygen. 
 
 Some at least of the strict anaerobic bacteria require 
 
CONDITIONS OF GROWTH. 55 
 
 for the full development of their life functions the 
 presence of fermentable substances, such as sugars, 
 from which they obtain oxygen. Among bacteria can 
 be found all gradations between those bacteria which 
 develop only in the presence of oxygen to those which 
 develop only in its absence. In so far as for any 
 variety the amount of oxygen present is unfavorable 
 there will be more or less restriction in some of the life 
 processes of these bacteria, such as pigment and toxin 
 production, spore formation, etc. It has also been 
 found that some, at least, of the aerobic bacteria can 
 be accustomed to grow without oxygen, and that some 
 of the anaerobics can be accustomed to grow with it. 
 
 Sulphur and phosphorus are two important food-stuffs 
 required by bacteria. Either calcium or magnesium 
 and sodium or potassium are also usually required for 
 bacterial growth. Iron is demanded by but few varie- 
 ties. 
 
 When we consider the more complex culture media, 
 either those naturally existing, such as blood-serum, 
 or those created by us for the cultivation of bacteria, 
 we find, beyond the necessary amount of soluble food- 
 stuffs, that the relative proportion of each form and 
 the total concentration are of great importance. It is, 
 nevertheless, true that very wide differences can exist 
 with but slight effect upon the development of bacteria, 
 the development of the bacteria usually ceasing through 
 the accumulation of deleterious substances in the cul- 
 ture media rather than through food exhaustion. 
 
 The reaction of the nutritive media is of very great 
 importance. Most bacteria grow best in those that are 
 slightly alkaline or neutral. Only a few varieties require 
 an acid medium, and none of these belong to the parasitic 
 
56 BACTERIOLOGY. 
 
 bacteria. An amount of acid or alkali insufficient to 
 prevent the development of bacteria may still suffice to 
 rob them of some of their most important functions, 
 such as the production of poison. The different effect 
 upon closely allied varieties of bacteria of a slight 
 excess of acid or alkali is sometimes made use of in 
 separating those which may be closely allied in many 
 other respects. 
 
 The influence of one species upon the growth of 
 another, either when the bacteria grow together or 
 follow one another, is very marked. The development 
 of one variety of bacteria in a medium causes that sub- 
 stance, in the majority of instances, to become less suit- 
 able for the growth of other bacteria. This isxdue 
 partly to the impoverishment of the food-stuff, but 
 more to the production of chemical substances or 
 enzymes, which are antagonistic not only to the growth 
 of the bacteria producing them, but to many other 
 varieties also; less frequently the changes produced by 
 one variety of bacteria in the food-stuff are favorable 
 for some other form. 
 
 For the growth of bacteria a suitable temperature is 
 absolutely requisite. For different varieties the most 
 favorable temperature varies, but for all a range of 
 about 2J C. above or below this most favorable point 
 covers the limits for their most vigorous growth. Few 
 bacteria grow well under 10 C. and few over 40 C. ; 
 2 C. is about the lowest temperature that any bacteria 
 have been found to grow and 70 C. the highest. 
 
 In many instances the temperature of the soil in 
 which the bacteria are deposited is the controlling 
 factor in deciding whether growth will or will not take 
 place. Thus nearly all parasitic hacleria require a 
 
CONDITIONS OF GROWTH. 57 
 
 temperature near that of the body for their develop- 
 ment, while many saprophytic bacteria can grow only 
 at much lower temperatures. Bacteria when exposed 
 to lower temperature than suffices for their growth, 
 while having their activities decreased, are not other- 
 wise injured; while exposure to higher temperatures 
 than allows of growth destroys the life of the bacteria. 
 The relations of the temperature to bacterial life and 
 death will be dealt with fully in a later chapter. 
 
CHAPTER III. 
 
 VITAL PHENOMENA OF BACTERFA MOTION, HEAT 
 AND LIGHT PRODUCTION CHEMICAL EFFECTS. 
 
 Motility. Many bacteria when examined under the 
 microscope are seen to exhibit active movements in 
 fluids. This motility is produced by the fine hair-like 
 flagella attached to all motile species. The movements 
 are of a varying character, being described as creeping, 
 waddling, rotary, undulatory, sinuous, snake-like, etc. 
 At one time they may be slow and sluggish, at another 
 so rapid that any detailed observation is impossible. 
 Some bacteria are very active in their movements, dif- 
 ferent individuals progressing rapidly in different direc- 
 tions, while with many it is difficult to say positively 
 whether there is any actual motility or whether the 
 organism shows only molecular movements so-called 
 " Brownian " movements a dancing, trembling mo- 
 tion possessed by all finely divided organic particles. 
 If in doubt in such cases it is best, wkeVe the matter is 
 of importance and one is skilled in the technique of 
 staining, to stain the organisms for flagella, and also to 
 examine them in a 0.1 per cent, bichloride of mercury 
 solution, when, if the movements continue, they are 
 purely molecular. Not all species of bacteria which 
 have flagella, however, exhibit at all times spontaneous 
 movements; such movements may be absent in certain 
 culture media and at too low or too high temperatures, 
 or of either an insufficient or excessive supply of oxygen. 
 
VITAL PHENOMENA OF BACTERIA. 59 
 
 Some chemical substances seem to exert a peculiar 
 attraction for bacteria, known as positive chemotaxis, 
 while others repel them negative chemotaxis. More- 
 over, all varieties are not affected alike, for the same 
 substances may exert on some bacteria an attraction 
 and on others a repulsion. Oxygen, for example, 
 attracts aerobic and repels anaerobic bacteria, and for 
 each variety there is a definite proportion of oxygen, 
 which most strongly attracts. The chernotaxic prop- 
 erties of substances are tested by pushing the open end 
 of a fine capillary tube, filled with the substance to be 
 tested, into the edge of a drop of culture fluid contain- 
 ing bacteria and examining the hanging drop under 
 the microscope. We are able thus to watch the action of 
 the bacteria and note whether they crowd about the tube 
 opening or are repelled from it. Substances showing 
 positive chemotaxis for nearly all bacteria are peptone, 
 urea, and very weak solutions of bichloride of mercury. 
 While among those showing negative chemotaxis are 
 alcohol and many of the metallic salts. 
 
 The Production of Light. Bacteria which have the 
 property of emitting light are quite widely distributed 
 in nature, particularly in media rich in salt, as in sea- 
 water, salt fish, etc. Many of these, chiefly bacilli and 
 spirilla, have been accurately studied. The emission of 
 light is a property of the living protoplasm of the bac- 
 teria, and is not usually due to the oxidation of any pho- 
 togenic substance given off by them ; at least only in two 
 instances has such substance been claimed to have been 
 isolated. Every agent which is injurious to the exist- 
 ence of the bacteria affects this property. Thus, cold 
 paralyzes them and interrupts their power of emit- 
 ting light. High temperature, acids, chloroform, etc., 
 
60 BACTERIOLOGY. 
 
 inhibit for a time or destroy this property. Living bac- 
 teria are always found in phosphorescent cultures; a 
 filtered culture free from germs is invariably non-phos- 
 phorescent; but while the organism cannot emit light ex- 
 cept during life, it can live without emitting light, as in 
 an atmosphere of carbonic acid gas, for instance. Most 
 organisms require, in order to be able to emit light, the 
 presence of peptone and oxygen, and many also need 
 carbon and nitrogen. They are best grown under free 
 access of oxygen in a culture medium prepared by 
 boiling fish in sea- water (or water containing 3 per 
 cent, sea-salt), to which 1 per cent, peptone, 1 per 
 cent, glycerin, and 0.5 per cent, asparagin are added. 
 Even in this medium the power of emitting light is 
 soon lost unless the organism is constantly trans- 
 planted to fresh m^dia. 
 
 Thermic Effects. The production of heat by bacteria 
 does not attract attention in our usual cultures because 
 of its slight amount, and even fermenting culture 
 liquids with abundance of bacteria cause no sensa- 
 tion of warmth when touched by the hand. Careful 
 tests, however, show that heat is produced. The 
 increase of temperature in organic substances when 
 stored in a moist condition, as tobacco, hay, manure, 
 etc., is one partly at least due to the action of bacteria. 
 Rabinowitsch suggests that very probably the high tem- 
 perature which is here exhibited is caused in part by the 
 so-called thermophilic bacteria, but there are no accurate 
 observations as to the true source of this heat. 
 
 Chemical Effects. The processes which bodies being 
 split up undergo depend, first, on the chemical nature 
 of the bodies involved and the conditions under which 
 they exist, and, secondly, on the varieties of bacteria 
 
VITAL PHENOMENA OF BACTERIA. 61 
 
 present. A complete description of these chemical 
 changes is at present impossible. Chemists can as yet 
 only enumerate some of the final substances evolved, 
 and describe, in a few cases, the manner in which they 
 were produced. Bacteria are able to construct their 
 body substance out of various kinds of nutrient mate- 
 rials and also to produce fermentation products or 
 poisons, and they are able to do these things either 
 analytically or synthetically with almost equal ease. 
 This ambidextrous metabolic power exists, according 
 to Hueppe, among bacteria to an extent known as yet 
 among no other living things. 
 
 In the chemical building up of their body substance 
 we can distinguish, as Hueppe concisely puts it, several 
 groups of phenomena: Polymerization, a sort of doub- 
 ling up of a simple compound; synthesis, a union of 
 different kinds of simple compounds into one or more 
 complex substances; formation of anhydride, by which 
 new substances arise from a compound through the loss 
 of water; and reduction or loss of oxygen, which is 
 brought about especially by the entrance of hydrogen 
 into the molecule. The breaking down of organic 
 bodies of complicated molecular structure into simpler 
 combinations takes place, on the other hand, through 
 the loosening of the bonds of polymerization; through 
 hyd ration or entrance of water into the molecule, and 
 through oxidation. 
 
 The chemical effects which take place from the action 
 of bacteria are greatly influenced by the presence or 
 absence of free oxygen. The access of pure atmos- 
 pheric oxygen makes the life processes of most bacteria 
 more easy, but is not indispensable when available 
 substances are present which can be broken up with 
 
62 BACTERIOLOGY. 
 
 sufficient ease. The standard of availability is very 
 different for different bacteria. Life processes carried 
 on without oxygen do not effect any profound molecular 
 changes in the organic material which is broken up; 
 but in order that the living organism may obtain the 
 requisite quantities of energy from this mode of life, 
 a proportionately large amount of material must be 
 superficially disintegrated. Therein lies the mechan- 
 ical foundation for the power of a small amount of fer- 
 ment to cause the production of much alcohol or lactic 
 acid, and that parasites which have invaded the living 
 body can generate intensely poisonous substances out 
 of the body proteids. 
 
 In the presence of oxygen the decomposition products 
 that are formed by the attack of the anaerobic bacteria 
 are further decomposed and oxidized by the aerobes; 
 they are thereby rendered, as a rule, inert, and conse- 
 quently harmless. Some bacteria have adapted them- 
 selves to the exclusive use of compound oxygen, using 
 those compounds from which oxygen can be obtained, 
 and others the obligatory anaerobes are able to live 
 only in the presence of free oxygen, The facts of anae'ro- 
 biosis are of great importance to technical biology and 
 to pathology. Since, under strictly anaerobic conditions, 
 any secondary oxidation of the primary decomposition 
 products is impossible, the latter accumulate without 
 formation of by-products. Many parasitic bacteria are 
 found to produce far more poison in the absence of air 
 than in its presence. 
 
 Organized and Unorganized Ferments. All the chem- 
 ical effects of bacteria are largely dependent upon the 
 composition of the culture media. Thus many species 
 of bacteria which in albuminous media produce no 
 
VITAL PHENOMENA OF BACTERIA. 63 
 
 visible change, when sugar is added decompose it, with 
 the production of gas. The term fermentation is dif- 
 ferently used by different authors. Some call every 
 kind of decomposition due to bacteria a fermentation, 
 speaking thus of the putrefactive fermentation of albu- 
 minous substances; others limit the term to the process 
 when accompanied by the visible production of gas; 
 others, again, take fermentation to mean only the 
 decomposition of carbohydrates, with or without gas- 
 production. 
 
 Fermentation may be defined as a chemical decom- 
 position of an organic compound, induced by living 
 organisms or substances contained within them (organ- 
 ized ferments), or by chemical substances thrown off 
 from the bacteria (unorganized or chemical ferments 
 or enzymes). In the first the action is due to the 
 growth of the organisms producing the ferment, 1 as 
 in the formation of acetic acid from alcohol by the 
 action of the vinegar-plant, and in the second the 
 enzyme causes a structural change without losing its 
 identity, as in digestion. These enzymes even when 
 present in the most minute quantities have the power 
 of splitting up or decomposing complex organic com- 
 pounds into simpler, more easily soluble and diffu- 
 sible molecules. We can only speak of chemical fer- 
 ments when it can be demonstrated that the fermenta- 
 tion continues in the absence of all living bacteria. 
 
 1 Buchner (Berichte d. deutsch. chem. Gesellsch., xxx. 117-124 and 1110- 
 1113) has shown that even in those cases of fermentation in which, until 
 lately, we have believed the organized cell itself was necessarily concerned 
 that the cell protoplasm squeezed from its capsule is able to cause the same 
 changes as the organized cells. This brings fermentation by unorganized and 
 organized ferments very closely together, the one being a substance thrown 
 off from the cell, the other a substance ordinarily retained in the cell. The 
 increase of both ceases with the death of the bacteria producing them. 
 
64 BACTERIOLOGY. 
 
 This may be accomplished by the addition of disinfect- 
 ants carbolic acid, chloroform, ether, etc. to the cul- 
 tures or by filtration. Ferments, like albuminoids, 
 are non-dialyzable. They withstand dry heat, but are 
 destroyed in watery solutions by a temperature of over 
 70 C. They are injured by acids, especially the 
 morganic ones, but are resistant to all alkalies. All 
 fermentation has for its object the acquisition by the 
 organism of a store of energy. This is accomplished 
 in either of the ways above mentioned. The simplest 
 and commonest example of decomposing fermentation 
 produced by an enzyme is that of sugar : 
 
 C 6 H 12 O 6 = 2C 2 H 6 O + 2CO 2 
 Grape-sugar. 2 Alcohol. 2 Carbon dioxide. 
 
 or, 
 
 C 6 H 12 6 = 2C 3 H 6 3 
 Grape-sugar. 2 Milk-sugar. 
 
 or, 
 
 CVH 12 O 6 = 3C 2 H 4 O 2 
 Grape-sugar. 3 Acetic acid. 
 
 Bacteria which develop in the absence of oxygen are 
 especially in need of this source of oxygen. Anaerobic 
 bacteria, for this reason, have the power of decomposing 
 sugar, while many facultative anaerobes are only capable 
 of producing fermentation when oxygen is excluded. 
 
 Opposite to this, and far less common, is oxidizing 
 fermentation, as in the production of acetic acid from 
 alcohol. Here the energy is acquired not by the de- 
 composition but by the oxidation of the alcohol. 
 
 The proteolytic or peptonizing ferments which are 
 somewhat analogous to pepsin and trypsin being capa- 
 ble of changing albuminous bodies into soluble and 
 diffusible substances are very widely distributed. 
 The liquefaction of gelatin, which is chemically allied 
 
VITAL PHENOMENA OF BACTERIA, 65 
 
 to albumin, is due to the presence of a proteolytic fer- 
 ment or trypsin. It is not pepsin, as pepsin acts only 
 in the presence of acid, and gelatin is liquefied with an 
 alkaline reaction only. The production of proteolytic 
 ferments by different cultures of the same varieties of 
 bacteria varies considerably, far more than is generally 
 supposed. Even among the freely liquefying bacteria, 
 such as the cholera spirillum and the staphylococcus, 
 poorly liquefying varieties have been repeatedly found. 
 These observations have detracted considerably from 
 the value in cultures of the property of liquefying 
 gelatin as a positive diagnostic characteristic. Most 
 conditions which are unfavorable to the growth of bac- 
 teria seem to interfere also with their liquefying power. 
 
 Certain bitter-tasting products of decomposition are 
 formed by liquefying bacteria in media containing albu- 
 min, as, for example, in milk. 
 
 Diastatic ferments convert starch into sugar. That 
 these are produced by bacteria is shown by mixing 
 starch paste containing 1 per cent, thymol with cultures 
 to which 1 to 2 per cent, thymol has been added, and 
 keeping the mixture for six to eight hours in the incu- 
 bating oven; then, on the addition of Fehling's solution 
 and heating, the reaction for sugar appears the red- 
 dish-yellow precipitate due to the reduction of the 
 copper. Bacteria may be directly tested for sugar also 
 by boiling potato-broth cultures and using the extract. 
 
 Inverting ferments that is, those which convert cane- 
 sugar into grape-sugar are of very frequent occurrence. 
 Bacterial invertin withstands a temperature of 100 C. 
 for more than an hour, and is produced in culture media 
 free from albumin. 
 
 Rennet ferments substances having the power of 
 
 5 
 
66 BACTERIOLOGY. 
 
 coagulating milk with neutral reaction, independent of 
 acids are found not infrequently among bacteria. The 
 B. prodigiosus, for instance, in from one to two days 
 coagulates to a solid mass milk which has been steril- 
 ized at 55 to 60 C. These ferments have not been 
 thoroughly investigated: they are probably present, 
 however, in all species of bacteria which coagulate milk 
 with the production of acid. 
 
 Fermentation yields products that are poisonous to 
 the ferment; hence fermentation ceases when the nu- 
 triment is exhausted or the fermentation is in excess. 
 Different kinds of fermentation obtain specific names, 
 according to product. Thus acetic, yielding acetic 
 acid; alcoholic or vinous, yielding alcohol; ammoniacal, 
 yielding ammonia; amylic, yielding amylic alcohol; 
 benzole, yielding benzoic acid; butyric, yielding butyric 
 acid; lactic, yielding lactic acid; and viscous, yielding 
 a gummy mass. 
 
 Pigment Production. Pigments have been little chem- 
 ically studied, but the recent investigations of Klein 
 and Migula, Thumm and Schneider, and others throw 
 some light on the subject. They have no known im- 
 portance in connection with disease, but are of interest 
 and have value in identifying bacteria. 
 
 RED AND YELLOW PIGMENTS. Of the twenty-seven 
 red and yellow bacteria studied by Schneider, almost 
 all produce pigments soluble in alcohol and insoluble 
 in water. The larger majority of these possess in com- 
 mon the property of being colored blue-green by sul- 
 phuric acid and red or orange by a solution of potash. 
 Though varying considerably in their chemical compo- 
 sition and in their spectra, they may be classified, for 
 the most part, among that large group of pigments 
 
VITAL PHENOMENA OF BACTERIA. 67 
 
 common to both the animal and vegetable kingdoms 
 known as lipochromes, and to which belong the pig- 
 ments of fat, yolk of egg, the carotin of carrots, tur- 
 nips, etc. 
 
 VIOLET PIGMENTS. Certain bacteria produce violet 
 pigments, also insoluble in water and soluble in alcohol, 
 but insoluble in ether, benzol, and chloroform. These 
 are colored yellow when treated in a dry state with sul- 
 phuric acid and ernerald-green with potash solution. 
 
 BLUE PIGMENTS are also produced by the so-called 
 fluorescent bacteria, along with a pigment named bac- 
 terio-fluorescein. In cultures the fluorescence is at first 
 blue; later, as the cultures become alkaline, it is green. 
 
 Numerous investigations have been made to deter- 
 mine the cause of the variation in the chrornogenic 
 function of bacteria. All conditions which are unfav- 
 orable to the growth of the bacteria decrease the pro- 
 duction of pigment, as cultivation in unsuitable media 
 or at too low or too high a temperature, etc. The B. 
 prodigiosus produce no pigment at 37 0., and when 
 transplanted at this temperature, even into favorable 
 media, the power of pigment production is gradually 
 lost. 
 
 Otherwise colorless species of bacteria sometimes pro- 
 duce pigments. Thus yellow to red colonies of the 
 pneutnococcus have been observed, and colored varie- 
 ties of the streptococcus pyogenes. Occasionally colored 
 and uncolored colonies of the same species of bacteria 
 may be seen to occur side by side in one plate culture, 
 as, for example, the staphylococcus pyogenes. 
 
 Alkaline Products and the Fermentation of Urea. Aero- 
 bic bacteria sometimes produce alkaline products from 
 albuminous substances in culture media free from sugar. 
 
68 BACTERIOLOGY. 
 
 Most species of bacteria produce acids in the presence 
 of sugar, which explains the fact that neutral or slightly 
 alkaline cultures become acid at first from the small 
 amount of sugar contained in the meat used for making 
 the media. When the sugar is used up the reaction 
 often becomes alkaline, as the production of alkalies 
 continues after the acid formation has ceased. The 
 substances producing the alkalinity in cultures are 
 chiefly ammonia, amine, and the ammonium bases. 
 
 The conversion of urea into carbonate of ammonia 
 affords special evidence of the production of alkaline 
 substances by bacteria: 
 
 CO(NH 2 ) 2 -f 2H 2 CO 3 (NH 4 ) 2 
 
 Urea. 2 Water. Ammonium carbonate. 
 
 Leube has isolated several organisms from putrefy- 
 ing urine which separate ammonia from urea. The 
 power of decomposing urea, however, is not wide-spread 
 among bacteria. Out of twenty-seven organisms studied 
 by Wariugton, only two were found to decompose urea. 
 Of sixty species investigated by Lehmaun, three only 
 developed the odor of ammonia from sterilized human 
 urine. 
 
 Ptomains Toxins. But beside ammonium carbonate, 
 a large number of basic crystalline substances have been 
 recognized, especially by Brieger, as products of bacterial 
 growth. These are now commonly known as ptoma'im, 
 or putrefactive alkaloids (from XTM/UX, putrefaction). 
 
 Nencki, and then later Brieger, Vaughan and others, 
 succeeded in preparing organic bases of a definite chem- 
 ical composition out of decomposing fluids meat, fish, 
 old cheese, and milk undergoing bacterial decomposition 
 as well as from pure bacterial cultures. Some of these 
 were found to exert a poisonous effect, and for a long time 
 
VITAL PHENOMENA OF BACTERIA. 69 
 
 were looked upon as the specific bacterial poisons, while 
 others were harmless. The poisons are particularly in- 
 teresting, since they may be present in the decomposing 
 cadaver (hence the name ptomai'n), and, in consequence, 
 have to be taken into consideration in questions of legal 
 medicine. They may be formed also in the living 
 human body, and, if not made harmless by oxidation, 
 may come to act therein as self-poisons or leuconiai'ns. 
 They are now known not to be the substances to which 
 are due the specific poisonous effects of bacteria which 
 are designated as toxins, and have entirely different 
 characteristics. 
 
 Many ptomaios are known already and the empirical 
 formula of each made out, and among them are some 
 whose exact chemical composition is established. The 
 first of these bodies to be separated was colloidin 
 (C 8 H U N), obtained byNencki from putrefying gelatin. 
 Another, trimethylamin (C 3 H 9 N = (CH 3 ) 3 N), gives an 
 odor like herring-brine. Especially interesting is the 
 substance cadaverin, which was separated by Brieger 
 from portions of decomposing dead bodies and from 
 cholera cultures, by reason of the fact that Ladenburg 
 prepared it synthetically and showed it to be penta- 
 methylenediamin [(NH 2 ) 2 (CH 2 ) 6 ]. The cholin group 
 is particularly interesting. Cholin itself (C 5 H 15 NO 2 ) 
 arises from the hydrolytic breaking-up of lecithin, the 
 fatty substance found in brain tissue and other nervous 
 tissue. 
 
 By the oxidation of cholin there can be produced the 
 non-poisonous betain or trirnethylglycocoll occurring in 
 beet-juice, and the highly toxic muscarin, found by 
 Schmiedeberg in a poisonous toadstool and by Brieger 
 in certain decomposing substances : 
 
70 BACTERIOLOGY. 
 
 C 5 H 15 N0 2 + 2 C 5 H 13 N0 3 + H 2 0. 
 
 Cholin, Betain. 
 
 C 5 H 15 N0 2 + O C 5 H 13 N0 3 
 
 Cholin. Muscarin. 
 
 The ptomai'n tyrotoxiu, obtained from cheese by 
 Vaughan, is apparently derived from butyric acid. 
 
 Pyocyanin (C 14 H 14 N 2 O), which produces the color of 
 blue or blue-green pus, and has been regarded by Led- 
 derhose as related to the coal-tar products, is a ptomainic 
 pigment. Similar bodies of a basic nature may be found 
 in the intestinal contents as the products of bacterial 
 decomposition. Some of these are poisons and can be 
 absorbed into the body, where they play the role of 
 self-poisons, or leucomai'ns. Some believe the symp- 
 toms designated as coma and tetany may be ascribed to 
 the absorption of substances of this nature. Since the 
 name ptomai'n was given to the poisonous products of 
 bacterial growth before these products were chemically 
 understood, and even now, when the name is restricted 
 to crystalline bodies, it is by many frequently applied 
 to all bacterial poisons, as in cases of poisoning due to 
 decomposing meat or sausage or to cheese or milk. 
 Instead of ptomai'ns these may be due to the poisonous 
 proteids or toxins. Such poisonous proteid bodies are 
 always formed in the beginning of decomposition pro- 
 cesses. Some of the ptomai'ns obtained by chemists 
 are due not to putrefactive changes but to the chemi- 
 cal methods used to obtain them. 
 
 The isolation of these substances can here be only 
 briefly referred to. According to Brieger's method, 
 which is the one now generally employed, the cultures 
 having a slight acid reaction (HC1) are boiled down, 
 filtered, and the filtrate concentrated to a syrupy con- 
 
VITAL PHENOMENA OF BACTERIA. 7] 
 
 sistency. This is then dissolved in 96 per cent, alcohol, 
 freed from albumin and other contamination by an 
 alcoholic solution of lead acetate, the lead precipitated, 
 the filtrate concentrated, and again precipitated by an 
 alcoholic solution of mercuric chloride, which forms a 
 double mercury compound with the ptomai'n. The 
 alcohol is evaporated by heat, the mercury separated 
 by sulphuretted hydrogen, and a double compound 
 formed with gold and platinum, the crystal lizability of 
 which permits of its purification; or the crystalline hydro- 
 chloride is directly obtained, and the free bases, which 
 are often liquid, separated by means of sodium hydrate. 
 
 Many of these ptomai'ns, like most vegetable alka- 
 loids when they are are set free by sodium or potassium 
 hydrate, are obtainable by agitation with ether in aque- 
 ous solution; but Brieger's method is preferable, because 
 many substances not taken up by ether are here ex- 
 tracted. 
 
 Complex Albuminoid Poisons Toxalbumins or Toxins. 
 These may be divided into two classes: 
 
 1. BACTERIAL PROTEINS (Buchner). By these are 
 understood poisonous substances of a proteid nature 
 produced by bacteria which are not affected by heat, 
 which are capable of producing fever (pyogenic) and 
 causing inflammation (phlogogenic), and which can be 
 obtained by boiling for several hours potato cultures 
 treated with an 0.5 per cent, solution of potassium 
 hydrate (about 50 volumes of potassium hydrate to 1 
 volume of bacterial substance). From the clear, fil- 
 tered liquid the proteins are precipitated by weak acid, 
 carefully added, and the precipitate washed and dried; 
 before use they can be dissolved in weak soda solution. 
 
 The best known protein is Koch's old tuberculin; 
 
72 BACTERIOLOGY. 
 
 mallein is another. According to Buchner and Romer, 
 all bacterial proteins are very similar in their action, 
 and Mathes maintains that deutero-alburuose, which is 
 obtained by the action of pepsin on albumin and has 
 no connection with bacteria, has an effect on tubercular 
 guinea-pigs somewhat similar to tuberculin. 
 
 Toxins Active Proteids. Tox ALBUMINS. Fraenkel 
 and Brieger, confirming the statements of previous in- 
 vestigators (Christmas, Roux and Yersin, and Hankin), 
 have shown that amorphous poisons having an intense 
 and often specific toxic action that is an effect similar 
 to that produced by infection with the living organism 
 may be precipitated from bouillon cultures by agents 
 precipitating albumin. They therefore called these 
 poisons "toxalburnins," and regarded them as being 
 analogous to the toxalbumins of vegetable origin, like 
 ricin from the castor-oil bean (Ricinis communis), 
 and abrin from the jequirity bean (Abras precatorius). 
 The majority of investigators, however, consider these 
 poisons to be "labile" 1 albuminous substances, which 
 are derived from the bacterial cells. Some have assumed 
 that they were similar to snake poisons or to the 
 enzymes. Like these substances, they are very sensi- 
 tive to the action of heat, chemical agents, light, etc. 
 
 Toxalbumins are obtained as crude substances by the 
 precipitation of the products of fully developed cultures 
 in bouillon under a vacuum, with absolute alcohol or 
 ammonium sulphate, after the culture fluid has been 
 freed from living germs by its passage through a por- 
 celain filter. When ammonium sulphate is used the 
 precipitate is freed from this reagent by dialysis 
 
 1 So-called "labile" substances are such as are proue to undergo chemical 
 changes or alterations of atomic structure ; unstable. 
 
VITAL PHENOMENA OF BACTERIA. 73 
 
 through parchment against running water, and after 
 concentration the substances are again precipitated by 
 absolute alcohol. Recently it has been found that zinc 
 chloride separates these bodies quantitatively, and that 
 the toxins may be obtained from this precipitate by 
 means of sodium phosphate (Brieger and Boer). 
 
 All along, however, some doubt has been expressed 
 as to whether these so-called toxalbumins were really 
 only obtainable by precipitation from albumin or 
 whether they had anything to do with albumin at 
 all. With regard to tetanus poison, Brieger and 
 Cohn have now succeeded in obtaining what they 
 consider an almost pure toxin from the crude poison by 
 means of acetate of lead and ammonia. This substance 
 gives a slight violet color with copper sulphate and 
 soda solution, but otherwise no albumin reaction; it 
 contains neither phosphorus nor sulphur, and is appar- 
 ently not an albuminous substance. The statement 
 previously made by Uschinsky that he had obtained 
 albuminoid tetanus and diphtheria poisons in culture 
 media devoid of albumin could not, heretofore, be con- 
 firmed, owing to the difficulty experienced by most 
 investigators in getting a sufficient growth of these 
 organisms on such media. Brieger and Cohu have 
 found that cholera spirilla produce a non-albuminous 
 poison in Uschinsky' s culture media (free from albu- 
 min); and now diphtheria toxin has been recognized to 
 be non-albuminous (Brieger and Boer). It is becoming 
 more and more customary to call proteid bacterial poisons 
 simply toxins , irrespective of their composition, and to 
 ignore the existence of the above- described crystallizable 
 toxins of simple constitution., 
 
 With regard to the other properties of these toxins, 
 
74 BACTERIOLOGY. 
 
 taking tetanus toxin as an example, it may be said that 
 in aqueous solution it is not coagulated by heat, but is 
 in time deprived of its poisonous qualities. The addi- 
 tion of small quantities of acids or alkalies to the solu- 
 tion, and the continued passage through it of carbon 
 dioxide or sulphuretted hydrogen, distinctly reduce its 
 toxicity. When exposed to light and air, either in a 
 dry state or in solution, the toxin deteriorates rather 
 rapidly. It withstands a temperature of 70 C. for 
 some time without being wholly destroyed ; higher 
 temperatures decompose it rapidly. When protected 
 from the light and air it is slowly converted into an in- 
 active substance; it is better preserved under absolute 
 alcohol, pure ether, and the like. The toxicity of the 
 purest tetanus toxin now obtainable is almost incred- 
 ible : 0.00005 milligramme of it kills a mouse of 15 
 grammes; a man of 150 pounds weight, if he were equally 
 susceptible, would be killed with 0.23 milligrammes. 
 It requires 30 to 100 milligrammes of strychnine to 
 kill a man under ordinary circumstances. The most 
 virulent diphtheria bacilli produce a specific poison 
 which does not fall far behind that of tetanus in power. 
 Sulphuretted Hydrogen. Sulphuretted hydrogen is a 
 very common bacterial product. Its presence is deter- 
 mined by pasting a piece of paper moistened with lead 
 acetate inside the neck of the flask containing the cul- 
 ture, closing the mouth with a cotton-wool stopper, and 
 over this again an India-rubber cap (black rubber free 
 from sulphur). The paper is colored at first brownish 
 and later black; repeated observation is necessary, as 
 the color sometimes disappears toward the end of the 
 reaction. Apparently negative results should not be 
 rashly accepted as conclusive. 
 
VITAL PHENOMENA OF BACTERIA. 
 
 Sulphuretted hydrogen may be formed: 
 
 1. From albuminous substances. This power, ac- 
 cording to Petri and Maassen, of forming sulphuretted 
 hydrogen, particularly in liquid culture media contain- 
 ing much peptone (5 to 10 per cent.) and no sugar, is 
 possessed, though in different degree, by all bacteria 
 examined by them; only a few bacteria form H 2 S in 
 bouillon in the absence of peptone, while about 50 per 
 cent, in media containing 1 per cent, peptone. 
 
 2. From powdered sulphur. All bacteria produce in 
 culture media to which pure powdered sulphur is added 
 considerably more H 2 S than without this addition. 
 Petri and Maassen suggest that this is due to the 
 nascent hydrogen produced by the bacteria. 
 
 3. From thiosulphates and sulphites. Studied par- 
 ticularly in yeast, but demonstrated also by Petri and 
 Maassen in several bacteria. 
 
 The presence of sugar in the culture does not affect 
 the production of H 2 S by bacteria, but saltpetre reduces 
 it, nitrites being formed. The absence of oxygen favors 
 the production of H 2 S. Light diminishes the develop- 
 ment of H 2 S by facultative anaerobes, sulphates being . 
 formed instead. 
 
 Reduction Processes. All bacteria, as we have seen, 
 possess the property of converting sulphur into sul- 
 phuretted hydrogen, for which purpose is required the 
 presence of nascent hydrogen. The following pro- 
 cesses depend also in part upon the action of nascent 
 hydrogen : 
 
 1. The reduction of blue litmus pigments, methylene- 
 blue, and indigo to colorless substances. The superficial 
 layer of cultures in contact with the air shows often no 
 reduction, only the deeper layers being affected. By 
 
76 BACTERIOLOGY. 
 
 agitation with access of air the colors may be again re- 
 stored, but at the same time, acid being formed, the 
 litmus pigment is turned red. According to Colin, the 
 property of reducing litmus belongs to all liquefying 
 bacteria, but some non-liquefying species also possess it. 
 
 2. The reduction of nitrates to nitrites and am- 
 monia. The first of these properties seems to pertain 
 to a great many bacteria at least Petri and Maassen 
 found in six species, grown in bouillon containing 2.5 
 to 5 per cent, peptone and 0.5 per cent, nitrate, that 
 almost all produced nitrite abundantly; once only was 
 ammonia observed. In a number of bacteria studied 
 by Rubner only one failed to produce nitrite. The 
 test for nitrites is made as follows : Two bouillon tubes 
 containing nitrates are inoculated, and, along with two 
 unirioculated tubes, are allowed to remain in the incu- 
 bator for several days; then to the cultures and control 
 test is added a small quantity of colorless iodide of 
 starch solution (thin starch-paste containing 0.5 per 
 cent, potassium iodide) and a few drops of pure sul- 
 phuric acid. The control tubes remain colorless or 
 become gradually slightly blue, while if nitrites are 
 present a dark blue or brown-red coloration is produced, 
 
 The demonstration of ammonia is made by the addi- 
 tion of Nessler's reagent to culture media free from 
 sugar. In bouillon, if ammonia be present, Nessler's 
 reagent is almost immediately reduced to black mer- 
 curous oxide. A strip of paper saturated with the 
 reagent can also be suspended over the bouillon tube, 
 or this can be distilled with the addition of magnesium 
 oxide and the distillate treated with Nessler's reagent. 
 A yellow to red coloration indicates the presence of 
 ammonia. Controls are necessary. 
 
VITAL PHENOMENA OF BACTERIA. 77 
 
 Aromatic Products of Decomposition. Many bacteria 
 produce aromatic substances as the result of their 
 growth. The best known of these are indol, skatol, 
 phenol, and ty rosin. Systematic investigations have 
 only been made with regard to the occurrence of indol 
 and phenol. 
 
 Test for Indol. To a bouillon culture, which should, 
 if possible, be not under eight days old and free from 
 sugar, is added half its volume of 10 per cent, sulphuric 
 acid. If in heating to about 80 C. a pink or bluish- 
 pink coloration is immediately produced it indicates the 
 presence of both indol and nitrites, the above-described 
 nitroso-indol reaction requiring the presence of both of 
 these substances for its successful operation. This is 
 the so-called " cholera-red reaction/' but it may be 
 applied to many other spirilla beside cholera. As a 
 rule, however, the addition of sulphuric acid alone is 
 not sufficient, and a little nitrite must be added; this 
 may be done later, the culture being first warmed with- 
 out ^nitrite, when if there is no reaction or a doubtful 
 one, 1 to 2 c.c. of a 0.5 per cent, solution of sodium 
 nitrite is added until the maximum reaction is obtained. 
 The addition of strong solutions of nitrite colors the 
 acid liquid brownish-yellow and ruins the test. 
 
 Out of sixty species examined by Lehmann, twenty- 
 three gave the indol reaction. Levandoosky states that 
 the color group in general, glanders, diphtheria, proteus 
 vulgaris, and most of the spirilla, are indol producers; 
 with the exception of the spirilla, these bacteria also 
 produce phenol. 
 
 Decomposition of Fats. Pure melted butter is not a 
 suitable culture medium for bacteria. The rancidity 
 of butter is brought about (1) ak the result of a purely 
 
 \ 
 
78 BACTERIOLOGY. 
 
 chemical decomposition of the butter by the oxygen of 
 the air under the influence of sunlight and (2) through 
 fermentation by the lactic acid of the milk-sugar left 
 in the butter. Fats are, however, attacked by bacteria 
 when mixed with gelatin and used as culture media, 
 with the consequent production of acid. 
 
 Putrefaction. By putrefaction is understood in com- 
 mon parlance every kind of decomposition due to bac- 
 teria which results in the production of malodorous 
 substances. Scientifically considered, putrefaction de- 
 pends upon the decomposition of complex organic 
 compounds, albuminous substances, and the like (glue, 
 albuminoid bodies), which are frequently first pepton- 
 ized and then further decomposed. Typical putrefac- 
 tion occurs only when oxygen is absent or scanty; the 
 free passage of air through a culture of putrefactive 
 bacteria an event which does not take place in natural 
 putrefaction very much modifies the process : first, 
 biologically, as the anaerobic bacteria are inhibited, 
 and then by the action of the oxygen on the products 
 or by-products of the aerobic and facultative anaerobic 
 bacteria. 
 
 As putrefactive products we have peptone, ammonia 
 and amines, leucin, tyrosin, and other amido substances. 
 Oxyfatty acids, indol, skatol, phenol, and, finally, sul- 
 phuretted hydrogen, mercaptan, carbonic acid, hydro- 
 gen, and, possibly, marsh-gas (H 4 C). 
 
 According to recent observations, nitrification is 
 produced by a small, special group of bacteria, culti- 
 vated with difficulty, which do not grow on our usual 
 culture media. From the investigations of Winograd- 
 sky it would appear that there are two common micro- 
 organisms present in the soil, one of which converts 
 
VITAL PHENOMENA OF BACTERIA. 79 
 
 ammonia into nitrites and the other converts nitrites 
 into nitrates. 
 
 Conversion of Nitrous and Nitric Acids into Free Nitrogen. 
 This process is performed by a number of bacteria. The 
 special nitrate- ferment ing bacteria, however, were first 
 accurately described by Barri and Stutzer. In their 
 exhaustive investigation they first isolated from horse- 
 manure two bacteria, neither of which was alone capable 
 of producing nitrogen from nitrates, but which together 
 in the presence of oxygen, but never without it entirely, 
 decomposed nitrates energetically. Later a second 
 denificating bacillus was found, B. denitrificans II., 
 which by itself was able to produce nitrogen from 
 nitrates. 
 
 The practical importance of these organisms is that 
 by their action large quantities of nitrates in the soil, 
 and especially in manure, may become lost as plant- 
 food by being converted into nitrogen. 
 
 Nitrogen Combination. The bacillus radicicola of 
 Beyerinck, which was isolated by him, has the power of 
 assimilating nitrogen from the air. This bacillus is found 
 in the small root-nodules of various leguminous plants 
 (pease, clover, etc.), and can be obtained from these in 
 cultures. Different varieties exist in different kinds 
 of legumes, each kind of legume apparently having a 
 special variety of bacteria adapted to it, and not every 
 variety is capable of producing nodules in all legumes. 
 There are certain " neutral " varieties, however, existing 
 free in the soil and not adapted to any special legume, 
 and these seem to be able to form nodules in different 
 legumes. 
 
 By the aid of these root-bacteria, which gain entrance 
 to the roots and there produce this nodular formation, 
 
80 BACTERIOLOGY. 
 
 the leguminous plants are enabled to assimilate nitrogen 
 from the atmosphere, thus yielding harvests of grain, 
 etc., which are highly nitrogenous, upon soils which are 
 naturally poor in nitrogen. This explains the reason 
 why poor, sandy soils become gradually fruitful when 
 pease, lupine and other varieties of legumes are grown 
 upon them and then turned under with the plough. It 
 is not known exactly how this assimilation of nitrogen 
 occurs, but it is assumed that the zoogloea-li ke bacteria, 
 called bacteroids, constantly observed in the nodules, 
 either alone or in a special degree, possess the property 
 of assimilating and. combining nitrogen. It seems, 
 moreover, to have been recently established that, in- 
 dependently of the assistance of the legumes, certain 
 nodule-bacteria exist free in the soil, which accumulate 
 nitrogen by absorbing it from the air (Stutzer). 
 
 Formation of Acids from Carbohydrates. Free acids 
 are formed by many bacteria in culture media contain- 
 ing sugar; the production of acid in ordinary bouillon 
 takes place on account of the presence of grape-sugar, 
 which is usually derived in small quantities from the 
 meat. 1 According to Theobald Smith, all anaerobic or 
 facultative anaerobic bacteria form acids from sugar; 
 the strict aerobic species do not, or so very slowly 
 that the acid is concealed by the almost simultaneous 
 production of alkali. The formation of acid occurs 
 sometimes with and sometimes without the production 
 of gas. Excessive acid production may cause the death 
 of the bacteria from the increase in acidity of the 
 culture media. 
 
 1 According to Theobald Smith, 75 per cent, of the beef ordinarily bought 
 in the markets contains appreciable quantities of sugar (up to 0.3 per cent.). 
 
VITAL PHENOMENA OF BACTERIA. 81 
 
 If after the sugar is consumed not enough acid has 
 been formed to kill the bacteria, a similar change in 
 reaction now takes place to that in ordinary culture 
 media in the absence of sugar viz., the acid is neu- 
 tralized gradually, and in the end the reaction becomes 
 alkaline. 
 
 Among the acids produced the most important is 
 lactic acid; also traces of formic acid, acetic acid, pro- 
 prionic acid, and butyric acid, and not infrequently 
 some ethyl-alcohol and aldehyde or ac'etone are formed. 
 Occasionally no lactic acid is present, and only the other 
 acids are formed. 
 
 Various bacteria, as yet incompletely 'studied, possess 
 the property of producing butyric acid and butyl-alcohol 
 from carbohydrates. 
 
 Some bacteria also seem to have the power of decom- 
 posing cellulose, found in the stomach and intestinal 
 contents of herbivorous animals and in marshy soils, 
 with the production of marsh-gas. 
 
 Formation of Gas from Carbohydrates and Other Ferment- 
 able Substances of the Fatty Series. The only gas pro- 
 duced in visible quantity in sugar-free culture media is 
 nitrogen. If sugar is vigorously decomposed by bac- 
 teria, as long as pure lactic acid or acetic acid is pro- 
 duced there may be no development of gas, as, for 
 instance, with the B. typhosus on grape-sugar; but 
 frequently there is much gas developed, especially in 
 the absence of air. About one-third of the acid-pro- 
 ducing species also develop gas abundantly, this con- 
 sisting chiefly of CO 2 , which, according to Smith, is 
 always mixed with H. Marsh-gas is seldom formed 
 by bacteria, with the exception of those decomposing 
 cellulose. 
 
 6 
 
82 
 
 BACTERIOLOGY. 
 
 In order to test the production of gas, a culture 
 medium composed of glucose-agar, containing about 
 1 per cent, grape sugar, may be used. At the end of 
 eight to twelve hours in the incubator (or twenty-four 
 hours 7 room-temperature) the agar will be seen to be 
 full of gas-bubbles or broken up into holes and fissures. 
 
 For the determination of the quantity and kind of 
 gas produced by a given micro-organism the fermenta- 
 tion tube recommended by Theobald Smith is the best. 
 This is a bent tube, constricted greatly at its lowest 
 portion (Eichorn's), supported upon a glass base, as 
 shown in Fig. 14. The graduation shown in the 
 
 FIG. 14. 
 
 Fermentation tube left side, ordinary tube on right side. 
 
 upright arm is not essential for ordinary laboratory 
 work. The tube is filled with a culture media consist- 
 ing of 1 per cent, glucose, peptone bouillon (without 
 air-bubbles), and sterilized in the steam sterilizer. It 
 is then inoculated with a loopful of a culture of the 
 organism in question, and observations taken: 
 
 1. If there is a turbidity produced in the open bulb 
 it indicates the presence of an aerobic species; if this 
 
VITAL PHENOMENA OF BACTERIA. 83 
 
 clouding occurs only in the closed arm, while the open 
 bulb remains clear, it is an anaerobic species. 
 
 2. The quantity of gas produced daily should be 
 marked on the upright arm; if the tube is graduated a 
 note of it is taken and the percentage calculated on the 
 fourth to the sixth day after gas-production has ceased. 
 
 3. A rough analysis of the gas produced may be 
 made as follows : Having signified by a mark on the 
 tube the quantity of gas produced, the open bulb is 
 completely filled with a 10 per cent, solution of soda, 
 the mouth tightly closed with the thumb, and the mix- 
 ture thoroughly shaken. After a minute or two all the 
 gas is allowed to rise to the top of the closed arm by 
 inclining and turning the tube, and then, removing the 
 thumb, the new volume of gas formed is permitted to 
 escape. That which passes off is carbonic acid gas; 
 the remainder is nitrogen, hydrogen, and marsh-gas. 
 
 For the quantitative determination of these gases 
 Hein pel's gas pipettes may be used. The principle of 
 the method is this : The hydrogen, mixed with oxygen 
 and passed over red-hot palladium asbestos, becomes 
 water, and thus disappears; the carburetted hydrogen 
 is converted into carbon-dioxide and is estimated as 
 such; the remainder is nitrogen. 
 
 Formation of Acids from Alcohol and Other Organic Acids. 
 It has long been known that the bacterium acetiand other 
 allied bacteria convert dilute solutions of ethyl-alcohol, 
 under the influence of oxidation, into acetic acid: 
 CH 3 + 2 + CH 3 + H 2 0. 
 
 CH 2 OH COOH. 
 
 The higher alcohols glycerin, dulcit, mannit, etc. 
 are also converted into acids glycerin, indeed, as 
 commonly as sugar. 
 
84 BACTERIOLOGY. 
 
 Finally, numerous results have been obtained from 
 the conversion of the fatty acids and their salts into 
 other fatty acids by bacteria. As a rule, the lime-salts 
 of lactic, malic, tartaric, and citric acids have been em- 
 ployed, these being converted in various acids by the 
 action of bacteria, such as butyric, priprionic, valeri- 
 anic, and acetic acids; also succinic acid, ethyl-alcohol, 
 and, more rarely, formic acid, have been produced. 
 Among the gases formed were chiefly CO 2 and H. 
 
 Thus Pasteur found that anaerobic bacteria convert 
 lactate of lime into butyric acid. 
 
CHAPTER IV. 
 
 THE RELATION OF BACTERIA TO DISEASE. 
 
 IN the preceding chapter our consideration has been 
 given largely to the chemical effects of bacteria on dead 
 organic substances. Here we have to consider the 
 growth of bacteria in living bodies and the results of 
 such development. While it is true that there is a 
 great difference between living and dead matter, and 
 that, therefore, the living animal cannot be considered 
 as merely a quantity of organic and inorganic material, 
 to be used for food for bacterial growth, still the fact 
 that bacteria do increase in the living body shows 
 that its tissues are under certain conditions a suitable 
 nutrient soil for their growth. In a sense, therefore, 
 we are warranted to consider the living body as we do 
 any other medium for bacterial growth, remembering, 
 however, that beside the chemical nature, temperature, 
 etc., of its tissues, micro-organisms have also to reckon 
 with the mysterious influence of life with which all 
 parts of the body are endowed. In the production of 
 disease by micro-organisms there are two main factors 
 involved viz., the power to elaborate poison and the 
 ability to multiply. No known variety of bacterial cell 
 has as a single organism the ability to produce enough 
 poison to do appreciable injury in the body, nor is there 
 any variety which if it multiplied in the body to the 
 full extent to which it is capable under favorable con- 
 
86 BACTERIOLOGY. 
 
 ditions will not produce disease. As already men- 
 tioned, bacteria even under similar conditions differ 
 enormously in the amount of poison which each organ- 
 ism produces and in their ability after gaining entrance 
 to multiply in the body. 
 
 To understands all the production of disease through 
 bacteria we must recognize that both the body invaded 
 and the bacteria which invade are living organisms. 
 They are in bulk, wide apart, but both have life. Just 
 as there are different races and species of animals, there 
 are different races and species among bacteria, and just 
 as the descendants of one animal species under changing 
 conditions gradually become diverse, so do the descend- 
 ants of one bacterial species. Considering these facts, 
 we can readily understand how all of bacteria do not 
 grow equally well in every variety of animal, nor even 
 find the body of the same animal always equally suit- 
 able. This is all the more apparent when we consider 
 that the study of bacteria in the more simple and 
 known conditions of artificial culture media has already 
 shown us how extremely sensitive many bacteria are to 
 slight chemical, thermal, and other changes. 
 
 Thus if we take specimens of diphtheria bacilli from 
 three different cases of diphtheria, we find that on grow- 
 ing them for several days in suitable bouillon one will 
 have produced poison in the culture fluid to such a 
 degree that one drop suffices to kill a large guinea- 
 pig; the second, grown in a similar manner, will kill 
 another animal of the same size with half a drop; 
 while the third will kill with one-tenth of a drop. 
 In other words, different varieties of diphtheria bacilli 
 under similar conditions have different toxin-producing 
 powers. 
 
RELATION OF BACTERIA TO DISEASE. 87 
 
 Let us now cultivate these same bacilli in bouillon, 
 which is a little too acid or a little too alkaline for their 
 maximum development, and we shall find that, while 
 all of them will grow, only the one which produced 
 the most toxin under favorable conditions will continue 
 to develop it, while the others will fail to produce 
 any specific poison. This shows that growth of bac- 
 teria may occur in the body and yet no specific poison 
 be produced, and that of the same species of bacteria 
 some varieties are capable of producing toxin under 
 less favorable circumstances than others. 
 
 Slight variations in the culture media are, moreover, 
 of great importance in aiding or inhibiting the growth 
 of bacteria. Thus the diphtheria bacillus grown in 
 neutral bouillon containing a little glucose will at first 
 thrive luxuriantly; but as the result of its growth fer- 
 mentation of the glucose takes place and acid is pro- 
 duced, which then inhibits the further development of 
 the bacillus. After a while, however, the glucose having 
 been entirely destroyed, acid formation ceases while 
 alkaline products continue to be formed, and thus render 
 the medium neutral or slightly alkaline again, and now 
 a vigorous growth again starts up. 
 
 The cultivation of the tetanus bacillus also furnishes 
 some interesting facts which illustrate the complicated 
 ways in which bacterial growth is hindered or assisted. 
 The tetanus bacillus, when placed in suitable media, 
 will not grow except in the absence of oxygen; but 
 place it under the same conditions, together with a 
 bacillus which actively assimilates oxygen, and the two 
 in association will grow in the presence of air. 
 
 The tubercle bacillus, when taken direct from an 
 animal, will only grow in a few selected media, such as 
 
88 BACTERIOLOGY. 
 
 blood-serum media, but later on it may be transplanted 
 to agar, and still later to bouillon. After the bacilli 
 have become accustomed to the bouillon they grow with 
 great luxuriance, but only when carefully floated on the 
 surface of the liquid. If submerged in the slightest 
 degree they will not grow. 
 
 Many bacteria which demand free access of oxygen 
 grow only in the superficial portion of the nutrient 
 agar jelly, where there is plenty of air. 
 
 It is evident, therefore, that for each variety of organ- 
 ism there are special conditions requisite for growth, 
 and that a temperature, degree of acidity, supply of 
 oxygen, immersion in fluid, etc., suitable for one may 
 be utterly unsuitable for another; that, still further, 
 when two organisms grow together one may so alter 
 some of these conditions as to render unsuitable ones 
 suitable, and vice versa. Since, therefore, bacteria vary 
 greatly as to the amount of toxin which they produce, 
 and their ability to develop under different conditions 
 outside of the body, we should certainly expect even 
 greater variations in the living bodies of men and ani- 
 mals where not only in different individuals, but even 
 in the same individual at different times, there is a 
 varying suitableness for such growth. 
 
 Let us now consider some of the facts which have 
 been observed concerning the growth of bacteria in the 
 body, and then endeavor, as far as possible, to explain 
 them. 
 
 In the first place, there are some bacteria which find 
 it impossible to grow in the living body. This is true 
 of the great mass of bacteria occurring in the air, water, 
 and soil. These bacteria cannot, therefore, produce 
 infectious diseases. Some of them, however, produce 
 
RELATION OF BACTERIA TO DISEASE. gg 
 
 poisons in foods, etc., which are absorbable, and which 
 when taken with food or drink can produce a chemical 
 intoxication. That they are really deleterious is shown 
 by the fact that if a sufficient quantity of their pure 
 cultures is injected into the tissues suppuration and ab- 
 scesses are produced by the toxic substances contained 
 within them. 
 
 Closely allied to the bacteria which cannot grow at 
 all in the bodies of warm-blooded animals are those 
 which are able to grow in or upon certain circum- 
 scribed areas only. Thus the diphtheria bacilli grow 
 upon the abraded mucous membranes of the respiratory 
 tract, but cannot develop in the blood or in the subcu- 
 taneous tissues. The cholera spirilla develop in the in- 
 flamed intestinal mucous membrane, but cannot grow 
 in the respiratory tract, blood, or tissues. The tetanus 
 bacilli develop in wounds of the subcutaneous tissues, 
 but cannot grow on the body-surface or in the blood. 
 
 Another group of bacteria find, indeed, certain regions 
 most suitable in their conditions for growth, but under 
 circumstances favorable for them are capable of more ex- 
 tensive growth. Thus the typhoid bacillus grows most 
 luxuriantly in the Peyer's patches and mesenteric glands, 
 but also invades the blood, spleen, and other regions. 
 The tubercle bacillus often remains localized in the 
 apex of a lung or a gland for years, but at any time 
 may invade many tissues of the body. The gonococcus 
 finds the mucous membrane of the genito-urinary tract 
 most suitable for its development, but also frequently is 
 capable of growth in the peritoneum and even some- 
 times in the general circulation. The pneumococcus 
 develops most readily in the lungs, but also invades the 
 connective tissues, serous membranes, and the blood. 
 
90 BACTERIOLOGY. 
 
 Still further removed from the saprophytic bacteria 
 are those which grow in the blood and most living 
 tissues as readily as in the most suitable artificial 
 media. Thus a streptococcus which has passed 
 through a number of animals or human beings will, 
 when introduced into the circulation or the tissues, 
 develop as rapidly and generally as in bouillon, and 
 produce death within twenty-four hours, every drop of 
 blood being crowded with bacteria. 
 
 Finally, there are bacteria which, in so far as we 
 know, find the bodies of human beings or animals the 
 only fit soil for their growth. These are the true 
 parasites. The leprosy bacillus grows only in man; 
 neither the food nor the conditions suitable for the de- 
 velopment of this micro-organism outside of the body 
 have as yet been discovered. The spirillum of relapsing 
 fever is another good example of this group. 
 
 Following rather closely the schematic separation of 
 bacteria according to their relation to disease we might 
 classify them as : 
 
 1. Strict saprophytes, or bacteria which grow readily 
 in suitable dead organic material, but not in the body 
 under ordinary conditions. 
 
 a. Bacteria which in their growth produce no sub- 
 stances which are poisonous to the body, or at least none 
 capable of absorption. 
 
 b. Bacteria which produce in their growth in dead 
 organic matter sufficient poisons to cause sickness if 
 they are absorbed into the animal body. 
 
 2. Facultative Saprophytes. These are bacteria which 
 can develop either as parasites or saprophytes. The 
 different varieties vary as to the amount of poison 
 which they produce. Some grow luxuriantly in dead 
 
RELATION OF BACTERIA TO DISEASE. 91 
 
 organic material under very diverse conditions, others 
 only under specially favorable conditions. In the body 
 they also vary some grow extensively in the blood, 
 while others are limited to one or more tissues, some 
 being widely disseminated throughout the body, while 
 others are localized in or upon a certain portion of it. 
 
 3. Strict parasites, or bacteria which, so far as we 
 know, grow only in the living animal or vegetable 
 organism. These again vary in the amount of poison 
 which they produce and in the local or general infec- 
 tion they give rise to. 
 
 Adaptation of Bacteria to the Soil upon which They are 
 Grown. Those bacteria which grow both in living and 
 dead substances vary from time to time as to their 
 readiness to develop in either the one or the other. 
 As a general rule, bacteria grown in any one medium 
 become more and more accustomed to that and other 
 media more or less analogous to it, while, on the other 
 hand, they are less easily cultivated on media widely 
 different from that in which they have developed. Thus 
 we have a culture of tubercle bacilli, which, after having 
 grown for three years in the bodies of guinea-pigs, will 
 no longer develop on dead organic matter, while a 
 bacillus which was obtained from the same stock, but 
 grown on bouillon for three years, will no longer de- 
 velop in the animal body. From the same stock, 
 therefore, two varieties have developed, the one being 
 now practically a saprophyte and the other a parasite. 
 
 The Local Effects Produced by Bacteria and their Prod- 
 ucts. Nearly all the forms of acute inflammation are 
 seen to follow the development of bacteria. Thus in- 
 flammation and serous exudation into the subcutaneous 
 tissues follow injections of the pneumococcus or anthrax 
 
92 BACTERIOLOGY. 
 
 bacillus. The development of the streptococcus or 
 pneumococcus in the endocardium or pleural cavity is 
 followed by a serous exudation, frequently with more or 
 less fibrin production. The formation of pus results, 
 more especially from the streptococcus, pneumococcus, 
 and staphylococcus; but also nearly all forms of bacteria, 
 when they accumulate in one locality, may produce 
 purulent inflammation. The colon, typhoid, and influ- 
 enza bacilli frequently cause the formation of abscesses. 
 
 Catarrhal inflammation, with or without pus, follows 
 the absorption of the products of many bacteria, such as 
 the gonococcus, pneumococcus, streptococcus, and in- 
 fluenza bacillus, etc. The hemorrhagic exudation seen 
 in pneumonia is due to the pneumococcus; it is observed 
 also in anthrax and other infections. Cell necrosis is 
 produced frequently by the products of the diphtheria 
 and of the typhoid bacilli and by those of other bac- 
 teria. Specific proliferative inflammation follows the 
 localization of the products derived from the tubercle 
 bacillus and the leprosy bacillus. 
 
 Not only can one species of bacteria produce several 
 forms of inflammation, but the same organism will vary 
 as to the kind or kinds of inflammation it will produce; 
 this depending, first, upon its own characteristics at the 
 time as to virulence, etc., and, second, upon the con- 
 ditions in the infected animal, such as its health and 
 power of resistance, the period of infection, and the 
 circumstances under which the animal remains. Such 
 variations, therefore, are in no case specific, for different 
 poisons will produce changes which appear identical. 
 
 The Manner in which Bacteria Produce Disease. The 
 actual mechanical presence of the bacteria is only of 
 importance when, as in septicaemia or pyiiemia, tliey exist 
 
RELATION OF BACTERIA TO DISEASE. 93 
 
 in such enormous numbers as to interfere mechanically 
 with the circulation or cause minute thrombi, and later 
 emboli, which finally produce infarction and abscesses 
 in different parts of the body. These dangerous effects 
 are chiefly due, first, to their alteration of the nutritive 
 substances in the body into others which are valueless, 
 and, second, to their production of substances which 
 are more or less directly poisonous. 
 
 A moment's consideration of the different changes 
 which take place in the tissues after the injection of 
 fine sterile sand and of an equal quantity of a dead 
 culture of the tubercle or typhoid bacillus would suf- 
 fice to convince any one that it was the poison produced 
 by the bacillus, and not its mechanical interference, 
 which caused disease. These poisonous products, as 
 already described in the previous chapter, can be 
 separated from the culture fluid in which the bac- 
 teria have grown or they can be extracted from their 
 bodies. These products without the bacteria them- 
 selves injected into animals cause essentially the same 
 lesions as are produced by the bacteria when they de- 
 velop in the animal body. When the body, as a whole, 
 is invaded by bacteria the abstraction from the body 
 of such substances as they consume exerts probably a 
 considerable influence; but even here it is the poisons 
 elaborated by bacteria from the body substances and 
 given up to the blood and tissue cells which are of most 
 importance. The substances contained in or produced 
 by the bacteria, with few exceptions, attract the leuco- 
 cytes, and when great masses of bacteria die suppura- 
 tion usually follows. 
 
 The General Symptoms Caused by Bacterial Poisons 
 Absorbed into the Circulation. Fever is produced under 
 
94 BACTERIOLOGY. 
 
 favorable conditions by all bacterial poisons. The first 
 requisite is that sufficient poison be absorbed; but, on the 
 other hand, it must not be absorbed with such rapidity as 
 to overwhelm the injected animal, for a moderate dose 
 may raise the temperature, while a very large dose 
 lowers it, as occurs sometimes when a very large sur- 
 face, such as the peritoneum, is suddenly involved. 
 
 Centanni 1 obtained through warmth and alcohol 
 from the bodies of bacteria a substance called pyro- 
 toxin, which was with difficulty dialyzed. From dif- 
 ferent bacteria not only the physiological but also the 
 chemical properties of the pyrotoxin were the same. 
 Not only did this cause fever, but also, when persisted 
 in, it produced emaciation, quickened heart-action, 
 apathy, dyspnoea, etc. 
 
 The bacterial poisons produce an increase in the 
 number of leucocytes and a lessening in the amount of 
 haemoglobin in the blood. The deleterious effects on 
 the nutrition are partly due to the direct effect of the 
 poison and partly to the diseased conditions of the 
 organs of the body, such as the spleen, kidney, and 
 liver. Degeneration of the nerve cells is frequently 
 noticed after infectious diseases; especially is this true 
 of diphtheria. Several bacterial poisons have been 
 found to produce convulsions; the best example of this 
 is the tetanus toxin. 
 
 The true bacterial poisons are, as already stated, 
 neither alkaloids nor albumins. Some of them, such 
 as the diphtheria and tetanus toxins, are peculiar in 
 their effects, while others, such as those produced by 
 the pneumococcus and streptococcus, can scarcely be 
 distinguished. They are destroyed by heat at 70 C. 
 
 1 Deutsche med. Wochenschrift, 1894, Nos. 7 and 8. 
 
RELATION OF BACTERIA TO DISEASE. 95 
 
 Bacteria also produce secondary poisons, which stand 
 a temperature of 100 to 120 C. 
 
 The Influence of Quantity in Infection. With bacteria 
 the number introduced has an immense influence upon 
 the probability of infection taking place. 
 
 If we introduce into a culture medium, which, like 
 the body, is only fairly suitable for growth, a few 
 bacteria, it is not improbable that they may all die; 
 whereas if a greater number are introduced, while there 
 will at first be a slight diminution of these, those that 
 die seem to neutralize the substances which were dele- 
 terious; then those bacteria which survive begin to in- 
 crease, and soon they multiply enormously. The same 
 is true for parasitic bacteria in the body. A few only 
 gaining entrance, they may die; a larger number being 
 introduced, some may or may not survive ; but if 
 a still greater quantity is injected it is almost certain 
 that there will be some surviving members, which, after 
 the destruction of antagonistic substances, and on be- 
 coming accustomed to their environment, will begin to 
 grow and produce disease. 
 
 With those bacteria whose virulence is great i. e., 
 those which are capable of growing with great ease in 
 the body fluids a very few organisms will produce 
 disease almost as quickly as a million, allowance only 
 being made for the short time required for the few 
 to become equal in number to the million. At the 
 other extreme of virulence, however, many millions 
 may have to be introduced to permit of the develop- 
 ment of any of the organisms in the body. With these 
 bacteria we are thus able to produce either no effect 
 whatever, a local effect, or in some cases a general sep- 
 ticaemia, by regulating the amount of infection intro- 
 
96 BACTERIOLOGY. 
 
 duced. In the majority of cases in man the number of 
 bacteria received is comparatively small; but by the 
 rupture of an abscess into a cavity or into the circula- 
 tion, or by the opening of the intestinal contents into the 
 peritoneum, the quantity introduced may be enormous. 
 
 The Degree of Virulence Possessed by Bacteria. Bac- 
 teria as found in nature differ, as has already been stated, 
 as to the amount of poison they produce and the ease and 
 rapidity with which they grow in any nutritive sub- 
 stance. Both of these properties not only vary greatly in 
 different members of the same species, but each variety 
 of bacteria may to a large extent be increased or dimin- 
 ished in virulence. The specific poisons produced by 
 bacteria can be best studied in diphtheria and tetanus. 
 We note, first, that different individual bacilli of diph- 
 theria and tetanus have, when freshly obtained, wide 
 variations in the amount of toxin which they produce 
 i.e., a diphtheria bacillus obtained from a case of diph- 
 theria will produce in suitable nutrient broth a poison 
 of such strength .that 1 c.c. will kill an average sized 
 guinea-pig, while the poison from another bacillus will 
 kill with a much less quantity, or 0.005 c.c. Further, 
 the bacilli obtained from some sources retain their power 
 of producing poison, when grown on artificial media, for 
 years unaltered, while others lose much of this in a few 
 months. This is equally true of the tetanus bacilli. 
 
 The power to produce toxin can be taken from bacilli 
 by growing them under adverse circumstances, such as 
 cultivation at the maximum temperature at which they 
 are capable of development. Some bacilli are easily 
 attenuated; others are robbed of their virulence only 
 with great difficulty. Increase of toxin-production is 
 more difficult, and it is only possible to obtain it to a 
 
RELATION OF BACTERIA TO DISEASE. 97 
 
 certain extent. The means usually employed are the 
 frequent replanting of cultures and their growth in cap- 
 sules placed in the bodies of susceptible animals. But 
 with all our efforts we are usually only able to restore 
 approximately the degree of toxin-formation which the 
 cultures originally possessed. The adaptation of bac- 
 teria to any nutritive substance, living or dead, so that 
 they will grow more readily, is more easily brought 
 about, provided they will grow at all. The streptococcus 
 from erysipelas and the pneumococcus from pneumonia 
 are typical of this class of bacteria. Inoculate a rabbit 
 with a few streptococci obtained from a case of human 
 sepsis, and, as a rule, no result follows ; inject a few 
 million, and usually a local induration or abscess ap- 
 pears ; but if one hundred million are administered 
 septicaemia develops. From this rabbit now inoculate 
 another, and we find that a dose slightly smaller suffices 
 to produce the same effect; in the next animal inoculated 
 from this still less is required, and so on, until in time, 
 with suitable cultures, a very minute number will surely 
 develop and produce death. The same increase in 
 virulence can be noted when septic infection is carried 
 in surgery or obstetrics from one human case to another. 
 By allowing bacteria to continue to develop under cer- 
 tain fixed conditions they become accustomed to them, 
 and less adapted for all that differ. 
 
 Somewhat distinct, again, from that class of bacteria 
 which multiply rapidly are those which, like the tubercle 
 and leprosy bacilli, develop slowly. Here increase of 
 virulence is shown, as before, by the production of dis- 
 ease through the introduction of very small numbers 
 into the body, but increase in rapidity of development 
 cannot progress except to within certain limits. A sin- 
 
 ' 7 
 
98 BACTERIOLOGY. 
 
 gle streptococcus may, through its rapid multiplication, 
 produce death in eighteen hours ; a single tubercle 
 bacillus, on the other hand, cannot produce sufficient 
 numbers in less than two weeks. The virulence of the 
 septicsemic class of bacteria is not at all the same when 
 measured in different animals, and it is largely for this 
 reason that the virulence in test animals does not 
 usually correspond with the severity of the case from 
 which the organism was derived. We should re- 
 member in this connection the varying power of resist- 
 ance in different animals and of the same individual 
 at different times. 
 
 Mixed Infection. The combined effect upon the tissues 
 of the products of two or more varieties of pathogenic 
 bacteria, and also of the influence of these different forms 
 on each other, are of great importance in the produc- 
 tion of disease. The infection from several different 
 organisms may occur at the same time, or one may fol- 
 low the other or others so-called secondary infection. 
 Mixed infection arises usually from the inoculation of 
 more than one variety of bacteria simultaneously. 
 Thus, an abscess is often due to several forms of 
 pyogenic cocci. If a wound is infected from such a 
 source the inflammation produced will probably be 
 caused by all the varieties present in the original in- 
 fection. Peritonitis following intestinal injuries must 
 necessarily be due to more than one organism. Thus, 
 whenever two or more varieties of bacteria are trans- 
 ferred to a new soil, mixed infection takes place if 
 more than one variety is capable of developing in that 
 locality. 
 
 Forms of infection which are allied to both mixed 
 and secondary infection are those occurring in the 
 
RELATION OF BACTERIA TO DISEASE. 99 
 
 mucous membranes of the respiratory and digestive 
 tract. In these situations pathogenic bacteria of slight 
 virulence are always present even in health. Thus 
 in the upper air-passages there are usually found strep- 
 tococci, staphylococci, andpneumococci. When through 
 a cold, or the invasion of another infective agent, as 
 the diphtheria bacillus, the epithelium of the mucous 
 membrane of the throat is injured or destroyed, the pyo- 
 genic cocci already present are now enabled in this dis- 
 eased membrane to grow, produce their poison, and 
 even invade deeper tissues. The intestinal mucous 
 membrane is invaded in a similar way by the colon 
 bacilli and other organisms after injury by the typhoid 
 bacilli or cholera spirilla. Generally speaking, all in- 
 flammations of the mucous membranes contain some of 
 the elements of mixed infection. Blood infection, on 
 the other hand, is usually due to one form of bacteria, 
 as even when several varieties are introduced, only 
 one, as a rule, is capable of development. The same 
 is true to a somewhat less extent of inflammation of 
 the connective tissue. The additional poison given off 
 by the associated bacteria aid infection by causing a 
 lowering of the vital resistance of the body. 
 
 The bacteria are also at times directly influenced by 
 the products of associated organisms. These may 
 affect them injuriously, as, for example, the pyogenic 
 cocci in anthrax; or they may be necessary to their 
 development, as in the case of anaerobic bacteria. JSTot 
 infrequently the tetanus bacilli or spores would not be 
 able to develop in wounds were it not for the presence 
 of aerobic bacteria introduced with them. This is shown 
 outside the body, where tetanus bacilli will not grow in 
 the presence of oxygen unless aerobic bacteria are asso- 
 
100 BACTERIOLOGY. 
 
 elated with them. Again, it is found that the associa- 
 tion of one variety with another may increase its viru- 
 lence. Thus Roux and Yersiu believe that they have es- 
 tablished the fact that streptococci and diphtheria bacilli 
 mutually increase each other's virulence. On the other 
 hand, the absorption of the products of certain bacteria 
 immunizes the body against the invasion of other bac- 
 teria, as shown by Pasteur that attenuated chicken 
 cholera cultures produce immunity against anthrax. 
 
 The Modes of Entrance of Infection. The various fluids 
 and tissues of the body differ greatly in their chemical 
 constituents, their reaction, their protection from in- 
 fection, their access to free oxygen, their temperature, 
 and in other less well-known respects. These varia- 
 tions are sufficient to render certain portions of the 
 body suitable for the growth of some bacteria and 
 unsuitable for others. This fact is of immense im- 
 portance in the transmission or prevention of disease. 
 Thus, for example, let us rub very virulent strepto- 
 cocci, typhoid bacilli, and diphtheria bacilli into an 
 abrasion on the hand. The typhoid bacillus produces 
 no lesion, the diphtheria bacillus but a very minute 
 infected area, but the streptococcus gives rise to a severe 
 cellulitis or fatal septicaemia. Now place the same 
 bacteria on an abrasion in the throat. The typhoid 
 bacillus is again harmless; the diphtheria bacillus pro- 
 duces inflammation, a pseudomembrane, and toxaemia, 
 and the streptococcus causes an exudate, an abscess, or 
 a septicaemia. Finally, introduce the same bacteria into 
 the intestines, and now it is the typhoid bacillus which 
 produces its characteristic lesions, while the strepto- 
 coccus and diphtheria bacillus are usually innocuous. 
 
 If we tried in this way all the parasitic bacteria we 
 
RELATION OF BACTERIA TO DISEASE. 1Q1 
 
 would find that certain varieties are capable of develop- 
 ing and thereby producing disease only on the mucous 
 membrane of the throat, others of the intestine, others 
 of the urethra ; some develop only in a wound or in 
 the blood, while others, again, under favorable condi- 
 tions, seem able to grow in or upon almost any region 
 of the body. 
 
CHAPTER V. 
 
 IMMUNITY. 
 
 THAT certain races of animals and men, and certain 
 individuals among these, are more refractory to disease 
 than others, is a fact which has long been known. 
 Experience and observation have taught us, further, 
 that the same individuals are at one time more resistant 
 to disease than at another. This inborn or spontaneous 
 refractory condition is termed natural immunity, in con- 
 tradistinction to that acquired by recovery from disease. 
 
 As in bacteria, we distinguish between the ability to 
 produce poison and the power to multiply in the body, 
 so here we may distinguish between immunity to poison 
 and immunity to the development of bacteria. 
 
 With regard to variations in susceptibility, certain 
 known facts have been ascertained. Thus, cold-blooded 
 animals are generally insusceptible to infection from 
 those bacteria which produce disease in warm-blooded 
 animals, and vice versa. This is readily explained by 
 the inability of the bacteria which grow at the tem- 
 perature of warm-blooded animals to thrive at the 
 temperature existing in cold-blooded animals. But dif- 
 ferences are observed not only between warm-blooded 
 and cold-blooded animals, but also between the several 
 races of warm-blooded animals. The anthrax bacillus 
 is very infectious for the mouse and guinea-pig, while 
 the rat is not susceptible to it unless its body resistance 
 
IMMUNITY. 103 
 
 is reduced by disease and the amount of infection is 
 great. The inability of a micro-organism to grow 
 in the body of an animal does not usually indicate, 
 however, an insusceptibility to its poison; thus, for in- 
 stance, rabbits are less susceptible than dogs to the effects 
 of the poison elaborated by the pneumococci, but these 
 bacteria develop much better in the former than in 
 the latter. Differences in susceptibility are sometimes 
 very marked among different varieties of the same race 
 of animals, as, for instance, between different kinds of 
 rats and pigeons to anthrax. In animals, as a whole, 
 it is noticed experimentally that the young of all species 
 are less resistant to infection than the older and larger 
 ones. 
 
 The difficulty experienced by the large majority of 
 bacteria in developing in the tissues of the healthy 
 body can be to a great extent removed by any cause 
 which lowers the general or local vitality of the tissues. 
 Among the causes which bring about such lessened re- 
 sistance of the body are hunger and starvation, bad 
 hygienic surroundings, exhaustion from overexertion, 
 exposure to cold, the deleterious effects of poisons, bac- 
 terial or other, acute and chronic diseases, vicious 
 habits, drunkenness, etc. Purely local injuries, such 
 as wounds, contusions, etc., also give sometimes a point 
 of entrance for infection, or at least a point of less re- 
 sistance, where the bacteria may develop and produce 
 local inflammation. This is noted in infection by the 
 tubercle and typhoid bacilli, pyogenic cocci, etc. 
 Local affections, such as endocarditis, may also afford 
 a weak spot for the bacteria to seize upon. The pres- 
 ence of foreign bodies in the tissues in like manner 
 predisposes them to bacterial invasion. Interference 
 
104 BACTERIOLOGY. 
 
 with free circulation of blood and retention in the body 
 of substances which should be eliminated also tend to 
 lessen the vitality. In these and other similar ways 
 animals which are otherwise refractory may acquire a 
 susceptibility to disease. 
 
 Immunization and Healing by Non-specific Means. Just 
 as all conditions which are deleterious to the body lessen 
 its power of resistance to bacterial invasion, so all con- 
 ditions which are favorable to it increase its resistance, 
 and thus aid in preventing and overcoming infection. 
 The internal use of antiseptics against bacteria has 
 not proved successful, for the reason that an amount 
 too small to inhibit bacterial growth is found to be 
 poisonous to the tissue cells. The efficacy of quinine 
 in malaria and mercury in syphilis is, possibly, an ex- 
 ception to the rule, but in both cases we are dealing 
 probably with animal parasites, not ordinary bacteria. 
 Such substances as nuclein and others contained in 
 blood-serum, when introduced into the body in consider- 
 able quantity, aid somewhat in inhibiting or preventing 
 the growth of many bacteria. Even bouillon, salt 
 solution, and small amounts of urine have a slight in- 
 hibitory action. The hastening of elimination of the 
 bacterial poisons by free intestinal evacuation and en- 
 couragement of the functions of the skin and kidneys 
 are also of some avail. The enzymes formed by certain 
 bacteria have been found to exert a slight bactericidal 
 action, not only on the germs which have directly or 
 indirectly produced them in the body, but also on other 
 varieties. JSTone of these enzymes are sufficiently pro- 
 tective to be of practical value nor equal in power to the 
 protective substances formed by the tissues from the 
 bacterial products. 
 
IMMUNITY. 105 
 
 The Use of Local Treatment in Inhibiting Bacterial In- 
 vasion. The total extirpation of the infected area by 
 surgical means, if thoroughly carried out, removes the 
 disease entirely; but, unfortunately, this procedure is 
 rarely possible. When incomplete it is frequently 
 helpful; but it may be harmful, for by creating and 
 exposing fresh wounded surfaces to infection it may 
 lead to the further development of the disease. Again, 
 it may be useless, for by removing only a portion of 
 the bacteria it may leave those which have already 
 reached the deeper tissues or blood to go on devel- 
 oping. In some cases, like anthrax and infection 
 from bites of rabid animals, total removal of the 
 virus is possible, either by the knife or thorough cauter- 
 ization, and will prevent a general infection. So also 
 in tetanus, the invasion being limited, surgical inter- 
 ference may be of great use by removing not only the 
 bacilli themselves but also that portion of their poison 
 which has not as yet been absorbed from the tissues. 
 The beneficial effects of opening an abscess, incising a 
 cellulitis, or cleansing and drainage of the uterine 
 cavity are well known. The retention of the poisonous 
 products of the bacteria and altered tissue substance 
 leads to their absorption, and thus lowers the tone of 
 the neighboring, and to a less extent of the general, 
 tissues in consequence of the poisoning. This enables 
 the bacteria to penetrate into tissues which would other- 
 wise resist them. The mechanical effect of pressure on 
 the walls of an abscess by its contents also aids the bac- 
 terial progress. Local bleeding and the application of 
 cold probably act by lessening tension. The application 
 of warmth hastens absorption, and so, when the infec- 
 tion is one which tends to localize, it acts favorably by 
 
106 BACTERIOLOGY. 
 
 accelerating the development and thus the disappear- 
 ance of the inflammation. A peculiar effect of opera- 
 tive interference is noticed in the frequently observed 
 beneficial result of laparotomy in tubercular peritonitis. 
 
 Antiseptic solutions have the power of cleansing and 
 rendering sterile the surfaces of a wound that is, of 
 preventing the introduction of infection. After infec- 
 tion has taken place, however, it is doubtful whether 
 antiseptic washing has much more direct influence 
 than simple cleansing, and it certainly can have no 
 bactericidal effect at any distance from the surface, 
 either direct or indirect. Certain infectious diseases 
 which are comparatively superficial are probably bene- 
 fited by antiseptic solutions, such as gonorrhoea, diph- 
 theria, and other inflammations of the mucous mem- 
 branes. Even here, however, it is impossible to do more 
 than disinfect superficially, and in some cases any 
 irritation of the tissues is apt to do more harm than 
 good. In the superficial lesions of syphilis and tuber- 
 culosis the local use of antiseptics is sometimes of 
 great value. In these diseases the irritant effects of the 
 antiseptics which stimulate the tissues may also be 
 beneficial. Roux has reported that certain specific 
 serums, just as certain enzymes, have some destructive 
 effect on the toxic substances of other species of bac- 
 teria; but this is a subject which has been as yet but 
 little investigated. 
 
 Specific immunity r , or a condition of the body which 
 prevents the development in it of one variety of micro- 
 organisms and renders it unaffected by their bacterial 
 poisons. The invasion of the body with more or less 
 serious results by most micro-organisms is followed 
 by a condition which for a variable period and to a 
 
IMMUNITY. 107 
 
 variable degree is deleterious to their further growth. 
 It also gives rise to substances which neutralize the 
 poisonous effects of the bacterial products. This im- 
 munity may take place in various ways : 
 
 1. Through recovery from disease naturally con- 
 tracted or from infection artificially produced. This 
 immunity may be slight, as after recovery from 
 erysipelas or pneumonia, marked for a short period of 
 time, as in diphtheria and typhoid fever, or prolonged, 
 as after scarlet fever or syphilis. 
 
 2. By the injection of the bacteria into tissues not 
 well suited to their development, as the injection of 
 typhoid bacilli or cholera spirilla into the subcutaneous 
 tissues. Here a mild local infection follows, with con- 
 siderable resulting immunity. 
 
 3. By the injection of micro-organisms attenuated 
 by heat, chemicals, or other means. In this case a 
 local or general infection of the animal is produced, 
 of moderate severity, as a rule, and the immunity is not 
 as marked and lasting as after recovery from a more 
 serious attack ; but it is, nevertheless, considerable. 
 The inoculation of sheep with the attenuated anthrax 
 bacillus and the use of vaccination in man are examples 
 of this method. 
 
 4. By the injection of the unaltered chemical con- 
 stituents of the dead bodies of bacteria and of the 
 chemical products which they elaborate and discharge 
 into the surrounding culture media during life. Smith 
 and Salmon proved that by repeated injections of the 
 filtered bouillon cultures of the hog-cholera bacillus a 
 considerable immunity may be produced against the in- 
 vasion of this bacillus. Similar results have followed 
 the injections of dead cultures of typhoid and anthrax 
 
108 BACTERIOLOGY. 
 
 bacilli and cholera spirilla, etc. After infection with 
 most parasitic bacteria the body resistance to the growth 
 of the same organism is greatly increased; in other in- 
 fections, however, it is but slightly augmented. 
 
 The protective substances held in solution in the 
 blood-serum are clearly apparent in their effects either 
 in preventing the increase of the bacteria or neutralizing 
 the toxic action of their products; chemically, however, 
 they are but little understood, and although some of 
 them have been shown to be to a large extent specific, 
 that is, they are far more efficient in protecting against 
 the special variety of bacteria which produced the infec- 
 tion than against any other, still we have no knowledge 
 of any chemical difference between them. The addition 
 of 0.5 per cent, of carbolic acid injures these substances 
 but slightly. At ordinary temperatures there is a 
 gradual deterioration in value, so that in from one to 
 six months they may become inert. Twenty hours' 
 exposure to a temperature of 60 C. does not destroy 
 them, but one hour at 70 C. does so almost totally. 
 Different protective substances differ as to the rapidity 
 with which they deteriorate. 
 
 Suitable animals after repeated infections gradually 
 accumulate in their blood considerable amounts of these 
 protective substances, so that very small amounts of 
 serum will inhibit the growth of the bacteria or neu- 
 tralize their products. Thus, 0.1 c.c. of a serum from 
 a horse frequently infected by the pneumococcus will 
 prevent the development in the body of a rabbit of one 
 hundred times the fatal dose of very virulent pneumo- 
 cocci, and a few times a fatal dose of less virulent ones, 
 the actual number as well as the virulence of the bac- 
 teria affecting the protective value of the serum. 
 
IMMUNITY. 109 
 
 These protective substances are found also in other 
 fluids of the body than in the blood; they occur, in- 
 deed, in the substance of all cells to a greater or less 
 extent. How much of this is simply in solution from 
 the serum, and where the substances are formed, is not 
 definitely known. 
 
 5. By the injection of the blood-serum of animals 
 which have previously passed through a specific disease 
 or have been inoculated with the bacterial products. 
 The first, probably, to think of the possibility of effect- 
 ing this was Raynaud, who, in 1877, showed that the 
 injection of large quantities of serum derived from a 
 vaccinated calf into an animal prevented its successful 
 vaccination. Hericourt, Richet, and others demon- 
 strated the same thing for other diseases. The results 
 obtained by Behring and Kitasato upon diphtheria and 
 tetanus, where, indeed, the serum prevented the action 
 of the poisons rather than the direct development of the 
 bacteria, gave a still greater impetus to these investiga- 
 tions. 
 
 The immunity produced by these substances affects 
 the entire body, as is only natural, since the blood into 
 which they are absorbed is distributed everywhere. 
 When the immunity is but slight, infection may take 
 place in the more sensitive regions and still be im- 
 possible in those tissues having more natural resistance. 
 If the serum is injected into other animals or man the 
 immunity is greatest immediately after absorption, and 
 then declines, being rather quickly (in several weeks or 
 months), almost entirely lost, so that repeated injections 
 are required to maintain the immunity. This is dis- 
 tinctly in contrast to the immunity acquired after the 
 introduction of bacterial products, where the tissues 
 
HO BACTERIOLOGY. 
 
 of the organism, in ways unknown, give out, in response 
 to the bacterial stimulus, inhibitory or antitoxic sub- 
 stances, or combine with the bacterial poisons to produce 
 them. Here immunity reaches its height a week or ten 
 days after the injection, and then continues for a week 
 or two, when it slowly declines again. The serum im- 
 munity is frequently called passive immunity and the 
 bacterial immunity active immunity. 
 
 If a greater quantity of protective substance is de- 
 sired in the blood than occurs after one infection, re- 
 peated injections of living or dead bacteria and their 
 products are given, the doses being administered at 
 short intervals and in sufficient amount to produce a 
 slight elevation of temperature and malaise. Then, as 
 soon as the animal returns to a normal condition, another 
 injection of slightly greater quantity is given. After 
 several months of such treatment the blood is withdrawn, 
 allowed to clot, and the serum then siphoned off asep- 
 tically and stored either with or without the addition of 
 preservatives. The serum is tested by mixing it with a 
 certain number of times the fatal dose of a culture or its 
 toxins whose virulence or toxicity is known, and then 
 injecting this under the skin, in the vein, or into the 
 peritoneum, according to the nature of the bacteria to 
 be tested. The main point is that some definite method 
 be carried out by which the relative value of the serum 
 can be judged in comparison with other serums. As a 
 rule, the value is stated in the number of fatal doses of 
 culture or toxin which a fraction of a cubic centimetre 
 of serum will prevent from destroying the animal. It 
 is well to remember, that with a living germ a mul- 
 tiple of a fatal dose is not as much more severe than a 
 single dose as the figure would suggest. One thousand 
 
IMMUNITY. HI 
 
 times a fatal dose of a very virulent micro-organism 
 will be neutralized by several times the amount of 
 serum which a single fatal dose requires, since in the 
 case of very virulent living bacteria whose virulence 
 is due to their ability to increase, it is not the organ- 
 isms which are introduced that kill but the millions 
 that develop from them. As a rule, the serum has to 
 be given before the bacteria introduced into the body 
 have multiplied greatly. After that period has elapsed 
 the serum usually fails to act, but some serums will 
 prevent further development even then. The immu- 
 nity conferred on a person from serum lasts from a 
 few days to several months, according to the amount 
 of serum injected. As in animals, it is strongest 
 immediately after absorption. An injection of bac- 
 terial poisons or the contraction of actual disease usu- 
 ally confers immunity from one to three weeks after 
 the infection, and lasts, according to the nature of the 
 infection, from one month to a year or more. The 
 serum loses all appreciable protective value as measured 
 in test animals in the usual doses before the person is 
 liable to infection. Repeated injections of serum con- 
 tinue this condition of immunity indefinitely. 
 
 The use of serums having specific protective proper- 
 ties has been tried both in animals and man as a pre- 
 ventive of infection. In susceptible animals injections 
 of some of the very virulent bacteria, as pneumococci, 
 streptococci, typhoid bacilli, and cholera spirilla, can 
 be robbed of all danger if small doses of their re- 
 spective serums are given before the bacteria have 
 increased to any great extent in the body. If given 
 later they are ineffective. For some bacteria, such 
 as tubercle bacilli, no serum has been obtained of suffi- 
 
112 BACTERIOLOGY. 
 
 cleat power to prevent infection. Through serums, 
 therefore, we can immunize against an infection, and 
 even stop one just commencing; but as yet we cannot 
 cure an infection which is already fully developed, 
 though even here there is reason to believe that we 
 may possibly prevent an invasion of the general system 
 from a diseased organ as by the pneumococcus from 
 an infected lung in pneumonia. On the whole, the 
 serums which simply inhibit the growth of bacteria 
 have not given, as observed in practice, conclusive 
 evidence of great value in already developed disease. 
 This is partly due to the difficulty to be discussed fully 
 later of determining early enough the exact nature of 
 the bacteria causing the infection. 
 
 Acquired Immunity to Poison. Although the serum 
 of animals which have been infected with any one of 
 many varieties of bacteria is usually both antitoxic and 
 bactericidal, still one of these protective substances may 
 be present almost alone; thus antitoxic substances are 
 present almost exclusively in animals injected with two 
 species of bacteria which produce powerful specific 
 poisons viz., the bacilli of diphtheria and tetanus. 
 When the toxins of either of these are injected in 
 small amounts the animals after complete recovery 
 are able to bear a larger dose without deleterious 
 effects, and these doses in the more suitable animals 
 can be gradually increased until a thousand times a 
 previously fatal dose may be administered without any 
 serious results whatever. To Behring and Kitasato we 
 owe the discovery that this protecting substance accu- 
 mulates to such an extent in the blood that very small 
 amounts of serum are sufficient to protect other animals 
 from the effects of the toxin. 
 
IMMUNITY. 113 
 
 Some other important parasitic bacteria produce 
 toxins and in the body antitoxins, but all to a far less 
 extent than those of tetanus and diphtheria. Follow- 
 ing them is the plague bacillus, and then, but far 
 behind, the cholera spirilla, the typhoid bacilli, the 
 streptococci, etc. These latter bacteria produce more 
 of the substances which inhibit bacterial growth than 
 of those which neutralize their toxins. 
 
 The effect of the antitoxin is to prevent the poison- 
 ous action of the toxin. It does not, so far as we 
 know, influence the cells after they have been injured 
 by the toxin; it is, therefore, a preventive rather than 
 a cure. \Ve find, experimentally, that a very much 
 smaller amount of antitoxin will neutralize a fatal dose 
 of toxin in an animal, if given before or at the same 
 time, than if given only shortly after it. An animal 
 already profoundly poisoned by the toxin is unaffected 
 by any amount of antitoxin. 
 
 The antitoxins of diphtheria and tetanus are gradu- 
 ally eliminated from the body after their injection or 
 after their production from toxin injections. After 
 the usual immunizing dose the duration of immunity 
 is only from two to six weeks, the period differing in 
 each individual. The elimination of the antitoxin 
 takes place partly through the urine and other secre- 
 tions, and it is partly destroyed in the body. An 
 animal which has been highly immunized will retain 
 considerable amounts of antitoxin for from two to four 
 months. 
 
 The antitoxins as contained in the serum are fairly 
 stable. The different antitoxins vary thus, that of 
 diphtheria is somewhat more stable than that of tetanus. 
 Kept aseptically in cold and dark storage, and pro- 
 
 8 
 
114 BACTERIOLOGY. 
 
 tected from access of air, the more resistant antitoxins 
 may be preserved sometimes for a year or two with 
 practically no deterioration in strength. At other 
 times, however, from unknown causes, they are gradu- 
 ally destroyed, so that there may be a loss of about 10 
 per cent, per month. A serum requires, therefore, to be 
 tested every few months if we wish to be assured of its 
 strength in antitoxin. Preservatives, such as carbolic 
 acid, trikresol, camphor, etc., alter antitoxins only very 
 slightly when in dilute solution, but in strong solution 
 they partially destroy them. Heat up to 62 C. does 
 not injure them greatly, but higher temperatures alter 
 them. In animals injected with diphtheria toxin 
 Atkinson has found that there is with the increase in 
 antitoxin an almost proportional increase in globulin. 1 
 He also found that the antitoxins behave like globulins 
 with the various reagents, being completely precipi- 
 tated by magnesium sulphate. NaCl, when added to 
 saturation to globulin solutions holding antitoxin, parti- 
 ally precipitates the globulin and the antitoxin. When 
 raised to 72 C. a series of precipitations are obtained 
 which contain at least the greater part of the antitoxin. 
 Whether this indicates that antitoxin is a form of 
 globulin or merely that it is similarly affected by many 
 reagents, and that the toxin in some way stimulates the 
 development of both, it is as yet impossible to say. 
 Although in a very rough way, the same animal pro- 
 duces antitoxin in direct proportion to the amount of 
 toxin injected so long as its condition remains good, yet 
 different animals of the same species give very varying 
 amounts from the same injections, some not giving one- 
 
 1 This work, carried out in the Research Laboratory of the Department of 
 Health of New York City, will appear in the Journ. of Exp. Med. in 1900. 
 
IMMUNITY. 115 
 
 fourth of that furnished by others. The antitoxin pro- 
 duced by a certain number of fatal doses of toxin will 
 neutralize many thousand times that amount. 
 
 Diphtheria and tetanus antitoxin are measured by the 
 protective power of the serum in which they are in 
 solution that is, the amount of serum required to pro- 
 tect susceptible animals from a certain number of fatal 
 doses. In diphtheria this is measured in units ; in 
 tetanus usually by the proportion which exists between 
 the amount of serum used and the animal's weight. For 
 detailed information, see under Diphtheria and Tetanus. 
 
 Antitoxins are absorbed to a very slight extent only 
 when taken by the mouth certainly less than 5 per 
 cent. They must, therefore, be introduced subcuta- 
 neously or intravenously to enter the body. The anti- 
 toxic serum does not act against the bacteria directly, 
 but by neutralizing their poison, it prevents them from 
 acting as irritants to the cells, and so the soil for the 
 growth of the bacteria becomes unsuitable, and they 
 cease to develop. The diphtheria bacilli grow perfectly 
 well iu their antitoxic serum. 
 
 The Elimination of the Bacteria and Their Products. 
 This takes place by the direct separation and removal 
 of the bacteria where there is access to the outside, 
 such as exists in the mucous membranes of the respi- 
 ratory, digestive, and urinary tracts, and from the 
 cutaneous surfaces, etc. The elimination of the bac- 
 teria and their products is almost a necessity where 
 there has been any great accumulation, if healing 
 occurs. When the bacteria have penetrated deeply 
 into the tissues, and continue steadily advancing, the 
 elimination from the surface is of little curative value, 
 as the number thrown off is so small in comparison 
 
116 BACTERIOLOGY. 
 
 with the number remaining. Occasionally the casting 
 off of the bacteria allows them to iufect other places, 
 as in some cases where laryngeal and intestinal tubercu- 
 losis follows pulmonary tuberculosis. We must bear in 
 mind, however, that infection in these regions may have 
 been produced through the lymph and blood channels. 
 
 In nearly all cases of infection the products of bacte- 
 rial growth are absorbed into the blood, and along with 
 them a few bacteria also, even when they do not repro- 
 duce themselves in it. The greater the extent of the 
 infection and the more deep-seated it is the greater is 
 the amount of absorption. The bacteria enter the blood, 
 according toKruse, by (1) passive eutrance through the 
 stomata of the capillary walls; (2) carriage into the 
 blood in the bodies of leucocytes; (3) growth of the 
 bacteria through the walls of the vessels; (4) transmis- 
 sion of the bacteria through the lymph-glands placed 
 between the lymph and bloodvessels. 
 
 When bacteria are abundant in the blood they become 
 fixed in the capillaries of one or all of the organs, espe- 
 cially of the liver, kidneys, spleen, and lungs, and then, 
 by means of the leucocytes, which penetrate the capil- 
 lary walls, or, directly, they pass into the tissues and 
 substance of the organs. They thus reach the lymph 
 channels and glands, or through the secretions gain 
 entrance into the gall-bladder, saliva, etc., or press 
 through the epithelium, as in the alveoli of the lungs; 
 more rarely they pass through the excretions into the 
 urine, as in typhoid fever, though some deny that this 
 can happen unless there is a previous inflammation of 
 the kidneys. The passage of bacteria through the 
 breast is important, from the fact that milk is so 
 largely used as food. Many observers have reported 
 
IMMUNITY. 117 
 
 the finding of tubercle bacilli in milk when the gland 
 itself was intact and the animal tubercular. Some 
 authorities have put its presence in milk, under these 
 circumstances, as high as 50 per cent, of the cases. 
 This, in our experience, is undoubtedly too high, and 
 probably these observers have been deceived by the 
 pseudo-tubercle bacilli. They are undoubtedly pres- 
 ent, however, in the milk of some animals in which 
 tubercular disease of the gland could not be demon- 
 strated. The finding of streptococci and staphylococci 
 is due probably in the majority of cases to the infections 
 taking place as the milk is voided, for the epithelium 
 at the outlet of the lacteal ducts is always infected with 
 staphylococci, and usually streptococci, which have often 
 been received from the mouth of the sucking infant. 
 
 Whether bacteria are eliminated from the blood by 
 the sweat is a mooted point. The skin is always the 
 seat of the staphylococcus and frequently of other bac- 
 teria, so that it is difficult to determine in any given 
 case the origin of the bacteria found in the sweat. 
 Many observers have reported the passage of bacteria 
 from the blood through the mucous membrane. So 
 long as the organs of secretion are not injured it is not 
 likely that many micro-organisms are eliminated from 
 the blood in this way. Bacteria are sometimes elimi- 
 nated through the urine, but here, as a rule, when 
 great numbers of organisms are found, it is due to 
 development in the bladder. Such removal, moreover, 
 has little if any beneficial effect; but, on the other hand, 
 may be a source of danger to others, as in typhoid 
 fever. The removal of the poisonous products of bac- 
 teria by the kidneys, intestines, etc., on the contrary, 
 is of great advantage to the organism. 
 
CHAPTER VI. 
 
 THEORIES OF INFECTION, IMMUNITY, AND RECOVERY. 
 
 THE tissues of the animal body under the normal 
 conditions of life are, as we have seen, unsuitable for 
 the growth of the great majority of the varieties of 
 bacteria. Indeed, only a very small number of the 
 parasitic bacteria find the conditions really satisfactory, 
 and even these must find a point of entrance into the 
 body. 
 
 In seeking to account for the difficulty which to a 
 greater or less extent all bacteria find to growing in 
 the tissues of the living body, we cannot find it either 
 in the insufficient or excessive concentration of the 
 nutritive substances, nor in the temperature, nor in the 
 reaction; for although some of these conditions may be 
 unsuitable for some bacteria, they are all suitable for 
 many, and thus cannot constitute the fundamental ex- 
 planation of either natural or acquired immunity. A 
 possible ground, for the inability of the bacteria to 
 invade living tissues, might be thought to be found in 
 the fact that the nutritious material in the living cells 
 is in a form which the bacteria cannot readily assimi- 
 late; but, if this be true to a certain extent, it does not 
 adequately explain why the bacteria do not develop in 
 the nutritious fluids, so abundant about and in the body 
 tissues, nor does it account for acquired immunity. We 
 are thus driven to the conclusion that the. body fluids 
 themselves contain substances which are directly dele- 
 
INFECTION, IMMUNITY, AND RECOVERY. H9 
 
 terious to the bacteria. As to the origin of these 
 substances, we may conceive that they may be either 
 regularly produced by the body cells, or by the fluids, 
 or by both, or that they may only be produced or at 
 least increased when bacteria invade the body. When 
 formed they may remain unaltered in the fluids or be 
 quickly eliminated or destroyed. It is probable that 
 more than one of these suppositions is actually true. 
 
 The bactericidal effect upon most bacteria of the body 
 fluids, noted by Nuttall in 1888, is now undisputed, 
 and is shown by the fact that bacteria when injected 
 into the blood usually soon die, and this destruction 
 may be so rapid that in a few hours none remain alive. 
 Even when bacteria survive and produce infection 
 there is for a time a decrease in the number living, 
 but this is soon followed by a progressive increase. 
 This fact can be observed not only by injecting bac- 
 teria into the blood and peritoneal cavity, but also when 
 the bacteria are placed in the animal body after being 
 enclosed in capsules. The bacteria are killed even if 
 they have previously grown outside the body in blood- 
 serum. Bacteria have also been injected into a vein 
 carefully ligated above and below, and here, without 
 coagulation, the blood exerts bactericidal properties. 
 The general germicidal effect of the blood-serum can 
 also be watched outside of the body. Here mixed 
 with it some species of bacteria die quickly, some 
 slowly, and some lose only a portion of their number, 
 those remaining alive after a time rapidly increasing. 
 The number of bacteria introduced is of great impor- 
 tance, for the serum with its contained substances seems 
 capable of destroying only a certain number, and after 
 that loses its bactericidal properties. 
 
120 BACTERIOLOGY. 
 
 If the bactericidal effect of the serum outside the 
 body always went hand-in-hand with the immunity of 
 the individual from which it was taken, the immediate 
 cause of immunity would be solved and our search be 
 directed to find the source and nature of these germi- 
 cidal substances; but this is not wholly the case, for 
 while in many instances it is so, in others it is as un- 
 doubtedly not true. We must, therefore, add to the 
 serum the activity of the cells, which produce constantly 
 the substances which are partly given up to the blood 
 and fluids of the body and partly retained in their own 
 bodies. This deleterious action of the blood in bac- 
 teria can be increased by infection. Some good ob- 
 servers have found that blood in animals naturally 
 immune to certain parasitic bacteria, which had little 
 or 110 bactericidal effect, became possessed of it after a 
 moderate infection; this seeming to indicate a protective 
 effort of the body cells to withstand bacterial invasion. 
 
 Concerning the nature of these non-specific protective 
 substances, named alexines by Buchner, we have as yet 
 little positive knowledge, but certain properties of them 
 are known. They are largely precipitated by a 40 per 
 cent, solution of sodium sulphate, but not by alcohol. 
 These substances would seem to belong to the so-called 
 living proteids, and resemble certain of the globulins 
 in their properties, but they are evidently extremely 
 complex in their nature. Many of them become inert 
 on standing for several months, even at low tempera- 
 tures, and after a few weeks at blood-heat. A tem- 
 perature above 62 to 70 C. soon totally destroys 
 them. Freezing does not affect them. A bactericidal 
 serum affects in a deleterious manner the red blood- 
 cells of a different species of animals. 
 
INFECTION, IMMUNITY, AND RECOVERY. 121 
 
 Their source must apparently be attributed to the cells, 
 but probably certain cells only produce them. The red 
 blood-cells, for instance, seem rather to destroy than to 
 increase them. The nuclein derived from the cells, al- 
 though it has a general bactericidal action, and may enter 
 into the alexines, yet as it has different properties it can- 
 not itself be one of these bodies. The cells which have 
 abundant nuclear substance, such as the leucocytes and 
 lymph-cells, seem especially to be a source of the alex- 
 ines. Buchner and others have found that through 
 the irritation of bacterial filtrates the leucocytes were 
 attracted in great numbers to the region of injection, 
 and that the fluid here, which was rich in leucocytes, 
 was more bactericidal than that of the blood-serum 
 elsewhere. The same fluid acted also more perfectly 
 when it contained numbers of leucocytes than when 
 they were filtered off. Substances similar to the alex- 
 ines are apparently derived from the leucocytes, and 
 their attraction to the injected area gives to that loca- 
 tion greater protective power. Some claim to have 
 demonstrated that along with increased leucocytosis 
 there is a general increase in the alexines in the blood, 
 still it has not yet been positively established that the 
 alexines are derived solely from the leucocytes, nor 
 from all leucocytes, and a mere increase in them does 
 not always mean an increase in alexines. The attrac- 
 tion between the leucocytes and the bacteria is due to 
 the chemical attraction between them and the bacterial 
 body substance and its poisons. Some chemical sub- 
 stances not derived from bacteria have this quality 
 also, called positive chemotaxis, while others repel the 
 leucocytes negative chemotaxis. The original theory 
 of Metchnikoff, that the leucocytes were the only actual 
 
 / 
 
122 BACTERIOLOGY. 
 
 protective bodies which warded off disease, and that 
 they did this by attacking the bacteria, was founded 
 on the observation that certain of the white cells pos- 
 sessed the power of taking up into themselves patho- 
 genic bacteria, and that they were there destroyed. It 
 was later observed that these cells had the property of 
 taking from the blood many lifeless foreign elements, 
 thereby keeping the blood-channels free of foreign par- 
 ticles. The question thereby arose as to whether these 
 cells engulfed and then killed the bacteria, or whether 
 perhaps other influences killed or injured them before 
 the cells took them up. The theory then became some- 
 what modified, more knowledge was obtained, and it is 
 now believed that the bacterial substances attract the 
 cells, and that when these cells are brought together the 
 general, and perhaps the specific, bactericidal property 
 of the blood in their neighborhood is thereby increased. 
 The death of the bacteria liberates this positive chemo- 
 taxic substance, and the disintegration of the white 
 blood-cells gives rise to the bactericidal bodies. Thus 
 we find that phagocytosis is most marked when the dis- 
 ease is on the decline or the infection mild, but that in 
 rapidly increasing progressive infection it is absent. 
 This would seem to indicate that the course of the in- 
 fection is often already determined before the leucocytes 
 become massed at the point of its entrance. The first 
 determining influence is given by the condition of the 
 tissues and the bactericidal substances contained in 
 them, and then, later, in cases where the infection is 
 checked, comes the additional bactericidal substance 
 given off by the attracted leucocytes. In so far as the 
 tissues themselves are unsuitable for the development 
 of bacteria they are sufficient to ward off infection, but 
 
INFECTION, IMMUNITY, AND RECOVERY. 123 
 
 in proportion as they are incapable of doing this they 
 are assisted by the substances contained in the leuco- 
 cytes. If the tissues are wholly adapted .for the 
 growth of the bacteria, neither they nor the leuco- 
 cytes, nor both combined, can furnish sufficient pro- 
 tective substances to prevent the bacterial increase. 
 The entrance of bacteria into the leucocytes, which is 
 not infrequent, may mean their destruction; but, on 
 the other hand, the bacteria may increase in the white 
 blood-cells and destroy them, and they may be killed 
 without entering the cells. The simple absorption by 
 the cells is not necessarily a destructive process. No 
 explanation can as yet be given of natural immunity 
 to bacterial poisons, except that it may be connected 
 with some general property of the tissue. There is far 
 less variation among different species in their resistance 
 to the bacterial poisons than in their suitability for the 
 growth of the living bacteria which produce them 
 Possibly certain organs, such as those which are rich 
 in nuclei n for example, the lymph-glands, the liver, 
 etc. may have some destructive power with regard to 
 poisons. The nature of the cell substance is known 
 to have much to do with its relations to certain poisons. 
 Thus the tetanus poison acts chiefly on the nerve cells 
 and leaves the others almost or altogether unaffected. 
 
 By what means are virulent bacteria enabled to in- 
 crease in the body, notwithstanding its protective powers, 
 when non-virulent organisms of the same species are 
 incapable of so doing? This is but little understood, 
 but experiment shows in the first place that both viru- 
 lent and non-virulent forms are equally resistant to 
 general destructive agencies; and, second, that the bac- 
 teria are capable of producing substances (lysines) which 
 
124 BACTERIOLOGY. 
 
 neutralize in some way the protective substances (alex- 
 ines). The virulence of bacteria would, therefore, de- 
 pend partly upon their ability to produce these lysines, 
 which act perhaps as the ferments upon the alexines, or 
 perhaps combine with them. That bacteria under certain 
 conditions form specific poisons, and under others, even 
 when they grow luxuriantly, do not, is clearly shown by 
 our experiments on the production of diphtheria toxin. 
 Here, as previously stated, it was found that when 
 the bouillon was either a little too alkaline or too acid, 
 though the bacilli grew rapidly, they did not produce 
 specific toxins. By growing the bacilli for a time in 
 such bouillon they eventually became able to develop 
 toxin in a soil in which they previously failed to do so. 
 Similar cultivation in the body may be assumed to 
 increase their ability to produce specific poison after a 
 while under what would at first be adverse conditions. 
 
 With regard to the increase and decrease of general, 
 and perhaps also of specific immunity, we have reason 
 to believe that as the protective substances are produced 
 by the living cells, anything which lowers the general 
 vitality must lessen the vitality of the cells, and thus 
 their ability to produce protective substances in the 
 amount possible in a normal condition. The attraction 
 of leucocytes to any point by some new infection might 
 increase the germicidal action of the tissues, and so in- 
 fluence the first infection. 
 
 Specific Immunity. The following theories have been 
 advanced concerning the nature of specific immunity: 
 The theory that a second infection is impossible because 
 the first used up substances which were necessary to the 
 growth of the bacteria is untenable for many reasons. 
 Thus it can be demonstrated that the injection of a 
 
INFECTION, IMMUNITY, AND RECOVERY. 125 
 
 small amount of specific serum, which robs the tissues 
 of nothing, produces the same immunity. Again, the 
 injection into the body of a sufficient number of patho- 
 genic bacteria gives rise to an infection in all cases. 
 
 The theory of Metchnikoff, that the leucocytes or 
 wandering cells of the body, after an infection with a 
 certain variety of bacteria, become influenced in some 
 way, so that they attack especially that form of infection 
 again and destroy the bacteria (phagocytosis), can no 
 longer be considered as more than a very partial ex- 
 planation, and can only be accepted by assuming as 
 proven a number of hypotheses. 
 
 The retention theory of Wernich and Chaveau, some- 
 what modified, has much to support it. The blood- 
 serum of animals recovering from an infection was found 
 to have changed chemically to such an extent as to be 
 capable of being demonstrated experimentally, and 
 these changes were shown to persist for a number 
 of weeks or months or even years. Similarly the serum 
 of immunized animals retains for a long time its immu- 
 nizing substances We are, therefore, compelled to ac- 
 cept the fact, that when an infection is passed through 
 there are more or less protective chemical substances 
 left in the blood, which remain there for a considerable 
 time. Kruse believes that these substances have the 
 power of neutralizing the bacterial poisons which are 
 given off by the bacteria upon their entrance into the 
 body, and of thus robbing them of their deleterious 
 effects on the alexines; the body fluids in this way 
 remaining unsuitable soil for the growth of the bac- 
 teria, the alexiues being bactericidal. If only a small 
 amount of antilysines are present some of the alexines 
 are destroyed and the bacteria are not all killed or 
 
126 BACTERIOLOGY. 
 
 weakened. Those remaining active are then further 
 acted upon by the alexines in the tissues and by the 
 substances given off by the leucocytes. If these pro- 
 tective substances are insufficient the infection is estab- 
 lished. R. Pfeiffer's experiments with cholera and 
 typhoid cultures injected into the peritoneal cavity of 
 the guinea-pig along with specific protective serum 
 showed that the bacteria were altered and destroyed by 
 the serum within a few minutes, just as if they had 
 been non-virulent bacteria, and this without the assist- 
 ance of the phagocytes. In this case non-virulent bac- 
 teria die because they produce no ly sines to destroy the 
 alexines, while those which are virulent do not thrive, 
 because although they produce lysines, the antilysines 
 in the serum destroy them, being thus acted upon by 
 alexines in like manner to the non-virulent bacteria. 
 
 As to the development of the specific protective sub- 
 stances, the most plausible theory seems to us to be that 
 they are formed by the activity of the cells from the 
 bacterial poisons, the lysiues. These substances are 
 stated by Pfeiffer and Marx to be most abundant in the 
 spleen, lymphatic glands, and bone-marrow. 
 
 Ehrlich and others believe antitoxin to be a portion 
 of the substance of certain cells, which, having been 
 stimulated by their effort to replace portions of their 
 substances destroyed by previous doses of toxin, have 
 reproduced it in excess. This cell substance, being 
 free in the fluids of the body, combines with the toxin, 
 and thus neutralizes it. But it is difficult by this 
 theory to explain many known facts, such as the one 
 that a fully neutralized mixture of toxin and antitoxin 
 is still capable of producing in the body more anti- 
 toxin. Others hold that the antitoxins, as the other 
 
INFECTION, IMMUNITY, AND RECOVERY. 127 
 
 protective substances, are always present in the body, 
 and that under special need, as when bacterial inva- 
 sion takes place, they are thrown out by the cells in 
 larger quantities, and this is especially true of the 
 special substances needed for existing infection. The 
 antitoxin then acts by fortifying the cells so that they 
 are enabled to resist the action of the toxin. In favor 
 of this view is the fact that the cells of certain ani- 
 mals are undoubtedly proof against these toxins, and 
 yet so far as chemistry in its present development can 
 detect, these cells are the same as similar but sensitive 
 cells in other animals. Another theory is that the 
 toxin, in some way in the body fluids, is changed into 
 antitoxin. This is made slightly plausible by the fact 
 that by the action of electricity there have been obtained 
 substances from toxins which are slightly antitoxic. 
 The practical point to remember is that whether or not 
 the theories are correct, there is no doubt that the pro- 
 tective substances exist. 
 
CHAPTER VII. 
 
 INFECTION. 
 
 THE spread of infection is influenced by: 1. The 
 number of species of animals subject to infection. 
 
 Many human infectious diseases do not occur in ani- 
 mals, and many animal infections are not found in man. 
 Thus, so far as we know, gonorrhoea, syphilis, measles, 
 smallpox, typhoid fever, etc., do not occur in animals 
 under ordinary conditions; while tuberculosis, anthrax, 
 glanders, hydrophobia, and some other diseases are 
 common to both man and animals. 
 
 2. The quantity of the infectious material thrown off 
 from the body and the prevalence of the disease. 
 
 In diphtheria, typhoid fever, cholera, pulmonary 
 tuberculosis, septic endometritis, influenza, and gonor- 
 rhoea enormous numbers of infectious bacteria are cast 
 off through the discharges from the mouth, intestines, 
 and genito urinary secretions, causing great danger of 
 infection. On the other hand, in tubercular perito- 
 nitis, cerebro- spinal meningitis, septic endocarditis, 
 gonorrhoeal rheumatism and the like, there is little or 
 no danger of infecting others, as few or no bacteria are 
 cast off. 
 
 3. The resistance of the infectious bacteria to the 
 deleterious effects of drying, light, heat, etc. 
 
 In this case the presence or absence of spores is of 
 the greatest importance. The spore-bearing bacilli, 
 
INFECTION. 129 
 
 such as tetanus, anthrax, etc., being able to withstand 
 destruction for a long time, retain their power of pro- 
 ducing infection for months or even years after elimi- 
 nation from the body. The bacteria which form no 
 spores show great variation in their resistance to out- 
 side influences. Some of these, such as the influenza 
 bacilli and the gonococci, the virus of syphilis and 
 hydrophobia, are extremely sensitive; the pneumococci, 
 cholera spirilla, glanders bacilli, etc., are a little hardier; 
 then follow the diphtheria bacilli, and after them the 
 typhoid and tubercle bacilli and the staphylococci. 
 
 4. The ability or the lack of ability to grow outside 
 of the infected tissues. 
 
 Such bacteria as the pneumococcus, tubercle, influ- 
 enza, and lebrosy bacilli do not develop, as far as we 
 know, outside of the body under ordinary conditions. 
 Others, like the diphtheria bacillus and the strepto- 
 coccus, may under certain conditions, as in milk in 
 warm places, develop and produce infection. Others, 
 again, such as the streptococcus and staphylococcus, 
 typhoid and anthrax bacillus, the cholera spirillum, 
 and some anaerobics, may develop under peculiar con- 
 ditions existing in water or soil. 
 
 While for the pathogenic bacteria, as a rule, the 
 saprophytes met with in the soil and water are antago- 
 nistic, yet in some cases and especially is this true of 
 the anaerobic bacteria they are helpful. Such bacilli 
 as tetanus are believed to require the associaton of an- 
 aerobic bacteria to permit of their development in the 
 soil in the presence of oxygen. 
 
 A large number of the infectious bacteria are able to 
 develop in or upon some portion of the skin or mucous 
 membrane, either after or before disease, and without 
 
 9 
 
130 BACTERIOLOGY. 
 
 causing infection. As complete a knowledge of these 
 facts as possible is necessary if we are to combat the 
 spread of infection. In the superficial layers of the 
 epithelium and on the surface of the skin we find the 
 different pyogenic cocci, which are capable of infecting 
 a wounded or injured part or causing inflammation in 
 the glands. Acne, the pustules in smallpox, the pus 
 on a burned surface, boils, etc., all come from the 
 pyogenic cocci. In surgical cases the skin has to be 
 as thoroughly disinfected as possible, to prevent the 
 formation of stitch-hole abscesses and wound- suppura- 
 tion. 
 
 In the secretion of the mucous membrane covering 
 the pharynx and nasopharynx there is always an abund- 
 ance of bacteria. In one hundred throats examined by 
 the writer in New York City, streptococci and staphylo- 
 cocci could be found in over 90 per cent., and pneumo- 
 cocci were very frequently discovered. Many other 
 varieties of bacteria, such as the influenza bacilli, are 
 probably often present in small numbers. In those con- 
 stantly in contact with cases of diphtheria, and in those 
 convalescent from diphtheria, virulent diphtheria bacilli 
 are frequently found in the throat. 
 
 After exposure to cold or injury of any kind, owing 
 to the presence of these bacteria, the persons harboring 
 them may develop tonsillitis, tonsillar abscess, or diph- 
 theria; or the bacteria may invade the bronchial mucous 
 membrane or the lungs. The diphtheria bacilli, and 
 perhaps other bacteria, transmitted to others may be- 
 come the source of infection to them, though the person 
 who spreads the infection may remain unaffected. 
 
 The stomach, on account of the acidity of its con- 
 tents, is'Vomparatively free from bacteria. The normal 
 
INFECTION. 131 
 
 intestines, on the other hand, contain great numbers of 
 bacteria. Among these the colon bacillus is constantly 
 present, and often the streptococcus and other patho- 
 genic bacteria. After typhoid fever the bacilli may 
 remain in the intestinal contents for weeks and in the 
 bladder and gall-bladder for months. The bacteria 
 swallowed to a considerable extent escape destruction 
 in the stomach, and thus appear in the intestines. Some 
 good observers have stated that bacteria can be absorbed 
 through the intestinal wall into the chyle and blood. 
 When the intestinal canal is injured, or its circulation 
 hindered by strangulation, etc., the bacillus coli and 
 some other bacteria may penetrate through the injured 
 walls and cause peritonitis or general infection. Under 
 certain conditions, as during the debility due to hot 
 weather, the bacteria in the intestines cause, through 
 their products, irritation, and in children even serious 
 intestinal inflammation. 
 
 The kidneys, bladder, and urethra may be the source 
 of infection and may give rise to disease in others. 
 Long after an acute gonorrhnea has passed gonococci 
 may remain in sufficient numbers to cause a new in- 
 flammation or produce infection in others. A cystitis 
 may run on chronically for years, and then suddenly 
 become acute or spread infection to the kidneys. After 
 typhoid fever the urine may contain abundant typhoid 
 bacilli for weeks and be little thought of as a source of 
 infection. A persistent gonorrhoeal vaginal infection 
 may lead to a gonorrhoeal endometritis, or salpingitis, or 
 peritonitis, under suitable conditions. The staphylococci 
 in the skin and the colon bacilli and pyogenic cocci in 
 the faecal discharges may also be carried into the uterus 
 and produce septic infection. 
 
132 BACTERIOLOGY. 
 
 Inherited Infection and Susceptibility to or Immunity 
 from Infection. The passage of bacteria from the 
 mother's blood through the placenta to the foetus has 
 been demonstrated for numerous bacteria, among the 
 most important of which may be mentioned the pneu- 
 mococcus, streptococcus, and tubercle bacillus. The 
 detection of the tubercle bacillus by Gartner and others 
 under these circumstances prevents us from denying the 
 possibility that tuberculosis developing in children may 
 have been due to infection taking place before birth. 
 The fact, however, that calves removed from tubercu- 
 lous cattle and fed on milk free from tubercle bacilli do 
 not develop tuberculosis, while those left with tubercu- 
 lous cattle become tuberculous, indicates that tubercu- 
 losis in man also is usually, at least, due to infection 
 after birth. The infection from spermatozoa is con- 
 ceivably possible in tuberculosis if the testicles are 
 affected; the same may be said of syphilis; but except 
 for syphilis, in which the nature of the infective agent 
 is unknown, we believe that such infection is, if ever 
 present, extremely rare. 
 
 Natural immunity pertains more to species than in- 
 dividuals, and such immunity is handed down by the 
 parents to their offspring. If the immunity of one or 
 both parents has been acquired by them during their 
 lifetime previous to the birth of the offspring the im- 
 munity conferred is slight or none at all. This is espe- 
 cially true of the male side. In the case of the female 
 parent another factor comes into play after the fructi- 
 fication of the ovum viz., the absorption of products 
 from the fluids of the mother, for the placenta is no 
 barrier to soluble substances. Thus, sheep which have 
 been immunized to anthrax have moderately immune 
 
INFECTION. 133 
 
 young. On the other hand, animals vaccinated with 
 cowpox have not been found to have immune offspring. 
 Toxins injected into the parents apparently do not pass 
 the placenta; but antitoxins do, giving thus a slight 
 transitory passive immunity. A slight immunity is 
 also given by immune mothers through their milk, a 
 small amount of antitoxic substance being absorbed. 
 
CHAPTER VIII. 
 
 THE EFFECT OF LIGHT, ELECTRICITY, PRESSURE, 
 AGITATION, DRYING, AND ASSOCIATION WITH OTHER 
 MICRO-ORGANISMS UPON BACTERIA. 
 
 VERY little is known about the influence of electricity 
 on bacteria. The majority of the observations hereto- 
 fore made on this subject would seem to indicate that 
 there is no direct action of the galvanic current on 
 bacteria; but the effect of heat and the electrolytic in- 
 fluence on the culture liquid may produce changes which 
 finally sterilize it. Gottstein and Spilker have recently 
 made experiments with an induction current from a 
 dynamo machine. They passed the current through 
 a spiral wire, which was wrapped around a test-tube 
 of glass containing the micro-organisms to be tested, 
 suspended in water. The bacillus prodigiosus, sus- 
 pended in sterilized distilled water and contained in 
 test-tubes having a capacity of 250 c.c., was subjected 
 to a current of 2.5 amperes -{- 1.25 volts for twenty- 
 four hours. The temperature did not go above 30 C. 
 No growth occurred when the organism tested was sub- 
 sequently planted in nutrient gelatin. It was found that 
 stronger currents were effective in a shorter time, but 
 in no case was sterilization effected in less than an hour. 
 These experiments, however, have not been confirmed. 
 
 Meltzer has shown that while slight agitation of 
 cultures of bacteria acts favorably on their develop- 
 
INFLUENCE OF LIGHT. 135 
 
 merit, the vitality of bacteria is destroyed by protracted 
 and violent shaking, which causes a molecular disinte- 
 gration of the cells. 
 
 D' Arson val and Charrin submitted a culture of bacil- 
 lus pyocyaneus to a pressure of fifty atmospheres under 
 carbonic acid. At the end of four hours cultures could 
 still be obtained, but the bacillus had lost its power of 
 pigment production. A few colonies were developed 
 after six hours 7 exposure to this pressure, but after 
 twenty-four hours no development occurred. 
 
 Influence of Light. A large number perhaps the 
 majority of bacteria are inhibited in growth by the 
 action of diffuse daylight, still more by that of direct 
 sunlight, and when the action is prolonged they lose 
 their power of developing when later placed in the dark. 
 
 In order to test the susceptibility of bacteria to 
 light, it is best, according to Buchner, to suspend a 
 large number of bacteria in nutrient gelatin or agar 
 and pour the media while still fluid in Petri dishes, 
 upon which has been pasted a strip of black paper on 
 the side upon which the light is to act. The action of 
 heat may be shut off by allowing the ray of light to 
 pass first through a layer of water or alum of several 
 centimetres' thickness. After the plates have been 
 exposed to the light for one-half, one, one and a half, 
 two hours, etc., they are taken into a dark room and 
 allowed to stand at 20 or 35 C., a sufficient length 
 of time to allow of growth, and then examined, to see 
 whether there are colonies anywhere except on the spot 
 covered by the paper; when the colonies exposed to the 
 light have been completely destroyed there is a sharply 
 defined region of the shape of the paper strip crowded 
 with colonies lying in a clear sterile field. 
 
136 BACTERIOLOGY. 
 
 Dieudonne, in experiments upon the bacillus pro- 
 digiosus, found that direct sunlight in March, July, and 
 August killed these bacilli in one and a half hours; in 
 November in two and a half hours. Diffuse daylight 
 in March and July restrained development after three 
 and a half hours 7 exposure (in November four and a 
 half hours), and completely destroyed vitality in from 
 five to six hours. Electric arc-light inhibited growth 
 in five hours and destroyed vitality in eight hours. 
 Incandescent light inhibited growth in from seven to 
 eight hours and killed in eleven hours. Similar results 
 have been obtained with B. coli, B. typhosus, and B. 
 anthracis. According to Koch, the tubercle bacillus is 
 killed by the action of direct sunlight in a time vary- 
 ing from a few minutes to several hours, depending 
 upon the thickness of the layer exposed and the season 
 of the year. Diffuse daylight also had the same effect, 
 although a considerably longer time of exposure was 
 required when placed close to a window, from five to 
 seven days. 
 
 Only the ultraviolet, violet, and blue rays of the 
 spectrum seem to possess bactericidal action; green 
 light is very much less so, red and yellow light not at 
 all. The action of light is apparently assisted by the 
 admission of air; anaerobic species, like the tetanus 
 bacillus, and facultative anaerobic species, such as the 
 colon bacillus, are able to withstand quite well the 
 action of sunlight in the absence of oxygen, the B. coli 
 intense direct sunlight for four hours. 
 
 According to Kichardson and Dieudonne, the mech- 
 anism of the action of light may be at least partially 
 explained by the fact that in agar plates exposed to 
 light for a short time (even after ten minutes 7 exposure 
 
INFLUENCE ONE SPECIES UPON ANOTHER. 137 
 
 to direct sunlight) hydrogen peroxide (H 2 O 2 ) is formed. 
 This is demonstrated by exposing an agar plate half- 
 covered with black paper, upon which a weak solution 
 of iodide of starch is poured, and over this again a 
 dilute solution of sulphate of iron; the side exposed 
 to the light turns blue-black. In gases containing no 
 oxygen, hydrogen peroxide is not produced, and the 
 light has no injurious effect. Access of oxygen also 
 explains the effect which light produces on culture 
 media which have been exposed to the action of sun- 
 light, as standing in the sun for a time, when after- 
 ward used for inoculation. The bacteria subsequently 
 introduced into such media grow badly far worse than 
 in fresh culture media which are kept in the shade. 
 
 Influence of One Species upon the Growth of Another. 
 While it is the custom of bacteriologists to have pure 
 cultures to work with, we should never forget that in 
 nature bacteria often occur in mixed cultures. If we 
 examine water, milk, or the contents of the intestines 
 of either sick or healthy persons we shall always find 
 several species of bacteria occurring together. This 
 admixture may, perhaps, seem to us at first merely 
 accidental, but on further investigation it will appear 
 that also in the department of bacteriology there exist 
 synergists and antagonists, or at least bacteria which 
 assist or oppose one another mutually or one-sidedly. 
 Nencki speaks of symbiosis and enantobiosix. 
 
 Gasse" has demonstrated experimentally the existence 
 of antagonisms by inoculating gelatin streak cultures of 
 various bacteria; it is found that many species will not 
 grow at all or only sparingly when in close proximity 
 to some other species. This antagonism, however, is 
 often only one-sided in character; for instance, the 
 
138 BACTERIOLOGY. 
 
 bacillus fluorescens putidus grows well when inoculated 
 between streaks of staphylococcus, but the latter micro- 
 coccus will not grow at all when inoculated between 
 cultures of the bacillus putidus, the growth of the 
 staphylococcus remaining scanty when the two species 
 are inoculated simultaneously. Again, when gelatin 
 or agar plates are made from two different species of 
 bacteria inoculated into the same tubes while liquid it 
 may be observed that only one of the two grows. A 
 third method of making this experiment is to simulta- 
 neously inoculate the same liquid medium with two 
 species, and then examine them later, both microscop- 
 ically and in thin plates; not infrequently the one spe- 
 cies may take precedence of the other, which it finally 
 overcomes entirely. The practical application of this 
 is to make only very thin plates for the estimation of 
 the number of bacteria or the isolation of pure cultures. 
 
 Finally, bacteria may oppose one another as antago- 
 nists in the animal body. As Emmerich has shown, 
 animals infected with anthrax may often be cured by a 
 secondary infection with the streptococcus. 
 
 The symbiotic or co-operative action of bacteria is of 
 still greater importance, of which the following examples 
 may be given: 
 
 1. Some bacteria thrive better in association with 
 other species than alone. Certain anaerobic species 
 grow even with the admission of air if only other 
 aerobic species are present (tetanus). 
 
 2. Certain chemical effects, as, for instance, the de- 
 composition of nitrates to gaseous nitrogen, cannot be 
 produced by many bacteria alone, but only when two 
 are associated. 
 
 3. In like manner it is observed that in a series of 
 
INFLUENCE OF NUTRITION AND MOISTURE. 139 
 
 soil bacteria none by itself may be pathogenic, but 
 when inoculated into animals in certain combinations 
 produce disease. 
 
 4. Slightly pathogenic species, such as attenuated 
 tetanus bacilli, for example, gain in virulence when 
 cultivated together with the proteus vulgaris. 
 
 Want of Nutrition and Water. When bacteria which 
 require much organic food for their development, and 
 these include most of the pathogenic species, are placed 
 in distilled water they soon die that is, within a few 
 days; even in sterilized well-water their life-duration 
 does not usually exceed eight to fourteen days, and they 
 rarely multiply. Instances, however, of much more 
 extended life under certain conditions are recorded. 
 Want of water affects bacteria in different ways. 
 Upon dried culture media development soon ceases; but 
 in media dried gradually at the room-temperature 
 (nutrient agar, gelatin, potato) they live often for a 
 longtime, even when there are no spores to account for 
 it. A shrunken residue of such cultures in bouillon has 
 often been found, after a year or more, to yield living 
 bacteria. The question as to how long the non-spore 
 bearing forms are capable of retaining their vitality 
 when dried on a cover glass or silk threads has been 
 variously answered. We know now that there are 
 many factors which influence the retention of vitality. 
 The following table of the results obtained by Sirena 
 and Alessi gives some idea of the extent and effect of 
 such influences. In the experiments silk threads were 
 saturated with bouillon cultures or aqueous suspensions 
 of the bacteria, and some then enclosed in tubes con- 
 taining sulphuric acid or calcium chloride, while others 
 were left exposed to various outside influences: 
 
140 
 
 BACTERIOLOGY. 
 
 Desiccation. 
 
 With sul- 
 phuric 
 acid, 
 killed at 
 end of 
 
 With 
 calcium 
 chloride, 
 killed at 
 end of 
 
 In incu- 
 bator at 
 37, 
 killed at 
 end of 
 
 In dry- 
 room, in 
 shade, 
 killed at 
 end of 
 
 In moist 
 room, 
 killed at 
 end of 
 
 Cholera spirilla . . . 
 
 1 day 
 
 Iday 
 
 1 day 
 
 1 day 
 
 12 days 
 
 B. of fowl cholera . . 
 
 2 days 
 
 1 " 
 
 1 " 
 
 5 days 
 
 59 " 
 
 B. typhosus 
 
 41 " 
 
 1 " 
 
 18 days 
 
 64 " 
 
 68 " 
 
 B. mallei 
 
 35 " 
 
 44 days 
 
 31 " 
 
 
 
 Diplococ. pneumonias . 
 
 114 " 
 
 31 " 
 
 131 " 
 
 164 " 
 
 192 " 
 
 The spirillum of Asiatic cholera is notorious for its 
 slight resistance to desiccation ; according to the ex- 
 haustive investigations of Koch and the above-men- 
 tioned observers, its life-duration is only from one to 
 five hours, depending upon the method of desiccation 
 employed. 
 
 The results of all investigators, however, would seem 
 to indicate that the greatest possible care must be exer- 
 cised in desiccation experiments to come to any positive 
 conclusions; but recently most astonishing results have 
 been obtained with regard to many species usually sup- 
 posed to be particularly sensitive to desiccation, showing 
 that under certain conditions they may retain their 
 vitality in a dry state for a very long time. Thus, 
 Koch found that cholera spirilla lived only a few hours 
 when dry; Kitasato determined their life-duration at 
 fourteen days at most; while Berckholz and various 
 French observers have found that they may, under 
 favorable conditions, live 150 to 200 days. The vary- 
 ing results sometimes reported by different observers in 
 such experiments may be explained by the fact that the 
 conditions under which they were made were different, 
 depending upon the desiccator used, the medium upon 
 
EFFECT OF OXYGEN. 141 
 
 which the cultures were grown and the use of silk 
 threads or cover-glasses. In all these experiments, of 
 course, it should be previously determined that in 
 spore-bearing species there are no spores present. 
 
 Behavior Toward Oxygen and Other Gases. As already 
 noted under the nutritious substances required by bac- 
 teria, it is customary to divide bacteria into three classes, 
 according to their behavior toward oxygen. 
 
 1 . Aerobic Bacteria. Growth only in the presence of 
 oxygen; the slightest restriction of air inhibits devel- 
 opment. Spore-formation especially requires the free 
 admission of air. 
 
 2. Anaerobic Bacteria. Growth and spore-formation 
 only in the total exclusion of oxygen. Among this class 
 of bacteria are the bacillus of malignant oedema, the 
 tetanus bacillus, the bacillus of symptomatic anthrax, 
 and many soil bacteria. Exposed to the action of oxy- 
 gen, the vegetative forms of these bacteria are readily 
 destroyed; these spores, on the contrary, are very re- 
 sistant. Anaerobic bacteria being deprived of oxygen 
 the chief source of energy supplied to the aerobic spe- 
 cies, by which they oxidize the nutritive substances in 
 the culture media they are dependent for their nutri- 
 tion upon decomposable substances, such as grape- 
 sugar, which on separating into two smaller molecules 
 alcohol and carbonic acid give out energy or heat. 
 Anaerobic bacteria, therefore, require for their cultiva- 
 tion, as a rule, media containing 1 to 2 per cent, of 
 glucose or some equivalent. 
 
 3. Facultative Aerobic and Facultative Anaerobic Bac- 
 teria. The greater number of aerobic bacteria, including 
 most of the pathogenic species, are capable of withstand- 
 ing, without being seriously affected, some restriction 
 
142 BACTERIOLOGY. 
 
 in the amount of oxygen admitted, and many, indeed, 
 grow equally luxuriantly in the partial exclusion of 
 oxygen. Life in the animal body, for example, as in 
 the intestines, necessitates existence with diminished 
 supply of oxygen. Pigment formation almost always 
 ceases with the exclusion of oxygen, but poisonous 
 products of decomposition are more abundantly pro- 
 duced (Hueppe). 
 
 It is important to note that, according to recent in- 
 vestigations, it has been shown that the aerobic devel- 
 opment of the anaerobes may be facilitated by the pres- 
 ence of living or dead aerobes. 
 
 It has also been observed not infrequently that certain 
 species which on their isolation at first showed more or 
 less anaerobic development that is, a preference to grow 
 in the depth of an agar stick culture, for instance 
 after a while seem to become strict aerobes, growing 
 only on the surface of the medium. This observation 
 proves that the simple fact of an organism showing 
 aerobic for anaerobic growth is not sufficient for its 
 separation into a distinct species. 
 
 While all facultative as well as strict anaerobes grow 
 well in nitrogen and hydrogen, they behave very differ- 
 ently toward carbonic acid gas. A large number of 
 these species do not grow at all, being completely in- 
 hibited in their development until oxygen is again 
 admitted for example, B. anthracis and B. subtilis 
 and other allied species. It has been found in some 
 species, as glanders and cholera, that the majority of 
 the organisms are quickly killed by CO 2 , while a few 
 offer a great resistance, rendering impossible complete 
 sterilization by means of this gas. Another group, 
 again viz., streptococcus and staphylococcus exhibits 
 
EFFECT OF CARBONIC ACID GAS. 143 
 
 a scanty growth; while a third group, like the B. 
 typhosus and B. prodigiosus, are not at all affected, 
 growing equally well as in the presence of oxygen, 
 and the liquefaction, even of gelatin, not being inter- 
 fered with; only, on account of the lack of oxygen, 
 there is no p : gment formation. Finally, a mixture of 
 one-fourth air to three-fourths carbonic acid gas seems 
 to have no injurious effect on bacteria which cannot 
 grow in an atmosphere of pure CO 2 . 
 
 Sulphuretted hydrogen in large quantity is a strong 
 bacterial poison, and even in small amount kills some 
 bacteria. 
 
CHAPTER IX. 
 
 EFFECT OF TEMPERATURE UPON BACTERIA. 
 
 IN judging the effect on bacteria of different agents 
 we have first to note the important fact that different 
 species of bacteria are differently influenced by the same 
 substances. Some bacteria thrive under conditions 
 which would destroy others, and they vary among 
 themselves in their powers of resistance to influences 
 which are deleterious to all. 
 
 Further, any species of bacteria will resist better 
 when under favorable conditions than under unfavor- 
 able ones. Bacteria also in recent cultures withstand 
 injury better than those in old cultures, so long as they 
 have not entered into the spore form. A ccor cK n g to 
 the amount of injury they have suffered, bacteria may 
 be only lamed in some of their functions or they may 
 be totally destroyed. Their loss of function may be 
 only temporary or permanent. 
 
 Every bacterial species makes certain demands on 
 the temperature of its culture media. Vegetative life 
 is possible within the limits of and 70 C. There 
 are some species, however, which grow at the lower and 
 others at the upper limit of these temperatures. The 
 maximum and minimum temperature for each indi- 
 vidual species lies about 30 C. apart. Bacteria have 
 been classified according to the temperatures at which 
 they develop, as follows : 
 
EFFECT OF TEMPERATURE UPON BACTERIA. 145 
 
 Psychrophilic Bacteria. Minimum at C., optimum 
 at 15 to 20 C., maximum at about 30 C. To this 
 class belong the water bacteria, such as the phosphor- 
 escent bacteria in sea-water. 
 
 Mesophilic Bacteria. Minimum at 10 to 15 C., 
 optimum at 37 C., maximum at about 45 C. To 
 this class belong all pathogenic bacteria, the conditions 
 for their pathogenic action in man requiring acclima- 
 tization to the temperature of the body. 
 
 Thermophilic Bacteria. Minimum at 40 to 49 C., 
 optimum at 50 to 55 C., maximum at 60 to 70 C. 
 This class includes many soil bacteria and almost ex- 
 clusively spore-bearing bacilli. According to Globig, 
 there are about thirty species of bacteria capable of de- 
 velopment at 60 C. and a few at 70 C. M.iguel has 
 described a certain bacillus thermophillm, which thrives 
 at from 42 to 72 C., its optimum being at 65 to 
 70 C., and found in cloacae, the contents of the intes- 
 tines, and in dirty water. Rabinowitsch has recently 
 described eight thermophilic facultative anaerobic spe- 
 cies, all spore-bearing, non-motile bacilli, the optimum 
 temperature of which is from 60 to 70 C., though 
 they grow slowly at from 34 to 44 C., and best on 
 anaerobic agar cultures. They are found widely dis- 
 tributed in the feces. 
 
 By carefully elevating or reducing the temperature 
 Dieudonn6 has succeeded in increasing the limits within 
 which a variety of bacteria will grow. Thus, anthrax 
 was gradually made to accommodate itself to a tem- 
 perature of 42 C., and pigeons, which are compara- 
 tively immune to anthrax, on account of their high 
 body temperature (42 C.), when inoculated with this 
 anthrax succumbed to the infection. DieudonnS also 
 
 10 
 
146 BACTERIOLOGY. 
 
 gradually acclimated anthrax to a temperature of 
 12 C., when it killed frogs kept at 12 C. We have 
 cultivated a very virulent diphtheria bacillus, so that 
 it will grow at 43 C. and produce strong toxin. 
 
 Bacterial growth is retarded by temperatures only a 
 little below the minimum of the species in question; 
 but they are not otherwise injured. Indeed, it is the 
 usual custom in laboratories to preserve bacteria 
 which die readily (such as streptococci) by keeping 
 them in the refrigerator at about 4 to 6 C , after cul- 
 tivation for two days at 20 C., as a means for retain- 
 ing their vitality without repeated transplantation. 
 Temperatures even far under* C. are only slowly 
 injurious to bacteria, different species being affected 
 with varying rapidity. Ordinarily, low temperatures, 
 though arresting the growth, do not destroy the vitality 
 of bacteria. This has been demonstrated by numerous 
 experiments in which they have been exposed for hours 
 in a refrigerating mixture at 18 C. They have 
 even been subjected by us to a temperature of 175 
 C. by immersing them in liquid air kept in an open 
 tube for two hours, and found to grow still when placed 
 in favorable conditions. 
 
 Temperatures from 5 to 10 C. over the optimum 
 affect bacteria injuriously in several respects. Varieties 
 are produced of diminished activity of growth, the 
 virulence and the property of causing fermentation are 
 decreased, and the power of spore-formation is gradu- 
 ally lost. These effects may predominate either in one 
 or the other direction. 
 
 If the maximum temperature is exceeded the organism 
 dies; the thermal death- point for the psychrophilic spe- 
 cies being about 37 C., for the mesophilic species about 
 
EFFECT OF TEMPERATURE UPON BACTERIA. 147 
 
 45 to 55 C., and for the thermophilic species about 
 75 C. There are no non-spore bearing bacteria which 
 when moist are able to withstand a temperature of 
 100 C. even for a few minutes. A long exposure to 
 temperatures between 80 and 100 has the same result 
 as a shorter one at the higher temperatures. Accord- 
 ing to Sternberg, ten minutes' exposure to moist heat 
 will at 52 C. kill the cholera spirillum, at 54 C. kill 
 the streptococcus, at 56 C. the typhoid bacillus, at 
 60 C. the gonococcus, and at 62 C. the staphylococ- 
 cus, the latter being about the most resistant of the 
 pathogenic organisms which have no spores. 
 
 When micro-organisms in a desiccated condition are 
 exposed to the action of heated dry air the temperature 
 required for their destruction is much above that re- 
 quired when they are in a moist condition or when they 
 are exposed to the action of hot water or steam. A 
 large number of pathogenic and non-pathogenic species 
 are able to resist a temperature of over 100 C. dry 
 heat for an hour. A temperature of 120 to 130 C. 
 maintained for one and a half hours is required to de- 
 stroy all bacteria, in the absence of spores, if dry heat 
 is used. 
 
 Spores are far more resistant to all injurious influ- 
 ences than vegetative forms. They retain their power 
 of germination for years without either nourishment or 
 water, and are much more indifferent to the action of 
 gases than bacilli, the spores of the anaerobic species 
 being especially resistant to the action of oxygen. 
 Spores possess a great power of resistance to both 
 moist and dry heat. Dry heat is comparatively well 
 borne, many spores resisting a temperature of over 
 130 C. The spores of bacillus anthracis and of 
 
148 BACTERIOLOGY. 
 
 bacillus subtilis require a temperature of 140 C. (dry 
 heat) maintained for three hours to insure their de- 
 struction. Moist heat at a temperature of 100 C., 
 either boiling water or free-flowing steam, destroys 
 the spores of known pathogenic bacteria within ten 
 minutes; certain non-pathogenic species, however, re- 
 sist this temperature for hours. Thus, Globig obtained 
 a bacillus from the soil, the spores of which required 
 five and a half to six hours' exposure to streaming 
 steam for their destruction. These spores survived 
 exposure for three-quarters of an hour in steam under 
 pressure at from 109 to 113 C. They were de- 
 stroyed, however, by exposure for twenty-five minutes 
 in steam at 113 to 116 C. and in two minutes at 
 127 C. 
 
 The resistance of spores to moist heat is tested by 
 suspending cover-glasses upon which the spores (an- 
 thrax) have been dried in little gauze bags in a boiling 
 steam sterilizer. The cover-glasses are removed from 
 minute to minute and laid upon agar plates, which are 
 then placed in the incubator at 37 C. Anthrax spores 
 are obtained by carefully removing sporulating streak 
 cultures on agar and heating the emulsion prepared 
 with a little water to 70 C. for five minutes. 
 
 In the practical application of steam for disinfecting 
 purposes it must be remembered that while steam under 
 pressure is more effective than streaming steam it is 
 scarcely necessary to give it the preference, in view of 
 the fact that all known pathogenic bacteria and their 
 spores are quickly destroyed by the temperature of 
 boiling water, and also that "superheated" steam is 
 less effective than moist steam. When confined steam 
 in pipes is " superheated " it has about the same germi- 
 
EFFECT OF TEMPERATURE UPON BACTERIA. 149 
 
 cidal power as hot, dry air at the same temperature. 
 Esmarch found that anthrax spores were killed in 
 streaming steam in four minutes, but were not killed 
 in the same time by superheated steam at a temperature 
 of 114 C. It should also be remembered that dry 
 heat has but little penetrating power. Koch and 
 Wolffhiigel found that registering thermometers placed 
 in the interior of folded blankets and packages of vari- 
 ous kinds did not show a temperature capable of kill- 
 ing bacteria after three hours' exposure in a hot-air 
 oven at 133 C. and over. 
 
 Fractional Sterilization (Tyndalization). Certain nutri- 
 ent media, such as blood-serum and the transudates of 
 the body cavities, as well as certain fluid food-stuffs, 
 such as milk, need at times to be sterilized, and yet 
 cannot be subjected to temperatures high enough to 
 kill spores without suffering injury. The property of 
 spores, when placed under suitable conditions, to germi- 
 nate into the non-spore bearing form, is here taken 
 advantage of by heating the fluids up to 55 to 70 C. 
 for one hour on each of six consecutive days. By this 
 means we kill, upon each exposure, all bacteria in the 
 vegetative form, and allow, during the intervals, for 
 the development of any still remaining in the spore 
 stage, or which have reproduced spores, to change again 
 into the vegetative form. Experience has shown 
 that, with but few exceptions, an exposure for six con- 
 secutive days will completely sterilize the fluids so 
 exposed. 
 
 Pasteurization. It is sometimes undesirable to expose 
 food, such as milk, to such a temperature as will destroy 
 spores, because of the deleterious effects of such high 
 temperatures, and yet where a partial sterilization is 
 
1 50 BA CTERIOL OGY. 
 
 necessary. Under these conditions we heat the food- 
 staffs for thirty minutes to such a temperature (70 C.) 
 as will kill the bacteria in the vegetative form, but 
 allow the spores to remain alive. Even this amount 
 of sterilization retards greatly the rapidity of putrefac- 
 tion and fermentation. 
 
CHAPTER X. 
 
 THE DESTRUCTION OF BACTERIA BY THE CHEMICALS. 
 
 MANY chemical substances when brought in contact 
 with bacteria unite with their cell substance. New 
 compounds are thus formed, and the life of the bacteria 
 and the disinfecting properties of the substances are usu- 
 ally destroyed. While in the vegetative stage bacteria 
 are much more easily killed than when in the spore 
 form, and their life processes are inhibited by substances 
 less deleterious than those required to destroy them. 
 
 Bacteria both in the vegetative and in the spore 
 form differ among themselves considerably in their 
 resistance to the poisonous effects of chemicals. The 
 reason for this is not as yet clear, but is apparently 
 connected with the structure and chemical nature of 
 their cell substance. 
 
 Chemicals are more poisonous at fairly high than at 
 a low temperature, and act more quickly upon bacteria 
 when they are suspended in fluids singly than when in 
 clumps. The increased energy of disinfectants at higher 
 temperatures indicates in itself a probability that a true 
 chemical reaction takes place. In estimating the ex- 
 tent of the destructive action of chemicals the follow- 
 ing degrees are usually distinguished: 
 
 1. The growth is not permanently interfered with, 
 but the pathogenic and zymogenic functions of the 
 organism are diminished attenuation. 
 
 2. The organisms are not able to multiply, but they 
 are not destroyed by antiseptic action. 
 
152 BACTERIOLOGY. 
 
 3. The vegetative development of the organisms is 
 destroyed, but not the spores incomplete sterilization. 
 
 4. Vegetative and spore-formation are destroyed. 
 This is complete sterilization or disinfection. 1 
 
 The methods employed for the determination of the 
 germicidal action of chemical agents on bacteria are, 
 briefly, as follows: 
 
 If it is desired to determine what is the minimum con- 
 centration of the chemical substance required to produce 
 complete inhibition of growth we proceed thus : A 10 
 per cent, solution of the disinfectant is prepared and 
 1 c.c., 0.5 c.c., 0.3 c.c., 0.1 c.c., etc., of this is added 
 to 10 c.c. of liquefied gelatin, agar, or bouillon, or, 
 more accurately, 10 c.c. minus the amount of solution 
 added, in so many tubes. The tubes then contain 1 per 
 cent., 0.5 per cent., 0.3 per cent., and 0.1 per cent, of 
 the disinfectant. The fluid med a in the tubes are 
 then inoculated with a platinum loopful of the test 
 bacteria. The melted agar and gelatin may be simply 
 shaken and allowed to remain in the tubes, and watched 
 as to whether any growth takes place, or the contents 
 of the tubes are poured out into Petri dishes, where the 
 development or lack of development of colonies and 
 the number can be observed. The same test can be 
 made with material containing only spores. 
 
 If it is desired to determine the degree of concentra- 
 tion required for the destruction of vegetative develop- 
 ment, the organism to be used is cultivated in bouillon, 
 and to each of a series of tubes holding in watery 
 solution different percentages of the disinfectant a 
 
 1 Disinfection strictly defined is the destruction of all organisms and their 
 products which are capable of producing disease. Sterilization is the destruc- 
 tion of all saprophytic as well as parasitic bacteria. Practically, however, 
 the two terms are used interchangeably as meaning the destruction of all 
 living bacteria. 
 
DESTE UCTION OF BA GTEEIA B Y CHEMICALS. 1 53 
 
 few drops of the culture from which all lumps have 
 been filtered are added. At intervals of one, five, ten, 
 fifteen, and thirty minutes, one hour, and so on, a small 
 platinum loopful of the mixture is taken from each tube 
 and inoculated into 10 c.c. of lukewarm gelatin, from 
 which plate cultures are made. The results obtained are 
 signified as follows : x per cent, of the disinfectant in 
 watery solution and at x temperature kills the organism 
 in twenty minutes, y per cent, kills in one minute, and 
 so on. If there be any doubt whether the trace of the 
 disinfectant carried over with the platinum loops may 
 have rendered the gelatin unsuitable for growth, thus 
 falsifying results, control cultures should be made with 
 vigorous bacteria in gelatin to which a similar trace of 
 the disinfectant has been added. 
 
 The disinfectant to be examined should always be 
 dissolved in an inert fluid, such as water; if on account 
 of its being difficultly soluble in water, it is necessary 
 to use alcohol for its solution, control experiments may 
 be required to determine the action of the alcohol on the 
 organism. Sometimes, as in the case of corrosive sub- 
 limate, the chemical unites with the cell substance to 
 form an unstable compound, which inhibits the growth 
 of the organism only while the union exists. In some 
 tests it is necessary to break up 'this union and note 
 then whether the organism is alive or dead. 
 
 In the above determinations the absolute strength of 
 the disinfectant required is considerably less when cul- 
 ture media rich in albumin are employed than when the 
 opposite is the case. Thus creoliu (Pearsons), when 
 bouillon is used as a culture medium, stops develop- 
 ment in the proportion of 1 to 15,000 or 1 to 5000; 
 when ox -serum is used only in the proportion of 1 to 
 
154 
 
 BACTERIOLOGY. 
 
 150 (Behring). Cholera spirilla grown in bouillon con- 
 taining no peptone or only 0.5 per cent, of peptone are 
 destroyed in half an hour by 0.1 per cent, of hydro- 
 chloric acid; grown in 2 per cent, peptone-bouillon 
 their vitality is destroyed in the same time on the 
 addition of 0.4 per cent. HC1. In any case the organ- 
 isms to be tested should all be treated in exactly the 
 same way and the results accompanied by a statement 
 of the conditions under which the tests were made. 
 
 The following table gives the results and methods 
 used in an actual experiment to test the effect of blood - 
 serum upon the disinfecting action of bichloride of 
 mercury and carbolic acid upon bacteria: 
 
 TEST FOR THE DIFFERENCE OF EFFECT OF BICHLORIDE OF 
 MERCURY AND CARBOLIC ACID SOLUTIONS ON ANTHRAX AND 
 TYPHOID BACILLI IN SERUM AND BOUILLON. 
 
 Time. 
 
 A. Serum . . . .2.5 c.c. 
 HgCl 2 sol. 1 : 1000 2.5 c.c. 
 Anthrax thread 
 
 B. Bouillon . . .2.5 c.c. 
 HgCl 2 sol. 1 : 1000 2.5 c.c. 
 Anthrax thread 
 
 C. Serum . . . .2.5 c.c. 
 Carbolic sol. 5 p.ct. 2.5 c.c. 
 Typhoid threads 
 
 D. Bouillon . . .2.5 c.c. 
 Carbolic sol. 5 p.ct. 2.5 c.c. 
 Typhoid threads 
 
 1 8 510208045 h U^ hr 2 s 
 
 Solution equals 
 
 1 in 2000 
 
 bichloride. 
 
 Same. 
 
 Solution equals 
 2% per cent, 
 carbolic acid. 
 
 Same. 
 
 Indicates total destruction of bacteria with no growth in media. 
 + Indicates lack of destruction of bacteria with growth in media. 
 
DESTR UCTION OF BACTERIA B Y CHEMICALS. 155 
 
 Pieces of sterile thread (one inch) were placed 
 in bouillon cultures of anthrax and typhoid bacilli 
 for ten minutes, then removed to Petri dishes, and 
 dried in the incubator for twenty-four hours. These 
 were then placed in serum and bouillon respectively 
 (2.5 c.c.). From each a control was taken. Then 2.5 
 c.c. HgCl 2 ( 1: 1000) and carbolic solution (5 per cent.) 
 was added to either, as shown in A, B, C, and D. 
 From each one thread was taken at varying periods of 
 time and planted in bouillon tubes. The threads from 
 A and B (HgCl 2 solution) were washed in sterile water, 
 then in a solution of ammonium sulphide (25 per cent.), 
 then in sterile water again, then in the bouillon. The 
 threads from the carbolic solution were washed in sterile 
 water before planting. 
 
 Observations: The serum seems to have an inhibi- 
 tory action with the bichloride solution, allowing a 
 growth up to forty-five minutes, while with bouillon 
 the action is much quicker, preventing a growth after 
 an exposure of one minute or over. With the carbolic 
 acid solution the serum seems to have made little or no 
 difference in the results. 
 
 Many substances which are strong disinfectants be- 
 come altered under the conditions in which they are 
 used, so that they lose a portion or all of their germ- 
 icidal properties; thus, quicklime and milk of lime are 
 disinfecting agents only so long as sufficient calcium 
 hydroxide is present. If this is changed by the carbon 
 dioxide of the air into carbonate of lime it becomes 
 harmless. Bichloride of mercury and many other 
 chemicals form compounds with many organic and 
 inorganic substances, which, though still germicidal, 
 are much less so than the original substances. 
 
156 BACTERIOLOGY. 
 
 A BRIEF DESCRIPTION OF SOME OF THE MORE 
 COMMONLY USED DISINFECTANTS. 
 
 Bichloride of Mercury. This substance, when present 
 in 1 part in 1,000,000 in nutrient gelatin or bouillon, 
 prevents the development of parasitic bacteria. In 
 water 1 part in 500,000 will kill many varieties in a 
 few minutes, but in bouillon twenty-four hours may 
 be needed. With organic substances its power is less- 
 ened, so that 1 part to 1000 may be required. Spores 
 are killed in 1 to 1000 watery solution within one hour. 
 Corrosive sublimate, as seen in the figures given above, 
 is less effective as a germicide in alkaline fluids con- 
 taining much albuminous substance than in watery 
 solution. In such fluids, beside loss in other ways, 
 precipitates of albuminate of mercury are formed 
 which are at first insoluble, so that a part of the mer- 
 curic salt does not really exert any action. In alkaline 
 solutions, such as blood, blood- serum, pus, tissue-fluids, 
 etc., the soluble compounds of mercury are converted 
 into oxides or hydroxides. The soluble compounds 
 can, of course, remain in solution only when there are 
 present sufficient quantities of certain bodies which 
 render solution possible. Bodies of this sort are espe- 
 cially the alkaline chlorides and iodides, and, above all, 
 sodium chloride and ammonium chloride. A very 
 simple way of preventing precipitation of the mer- 
 cury, then, is to add a suitable quantity of common 
 salt to the corrosive sublimate. Those compounds of 
 mercury which, like the cyanides, are not precipitated 
 with alkalies, because they at once form double salts, 
 require no addition of salt. 
 
 For ordinary use, where corrosive sublimate is em- 
 
DESTR UCTION OF BA CTEEIA B Y CHEMICALS. 1 57 
 
 ployed, solutions of 1 to 500 and 1 to 1000 will suffice, 
 when brought in contact with bacteria in that strength, 
 to kill the vegetative forms within fifteen minutes, 
 the stronger solution to be used when much organic 
 matter is present. 
 
 Biniodide of Mercury. This salt is very similar in 
 its effects to the bichloride. It is even somewhat more 
 powerful. 
 
 Nitrate of Silver. Nitrate of silver in solution has 
 about one-fourth the value of the bichloride of mer- 
 cury as a disinfectant, but nearly the same value in 
 inhibiting growth. 
 
 Sulphate of Copper. This salt has about 5 per cent, 
 of the value of mercuric chloride. 
 
 Sulphate of Iron. This is a very feeble disinfectant. 
 
 Sodium Compounds. A 30 per cent, solution of NaOH 
 kills anthrax spores in about ten minutes, and in 4 per 
 cent, in about forty-five minutes. Sodium carbonate 
 kills spores with difficulty even in concentrated solution, 
 but at 85 C. it kills spores in from eight to ten min- 
 utes. A 5 per cent, solution kills in a short time the 
 vegetative forms of bacteria. Even ordinary soapsuds 
 have a slight bactericidal as well as a marked cleansing 
 effect. The bicarbonate has almost no destructive effect 
 on bacteria. 
 
 Calcium Compounds. Calcium hydroxide Ca(OH) 2 is a 
 powerful disinfectant; the carbonate, on the other hand, 
 is almost of no effect. A 1 per cent, watery solution of 
 the hydroxide kills bacteria which are not in the spore 
 form within a few hours. A 3 per cent, solution kills 
 typhoid bacilli in one hour. A 20 per cent, solution 
 added to equal parts of feces or other filth and mixed 
 with them will completely sterilize them within one hour. 
 
158 BACTERIOLOGY. 
 
 The Effect of Acids. An amount of acid which equals 
 40 c.c. of normal hydrochloric acid per litre is sufficient 
 to prevent the growth of all varieties of bacteria and 
 to kill many. Twice this amount destroys most bacteria 
 within a short time. The variety of acid makes little 
 difference. Bulk for bulk, the mineral acids are more 
 germicidal than the vegetable acids, but that is because 
 their molecular weight is so much less. A 1 to 500 
 solution of sulphuric acid kills typhoid bacilli within 
 one hour. Hydrochloric acid is about one-third weaker, 
 and acetic acid somewhat weaker still. Citric, tartaric, 
 malic, formic, and salicylic acids are similar to acetic 
 acid. Boric acid destroys the less resistant bacteria in 
 2 per cent, solution and inhibits the others. 
 
 GASEOUS DISINFECTANTS. 
 
 The germicidal action of gases is much more active 
 in the presence of moisture than in a dry condition. 
 
 Numerous experiments have been made with sulphur 
 dioxide gas (SO 2 ), owing to the fact that it has been so 
 extensively used for the disinfection of hospitals, ships, 
 apartments, clothing, etc. This gas is a much more 
 active germicide in a moist than in a dry condition; 
 due, no doubt, to the formation of the more active disin- 
 fecting agent sulphurous acid (H 2 SO 3 ). In a pure state 
 anhydrous sulphur dioxide does not destroy spores, and 
 is not certain to destroy bacteria not in spore form. 
 Sternberg has shown that the spores of the bacillus 
 anthracis and bacillus subtilis are not killed by contact 
 for some time with liquid SO 2 (liquefied by pressure). 
 Koch found that various species of spore-bearing bacilli 
 
titiSTR UOTION OF BACTERIA S Y CHEMICALS. 159 
 
 exposed for ninety-six hours in a disinfecting chamber 
 to the action of SO 2 in the proportion of from 4 to 6 
 per cent, by volume were not destroyed. In the ab- 
 sence of spores, however, the anthrax bacillus in a 
 moist condition, attached to silk threads, was found 
 by Sternberg to be destroyed in thirty minutes in an 
 atmosphere containing 1 volume per cent. As the re- 
 sult of a large number of experiments with SO 2 as a 
 disinfectant it has been determined that an " exposure 
 for eight hours to an atmosphere containing at least 
 4 volumes per cent, of this gas in the presence of moist- 
 ure 77 will destroy most if not all of the pathogenic 
 bacteria in the absence of spores. 
 
 Peroxide of Hydrogen (H 2 O 2 ) is an energetic disin- 
 fectant, and in 2 per cent, solution (about 40 per cent, 
 of the ordinary commercial article) will kill the spores 
 of anthrax in from two to three hours. A 20 per cent, 
 solution of a good commercial hydrogen peroxide solu- 
 tion will quickly destroy the pyogenic cocci and other 
 spore-free bacteria. It combines with organic matter, 
 becoming inert. It is prompt in its action and not 
 poisonous, but apt to deteriorate if not properly 
 kept. 
 
 Chlorine is a powerful gaseous germicide, owing its 
 activity to its affinity for hydrogen and the consequent 
 release of nascent oxygen when it comes in contact with 
 micro-organisms in a moist condition. It is, there- 
 fore, a much more active germicide in the presence of 
 moisture than in a dry condition. Thus, Fischer and 
 Proskauer found that dried anthrax spores exposed for 
 an hour in an atmosphere containing 44.7 per cent, of 
 dry chlorine were not destroyed; but if the spores were 
 previously moistened and were exposed in a moist 
 
160 BACTERIOLOGY. 
 
 atmosphere for the same time, 4 per cent, was effective, 
 and when the time was extended to three hours, 1 per 
 cent, destroyed their vitality. The anthrax bacillus, 
 in the absence of spores, was killed by exposure in a 
 moist atmosphere containing 1 part to 2500 for twenty- 
 four hours. 
 
 In watery solutions 0.2 per cent, kills spores within 
 five minutes and the vegetative forms almost imme- 
 diately. 
 
 Chloride of Lime. The efficacy of chloride of lime 
 depends on the chlorine it contains in the form of 
 hypochlorites. A solution in water of 0.5 to 1 per 
 cent, of chloride of lime will kill most bacteria in one 
 to five minutes. A 5 per cent, solution usually de- 
 stroys spores within one hour. 
 
 Bromine and iodine are of about the same value as 
 chlorine for gaseous disinfectants, in the moist condition; 
 but, like chlorine, they are not applicable for general 
 use in house disinfection, owing to their poisonous and 
 destructive properties; they have a use in sewers and 
 similar places. 
 
 Trichloride of iodine in 0.5 per cent, solution de- 
 stroys the vegetative forms of bacteria in five minutes. 
 
 ORGANIC DISINFECTANTS. 
 
 Alcohol in 10 per cent, solution inhibits the growth 
 of bacteria; absolute alcohol kills bacteria in the vege- 
 tative form in from several to twenty-four hours. 
 
 Formaldehyde. Formaldehyde, or formic-aldehyde, 
 was isolated by von Hoffmann in 1867, who obtained 
 it by passing the vapors of methyl-alcohol mixed with 
 air over finely divided platinum heated to redness. 
 
DESTR UCTION OF BA CTEEIA B Y CHEMICALS. 161 
 
 The methyl-alcohol is oxidized and produces formal- 
 dehyde as follows: 
 
 CH 2 OH + O = CH 2 O H 2 O. 
 
 Formaldehyde is a gaseous compound having the 
 chemical formula CH 2 O and possessed of an extremely 
 irritating odor. At a temperature of 68 F. the gas 
 is polymerized that is to say, a second body is formed, 
 composed of a union of two molecules of CH 2 O. This 
 is known as a paraformaldehyde, and is a white, soapy 
 body, soluble in boiling water and alcohol; it exists in 
 the solution of commerce a clear, watery liquid con- 
 taining from 33 to 40 per cent, of the gas and 10 to 20 
 per cent, of methyl-alcohol, its chief impurity. If the 
 commercial solution ordinarily known in the trade as 
 "formalin" is evaporated or concentrated above 40 
 per cent., paraformaldehyde results; and when this is 
 dried in vaccuo over sulphuric acid a third body 
 trioxymethylene is produced, consisting of three mole- 
 cules of CH 2 O. This is a white powder, almost soluble 
 in water or alcohol, and giving off a strong odor of 
 formaldehyde. The solid polymers of formaldehyde, 
 when heated, are again reduced to the gaseous condi- 
 tion; ignited, they finally take fire and burn with a 
 blue flame, leaving but little ash. 
 
 Formaldehyde has an active affinity for many organic 
 substances, and forms with some of them definite chem- 
 ical combinations. It combines readily with ammonia 
 to produce a compound called ammoniacal-aldehyde, 
 which possesses neither the odor nor the antiseptic 
 properties of formaldehyde. This action is made use 
 of in neutralizing the odor of formaldehyde when it is 
 desired to dispel it rapidly after disinfection. Formal- 
 
 11 
 
162 BACTERIOLOGY. 
 
 dehyde also forms combinations with certain aniline 
 colors viz., fuchsin and safranin the shades of which 
 are thereby changed or intensified. These are the only 
 colors, however, which are thus affected, and as they 
 are seldom used in dyeing, owing to their liability to 
 fade, this effect is of little practical significance. The 
 most delicate fabrics of silk, wool, cotton, fur, leather 
 etc., are unaffected in texture or color by formaldehyde. 
 Iron and steel are attacked, after long exposure, by the 
 gas, and more so by its solution; but copper, brass, 
 nickel, zinc, silver, and gilt work are not at all acted 
 upon. Formaldehyde unites with nitrogenous products 
 of decay fermentation or decomposition forming true 
 chemical compounds, which are odorless and sterile. It 
 is thus a true deodorizer in that it does not replace one 
 odor by another more powerful, but forms new chemical 
 compounds which are odorless. Formaldehyde has a 
 peculiar action upon albumin, which it transforms into 
 an insoluble and indecomposable substance. It ren- 
 ders gelatin insoluble in boiling water and most acids 
 and alkalies. It is from this property of combining 
 chemically with the albuminoids forming the protoplasm 
 of bacteria that formaldehyde is supposed to derive its 
 bactericidal powers. Formaldehyde is an excellent pre- 
 servative of organic products. It has been proposed to 
 make use of this action for the preservation of meat, 
 milk, and other food products; but, according to Trillat 
 and other investigators, formaldehyde renders these sub- 
 stances indigestible and unfit for food. It has been 
 successfully employed, however, as a preservative of 
 pathological and histological specimens. 
 
 There are no exact experiments recorded of the 
 physiological action of formaldehyde on the human 
 
DESTR UCTION OF BA CTERIA B Y CHEMICALS. 163 
 
 subject when taken internally. Slater and Rideal 1 
 report that a 1 per cent, solution has been taken in 
 considerable quantity without serious results; and tri- 
 oxymethylene has been given in doses up to 90 grains 
 as an intestinal antiseptic. The vapors of formalde- 
 hyde are extremely irritating to the mucous membrane 
 of the eyes, nose, and mouth, causing profuse lachry- 
 mation, coryza, and flow of saliva. Aronson reports 
 that in many of his experiments rabbits and guinea- 
 pigs allowed to remain for twelve and twenty-four 
 hours in rooms which were being disinfected with for- 
 maldehyde gas were found to be perfectly well when 
 the rooms were opened. On autopsy the animals 
 showed no injurious effects of the gas. Others have 
 noticed that animals, such as dogs and cats, which 
 have accidentally been confined for any length of time 
 in rooms undergoing formaldehyde disinfection occa- 
 sionally died from the effects of the gas. Many 
 observers, however, have reported that insects, such as 
 roaches, flies, and bedbugs, are not, as a rule, affected. 
 The result of these observations would seem to indicate 
 that although formaldehyde is comparatively non-toxic 
 to the higher forms of animal life, nevertheless a cer- 
 tain degree of caution should be observed in the use of 
 this agent. 
 
 The results of numerous experiments have shown 
 that in the air, 2.5 per cent, by volume of the aqueous 
 solution, or 1 per cent, by volume of the gas, are suffi- 
 cient to destroy fresh virulent cultures of the common 
 pathogenic bacteria in a few minutes. The researches 
 of Pottevin and Trillat have shown that the germicidal 
 
 i Lancet, April 21, 1894. 
 
164 BACTERIOLOGY. 
 
 power of the gas depends not only upon its concentra- 
 tion, but also upon the temperature and the condition 
 of the objects to be sterilized. As with other gaseous 
 disinfectants viz., sulphur dioxide and chlorine it has 
 been found that the action is more rapid and complete 
 at higher temperatures i. e., at 35 to 45 C. (95 to 
 120 F.) and when the test objects are moist than at 
 lower temperatures and when the objects are dry. Still 
 it has been repeatedly demonstrated by actual experi- 
 ment in rooms that it is possible to disinfect the surface 
 of apartments and articles contained in them, under the 
 conditions of temperature and moisture ordinarily 
 existing in rooms, by an exposure of a few hours to 
 a saturated atmosphere of formaldehyde gas. 
 
 Stahl l has shown that bandages and iodoform gauze 
 can be kept well sterilized by placing in the jars con- 
 taining them pieces of <( formolith," a preparation of 
 paraformaldehyde in tablet form containing 50 per 
 cent, of formaldehyde. The same experimenter has 
 also succeeded in making carpets and articles of cloth- 
 ing germ-free by spraying them with 0.5 to 2 per cent, 
 solution of formaldehyde for fifteen to twenty minutes 
 without the color of the fabrics being in any way 
 affected. The investigations of Trillat, Aronson, Pot- 
 tevin, and others have shown that a concentration of 
 f * ne aqueous solution (40 per cent.), equal to 
 ^ P ure formaldehyde, was safe and sufficiently 
 powerful to retard bacterial growth. 
 
 A 2 per cent, watery solution of formalin destroys 
 the vegetative forms of bacteria within five minutes. 
 In our experiments formalin has upon the vegetative 
 
 1 Pharmaceutische Zeitung, No. 22, 1893. 
 
DESTR UCTION OF BA CTEEIA B Y CHEMICALS. 165 
 
 forms about one-third the strength of pure carbolic 
 acid. 
 
 Chloroform. This substance, even in pure form, does 
 not destroy spores, but it does bacteria in vegetative 
 form, even in 1 per cent, solution. Chloroform is used 
 practically in sterilizing and keeping sterile blood-serum, 
 which can be used later for culture purposes by driving 
 off the chloroform. 
 
 lodoform. This substance has but very little destruc- 
 tive action upon bacteria; indeed, upon most varieties 
 it has no appreciable effect whatever. When mixed 
 with putrefying matter, wound discharges, etc., the 
 iodoform is reduced into soluble iodine compounds, 
 which partly act destructively upon the bacteria and 
 partly unite with the poisons already produced. 
 
 Carbolic Acid (C 6 H 5 OH). A solution having 1 part 
 to 1000 inhibits the growth of bacteria; 1 part to 400 
 kills the less resistant bacteria, and 1 part to 100 kills 
 the remainder. A 5 per cent, solution kills the less 
 resistant spores within a few hours and the more 
 resistant in from one day to four weeks. A slight 
 increase in temperature aids the destructive action; 
 thus, even at 37.5 spores are killed in three hours. 
 A 3 per cent, solution kills streptococci, staphylococci, 
 anthrax bacilli, etc., within one minute. Carbolic acid 
 loses much of its value when in solution in alcohol or 
 ether. An addition of 0.5 HC1 aids its activity. 
 Carbolic acid is so permanent and so comparatively 
 little influenced by albumin that it is rightly widely 
 used in practical disinfection even in places of more 
 powerful substances. 
 
 Cresol [C 6 H 4 (CH 3 )OH] is the chief ingredient of the 
 so-called " crude carbolic acid." This is almost in- 
 
166 BACTERIOLOGY. 
 
 soluble in water, and has, therefore, little value. 
 Many methods are used for bringing it into solution 
 so as to make use of its powerful disinfecting prop- 
 erties. With equal parts of crude sulphuric acid it is 
 a powerful disinfectant, but it is, of course, strongly 
 corrosive. An alkaline emulsion of the cresols and 
 other products contained in " crude' 7 carbolic acid 
 with soap is called creolin. It is used in 1 to 5 per 
 cent, emulsions. It is fully as powerful as pure car- 
 bolic acid. Lysol is similar to creolin, except that it 
 has more of the cresols and less of the other products. 
 It and creolin are of about the same value. 
 
 Tricresol is a refined mixture of the three cresols 
 (meta-, para-, and ortho-). It is soluble in water to 
 the extent of 2.5 per cent., and is about three times 
 the strength of carbolic acid. 
 
 Aniline Dyes. Some of these colors possess marked 
 germicidal qualities. According to observers, methyl- 
 violet (pyoktanin) and malachite-green destroy the 
 typhoid bacillus in bouillon cultures in the proportion 
 of 1 to 200 in two hours'* exposure, and the pyogenic 
 cocci in less. In 1 to 100,000 solutions they are said 
 to retard the development of bacteria. 
 
 Oil of turpentine, 1 to 200, prevents the growth of 
 bacteria. 
 
 Camphor has very slight antiseptic action. 
 
 Creosote in 1 to 200 kills many bacteria in ten min- 
 utes; 1 to 100 failed to kill tubercle bacilli in twelve 
 hours. 
 
 Essential oils: Cardiac and Meumir found that the 
 essences of cinnamon, cloves, thyme, and others killed 
 typhoid bacilli within one hour. Sandal- wood required 
 twelve hours. 
 

 DESTR UCTION OF BA CTERIA B Y CHEMICALS. 167 
 
 Thymol and eucalyptol have about one-fourth the 
 strength of carbolic acid (Behring). 
 
 Oil of peppermint in 1 to 100 solution prevents the 
 growth of bacteria. 
 
 TABLE OF ANTISEPTIC VALUES. 1 
 
 Alum . . -. 
 
 1 : 222 
 
 Mercuric chloride 
 
 1 14300 
 
 Aluminium acetate , . 
 Ammonium chloride . 
 Boric acid . . 
 
 1 : 6000 
 1 :9 
 1 : 143 
 
 Mercuric iodide . 
 Potassium bromide . 
 Potassium iodide 
 
 1 40000 
 1 10 
 1 10 
 
 Calcium chloride 
 Calcium hypochiorite 
 Carbolic acid . . . 
 Chloral hydrate . . 
 Cupric sulphate . 
 Ferrous sulphate . . . 
 Formaldehyde (40 per cent.) 
 Hydrogen peroxide . 
 
 1:25 
 1 :1000 
 1: 333 
 1 :107 
 1 : 2000 
 1 : 200 
 1 : 10000 
 1 : 20000 
 
 Potassium permanganate . 
 Pure formaldehyde . 
 Quinin sulphate . 
 Silver nitrate 
 Sodium borate 
 Sodium chloride . 
 Zinc chloride 
 Zinc sulphate 
 
 1 300 
 1 25000 
 1 800 
 1 12500 
 1 14 
 1 6 
 1 500 
 1 20 
 
 1 These figures are approximately correct, and represent the percentage of 
 disinfectant required to be added to a fluid containing considerable organic 
 material, in order to permanently inhibit any bacterial growth. 
 
CHAPTEE XI. 
 
 PRACTICAL DISINFECTION AND STERILIZATION (HOUSE, 
 PERSON, INSTRUMENTS, AND FOOD) STERILIZATION 
 OF MILK FOR FEEDING INFANTS. 
 
 DISINFECTANTS AND METHODS OF DISINFECTION 
 EMPLOYED IN THE HOUSE AND SICK-ROOM. 
 
 Disinfection and Disinfectants. 
 
 SUNLIGHT, pure air, and cleanliness are always very 
 important agents in maintaining health and in protect- 
 ing the body against many forms of illness. When, 
 however, it becomes necessary to guard against such 
 special dangers as accumulated filth or contagious dis- 
 eases, disinfection is essential. In order that disinfec- 
 tion shall afford complete protection it must be thorough, 
 and perfect cleanliness is better, even in the presence of 
 contagious disease, than filth with poor disinfection. 
 
 Since all forms of fermentation, decomposition, and 
 putrefaction, as well as the infectious and contagious 
 diseases, are caused by micro-organisms, it is the object 
 of disinfection to kill these. Decomposition and putre- 
 faction should at all times be prevented by the imme- 
 diate destruction or removal from the neighborhood of 
 the dwelling of all useless putrescible substances. In 
 order that as few articles as possible shall be exposed to 
 the germs causing the contagious diseases and thus be- 
 come carriers of infection, it is important that all arti- 
 cles not necessary for immediate use in the care of the 
 
DISINFECTION AND STERILIZATION. 169 
 
 sick person, especially upholstered furniture, carpets, 
 and curtains, should be removed from the room before 
 placing in it the sick person. 
 
 Agents for Cleansing and Disinfection. 
 
 Too much emphasis cannot be placed upon the im- 
 portance of cleanliness, both as regards the person and 
 the dwelling, in preserving health and protecting the 
 body from all kinds of infectious disease. Sunlight 
 and fresh air should be freely admitted through open 
 windows, and personal cleanliness should be attained 
 by frequently washing the hands and body. 
 
 Cleanliness in dwellings, and in all places where men 
 go, may, under ordinary circumstances, be well main- 
 tained by the use of the two following solutions : 
 
 1. Soapsuds Solution. For simple cleansing, or for 
 cleansing after the methods of disinfection by chemicals 
 described below, one ounce of common soda should be 
 added to twelve quarts of hot soap (soft soap) and water. 
 
 2. Strong Soda Solution. This, which is a stronger 
 and more effective cleansing solution and also a feeble 
 disinfectant, is made by dissolving one-half pound of 
 common soda in three gallons of hot water. The solu- 
 tion thus obtained should be applied by scrubbing with 
 a hard brush. 
 
 When it becomes necessary to arrest putrefaction or 
 to prevent the spread of contagious diseases by surely 
 killing the living germs which cause them, more powerful 
 agents must be employed than those required for simple 
 cleanliness, and these are commonly called disinfectants 
 The following are some of the most reliable ones: 
 
 3. Heat. Complete destruction by fire is an abso- 
 lutely safe method of disposing of infected articles of 
 
170 BACTERIOLOGY. 
 
 small value, but continued high temperatures not as 
 great as that of fire will destroy all forms of life; thus, 
 boiling or steaming in closed vessels for one-half hour 
 will absolutely destroy all disease germs. 
 
 4. Carbolic Acid Solution. Dissolve six ounces of 
 carbolic acid in one gallon of hot water. This makes 
 approximately a 5 per cent, solution of carbolic acid, 
 which, for many purposes, may be diluted with an 
 equal quantity of water. The commercial " crude car- 
 bolic acid" should not be used, as it does not readily 
 enter into solution. Care must be taken that the pure 
 acid does not come in contact with the skin. 
 
 5. Bichloride Solution (bichloride of mercury or cor- 
 rosive sublimate). Dissolve sixty grains of pulverized 
 corrosive sublimate and two tablespoonfuls of common 
 salt in one gallon of hot water. This solution must be 
 kept in glass, earthen, or wooden vessels (not in metal 
 vessels). For safety it is well to cover the solution. 
 
 The carbolic and bichloride solutions are very pois- 
 onous when taken by the mouth, but are harmless when 
 used externally. 
 
 6. Milk of Lime. This mixture is made by adding 
 one quart of dry, freshly slaked lime to four or five 
 quarts of water. (Lime is slaked by pouring a small 
 quantity of water on a lump of quicklime. The lime 
 becomes hot, crumbles, and as the slaking is completed 
 a white powder results. The powder is used to make 
 milk of lime.) Air-slaked lime (the carbonate) has no 
 value as a disinfectant. 
 
 7. Dry Chloride of Lime. This must be fresh and 
 kept in closed vessels or packages. It should have 
 the strong, pungent odor of chlorine. 
 
 8. Formalin. Add one part of formalin to ten of 
 
DISINFECTION AND STERILIZATION. 171 
 
 water. This equals in value the 5 per cent, carbolic 
 acid solution. 
 
 9. Creolin, Tricresol, and Lysol are of about the same 
 value as pure carbolic acid. 
 
 The proprietary disinfectants, which are so often 
 widely advertised and whose composition is kept secret, 
 are relatively expensive and often unreliable and ineffi- 
 cient. It is important to remember that substances 
 which destroy or disguise bad odors are not necessarily 
 disinfectants and that there are very few disinfectants 
 that are not poisonous when taken internally. 
 
 [NOTE. The cost of the carbolic solution is much 
 greater than that of most of the other solutions, but 
 except for the disinfection of the skin, which in some 
 persons it irritates, generally is to be much preferred 
 by those not thoroughly familiar with disinfectants, as 
 it does not deteriorate, and is rather more uniform in 
 its action than some of the other disinfectants.] 
 
 Methods of Disinfection in Infectious and Contagious 
 Diseases. 
 
 The diseases to be commonly guarded against, outside 
 of surgery, by disinfection are scarlet fever, measles, 
 diphtheria, tuberculosis, smallpox, typhoid and typhus 
 fever, yellow fever, and cholera. 
 
 1. Hands and Person. Dilute the carbolic solution 
 with an equal amount of water or use the bichloride 
 solution without dilution. Hands soiled in caring for 
 persons suffering from ontagious diseases, or soiled 
 portions of the patient's body, should be immediately 
 and thoroughly washed with one of these solutions and 
 then washed with soap and water, and finally immersed 
 
172 BACTERIOLOGY. 
 
 again in the solutions. The nails should always be 
 kept perfectly clean. Before eating the hands should 
 be first washed in one of the above solutions, and then 
 thoroughly scrubbed with soap and water by means of 
 a brush. 
 
 2. Soiled Clothing, Towels, Napkins, Bedding, etc., 
 should be immediately immersed in the carbolic solu- 
 tion, in the sick-room, and soaked for one or more hours. 
 They should then be wrung out and boiled in the soap- 
 suds solution for one hour. Articles such as beds, 
 woollen clothing, etc., which cannot be washed, should 
 at the end of the disease be referred to the Health De- 
 partment, if such is within reach, for disinfection or 
 destruction ; or if there is no public disinfection, these 
 goods should be thoroughly exposed to formaldehyde 
 gas, as noted later. 
 
 3. Food and Drink. Food thoroughly cooked and 
 drinks that have been boiled are free from disease germs. 
 Food and drinks, after cooking or boiling, if not immedi- 
 ately used, should be placed when cool in clean dishes 
 or vessels and covered. In the presence of an epidemic 
 of cholera or typhoid fever, milk and water used for 
 drinking, cooking, washing dishes, etc., should be 
 boiled before using, and when cholera is prevalent all 
 persons should avoid eating uncooked fruit, fresh vege- 
 tables, and ice. Instead of boiling milk may be heated 
 to 80 C. for one-half hour. 
 
 4. Discharges of all Kinds from the Mouth, Nose, Blad- 
 der, and Bowels of patients suffering from contagious 
 diseases should be received into glass or earthen vessels 
 containing the carbolic or bichloride of mercury solu- 
 tion, or milk of lime, or they should be removed on 
 pieces of cloth, which are immediately immersed in one 
 
DISINFECTION AND STERILI2A TION. 1 73 
 
 of these solutions. Special care should be observed to 
 disinfect at once the vomited matter and the intestinal 
 discharges from cholera patients. In typhoid fever 
 the urine and the intestinal discharges, and in diph- 
 theria, measles, and scarlet fever the discharges from 
 the throat and nose, all carry infection, and should be 
 treated in the same manner. The volume of the solu- 
 tion used to disinfect discharges should be at least twice 
 as great as that of the discharge. After standing for 
 an hour or more the disinfecting solution with the dis- 
 charges may be thrown into the water-closet. Cloths, 
 towels, napkins, bedding, or clothing soiled by the dis- 
 charges must be at once placed in the carbolic solution 
 and the hands of the attendants disinfected, as described 
 above. In convalescence from measles and scarlet fever 
 the scales from the skin are also carriers of infection. 
 To prevent the dissemination of disease by means of 
 these scales the skin should be carefully washed daily in 
 warm soap and water. After use the soapsuds should 
 be disinfected and thrown into the water-closet. 
 
 Masses of feces are extremely difficult to disinfect 
 except on the surface, for it takes disinfectants such as 
 the carbolic acid solution some twelve hours to pene- 
 trate to their interior. If fecal masses are to be thrown 
 into places where the disinfectant solution covering 
 them will be washed off, it will be necessary to be cer- 
 tain that the disinfectant has previously penetrated to 
 all portions and destroyed the disease germs. This can 
 be brought about by stirring them up with the disinfec- 
 tant and allowing the mixture to stand for one hour, or 
 by washing them into a pot holding soda solution which 
 is already at the boiling temperature, or later will be 
 brought to one. 
 
174 BACTERIOLOGY. 
 
 5. The Sputum from Consumptive Patients. The im- 
 portance of the proper disinfection of the sputum from 
 consumptive patients is still underestimated. Con- 
 sumption is an infectious disease, and is always the 
 result of transmission from the sick to the healthy or 
 from animals to man. The sputum contains the germs 
 which cause the disease, and in a large proportion of 
 rases is the source of infection. After being discharged, 
 unless properly disposed of, it may become dry and pul- 
 verized and float in the air as dust. This dust con- 
 tains the germs, and is a common cause of the disease, 
 through inhalation. In all cases, therefore, the sputum 
 should be disinfected when discharged. It should be 
 received in covered cups containing the carbolic or milk 
 of lime solution. Handkerchiefs soiled by it should be 
 soaked in the carbolic solution and then boiled. Dust 
 from the walls, mouldings, pictures, etc., in rooms that 
 have been occupied by consumptive patients, where the 
 rules of cleanliness have not been carried out, contain 
 the germs and will produce tuberculosis in animals when 
 used for their inoculation ; therefore, rooms should be 
 thoroughly disinfected before they are again occupied. 
 If the sputum of all consumptive patients were de- 
 stroyed at once when discharged a large proportion of 
 the cases of the disease would be prevented. 
 
 6. Closets, Kitchen and Hallway Sinks, etc. The closet 
 should never be used for infected discharges until they 
 have been thoroughly disinfected, if it can be avoided; 
 if done, one pint of carbolic solution should be poured 
 into the pan (after it is emptied) and allowed to remain 
 there. Sinks should be flushed at least once daily. 
 
 7. Dishes, Knives, Forks, Spoons, etc., used by a patient 
 should, as a rule, be kept for his exclusive use and not 
 
DISINFECTION AND STERILIZATION. 175 
 
 removed from the room. They should be washed first 
 in the carbolic solution, then in boiling hot soapsuds, 
 and finally rinsed in hot water. These washing fluids 
 should afterward be thrown into the water-closet. The 
 remains of the patient's meals may be burned or thrown 
 into a vessel containing the carbolic solution or milk of 
 lime, and allowed to stand for one hour before being 
 thrown away. 
 
 8. Rooms and Their Contents. Rooms which have 
 been occupied by persons suffering from contagious 
 disease should not be again occupied until they have 
 been thoroughly disinfected. For this purpose either 
 careful fumigation with formaldehyde gas or sulphur 
 should be employed, or this combined with the following 
 procedure : Carpets, curtains, and upholstered furniture 
 which have been soiled by discharges, or which have 
 been exposed to infection in the room during the illness, 
 will be removed for disinfection to chambers where they 
 can be exposed to formaldehyde gas and moderate 
 warmth for twelve to twenty-four hours, or to steam. 
 Woodwork, floors, and plain furniture will be thor- 
 oughly washed with the soapsuds and bichloride solu- 
 tions. 
 
 9. Rags, Cloths, and Articles of Small Value, which 
 have been soiled by discharges or infected in other 
 ways, should be boiled or burned. 
 
 10. In Case of Death, the body should be completely 
 wrapped in several thicknesses of cloth wrung out of 
 the carbolic or bichloride solution, and when possible 
 placed in a hermetically sealed coffin. 
 
 It is important to remember that an abundance of 
 fresh a//-, sunlight, and absolute cleanliness not only 
 helps protect the attendants from infection and aid in 
 
176 BACTERIOLOGY. 
 
 the recovery of the sick, but directly destroys the bac- 
 teria which cause disease. 
 
 Methods of Cleanliness and Disinfection to Prevent the 
 Occurrence of Illness. 
 
 1. Water-closet Bowls and all Receptacles for Human 
 Excrement should be kept perfectly clean by frequent 
 flushing with a large quantity of water, and as often as 
 necessary disinfected with the carbolic or bichloride solu- 
 tions. The woodwork around and beneath them should 
 be frequently scrubbed with the hot soapsuds solution. 
 
 2. Sinks and the Woodwork Around and the Floor 
 Beneath them should be frequently and thoroughly 
 scrubbed with the hot soapsuds solution. 
 
 3. School Sinks. School sinks should be thoroughly 
 flushed with a large quantity of water at least twice 
 daily, and should be carefully cleaned twice a week or 
 oftener by scrubbing. Several quarts of the carbolic 
 solution should be frequently thrown in the sink after 
 it has been flushed. 
 
 4. Cesspools and Privy Vaults. An abundance of 
 milk of lime or chloride of lime should be thrown into 
 these daily, and their contents should be frequently 
 removed. 
 
 5. Cellars and Rooms in Cellars are to be frequently 
 whitewashed, and, if necessary, the floors sprinkled 
 with dry chloride of lime. Areas and paved yards 
 should be cleaned, scrubbed, and, if necessary, washed 
 with the bichloride solution. Street gutters and drains 
 should be cleaned, and, when necessary, sprinkled with 
 chloride of lime or washed with milk of lime. 
 
 6. Air-shafts. Air-shafts should be first cleaned 
 thoroughly and then whitewashed. To prevent ten- 
 
DISINFECTION AND STERILIZATION. 177 
 
 ants throwing garbage down air-shafts it is sometimes 
 advisable to put wire netting outside of windows open- 
 ing on shafts. Concrete or asphalt bottoms of shafts 
 should be cleaned and washed with the bichloride solu- 
 tion or sprinkled with chloride of lime. 
 
 7. Hydrant Sinks, Garbage Receptacles, and Garbage 
 and Oyster-shell Shutes and Receptacles should be 
 cleaned daily and sprinkled with dry chloride of lime. 
 
 8. Refrigerators and the Surfaces Around and Beneath 
 Them, Dumb-waiters, etc., may be cleaned by scrubbing 
 them with the hot soapsuds solution. 
 
 9. Traps. All traps should be flushed daily with an 
 abundance of water. If at any time they become foul 
 they may be cleaned by pouring considerable quantities 
 of the hot strong soda solution into them, followed by 
 the carbolic solution. 
 
 10. Urinals and the Floors Around and Underneath 
 them should be cleaned twice daily with the hot soap- 
 suds solution, and in addition to this, if offensive, they 
 may be disinfected with the carbolic solution. 
 
 11. Stable Floors and Manure Vaults. Stable floors 
 should be kept clean and occasionally washed with the 
 hot soapsuds or the hot strong soda solution. Pow- 
 dered fresh chloride of lime or formalin may be used 
 in manure vaults. 
 
 12. Vacant Rooms should be frequently aired. 
 
 13. The Woodwork in School-houses should be scrubbed 
 weekly with hot soapsuds. This refers to floors, doors, 
 door-handles, and all woodwork touched by the scholars 7 
 hands. 
 
 14. Spittoons in all Public Places should be emptied 
 daily and washed with the hot soapsuds solution, after 
 which a small quantity of the carbolic solution or milk 
 
 12 
 
178 BACTERIOLOGY. 
 
 of lime should be put in the vessel to receive the expec- 
 toration. 
 
 15. Elevated and Surface Cars, Ferry-boats, and Public 
 Conveyances. The floors, door-handles, railings, and 
 all parts touched by the hands of passengers should be 
 washed frequently with the hot soapsuds solution. Slat- 
 mats from cars, etc., should be cleaned by scrubbing 
 with a stiff brush in the hot soapsuds solution. 
 
 Use of Bromine Solution as a Deodorant. Slaughter- 
 houses, butchers' ice-boxes and wagons, trenches, excava- 
 tions, stable floors, manure-vaults, dead animals, offal, 
 offal docks, etc., may be deodorized by a weak solution 
 of bromine, which is a valuable agent for this purpose. 
 The bromine solution, however, is only temporary in 
 its action, and must be used repeatedly. It should be 
 applied by sprinkling. Although somewhat corrosive 
 in its action on metals, it is otherwise harmless. 
 
 The solution of bromine must be prepared with great 
 care, as the pure bromine from which it is made is dan- 
 gerous. It is very caustic when brought in contact with 
 the skin; it is volatile and its fumes are very irritating 
 when inhaled. To prepare the solution an ounce bottle 
 of liquid bromine is dropped into three gallons of water, 
 and broken under the water and thoroughly stirred. 
 
 The Practical Employment of Formaldehyde and Sulphur 
 Dioxide Gases in the surface disinfection of rooms and the 
 disinfection of goods which would be injured by heat. 
 Formaldehyde gas has so recently come into use, and is 
 for many purposes so valuable, that the description of 
 methods employed to generate and use it will be given 
 in detail. 
 
 If we consider now the practical application of 
 formaldehyde gas for purposes of disinfection we find 
 
DISINFECTION AND STERILIZATION. 179 
 
 that its destructive action on micro-organisms depends 
 upon a number of factors, the chief of which are its 
 concentration in the surrounding atmosphere, the length 
 of the contact, the existing temperature, the accompany, 
 ing moisture, and the nature of the organism. 
 
 The necessary concentration of the gas in the sur- 
 rounding atmosphere to kill the micro-organisms varies 
 with each species, for some resist chemical agents much 
 more than others, and also with the freedom of access 
 of the gas to the bacteria, for if they are under cover 
 or within fabrics a greater amount of gas must be gen- 
 erated than if they are freely exposed. 
 
 For purely surface disinfection, when the less resistant 
 bacteria or other micro-organisms are to be destroyed, 
 there will be required, according to the method used, 
 6 to 10 ounces of formalin of full strength, or its equiv- 
 alent, to 1000 cubic feet. 
 
 For the destruction of the more resistant, but non- 
 spore bearing forms, such as typhoid fever or tubercle 
 bacilli, at least twelve ounces of formalin should be used. 
 The gas penetrates through fabrics with difficulty, and 
 to pass through heavy goods the concentration of the 
 gas must be doubled and heat added. 
 
 The Value of Moisture. At first it was thought that 
 formaldehyde gas acted more effectually in a dry atmos- 
 phere, but further investigation has proved that although 
 it does destroy bacteria with the amount of moisture 
 usually present in the air, and contained in their own 
 substance, yet it acts much more powerfully and cer- 
 tainly when additional moisture is present, and best 
 when present up to the point of saturation. The 
 actual spraying of walls and goods to be disinfected 
 with water is even more efficacious. 
 
180 BACTERIOLOGY. 
 
 A fairly high /temperature but one still below that 
 which would injure delicate fabrics increases not only 
 the activity of formaldehyde gas but also its penetra- 
 tive power, and for heavy goods it is essential. The 
 production of a partial vacuum in the chambers be- 
 fore the introduction of the formaldehyde gas still 
 further assists its penetration. 
 
 The length of exposure necessary for complete dis- 
 infection depends upon the nature of the disease for 
 which it is carried out the penetration required, the 
 concentration of the gas used, the amount of moisture 
 in the air, the temperature of the air, and the size and 
 shape of the room. For surface disinfection in rooms, 
 when as much as 12 ounces of formalin are used for 
 each 1000 cubic feet, five hours' exposure is amply 
 sufficient, most bacteria being killed within the first 
 few minutes. For the destruction of micro-organisms 
 protected by even a layer of thin covering, double the 
 formalin and double the time of exposure should be 
 allowed, and even then the killing of many species of 
 non-spore bearing bacteria cannot be counted upon 
 in ordinary rooms. When absolutely complete disinfec- 
 tion is demanded, where penetration of gas is required, 
 the goods must be placed in chambers where moderate 
 heat can be added and all leakage of gas prevented. 
 
 Various forms of apparatus can be properly employed 
 to liberate formaldehyde gas for purposes of disinfec- 
 tion, as each of these is lauded by its maker as the 
 best; it may be of interest to give the results obtained 
 by us from those in most common use. There are two 
 essentials to any good method namely, that the for- 
 maldehyde gas is given off quickly; and that there is 
 no great loss by deterioration of the formalin. 
 
DISINFECTION AND STERILIZATION. 181 
 
 From Wood Alcohol. A number of lamps have been 
 devised, all very much on the same principle, though 
 varying somewhat in mechanical construction, which 
 bring about the incomplete oxidation of methyl-alcohol 
 by passing the vapors mixed with air over the incan- 
 descent metal. Although disinfection can be carried 
 out by the best of these lamps, in our experience none 
 of them up to the present time are satisfactory or eco- 
 nomical. They may be very useful as deodorizers in 
 the sick-room or other places. 
 
 In spite of present failures, it is, however, probable 
 that in the future this method may become practicable. 
 
 From Formochloral by the Trillat System. This sys- 
 tem consists in heating, under three atmospheres of 
 pressure, a solution of formaldehyde gas in water 
 mixed with 30 per cent, of calcium chloride, known 
 as " forrnochloral," to a temperature of 135 C. 
 (255 F.). It is claimed for this method of pro- 
 ducing the gas from formochloral that the polymeri- 
 zation of the formaldehyde is prevented, which would 
 otherwise take place if a solution of formaldehyde were 
 evaporated under ordinary conditions, and that thereby 
 the whole of the formaldehyde is obtained in the gas- 
 eous state. The addition of any neutral salt aids the 
 process, it is said, but calcium chloride is the best. 
 The results with this apparatus have been satisfactory, 
 but not more so than by other methods. The appa- 
 ratus is expensive and heavy. 
 
 From Formalin by the New York Sanitary Construction 
 Company's System. This system consists in heating 
 the ordinary commercial formalin to a temperature of 
 about 1000 F. in an incandescent copper coil or 
 chamber, and allowing the vapors to pass off freely. 
 
182 BACTERIOLOGY. 
 
 It is claimed for this method that the degree of heat 
 necessary to break up the polymerized products formed 
 is supplied, and thus a loss of formaldehyde is pre- 
 vented. A further action of the intense heat in the 
 copper tube on the solution is to partially convert the 
 methyl-alcohol contained in commercial formalin into 
 formaldehyde gas by partial oxidation, thereby pre- 
 venting the formation of methylal and increasing the 
 amount of formaldehyde. 
 
 The apparatus consists of a closed receiver of copper 
 holding about a gallon, a coil of copper pipe attached 
 at one end to the bottom of the receiver, and, like the 
 preceding apparatus and that made by Lentz, at the 
 other, by means of a suitable connection (rubber tube 
 with gutta-percha or metallic mouth-piece), with the 
 room or apartment to be disinfected, and a heating 
 lamp (Swedish lamp or Bunsen burner). In opera- 
 tion the desired quantity of formalin is placed in the 
 receiver and the receiver is closed. The lamp is 
 lighted and the coil brought to a red heat. The valve 
 is then opened and the solution contained in the receiver 
 is allowed to pass down and into the coil in a fine 
 stream. Upon coming in contact with the heated 
 metal the formaldehyde solution is instantly decom- 
 posed, and the liberated gas is further purified as it 
 progresses through the incandescent coil. The re- 
 sults with this apparatus have been as good as those 
 obtained by the Trillat or Lentz systems. The 
 apparatus is liable to get out of order, in that the 
 valve is apt to become clogged and so stop the flow 
 of formalin until freed by a wire supplied for the 
 purpose. 
 
 A great improvement in this apparatus has recently 
 
DISINFECTION AND STERILIZATION. 183 
 
 been made by the originator of the previous apparatus, 
 Mr. Taylor, partly through our suggestions. 
 
 In the new form (Fig. 15) the formalin is first boiled 
 in the large chamber and passes as vapor through the 
 tube connecting B and C. In C it is superheated and 
 passes out the tube D into the room. In this apparatus 
 
 FIG. 15. 
 
 Formaldehyde apparatus. 
 
 there is nothing to get out of order, and it operates 
 quickly. Up to the present time this is the most prac- 
 tical apparatus we have met with, when the initial cost, 
 about $25.00, is not an objection. It is handled by 
 H. K. Mulford Company, Philadelphia. In all forms 
 of apparatus where formalin is used the large receiving 
 
184 BACTERIOLOGY. 
 
 chamber should be washed out from time to time with 
 hot water, to remove any deposit there may be. 
 
 From Trioxymethylene by Schering's System. This sys- 
 tem consists in heating the solid polymer of formalde- 
 hyde (trioxymethylene) in a lamp specially constructed 
 for the purpose by the Chemische Fabrik auf Actien, 
 in Berlin. The trioxymethylene is used in the form of 
 compressed tablets or pastilles, as being more convenient 
 for use. Each pastille contains the equivalent of 100 
 per cent, of formaldehyde gas, according to the manu- 
 facturers, and weighs 1 gramme. 
 
 The mode of using the apparatus is very simple: 
 The disinfector is placed upon a sheet of iron on the 
 floor of the room to be disinfected. From 100 to 250 
 pastilles can be evaporated at a time in the apparatus. 
 For the production of greater quantities of formalde- 
 hyde vapor several of these outfits may be used 
 together. The lamp is filled with ordinary or wood 
 alcohol, about twice as many cubic centimetres of the 
 alcohol being employed as there are pastilles to be evap- 
 orated. The wicks should project but little above the 
 necks of the burners, or the apparatus may get too hot 
 and ignite the pastilles. The vessel is charged with 
 formalin pastilles and the disinfector placed over the 
 lighted spirit lamp. The lamp is then allowed to burn 
 out in the closed room. One hundred pastilles are con- 
 sidered to be sufficient for the disinfection of 1000 cubic 
 feet of space. Lately, a small steam boiler has been 
 added to the apparatus, for the purpose of furnishing 
 sufficient moisture with the gas. The results obtained 
 by us in superficial disinfection, when from 150 to 200 
 pastilles have been used to each 1000 cubic feet, have 
 been good. The great advantage of the method is in the 
 
DISINFECTION AND STERILIZATION. 185 
 
 small cost of the apparatus, $3.00, and the avoidance 
 of the danger of deterioration, which is present to some 
 extent in formalin. Smaller lamps are very useful for 
 the deodorization of rooms. 
 
 From Formalin to which Glycerin has been Added. 
 A very convenient apparatus of somewhat greater cost 
 than that of Sobering' s is prepared by Charles Lentz & 
 Sons, of Philadelphia. To the formalin is added 10 
 per cent, of glycerin, and the mixture is simply boiled 
 iu a suitable copper vessel, the steam and formaldehyde 
 gas passing off by a tube. This is a very serviceable 
 apparatus. When it is attempted to vaporize the for- 
 malin too rapidly part of it passes over in fluid form, 
 and is thus wasted. 
 
 With a slightly greater amount of formalin than that 
 used in the high temperature autoclave and heated tube 
 or chamber methods the results seem to be equally as 
 good. The apparatus is very easy to use, and not liable 
 to get out of order. 
 
 Similar forms of apparatus are also employed, when 
 instead of glycerin the formalin is mixed with an 
 equal quantity of water. The water is for the purpose 
 of giving additional moisture to the air, and, at the 
 same time, like the glycerin, to prevent the change of 
 formaldehyde into inert substances. A still simpler 
 method is to hang sheets in a room and throw on them 
 six to twelve ounces of formalin for each 1000 cubic 
 feet, and leave for six hours. If the room is tightly 
 sealed very fair superficial disinfection will take 
 place. 
 
 As a result of the investigations undertaken in the 
 department of health laboratories on the use of form- 
 aldehyde as a disinfectant, and a consideration of the 
 
186 BACTERIOLOGY. 
 
 work of others, the conclusions reached by us may be 
 summarized as follows : 
 
 1. Disinfection of Infected Dwellings. 
 
 Exposed surfaces of walls, * carpets, hangings, etc., 
 in rooms may be superficially disinfected by means 
 of formaldehyde gas. All apertures in the rooms should 
 be tightly closed and from 6 to 12 ounces of formalin 
 or its equivalent used to generate the gas for each 1000 
 cubic feet. The time of exposure should be not less 
 than four hours, and a suitable apparatus should be 
 employed. The temperature of the apartment should 
 be as high as possible, and certainly not below 52 F. 
 When generated very rapidly the formaldehyde gives 
 much better results than when giveu off slowly. 
 
 Under these conditions spore-free bacteria and the 
 contagion of the exanthemata are surely destroyed when 
 freely exposed to the action of the gas. Spore-bearing 
 bacteria are not thus generally destroyed; but these 
 latter are of such rare occurrence in disease, that in 
 house disinfection they may usually be disregarded, 
 and, if present, special measures can be taken. 
 
 The penetrative power of formaldehyde gas in the 
 ordinary room, at the usual temperature, even when 
 used in double the strength necessary for surface disin- 
 fection, is extremely limited, not passing, as a rule, 
 through more than one layer of cloth of medium thick- 
 ness. Articles, therefore, such as bedding, carpets, 
 upholstery, clothing, and the like, should, when pos- 
 sible, be subjected to steam, hot air, or formaldehyde 
 disinfection in special chambers constructed for the 
 purpose. If not, they must be thoroughly exposed on 
 all sides. 
 
DISINFECTION AND STERILIZATION. 187 
 
 2. Disinfection of Bedding, Carpets, Upholstery, Etc. 
 
 Bedding, carpets, clothing, etc., which would be in- 
 jured by steam, may be disinfected by means of formal- 
 dehyde gas in an ordinary steam disinfecting chamber, 
 the latter to be provided with a heating and if pos- 
 sible a vacuum apparatus and special apparatus for 
 generating the gas. Where penetration through heavy 
 articles is required the gas should be used in the pro- 
 portion of not less than the amount derived from 30 
 ounces of formalin for each 1000 cubic feet, the time 
 of exposure to be not less than eight hours and the 
 temperature of the chamber not below 110 F. 
 
 In order to insure complete sterilization of the articles 
 they should be so placed as to allow of a free circulation 
 of the gas around them that is, in the case of bedding, 
 clothing, etc., these should either be spread out on per- 
 forated wire shelves or loosely suspended in the cham- 
 ber. The aid of a partial vacuum facilitates the opera- 
 tion. Upholstered furniture and articles requiring much 
 space should be placed in a large chamber, or, better, in 
 a room which can.be heated to the required temperature. 
 
 The most delicate fabrics, furs, leather, and other 
 articles, which are injured by steam, hot air at 230 F., 
 or other disinfectants, are unaffected by formaldehyde. 
 
 3. Disinfection of Books. 
 
 Books may be satisfactorily disinfected by means of 
 formaldehyde gas in a special room, or in the ordinary 
 steam chamber, as above described, and under the same 
 condition of volume of gas, temperature, and time of 
 exposure. The books should be arranged to stand as 
 widely open as possible upon perforated wire shelves, 
 
188 BACTERIOLOGY. 
 
 set about one or one and a half feet apart in the chamber. 
 A chamber having a capacity of 200 to 250 cubic feet 
 would thus afford accommodation for about one hundred 
 books at a time. 
 
 Books, with the exception of their surfaces, cannot 
 be satisfactorily disinfected by formaldehyde gas in the 
 book-cases of houses and libraries, or anywhere except 
 in special chambers constructed for the purpose, because 
 the conditions required for their thorough disinfection 
 cannot otherwise be complied with. 
 
 The bindings, illustrations, and print of books are in 
 no way affected by the action of formaldehyde gas. 
 
 4. Disinfection of Carriages, Etc. 
 
 Carriages, ambulances, cars, etc., can be easily disin- 
 fected by having built a small, tight building, in which 
 they are enclosed and surrounded with formaldehyde 
 gas. Such a building is used for disinfecting ambu- 
 lances in New York City. With the apparatus there 
 employed a large amount of formalin is rapidly vapor- 
 ized, and superficial disinfection is completed in thirty 
 minutes. 
 
 5. Advantages of Formaldehyde Gas over Sulphur Dioxide 
 for the Disinfection of Dwellings. 
 
 Formaldehyde gas is superior to sulphur dioxide as 
 a disinfectant for dwellings, first, because it is more 
 efficient in its action; second, because it is less inju- 
 rious in its effects on household goods; third, because 
 when necessary it can easily be supplied from a gen- 
 erator placed outside of the room and watched by an 
 attendant, thus avoiding in some cases danger of fire. 
 
 Apart from the cost of the apparatus and the greater 
 
DISINFECTION AND STERILIZATION. 189 
 
 time involved, formaldehyde gas, generated from com- 
 mercial formalin, is not much more expensive than 
 sulphur dioxide viz., fifteen to thirty cents per 1000 
 cubic feet against ten cents with sulphur. Therefore, 
 we believe that formaldehyde gas is the best disinfect- 
 ant at present known for the surface disinfection of 
 infected dwellings. For heavy goods it is far inferior 
 in penetrative power to steam; but for the disinfection 
 of fine wearing apparel, furs, leather, upholstery, books, 
 and the like, which are injured by great heat, it is, 
 when properly employed, better adapted than any other 
 disinfectant now in use. 
 
 Sulphur Dioxide in House Disinfection. Four pounds 
 of sulphur should be burned for every 1000 cubic feet. 
 The sulphur should be broken into small pieces and 
 put in a pan sufficiently large not to allow the melted 
 sulphur to overflow. This pan is placed in a much 
 larger pan holding a little water. The cracks of the 
 room should be carefully pasted up and the door, after 
 closing, also sealed. Upon the broken sulphur is poured 
 three to four ounces of alcohol and the whole lighted 
 by a match. The alcohol is not only for the purpose 
 of aiding the sulphur to ignite, but also to add moisture 
 to the air. An exposure of eight to twelve hours should 
 be given. 
 
 Sulphur fumigation carried out as above indicated is 
 not as efficient as formaldehyde fumigation, but seems 
 to suffice for surface disinfection for diphtheria and the 
 exanthemata. All heavy goods should be removed 
 for steam disinfection if there is any possibility of the 
 infection having penetrated beneath their surface. If 
 there is no place for steam disinfection their surfaces 
 should be thoroughly exposed to fumigation and then to 
 
190 BACTERIOLOGY. 
 
 the air and sunlight. In many cases when cleanliness 
 has been observed, surface disinfection of halls, bedding, 
 and furniture may be all that will be required. 
 
 There is always a very slight possibility of a deeper 
 penetration of infection than that believed to have 
 occurred; it is, therefore, better to be more thorough 
 than is considered necessary rather than less. 
 
 Sulphur dioxide without the addition of moisture has, 
 as already stated under the consideration of disinfect- 
 fants, very little germicidal value upon dry bacteria. 
 
 Public Steam Disinfecting Chambers. 
 
 These should be of sufficient size to receive all neces- 
 sary goods, and may be either cylindrical or rectangular 
 in shape, and are provided with steam-tight doors open- 
 ing at either end, so that the goods put in at one door 
 may be removed at the other. When large the doors 
 are handled by convenient cranes and drawn tight by 
 drop-forged steel eye-bolts swinging in and out of slots 
 in the door frames. The chambers should be able to 
 withstand a steam-pressure of at least one- half an at- 
 mosphere, and should be constructed with an inside 
 jacket, either in the form of an inner and outer shell or 
 of a coil of pipes. This jacket is filled with steam dur- 
 ing the entire operation, and is so used as to bring the 
 goods in the disinfecting chamber up to the neighbor- 
 hood of 220 F. before allowing the steam to pass 
 in. This heats the goods, so that the steam does not 
 condense on coming in contact with them. It is an 
 advantage to displace the air in the chamber before 
 throwing in the steam, as hot air has far less germicidal 
 value than steam of the same temperature. To do this, 
 a vacuum pump is attached to the piping, whereby a 
 
DISINFECTION AND STERILIZATION. 191 
 
 vacuum of fifteen inches can be obtained in the cham- 
 ber. The steam should be thrown into the chamber in 
 large amount, both above and below the goods, and the 
 excess should escape through an opening in the bottom 
 of the chamber, so as to more readily carry off with it 
 any air still remaining. The live steam in the cham- 
 ber should be under a pressure of two to three pounds, 
 so as to increase its action. 
 
 To disinfect the goods, we place them in the chamber, 
 close tight the doors, and turn the steam into the jacket. 
 After about ten minutes, when the goods have become 
 heated, a vacuum of ten to fifteen inches is produced, 
 and then the live steam is thrown in for twenty min- 
 utes. The steam is now turned off, a vacuum is again 
 formed, and the chamber again superheated. The 
 goods are now thoroughly disinfected and dry. In 
 order to test the thoroughness of any disinfection, or any 
 new chamber maximum, thermometers are placed, some 
 free in the chamber and others surrounded by the 
 heaviest goods. It will be found that, even under a 
 pressure of three pounds, live steam will require ten 
 minutes to penetrate heavy goods. 
 
 The Disinfection of Hands, Instruments, Ligatures, and 
 Dressings for Surgical Operations. 
 
 Instruments. All instruments, except knives, after 
 having been thoroughly cleansed, are boiled for 'three 
 minutes in a 1 per cent, solution of washing soda. 
 Knives, after having been thoroughly cleansed, are 
 washed in sterile alcohol and wiped with sterile gauze 
 and then put into boiling soda solution for one minute. 
 This will not injure their edges to any great extent. 
 
 Gauze. Gauze is sterilized by moist heat either in 
 
192 BACTERIOLOGY. 
 
 an Arnold steam sterilizer for one hour or in an auto- 
 clave for thirty minutes. It is placed in a perforated 
 cylinder or wrapped in clean towels before putting in 
 the sterilizer, and only opened at the operation. 
 
 lodoform gauze is best made by sprinkling sterile 
 iodoform on plain gauze sterilized as described above. 
 
 Ligatures Catgut. Boil for one hour in alcohol under 
 pressure at about 97 C. It is often put in sealed glass 
 tubes, which are boiled under pressure. These remain 
 indefinitely sterile. The alcohol does not injure the 
 catgut. If desired, the catgut can be washed in ether 
 and can be soaked a short time in bichloride before 
 heating in alcohol. Boeckman, of St. Paul, suggested 
 wrapping the separate strands of catgut in paraffin 
 paper and then heating for three hours at 140. This 
 procedure prevents the drying out of the moisture and 
 fat from the catgut, so that it remains unshrivelled and 
 flexible after its exposure. Darling, of Boston, tested 
 this method and found it satisfactory. Dry formalde- 
 hyde gas does not penetrate sufficiently, and is not reli- 
 able. Silver wire, silk, silkworm-gut, rubber tubing, 
 and catheters are boiled the same as the instruments. 
 
 The Skin of the Patient. This is washed thoroughly 
 with soap and water, then with alcohol, and finally with 
 1 : 1000 bichloride. A soap poultice is now placed on 
 for six to twelve hours, and after its removal the skin 
 is covered with a gauze compress previously moistened 
 with a 1 : 1000 bichloride of mercury solution. At the 
 operation the skin is washed off with alcohol and then 
 with the bichloride of mercury solution. 
 
 The Hands. Furbinger's method, slightly modified, 
 is now much used, and gives good results : The hands 
 are washed in hot soap and water for five minutes, 
 
DISINFECTION A ND STERILIZA TION. 1 93 
 
 using the nail-brush. They are then soaked in alcohol 
 for one minute and scrubbed with a sterile brush. 
 They are finally soaked in a 1 : 1000 bichloride of mer- 
 cury solution for three minutes. 
 
 Sterilized rubber gloves are now being used more 
 and more in operations. The gloves can be sterilized 
 by being left for one minute in boiling 1 per cent, soda 
 solution, or they can be sterilized by steam. 
 
 The surgeon's gowns and caps are sterilized by steam. 
 Mucous membranes, as those of the mouth and throat, 
 are cleansed by a solution consisting of equal parts of 
 peroxide of hydrogen and lime-water. In the nostrils 
 it is better to employ the milder solutions, such as 
 diluted DobelPs or listerine. These are also used in 
 the mouth instead of the peroxide. 
 
 The vagina is swabbed out thoroughly with sterile 
 warm soap and water and then irrigated with a 2 per 
 cent, carbolic acid or a 1 : 1000 bichloride of mercury 
 solution. 
 
 Hypodermic syringes and other syringes are steril- 
 ized by drawing up into them boiling water a number 
 of times and then finally a 5 per cent, solution of car- 
 bolic acid, the acid after three minutes to be washed 
 out by boiling water. If cold water is used the car- 
 bolic solution should remain in the barrel for ten min- 
 utes. Great care should be taken to wash out all pos- 
 sible matter before using the carbolic acid to sterilize. 
 Syringes made entirely of glass or of glass and asbestos 
 can be boiled in soda solution. 
 
 THE STERILIZATION OF MILK. 
 
 Bacteria when allowed to develop in milk produce 
 fermentation (souring) and render the milk unfit to 
 
 13 
 
194 BACTERIOLOGY. 
 
 be used as an article of food, especially for infants. 
 Milk as it reaches the city contains enormous num- 
 bers of germs, and these will produce fermentation, 
 even though the milk is kept on ice. Unclean vessels 
 hasten this process. No matter how good milk may 
 be in the morning, when comparatively fresh, toward 
 evening, unless it has been partly or completely steril- 
 ized, it may be dangerous to an infant, and may, espe- 
 cially in summer, cause fatal illness, even though it 
 still tastes sweet. 
 
 Complete sterilization destroys all the germs in milk, 
 and so prevents permanently fermentative changes. By 
 partial sterilization most of the germs which are not in 
 the spore form may be destroyed, so that the milk will 
 remain wholesome for at least twenty -four hours in the 
 warmest weather. 
 
 Milk is best sterilized by steam, for nearly all chem- 
 icals, such as boric acid, salicylic acid, and formalin, 
 make the milk less digestible, and, as a rule, unfit for 
 food. It may be sterilized at a high or low tempera- 
 ture that is, at the boiling temperature (212 F.) or 
 at a lower degree of heat, obtained by modifying the 
 steaming process. 
 
 It has been found that milk sterilized at a high tem- 
 perature (212 F.) is not desirable for prolonged use, as 
 the high temperature causes certain changes in the milk, 
 which make it less suitable as a food for infants. These 
 changes are almost altogether avoided if a temperature 
 below 80 C. is used. It is recommended, therefore, 
 that the lowest temperature be used for partial sterili- 
 zation, which will keep the milk wholesome for twenty- 
 four hours in the warmest weather and kill the tubercle, 
 typhoid, and other non-spore-bearing bacilli. Raising 
 
DISINFECTION AND STERILIZATION. 195 
 
 the milk to a temperature of 70 C. for fifteen minutes 
 or 80 C. for twelve minutes will accomplish this. 
 One of the many forms of apparatus is the following: 
 
 (a) A tin pail or pot, about ten inches deep by nine 
 inches in diameter, provided with the ordinary tin 
 cover, which has been perforated with eight holes, each 
 an inch in diameter. 
 
 (6) A wire basket, with eight nursing bottles (as sold 
 in the shops for this purpose). 
 
 (c) Rubber corks for the bottles and a bristle brush 
 for cleaning them. 
 
 Directions (Koplik). Place the milk, pure or diluted 
 (as the doctor may direct), in the nursing-bottles and 
 place the latter in the wire basket. Put only sufficient 
 milk for one nursing in each bottle. Do not cork the 
 bottles at first. 
 
 Having previously poured about two inches of water 
 in the tin pail or pot and brought it to the boiling- 
 point, lower the basket of nursing bottles slowly into 
 the pot. Do not allow the bottles to touch the water 
 or they will crack. Put on the perforated cover and 
 let the steaming continue for ten minutes; then remove 
 the cover and firmly cork each bottle. After replacing 
 the cover, allow the steaming to continue for fifteen 
 minutes. The steam must be allowed to escape freely 
 or the temperature will rise too high. 
 
 The process of sterilization is now completed. Place 
 the basket of bottles in a cool, dark place or in an ice- 
 chest. The bottles must not be opened until just before 
 the milk is to be used, and then it may be warmed by 
 plunging the bottle in warm water. If properly 
 prepared the milk will taste but little like boiled 
 milk. 
 
196 BACTERIOLOGY. 
 
 The temperature attained under the conditions stated 
 above will not exceed in extreme cases 188 F. (87 C.). 
 
 Milk should be sterilized when it is as fresh as pos- 
 sible, and only sufficient milk for twenty-four hours 
 should be sterilized at one time. If, after nursing, the 
 infant leaves some milk in the bottle, this should be 
 thrown away. 
 
 Care of the Bottles is Important. After nursing,, the 
 bottles should be filled with a strong solution of wash- 
 ing soda, allowed to stand twenty-four hours, and then 
 carefully cleaned with a bristle (bottle) brush. The 
 rubber corks and nipples should be boiled after using 
 in strong soda solution for fifteen minutes and then 
 rinsed and dried. 
 
 After sterilizing milk should never be put into unster- 
 ilized bottles, as this will spoil it. 
 
 A different but admirable method is the one devised 
 by Dr. Freeman. 1 Here a pail is filled to a certain 
 mark with water and then placed on the stove until the 
 water boils. It is then removed, and immediately a 
 milk-holder, consisting of a series of zinc cylinders, is 
 lowered with its milk bottles partially full of milk. 
 The cover is again applied. The heat of the outside 
 water raises the temperature of the milk in ten minutes 
 to 75 C. (167 F.), and holds it nearly at that point 
 for some time. 2 After twenty minutes the milk is re- 
 moved, placed in cold water, and quickly cooled. The 
 milk is kept in the ice-chest until used. 
 
 1 Agent for Pasteurizer, James Dougherty, 411 W. 59th St. 
 
 2 A temperature of 75 C. is advised in Pasteurizing milk, instead of 65 C., 
 which would ordinarily suffice to kill all bacteria free of spores, because of 
 the fact pointed out by Theobald Smith, that the bacteria embedded in the 
 pellicle which forms on the surface are more resistant than those surrounded 
 by fluid. 
 
CHAPTER XII. 
 
 THE PREPARATION, STAINING, AND MICROSCOPICAL 
 EXAMINATION OF BACTERIA. 
 
 As the purpose of this book is to give, outside of 
 special methods devised for purposes of diagnosis and 
 the development of curative serums, only such descrip- 
 tions of technique as are necessary to students in their 
 laboratory courses, or to physicians in the very simple 
 examinations which they will be able to carry on in 
 their private offices, readers are referred to works 
 such as Sternberg's or Abbott's for fuller descriptions 
 of the apparatus and technique used in bacteriological 
 research. 
 
 Since bacteria are present to a greater or less extent 
 in the air, earth, and water around us, on our bodies, 
 clothes, and all surrounding objects, it follows that 
 when we begin to examine substances for bacteria the 
 first requisite is that all the materials we use must be 
 free and kept free from bacteria, both living and dead, 
 otherwise we cannot tell whether those we detect are 
 in the substances examined or only in the materials we 
 have used in the investigation. 
 
 Additional care has to be taken when we study in- 
 fection in the living body, for in the skin and mucous 
 membranes there are not only abundant bacteria but 
 varieties similar to those which produce disease, so that 
 if we do not use the greatest precautions we will con- 
 taminate our material with these bacteria and get utterly 
 
198 BACTERIOLOGY. 
 
 misleading results. After death we have an added dif- 
 ficulty, in that even the blood and body tissues become 
 invaded by bacteria from the intestines and elsewhere, 
 so that bacteria actually present in the diseased tissues 
 may have had no connection with the disease under in- 
 vestigation. Whenever bacteria are found, therefore, 
 the methods carried out in the investigation should be 
 most carefully examined, to see if some error in tech- 
 nique has not been committed. The aim of the 
 bacteriological examination of any material is to de- 
 termine whether bacteria are present or not, and, if 
 present, to ascertain their number and distribution, and, 
 if possible, their species. This is accomplished chiefly 
 by means of two methods viz., the direct examina- 
 tion with the microscope of cover glass preparations 
 and the results of cultures made from the material. 
 Sometimes animal inoculations are also employed. 
 
 The direct microscopical examination of suspected sub- 
 stances for bacteria can be made either with or without 
 staining. Unstained, the bacteria are examined, to note 
 their motility, their form, and their general arrangement: 
 but for more exact study, they can be so much better 
 observed when stained that this step is always advisable. 
 A cover-glass preparation is made as follows : A 
 very small amount of the blood, pus, discharges from 
 mucous membranes, culture fluids, or other material to 
 be examined is removed by means of a sterile swab or 
 platinum loop and smeared undiluted in a thin film 
 over a clean, thin cover-glass. 1 From cultures on solid 
 
 1 To render new cover-slips clean and free from grease, place them in strong 
 nitric acid for a few hours, then rinse them olF in water, then in alcohol, 
 then in ether. Place them finally for keeping in alcohol, to which a little 
 ammonia has been added. When used wipe with soft clean handkerchief. 
 If old cover-slips are used boil first in soda solution. 
 
MICROSCOPICAL EXAMINATION. 199 
 
 media, however, on account of the abundance of bacteria 
 in the material, a little of the growth is diluted by add- 
 ing it to a tiny drop of clean water which has been 
 previously placed on the cover-glass. The amount of 
 dilution is learned after a few trials. It is best to add 
 to the drop just enough to make a perceptible cloudi- 
 ness. The mixture is then smeared over the cover- 
 glass. From whatever source derived, the film is al- 
 lowed to dry at the usual air temperature, and then, in 
 order to fix the film with its contained bacteria to the 
 glass, the latter is passed three times by a rather slow 
 movement through the Bunsen or alcohol flame. In- 
 stead of this method, the film may be fixed to the glass 
 by placing it in absolute alcohol for a few minutes. 
 The smear thus prepared is usually stained either 
 by the simple addition of a solution of an aniline dye, 
 for from one to five minutes, or by one of the more 
 complicated special stains described later, such as that 
 of Gram or that used for the tubercle bacillus, where 
 the ability of the bacteria to retain their stain when 
 placed in decolorizing solutions is tested. When the 
 stain is to be hastened or made more intense the dye is 
 used warm. For ordinary staining the bacteria are 
 simply covered completely by the cold staining fluid. 
 The cover-glass, with the charged side uppermost, may 
 either rest on the table or be held by some modifica- 
 tion of Cornet's forceps. When the solution is to be 
 warmed the cover-glass may be floated, smeared side 
 down, upon the fluid contained in a porcelain dish rest- 
 ing on a wire mat, supported on a stand, or it may be 
 held in the Cornet forceps. The fluid in both the dish 
 and on the cover-glass should be carefully warmed, so 
 as to steam without actually boiling. The cover-glass 
 
200 BACTERIOLOGY. 
 
 should be kept completely covered with fluid. The 
 bacteria having now been stained, the cover-glass is 
 grasped in the forceps and thoroughly washed in clean 
 water and then dried, first between layers of filter-paper 
 and then in the air. A drop of balsam or water is now 
 placed on a glass slide and the cover-glass placed upon 
 it with the bacterial side down. The preparation is 
 now ready for microscopical examination. 
 
 The Preparation of Sections. Occasionally it is of value 
 to examine the bacteria as they are in the tissues them- 
 selves. These should be obtained as soon as possible 
 after death, so as to prevent any post-mortem changes 
 or increase of the bacteria in them. From the properly 
 selected spots small portions, not larger than one cubic 
 centimetre, are removed and placed in absolute alcohol 
 to harden. These portions, when of nearly the con- 
 sistency of fresh, solid rubber, are removed, and, if the 
 nature of the tissues will allow, fastened to corks or 
 pieces of hard rubber by mucilage. After dry ing, the 
 specimens are replaced in alcohol for twenty-four hours 
 and then cut into thin sections with the microtome. 
 Sometimes the tissues do not become sufficiently dense 
 by this simple method; they must then undergo the 
 process of embedding in celloidin or paraffin. 
 
 The Ordinary Staining Solutions. Simple staining is 
 used for the demonstration of bacteria in general, and 
 is also useful for gaining an idea of the other elements in 
 the preparation. The solutions commonly employed in 
 staining bacteria are the watery solutions of basic aniline 
 dyes fuchsin, gentian-violet, and methylene - blue. 
 These solutions may either be prepared by dissolving 
 the dyes directly in water, or, more usually, by having 
 stock saturated alcoholic solutions of them, from which 
 
MICROSCOPICAL EXAMINATION. 201 
 
 we can take from time to time the amount necessary to 
 make up the watery solutions for use. The stock satu- 
 rated alcoholic solutions are made by pouring into a bot- 
 tle enough of the dye in substance to fill them to about 
 one-quarter of their capacity. The bottle should then 
 be filled with alcohol, tightly corked, well shaken, and 
 allowed to stand for twenty-four hours. If at the end 
 of this time all the staining material has been dis- 
 solved, more should be added, the bottle being again 
 shaken and allowed to stand for another twenty-four 
 hours. This must be repeated until a permanent sedi- 
 ment of undissolved coloring-matter is seen upon the 
 bottom of the bottle. This will then be labelled t( satu- 
 rated alcoholic solution, 77 of whatever dye has been 
 employed. The alcoholic solutions are not themselves 
 employed for staining purposes. The solution for use 
 is made by filling a small bottle three-fourths with dis- 
 tilled water, and then adding the concentrated alcoholic 
 solution of the dye, little by little, until one can just 
 see through the solution. Care must be taken that the 
 color does not become too dense. Small wooden cases 
 come prepared for holding about one-half dozen bottles 
 of the staining solutions. This number will answer 
 for all routine purposes of the student or physician. 
 
 For certain bacteria, which stain only imperfectly 
 with these solutions, it is necessary to employ some 
 agent that will increase the penetrating action of the 
 dyes. We have learned that the addition to a solu- 
 tion of a small quantity of alkaline substance, or by 
 dissolving the staining materials in strong watery solu- 
 tions of either aniline oil or carbolic acid, instead of 
 simple water, will accomplish this. Of the solutions 
 thus prepared there are three in common use: Loeffler's 
 
202 BACTERIOLOGY. 
 
 alkaline methylene-blue solution, the Koch-Ehrlich 
 aniline water solution, of either fuchsin, gentian-violet, 
 or methylene-blue, and Ziehl's solution of fuchsin in 
 carbolic acid. These solutions are as follows : 
 
 Loeffler's Alkaline Methylene-blue Solution. This con- 
 sists of concentrated alcoholic solution of methylene- 
 blue, 30 c.c.; caustic potash in one ten thousandth 
 solution, 100 c.c. 
 
 Koch-Ehrlich Aniline Water Solution of Fuchsin or 
 Gentian-violet is prepared as follows: To about 100 
 c.c. of distilled water, aniline oil is added, drop by 
 drop, until it has an opaque appearance, the solution 
 being thoroughly shaken after the addition of each drop. 
 It is then filtered into a beaker through moistened 
 filter-paper until the filtrate is perfectly clear. To 
 100 c.c. of the filtrate add 10 c.c. of absolute alcohol 
 and 11 c.c. of the concentrated alcoholic solution of 
 either fuchsin, methylene-blue, or gentian- violet. 
 
 Ziehl's Carbolic Fuchsin Solution. Distilled water, 
 100 cc. ; carbolic acid (crystalline), 5 grams; absolute 
 alcohol, 10 c.c ; fuchsin, 1 gram; or it may be pre- 
 pared by adding to a 5 per cent, watery solution of 
 carbolic acid the saturated alcoholic solution of fuchsin 
 until a metallic lustre appears on the surface of the 
 fluid. 
 
 The last two methods, combined with heating, are 
 used to stain the bacteria intensely, so that the more 
 resistant of them may retain their color when exposed 
 to decolorizing agents. When so treated certain of 
 the bacteria will retain their color, even when exposed 
 to very strong decolorizers. The bacilli of tuberculosis 
 and of leprosy are examples. They are both difficult 
 to stain, but when once stained are equally resistant to 
 
MICROSCOPICAL EXAMINATION. 203 
 
 give up their stain. The details of staining tubercle 
 bacilli will be found under Tuberculosis. 
 
 Another differential method of staining which is 
 very commonly employed is that known as Gram's 
 method. In this method the objects to be stained 
 are covered with the aniline gentian- violet solution. 
 After remaining in this for a few minutes they are 
 immersed in an iodine solution, composed of iodine, 1 
 grain; potassium iodide, 2 grains; distilled water, 300 
 c.c. In this they remain for from one to two minutes. 
 They are then transferred to alcohol and thoroughly 
 rinsed. If the cover-glass as a whole still shows a 
 violet color, it is again treated with the iodine solu- 
 tion, followed by alcohol, and this is continued until 
 no trace of violet color is visible to the naked eye. 
 They may then be washed in water and examined, or 
 a contrasting color of carmine or Bismarck brown may 
 be given them. This method is particularly useful in 
 demonstrating the capsule which is seen to surround 
 some bacteria particularly the pneumococcus and 
 also in differentiating between varieties of bacteria, for 
 some do and others do not retain their stain when put 
 in the iodine solution for a suitable time. 
 
 Another method of demonstrating the capsule is the 
 glacial acetic acid method, as described by Welch: 
 1. Cover the preparation with glacial acetic acid for a 
 few seconds. 2. Drain off and replace with aniline 
 gentian-violet solution; this is to be repeatedly added 
 until all the acid is replaced. 3. Wash in 2 per cent, 
 solution of sodium chloride and mount in the same. 
 
 Staining the Spores. We have already noted that 
 during certain stages in the growth of a number of 
 bacteria spores are formed which refuse to take up color 
 
204 BA CTERIOL OGY. 
 
 when the bacteria are stained in the ordinary manner. 
 Special stains have been devised for causing the color to 
 penetrate through the resistant spore. Thus in Abbott's 
 method the cover-slip after having been prepared in the 
 usual way is covered with a dye and held over the Bun- 
 sen flame until the fluid steams. This is continued for 
 one or two minutes. It is then washed and dipped in a 
 decolorizing acid solution, such as a 2 per cent, alco- 
 holic solution of nitric acid, until all visible color has 
 disappeared, then it is washed off and dipped for ten sec- 
 onds in a solution containing 10 parts saturated alco- 
 holic solution of eosin and 90 of water. The bacilli 
 will then be rose-colored and the spores blue. Some- 
 times, however, the spores refuse to take the stain in 
 this manner. We then can adopt Moeller's method, 
 which is designed still further to favor the penetration 
 of the coloring-matter through the spore membrane. 
 He macerates the spores in a solution of chromic acid 
 before staining them. The prepared cover-slip is held 
 for two minutes in chloroform, then washed off in water, 
 then placed from one-half to three minutes in a 5 per 
 cent, solution of chromic acid, again washed off in 
 water, and now restained by adding to it carbolic 
 fuchsin, which is steamed for several minutes. The 
 staining fluid is then washed off and the preparation 
 decolorized in a 3 per cent, solution of hydrochloric 
 acid or a 5 per cent, solution of sulphuric acid. The 
 preparation is finally stained for a minute in methyleue- 
 blue solution. The spores will be red and the body of 
 the cells blue. The different spores vary greatly in 
 the readiness with which they take up the dyes, and 
 we have, therefore, to experiment with each variety 
 as to the length of time they should be exposed to the 
 
MICROSCOPICAL EXAMINATION. 205 
 
 maceration of the chromic acid. Even under the best 
 conditions it is almost impossible to stain some spores. 
 
 Staining Flagella. For the demonstration of flagella, 
 which are possessed by all motile bacteria, we are in- 
 debted to Loeffler. The special stains devised by him, 
 and also the one devised by Van Ermengem, are those 
 usually employed. 
 
 Bunge's modification of Loeffler 7 s method is carried 
 out as follows: Cover-glasses which have been most 
 carefully cleaned are covered by a very thin smear of 
 an eighteen-hours' old culture of the motile organism 
 to be examined. After drying in the air and passing 
 three times through the flame the smear is treated with 
 a mordant solution, which is prepared as follows: To 
 3 parts of saturated alum solution add 1 part of a solu- 
 tion of liquor ferri sesquichloride, of the strength of 
 1:20 of distilled water. To 10 c.c. of this mixture 
 add 1 c.c. of a concentrated watery solution of fuchsin. 
 This mordant should be allowed to stand for several 
 days before using. After preparing the cover-slip with 
 all precautions necessary to cleanliness the filtered mor- 
 dant is allowed to act cold for five minutes, after which 
 it is slightly warmed and then washed off. After dry- 
 ing the smear is faintly stained with the carbol fuchsin 
 solution and then washed off, dried, and mounted. 
 
 Frequently the flagella appear well stained, but often 
 the process has to be repeated a number of times before 
 success is arrived at. 
 
 The Preservation of Specimens. Dry unstained or 
 stained preparations of bacteria keep indefinitely if 
 mounted in Canada balsam, cedar oil, or dammar lac; 
 they tend to gradually fade, but may be preserved for 
 many months or years. 
 
206 
 
 BACTERIOLOGY. 
 
 The Microscopical Examination of Bacteria. 
 
 The Different Parts of the Microscope. A complete 
 instrument usually has four oculars, or eye-pieces (A), 
 which are numbered from 1 to 4, according to the 
 
 FIG. 16. 
 
 G 
 
 Microscope. 
 
MICROSCOPICAL EXAMINATION. 207 
 
 amount of magnification which they yield. Numbers 
 2 and 4 are most useful for bacteriological work. The 
 objective (B) the lens at the distal end of the barrel 
 serves to give the main magnification of the object. For 
 stained bacteria the 1/12 achromatic oil immersion lens 
 is regularly employed; except for photographic purposes 
 the apochromatic lenses are not needed. Even here 
 they are not indispensable. A 1/10 or 1/16 may at 
 times be useful but hardly necessary; a No. 4 ocular and 
 a 1/12 lens give a magnification of about 1000 diame- 
 ters. (Fig. 17.) For unstained bacteria we employ 
 
 FIG. 17. 
 
 Anthrax bacilli and blood-cells. X 1000 diameters. 
 
 either the 1/12 immersion or 1/7 dry lens, according to 
 the purpose for which we study the bacteria; for the 
 examination of colonies where, as a rule, we do not 
 wish to see individual bacteria but only the general 
 appearance of whole groups, we use lenses of much 
 lower magnification. (Fig. 18.) 
 
 The stage (C) the platform upon which the .object 
 rests should be large enough to support the Petri 
 
208 BACTERIOLOGY. 
 
 plates if culture work is to be done. The iris dia- 
 phragm (D), which is now regularly used in bacterio- 
 logical work, opens and closes like the iris of the eye, 
 and so controls the amount of light. Its opening is 
 diminished or increased by moving a small arm, which 
 is underneath the stage, in one or another direction. 
 The reflector placed beneath the stage serves to direct 
 the light to the object to be examined. It has two sur- 
 faces one concave and one convex. The coarse ad- 
 
 FIG. 18. 
 
 Colonies of diphtheria bacilli. X 200 diameters. 
 
 justment (F) is the rack-and-pinion arrangement by 
 which the barrel of the microscope can be quickly 
 raised or lowered. It is used to bring the bacteria 
 roughly into focus. The fine adjustment (G) serves to 
 raise and lower the barrel very slowly and evenly, and 
 is used for the exact study of the bacteria when high- 
 power lenses are used. For the microscopical study of 
 bacteria it is essential that we magnify the bacteria as 
 much as possible and still have their definition clear and 
 
MICROSCOPICAL EXAMINATION. 209 
 
 sharp. It is essential, therefore, that the microscope be 
 provided with an oil immersion system and a substage 
 condensing apparatus. In using the oil immersion lens 
 a drop of oil of the same index of refraction as the 
 glass is placed upon the face of the lens, so as to con- 
 nect it with the cover-glass when the bacteria are in 
 focus. There is thus no loss of light through deflection, 
 as is the case in the dry system. 
 
 The Substage Condensing Apparatus (H) is a system of 
 lenses situated beneath the central opening of the stage. 
 It serves to condense the light passing through the re- 
 flector to the object in such a way that it is focussed 
 upon the object, thus furnishing the greatest amount of 
 luminosity. Between the condenser and the reflector is 
 placed an adjustable diaphragm, the aperture of which 
 can be regulated, as circumstances require, to permit 
 of either a very small or a very large amount of light 
 passing to the object. 
 
 The Examination of Bacteria in the Hanging Drop. 
 It is often valuable to observe bacteria alive, so as to 
 study them under natural conditions. We can thus 
 note the method and rate of their multiplication, 
 
 FIG. 19. 
 
 Hollow slide with cover-glass. 
 
 whether they move and produce spores, and whether 
 or not they clump or disintegrate with specific serums. 
 For this special slides and methods are desirable. The 
 usual form is one in which there is ground out on one 
 surface a hollow having a diameter of about half an 
 inch. (Fig. 19.) According to the purpose for which 
 
 14 
 
210 BACTERIOLOGY. 
 
 the hanging drop is to be studied, sterilization of the 
 slide and cover-glass may or may not be necessary. 
 The technique of preparing and studying the hanging 
 drop is as follows : The surface of the glass around the 
 hollow in the slide is smeared with a little vaseline or 
 other inert substance. This has for its purpose both 
 the sticking of the cover-glass to the slide and the pre- 
 vention of evaporation in the drop placed in the little 
 chamber, which is to be formed between the cover- 
 glass, when placed over the hollow, and the slide. 
 For the purpose of studying the bacteria we place, if 
 they are in fluids, simply a platinum loopful upou the 
 centre of the cover-glass and then invert it by means of 
 a slender pair of forceps over the hollow in the slide, 
 being very careful to have the bacteria over the very 
 centre of the space. If the bacteria, on the contrary, 
 are growing on solid media, or are obtained from thick 
 pus or tissues from organs, they are mixed with a suit- 
 able amount of bouillon or sterile physiological salt 
 solution either before or after being placed upon the 
 cover glass. If we wish to observe the bacteria under 
 natural conditions we must keep the tiny drop of fluid 
 at the proper temperature for the best growth of the 
 bacteria. If, however, we simply wish to observe their 
 form and arrangement this is not necessary. In the 
 study of living bacteria we often wish to observe their 
 grouping and motion rather than their individual char- 
 acters, and so use less magnification than for stained 
 bacteria. In studying unstained bacteria and tissues 
 we shut off as large a portion of the light with our 
 diaphragm as is compatible with distinct vision, and 
 thus favor contrasts which appear as lights and shadows, 
 due to the differences in light transmission of the dif- 
 
MICROSCOPICAL EXAMINATION. 211 
 
 ferent materials under examination. It is necessary 
 to remember that they are seen with difficulty, and that 
 we are very apt, unless extremely careful in focussing, 
 to allow the lens to go too far, and so come upon the 
 cover-glass, break it, destroy our preparation, and, if 
 examining parasitic bacteria, infect the lens. This may 
 be avoided by first finding the hanging drop with a low 
 power lens and thus exactly centre it. The lens of 
 higher magnification is now very gradually lowered, 
 while at the same time gently moving the slide back 
 and forth to the slightest extent possible with the left 
 hand. If any resistance is felt raise the lens, for it has 
 gone beyond the point of focus and is touching the 
 cover-glass. 
 
CHAPTER XIII. 
 
 BACTERIOLOGICAL TECHNIQUE Continued. 
 
 THE CULTIVATION OF BACTERIA. 
 
 IN order to determine the number of living bacteria 
 in any substance and their nature we have to cultivate 
 and isolate them. 
 
 The Most Common of the Nutrient Media Used for the 
 Growth of Bacteria. 
 
 All of these must have, as noted earlier, food con- 
 taining the necessary carbon, nitrogen, and mineral 
 substances in a form easily assimilated and in the 
 proper concentration. The pathogenic bacteria nearly 
 all require for good growth peptone, albumins, and 
 sugar. For each kind the proper food must be found 
 through experimentation, as slight alterations may 
 make a great difference. 
 
 Physicians will find it, as a rule, convenient to 
 purchase their media already prepared from some of 
 the reliable firms that deal in bacteriological products. 
 Special media, such as those employed for isolation and 
 identification of the typhoid bacillus and gonococcus, 
 will be found described along with those bacteria. For 
 those who may wish to make their own, we will de- 
 scribe here those in common use: 
 
 Nutrient Bouillon or Broth. One part of finely chopped 
 fresh, lean meat is macerated in two parts of water and 
 
BACTERIOLOGICAL TECHNIQUE. 
 
 213 
 
 put in an ice-chest for from eighteen to twenty-four 
 hours. The infusion is strained, when cold, through a 
 fine cheese-cloth, and to the clear filtrate 1 per cent, of 
 peptone and 0.5 per cent, of sodium chloride are added. 
 The medium is then warmed for some minutes until the 
 peptone is dissolved, and then exposed to live steam 
 either without pressure in the Arnold steam sterilizer 
 (Fig. 20) for thirty minutes, or in the autoclave (Fig. 
 
 FTG. 20. 
 
 STERILIZING CHAMBER 
 
 U A 
 
 Arnold steam sterilizer. 
 
 21) at one atmosphere of pressure for fifteen minutes, 
 or boiled over a free flame for ten minutes. While 
 still hot it is filtered through filter-paper or through 
 absorbent cotton, and the reaction is tested and suf- 
 ficient hydrochloric acid or sodium hydroxide added 
 
214 
 
 BACTERIOLOGY. 
 
 to give it the desired reaction, which is for most bac- 
 teria slightly alkaline to litmus. If the fluid is clear 
 it is put into flasks and tubes and sterilized; if not 
 clear, the white of one or two eggs is added to the 
 fluid after cooling it down to about 55 C. After 
 
 FIG. 21. 
 
 Autoclave for sterilization with live steam under pressure. 
 
 thoroughly mixing the eggs the bouillon is boiled 
 briskly for a few minutes and then again filtered and 
 distributed in flasks and tubes and put in the Arnold 
 sterilizer for one hour on each of two consecutive days, 
 or in the autoclave for twenty minutes for sterilization. 
 Instead of meat 2 to 4 grammes of Liebig's or some 
 other meat extract are added to each litre of water. 
 For most purposes the extract is as good as the fresh 
 meat, but for toxin production it is inferior. 
 
BACTERIOLOGICAL TECHNIQUE. 215 
 
 Fermentation broth is made usually by adding 1 per 
 cent, of glucose to the above. For accurate work the 
 meat sugars are first extracted by allowing the colon 
 bacillus to grow in the broth over night. The bouillon 
 is then sterilized and the peptone and salt added, and 
 the process already given gone through with. 
 
 Fermentation bouillon is usually placed in a tube of 
 special construction, known as a fermentation tube (see 
 Fig. 14, p. 82). This is essentially a tube 1.5 cm. in 
 diameter, bent at an acute angle, closed at one end, and 
 provided with a bulb at the other end, which latter 
 should be large enough to receive all the fluid in the 
 closed branch should gas in any considerable quantity 
 collect there. The tube also serves a most important end 
 in giving information as to the aerobic and anaerobic 
 growth of the species under consideration, for the con- 
 necting tube being constricted serves to prevent, to a 
 great degree, the entrance of oxygen of the air into the 
 closed branch, and the free oxygen in the medium is 
 driven out by the heat during sterilization; from which 
 it may be seen that growth in the bulb is aerobic and 
 growth in the closed branch is anaerobic. For the study 
 of fermentation alone small tubes may be inverted into 
 larger ones or tubes may be bent on themselves. 
 
 Nutrient Gelatin. To the bouillon already prepared 
 as described add 10 per cent, of sheet gelatin and neu- 
 tralize. Add the whites of two eggs for each litre and 
 boil for a few minutes. Filter, place in tubes or flasks, 
 and sterilize. Instead of adding gelatin to bouillon 
 already prepared, it may be added to the meat infusion 
 at the same time the peptone and salt were added in 
 preparing nutrient bouillon as just described. 
 
 Nutrient Agar. This is prepared by adding to stock 
 
216 BACTERIOLOGY. 
 
 bouillon 1 or 2 per cent., as desired, of thread agar, 
 melting it by placing over a free flame or in the auto- 
 clave or steam sterilizer. When the agar is brought 
 into solution over a free flame there may be consider- 
 able loss of fluid by evaporation. This should be com- 
 pensated for by adding additional water before boiling. 
 Agar may be added directly to the meat infusion along 
 with the peptone and salt. Indeed, this is an advan- 
 tage, as agar-agar is very difficult to bring into solution, 
 and is not injured in the least by prolonged boiling. 
 Glycerin agar is simply nutrient agar plus 3 to 6 per 
 cent, of glycerin. It is added to the hot nutrient agar 
 just previous to putting it in the flasks. Nutrient agar 
 begins to thicken at a fairly high temperature, and 
 should be filtered as hot as possible. When small 
 amounts are made it is well to place the filter and re- 
 ceiving-flask in the sterilizer while filtering. 
 
 Milk. This fluid is a good culture medium for most 
 pathogenic bacteria. It should be obtained as fresh as 
 possible, so that but little bacterial change has occurred. 
 It is first put in a steam sterilizer for fifteen minutes 
 and then put in the ice-chest for twelve hours, to allow 
 the cream to rise. The milk is then siphoned off from 
 below the cream into a flask and its reaction tested. A f ter 
 correction it is put in tubes or flasks and sterilized. 
 
 Potatoes. Potatoes are used for some special pur- 
 poses. The potatoes may after thorough scrubbing and 
 removal of "eyes" be soaked in bichloride of mer- 
 cury (1 : 1000) for twenty minutes, and then sterilized 
 on three consecutive days for one-half hour in the 
 steam sterilizer. To use they are cut in thick slices and 
 put in deep Petri dishes. For more careful work the 
 potatoes are first cut into proper sizes for tubes or 
 
BACTERIOLOGICAL TECHNIQUE. 217 
 
 dishes, and then soaked for from twelve to eighteen 
 hours in running water; this removes excessive acidity; 
 they are then placed in test-tubes and sterilized by steam 
 on two consecutive days. 
 
 Blood-serum and Ascitic Fluid with and without the 
 Addition of Bouillon. Blood-serum is used in the fluid 
 state, semi-solid, and firmly coagulated. It is used 
 alone, with 66 per cent, of bouillon and with 25 per 
 cent, of bouillon plus 1 per cent, of glucose. Ascitic, 
 pleuritic, and hydrocele fluids are also used alone, with 
 bouillon, or with nutrient agar. 
 
 The Correction of the Reaction in Media. 
 
 Formerly it was customary to use litmus-paper as 
 the indicator in neutralizing media, adding soda solu- 
 tion until the mixture turned the red litmus slightly 
 blue, and the blue litmus just a tinge less blue. This 
 is still the best method for those who are only going 
 to cultivate the common pathogenic bacteria for diag- 
 nostic purposes or for the development of toxin. Most 
 parasitic bacteria which grow at all on artificial culture 
 media develop best in them when they have a slightly 
 alkaline reaction to litmus. If a greater alkalinity is 
 desired a certain number of c.c. of normal soda solu- 
 tion can be added for each litre; if an acidity is desired, 
 normal hydrochloric acid solution is added. 
 
 Many bacteriologists consider that litmus is not deli- 
 cate enough to be entirely satisfactory, especially when 
 experiments are to be reported or exactly repeated. 
 For these purposes phenolphthalein has been generally 
 selected. A little experience will show that different 
 indicators not only differ in delicacy, but that they 
 react differently to different substances. 
 
218 BACTERIOLOGY. 
 
 A litre of bouillon becomes on the addition of 1 per 
 cent, of peptone more alkaline to litmus, but decidedly 
 more acid to phenolphthalein. We have, therefore, 
 especially with the latter substance, to find by growing 
 the bacteria just what reaction we want, and then test 
 the fluid with phenolphthalein as the indicator. With 
 exactly similar materials we can exactly reproduce at 
 any time in the future the same reaction, but with dif- 
 ferent materials this would be impossible. A bou- 
 illon which contains 1 per cent, of peptone and reacts 
 neutral to litmus is about 15 points acid to phenolph- 
 thalein that is, 15 c.c. of normal soda solution must 
 be added per litre to make the bouillon neutral. 
 
 When phenolphthalein is used we must have accu- 
 rately standardized solutions of caustic soda and hydro- 
 chloric acid. The test is carried out as follows : To 
 10 c.c. of the hot nutrient bouillon add one drop of a 
 1 : 300 solution in alcohol of phenolphthalein; into this 
 is dropped slowly a 4 per cent, solution of caustic soda 
 until a faint rose-tint appears. This indicates the be- 
 ginning of an alkaline reaction. To make a litre neu- 
 tral we would add 100 times as much of the decinormal 
 solution of caustic soda as was required to make 10 c.c. 
 neutral. As a rule, we use 1 per cent, peptone bou- 
 illon of such an acidity that 15 c.c. of normal soda 
 solution must be added to each litre to make it neutral. 
 
 The Sterilization of Different Media. 
 
 Flasks and tubes of nutrient broth and agar are 
 easily sterilized by placing them in an Arnold steam 
 sterilizer (Fig. 20) for from fifteen minutes to one hour, 
 according to the bulk of the fluid, upon two or three 
 consecutive days. They can also be even more cer- 
 
BACTERIOLOGICAL TECHNIQUE. 219 
 
 tainly sterilized by putting them in an autoclave (Fig. 
 21) at 110 C. for from fifteen to thirty minutes on 
 two consecutive days. 
 
 Gelatin is sterilized in the same manner except, as 
 already stated, the shorter times are used. Pro- 
 longed heating destroys the congealing properties of the 
 gelatin. 
 
 Blood-serum may be sterilized by fractional sterili- 
 zation and remain fluid, or may be rendered solid by 
 the degree of heat used in sterilizing. 
 
 For the sterilization of fluid serum it is requisite that 
 it be exposed to a temperature of from 62 to 66 C. 
 for one hour on each of six consecutive days. The 
 best apparatus for obtaining and maintaining this tem- 
 perature (about 65 C.) is a small and well-regulated 
 incubator or chamber surrounded by a water space, 
 into which the tubes and flasks containing serum are 
 to be put each day and in which they are to be left for 
 the prescribed time after having been warmed to the 
 desired temperature. 
 
 Serum may be solidified and still remain translucent 
 at a temperature of 76 C., but when heated to a higher 
 degree a more definite coagulation takes place, and the 
 medium becomes opaque. Care must be taken in coagu- 
 lating blood-serum at the higher temperatures to run 
 the temperature up slowly and not to heat above 90 C. 
 until the serum has firmly coagulated; for unless these 
 precautions are taken ebullition is likely to occur, which 
 will lead to the formation of bubbles and an uneven- 
 ness of the surface upon which growth is to be obtained 
 and studied. Serum may be solidified at the tempera- 
 tures mentioned in an incubator, water-oven, or even 
 in an Arnold steam sterilizer, with the top covered by 
 
220 BACTERIOLOGY. 
 
 a cloth instead of the usual lid, and when coagulated 
 firmly (90 C.) the tubes and their contents may, on 
 the following day, be sterilized in streaming steam at 
 100 C. without danger of the subsequent formation 
 of bubbles. Koch's serum coagulator (Fig. 22) is, 
 however, the most convenient apparatus. 
 
 Serum may be preserved by placing it in flasks 
 which, after the addition of 5 per cent, of chloroform, 
 are sealed. When it is to be used it is filled into 
 
 FIG. 22. 
 
 Blood-serum coagulator. 
 
 sterilized culture (test) tubes and sterilized by exactly 
 the same methods as are employed in sterilizing fresh 
 serum. The chloroform, being volatile, tends to dis- 
 appear at ordinary temperatures, but is quickly and 
 surely driven off at the temperatures used in steril- 
 izing. 
 
 Serum may be efficiently sterilized, when great care 
 is used, by passing it through a Pasteur or Berkefeld 
 filter, under pressure. When so treated the fluid is 
 very clear and light-colored. 
 
 Flasks, Dishes, Tubes, etc., Used for the Preservation of 
 Media and for other Bacteriological Purposes. The nutri- 
 
BACTERIOLOGICAL TECHNIQUE. 
 
 221 
 
 ent media are stored in large quantities in round or flat- 
 bottomed Erlenmeyer flasks (Fig. 23). From these, as 
 needed, glass tubes (Fig. 24) are filled. Glass dishes 
 with covers (Petri dishes, Fig. 27) and flat flasks are 
 used for growing bacteria in or upon thin layers of 
 media. When small amounts of media are taken fre- 
 quently from flasks, Pasteur's flasks (Fig. 25) are of 
 
 FIG. 24. 
 
 FIG. 23. 
 
 FIG. 25. 
 
 Erlenmeyer flask. 
 
 Pasteur flask. 
 
 Glass test-tubes. 
 
 great convenience. They consist of a flask with a 
 ground-glass neck, over which fits a cap. This cap 
 may or may not terminate, as desired, in a narrow 
 tube, which is plugged with cotton. The cap keeps 
 the edges of the flask free from bacteria and prevents 
 the cotton from sticking. 
 
222 BACTERIOLOGY. 
 
 The Methods of Obtaining and Studying Pure Cultures of 
 Single Species of Bacteria. 
 
 In order to study bacteria, both in culture media 
 and in the living body, we must separate those devel- 
 oped from one organism from all others and study 
 them by themselves in pure cultures. In order to do 
 this we have to take the greatest precautions to insure 
 that the materials that we make use of for the growth of 
 bacteria, the flasks and tubes that hold these materials, 
 and the instruments with which we transfer the bac- 
 teria are sterile. We also carefully try to prevent any 
 bacteria entering from the air or elsewhere. 
 
 The Cleansing and Sterilization of Apparatus. 
 
 In bacteriological work sterilization is practically 
 always done by means of dry and moist heat, for no 
 antiseptic substances can be allowed to remain in any 
 of the media used for the growth of bacteria or on any 
 of the apparatus which would come in contact with 
 them, as such substances would inhibit the growth of 
 the bacteria which we desired to study. 
 
 The platinum wires and loops used in transferring 
 bacteria are sterilized by holding them for a moment 
 until red-hot in a gas or alcohol flame. They should 
 not be used until time enough has elapsed for them to 
 cool sufficienly not to injure the bacteria touched by 
 them. Knives, instruments, etc., are, after thorough 
 cleansing, placed in boiling 1 per cent, soda solution 
 for three to five minutes. Hypodermic needles are 
 sterilized by boiling in soda solution, or, when this is 
 impossible, they are first frequently rinsed with boil- 
 ing or with very hot water and then filled with a 5 per 
 
BACTERIOLOGICAL TECHNIQUE. 
 
 223 
 
 cent, carbolic acid solution for at least thirty minutes 
 and then rinsed again with sterile water. New tubes 
 and flasks sometimes require to be washed in a 2 per 
 cent, solution of nitric acid, so as to remove any free 
 alkali which may be present. They are finally thor- 
 oughly rinsed in pure water. Old tubes, flasks, and 
 other glassware are boiled for about thirty minutes in a 
 5 per cent, solution of washing soda and then thoroughly 
 rinsed off with water until perfectly clean. If neces- 
 
 FIG. 26. 
 
 Dry heat sterilizer. 
 
 sary, any dirt clinging to the insides of the flasks 
 and tubes can be removed by bristle brushes or suit- 
 able swabs. After the tubes and flasks have been 
 thoroughly cleaned they are plugged loosely with ordi- 
 nary cotton batting, or, if that is not at hand, the more 
 expensive absorbent cotton. The tubes and flasks with 
 their cotton plugs and all other glassware are sterilized 
 by dry heat at 150 C. for one hour (Fig. 26). 
 
 The sterile tubes and flasks are filled with the media, 
 when small quantities are used, by means of a glass 
 funnel. The main precaution to be observed is not to 
 let the media soil the neck of the tubes and flasks, as 
 
224 BACTERIOLOGY. 
 
 this would cause the fibres of the cotton plugs to adhere 
 to the sides of the tubes when the media dried and 
 make it difficult to remove the plugs wholly when we 
 wished to inoculate the contents of the tubes. 
 
 The tubes and flasks, plugged with sterile cotton 
 and full of media, are put in the steam sterilizer for 
 one half hour on three consecutive days, or in the 
 autoclave for twenty minutes for two consecutive days. 
 A portion of the tubes containing nutrient agar are 
 laid in a slanted position before cooling, after the final 
 sterilization, so that a larger surface may be obtained. 
 
 Technique of Making Plate Cultures. 
 
 When we make cultures from any material, we are 
 very apt to find that instead of one variety of bacteria 
 only there are a number present. If such material 
 is placed in fluid media contained in test-tubes, we find 
 that the different varieties all grow together and be- 
 come hopelessly mixed. When, on the other hand, the 
 bacteria are placed on solid media they develop about the 
 spot where they were inoculated. If different varieties, 
 however, are placed too near together, they overgrow 
 one another ; it is thus advisable to have a greater sur- 
 face of nutrient material than is given on the slanted 
 surface of nutrient agar or blood-serum contained in 
 test-tubes. This need is met by pouring the media 
 while warm on flat, cool, glass plates or into shallow 
 dishes. In making plate cultures two methods are 
 carried out. In the first the material with its contained 
 bacteria is scattered throughout the fluid before it 
 hardens; in the second it is streaked over the surface of 
 the medium after it has solidified. Nutrient agar and 
 nutrient gelatin, the two substances used for plate 
 
BACTERIOLOGICAL TECHNIQUE. 225 
 
 cultures, differ in two essential points, which cause 
 some difference in their uses. Nutrient 1 per cent, 
 agar melts at a high temperature and begins to thicken 
 at about 36 C. It is not liquefied by bacterial fer- 
 ments. Nutrient 10 per cent, gelatin melts at the low 
 temperature of about 23 C. and solidifies at a point 
 slightly below that. It is liquefied by many bacterial 
 ferments. When we wish to inoculate fluid nutrient 
 agar for plate cultures we have to take great care that 
 in cooling it to a point which will not injure the bac- 
 teria, about 41 C., we do not allow it to cool too much 
 and thus solidify and prevent our pouring it into the 
 plates. To prevent this, when a number of tubes are 
 to be inoculated they are placed while still hot in a 
 basin of water which has been heated to about 45 C. 
 When the temperature of the agar in one of the tubes, 
 
 FIG. 27. 
 
 Petri dish. 
 
 as tested by a thermometer, has fallen to 40 the water, 
 milk, feces, bacterial culture, or other substances to be 
 tested are added to the other tubes in whatever quantity 
 is thought to be proper. After inoculation the con- 
 tents of the tubes are thoroughly shaken and poured 
 out quickly into round, flat-bottomed glass dishes (Fig. 
 27), the covers of which are removed for the required 
 time only. The bacteria are now scattered throughout 
 the fluid, and as it quickly solidifies they are fixed 
 wherever they happen to be, and thus as each individual 
 
 15 
 
226 BACTERIOLOGY. 
 
 multiplies clusters are formed about it at the spot where 
 it was fixed at the moment of solidification. The num- 
 ber of colonies of bacteria (Fig. 28) thus indicate to 
 us roughly the number of living bacteria in the quan- 
 tity of fluid added to the liquid agar. Nutrient gelatin 
 is used exactly as agar, except that, as it does not 
 congeal until cooled below 22 C., we have no fear of 
 
 FIG. 28. 
 
 Photograph of a large number of colonies developing in a layer of gelatin 
 contained in a Petri dish. Some colonies are only pin point in size ; some as 
 large as a pencil. The colonies here appear in their actual size. 
 
 its cooling too rapidly. In order not only to count 
 the number of colonies which develop, but also to 
 obtain a characteristic growth, it is desirable not to 
 have them too near together. As it is impossible to 
 determine accurately the number in any suspected fluid, 
 it is usual to make a set of four different plates, to each 
 of which a different amount of material is added, so that 
 
BACTERIOLOGICAL TECHNIQUE. 227 
 
 some one of the four will have the required number of 
 colonies. In the first tube we place an amount which we 
 believe will surely contain sufficient and probably too 
 many bacteria. To the second tube we add 10 per cent, 
 of the amount added to the first, and to the third 10 
 per cent, of the second, and to the fourth 10 per cent, 
 of the third. Thus if the first contained 60,000 colo- 
 nies, the second would have 6000 (Fig. 28), the third 
 600, and the fourth 60. If, on the other hand, the first 
 contained but 60, the second would have about 6, and 
 the remaining two would probably contain none at all. 
 When there are many colonies present the dishes are 
 covered by a glass plate (Fig. 29), ruled in larger and 
 
 FIG. 29. 
 
 WolffhiigePs apparatus for counting colonies. 
 
 smaller squares. With a hand lens the colonies in a 
 certain number of squares are counted and then the 
 number for the whole contents estimated. 
 
 When the material to be tested is crowded with bac- 
 teria it is often best to make an emulsion of a portion 
 of it, and use this rather than the original substance 
 for making the cultures. 
 
 Measured quantities of the diluted material can be 
 transferred most accurately through a sterilized long 
 glass pipette graduated in one-hundredth cubic centi- 
 metres, or, more roughly, by a platinum loop of known 
 size. 
 
228 BACTERIOLOGY. 
 
 The nutrient agar-agar is frequently used in a dif- 
 ferent manner. A small quantity is poured into the 
 Petri dish and allowed to harden. The substance to 
 be tested bacteriologically, or a dilution of it, is then 
 streaked by means of a platinum loop lightly over its 
 surface. While in the former method most of the bac- 
 teria developed under the surface, here all develop upon 
 it. This is an advantage, as many forms of bacteria 
 develop more characteristically on the surface than in 
 the midst of the media, and it is easier to remove them 
 free from other bacteria with the platinum needle. The 
 method of using glass plates upon a cooling stage has 
 now been practically given up for the more convenient 
 one of Petri dishes. In warm weather the dishes should 
 be cooled before using, so as to harden quickly the agar 
 or gelatin that is poured into them. 
 
 An old method, which is still sometimes used to find 
 the number of living bacteria, is, instead of pouring 
 out the media which has been inoculated, to congeal it 
 on the sides of the test-tube. This is best done by 
 laying the tube flat on its side on a cake of ice and 
 rotating it. Tubes come especially formed for this by 
 having a slight neck, which prevents the media run- 
 ning up to the plugged end of the tube. This method, 
 Esmarch's, is used only when the Petri dishes are not 
 obtainable or cannot easily be transported. 
 
 The Study of Colonies in Plate Cultures in Nutrient Agar. 
 
 The plates should be removed after twelve to twenty- 
 four hours' growth at blood temperature and after one 
 to three days at 70. The special time allowed varies 
 according to the rapidity of the growth of the varieties 
 developing, thus bacteria, such as the streptococci and 
 
BACTERIOLOGICAL TECHNIQUE. 229 
 
 influenza bacilli, reach the maximum development of 
 their colonies in from ten to sixteen hours, while others 
 continue to spread for several days. If we wait too long 
 where numerous varieties of bacteria are growing the 
 colonies of heavier growth may cover up the finer and 
 more delicate ones. As a rule, the younger colonies 
 are more characteristic, except where the development 
 of pigment is sought. 
 
 FIG. 30. 
 
 Two surface colonies of diphtheria bacilli upon agar. X 500 diameters. 
 
 The colonies are first examined with the eye (Fig. 28), 
 then with a low magnification, and then again at from 
 400 to 500 diameters (Fig. 30). We note everything 
 we can about them, such as their size, border, density, 
 color, and granular appearance. At the higher mag- 
 nification we begin to detect the individual bacteria. 
 After studying the colonies we remove a few of the bac- 
 teria from one or more of them by touching them with 
 
230 BACTERIOLOGY. 
 
 the tip of a sterile platinum needle, and thus transfer 
 them to a cover-glass for microscopical examination, or 
 to new media where they may develop in pure cultures 
 and show their growth characteristics. 
 
 The Study of Plate Cultures in Gelatin Media. 
 
 The gelatin media have one marked characteristic to 
 be noted which never occurs upon agar namely, some 
 of the colonies will be lying in or surrounded by slightly 
 opaque fluid, due to the liquefaction of the gelatin. 
 (See paler and larger colonies in Fig. 28.) In using 
 nutrient gelatin one must always remember not to allow 
 it to stay where the temperature is over 20 C., for if 
 that happens the media will melt, nor must the lique- 
 fying colonies be allowed to grow for too long a time, 
 or the entire media will become fluid. 
 
 FIG. 81. 
 
 Stab cultures of three cholera spirilla in gelatin, showing in upper portion 
 of growth considerable liquefaction of nutrient gelatin. 
 
 Pure Cultures. If we transfer without contamination 
 bacteria from a colony formed from a single organism 
 
BACTERIOLOGICAL TECHNIQUE. 231 
 
 to new media, and these grow, we have what we call a 
 pure culture of that variety. When these are trans- 
 ferred to the solid media we call the growth which 
 takes place from smearing the bacteria over the surface, 
 a surface or smear culture, and that formed in the 
 depth of the media by plunging the needle carrying 
 the bacteria into it, a stab culture. (Fig. 31.) 
 
 In transferring bacteria from one tube to another we 
 slant the tubes so that no dust may fall within and 
 contaminate with other bacteria the special variety we 
 wish to transplant. The greatest care must be taken 
 that the sterilized platinum needle used to transfer the 
 bacteria is not infected by touching any non-sterile 
 matter. Even with our utmost care bacteria will from 
 time to time pass from the air or edges of our tubes 
 into the culture media, and thus possibility of contami- 
 nation must always be kept in mind. When it occurs 
 upon solid media we, as a rule, easily detect it, for we 
 notice the growth at some point of bacteria of different 
 colony characteristics; but in fluid media, on account of 
 the complete mingling of the bacteria, we are not so apt 
 to notice the additional growth. 
 
 Incubators. In order to have a constant and proper 
 temperature for the growth of bacteria, forms of appa- 
 ratus called incubators have been devised. These (Fig. 
 32) consist in their simplest form of an inner air cham- 
 ber surrounded by a double copper wall containing water. 
 The apparatus externally is lined with asbestos, to pre- 
 vent radiation. It is supplied with doors and with 
 openings for thermometers and a thermo-regulator. The 
 ther mo-regulators are of various kinds; those in most 
 use depend upon the expansion or contraction of the 
 fluid in a bulb (A, Fig. 33), which rests within the 
 

 232 
 
 BACTERIOLOGY. 
 
 water-jacket, to lessen or increase the space between 
 the surface of the mercury, B, and the inner tube, D, 
 thus allowing of the passage of a greater or less quan- 
 tity of gas to the burner through the tube D (Fig. 33). 
 
 FIG. 33. 
 
 FIG. 32. 
 
 Small incubator. 
 
 Thermo-regulator. 
 
 Other forms depend upon the contraction or expansion 
 of metal, or the use of the electric current to control 
 the flow of the 2;as. 
 
 O 
 
 The temperature in the air chamber is kept above 
 that of the surrounding air by means of a gas flame 
 regulated as above described, or, when that cannot be 
 obtained, a lamp. 
 
 The temperature is reduced by passing a stream of 
 cool water through the water chamber, which is itself 
 regulated. When very accurate investigations are to be 
 
BACTERIOLOGICAL TECHNIQUE. 233 
 
 made a gas-pressure regulator is added to the thermo- 
 regulator. Incubators are also both warmed and regu- 
 lated by electricity. 
 
 In emergencies a culture may be developed at the 
 blood temperature by placing it in water contained in 
 a small vessel, which itself is contained in a larger 
 vessel, also filled. By adding a little hot water from 
 time to time the temperature can readily be kept be- 
 tween 34 and 38 C., which is sufficiently uniform for 
 bacteria such as the diphtheria bacilli to grow. 
 
 As a temporary expedient during the night, when 
 haste is necessary, it is possible, when the culture medium 
 is solid and within a strong glass tube or metal case, 
 to make use of the body heat by putting it under the 
 clothing next to the body and sleeping upon it. Natu- 
 rally, this should only be done when other means fail. 
 Several times, when in the country, this method has 
 enabled the writer to obtain a growth of diphtheria 
 bacilli over night, and thus get important information, 
 when otherwise it would have been impossible. 
 
 Culture Methods for Anaerobic Bacteria. 
 
 Anaerobic bacteria will scarcely be cultivated except 
 in bacteriological laboratories, where the technique is 
 already understood, so that the methods employed in 
 their culture will only be touched on here. A simple 
 device is that of Koch, who placed a thin strip of 
 sterile mica upon the still fluid agar or gelatin in the 
 Petri dish, which had already been inoculated. After 
 the solidification of the media the portion under the 
 mica is excluded from the air and anaerobic growth can 
 develop. A second simple method (Liborius) is to fill 
 the tubes with media fuller than usual and to inoculate 
 
234 BACTERIOLOGY. 
 
 the bacteria deep down to near the bottom of the tubes 
 while the media are still semi-solid. An anaerobic 
 growth will take place in the lower part of the tube. 
 In a similar way the closed arm of the fermentation 
 tube will suffice for anaerobic growth, if the opening 
 connecting it with the open bulb is quite small. In 
 the more complicated methods the plates or tubes are 
 placed in jars (Fig. 34), in which the oxygen is dis- 
 
 FIQ. 34. 
 
 Jar for anaerobic cultures. 
 
 placed by a stream of hydrogen developed by the Kipp 
 apparatus through the action of pure granulated zinc 
 and a pure 25 per cent, solution of sulphuric acid. 
 When all the oxygen has been displaced the jars are 
 sealed by rotating the stopper. In another method the 
 oxygen is extracted by a mixture of pyrogallic acid and 
 caustic potash. To each 100 c.c. of air space in the jar 
 1 gramme of pyrogallic acid and 10 c.c. of 6 per cent, 
 solution of potassium hydroxide are added and the jars 
 immediately sealed. When spores are present, a simple 
 method suggested, I believe, by McFarland can be 
 successfully employed. Vessels plugged with stoppers 
 
BACTERIOLOGICAL TECHNIQUE. 235 
 
 perforated by glass tubes drawn to a point are filled to 
 such a height that when the fluid is heated to 80 C. 
 it will just fill them. They are inoculated when the 
 bouillon is at about 60 C., heated to 80 C., and then 
 sealed by closing the tube's point by means of a flame. 
 After inoculating and heating, instead of sealing the 
 glass tube a sterile rubber cork can be inserted. If 
 much fermentation is expected, the cork should be 
 clamped or tied to the bottle, so that it will not blow 
 out. One advantage of this method is that any con- 
 taminating organisms which have no spores will be 
 killed. When sealed the bottles should be cooled and 
 then placed in the incubator. 
 
CHAPTER XIY. 
 
 THE USE OF ANIMALS FOR DIAGNOSTIC AND TEST 
 PURPOSES. 
 
 SUITABLE animals are necessarily employed for many 
 bacteriological purposes. Thus they may be used as a 
 soil for bacterial growth, when, as in the case of tubercle 
 bacilli, the bacteria will not develop in the dead culture 
 media. For this reason material suspected to contain 
 tubercle bacilli is injected into rabbits or guinea-pigs, 
 with the knowledge that, if present, although in too 
 small numbers to be detected by microscopical or cul- 
 ture methods, they will develop their lesions in the 
 animal's bodies, and thus reveal themselves. The same 
 may be true of glanders and anthrax bacilli and of 
 other bacteria. Again, animals are used to test the 
 virulence of organisms, where, as in the case of diph- 
 theria, we have very virulent, attenuated, and non- 
 virulent bacilli of, so far as we know, identical cultural 
 characteristics. Here the injection of a susceptible 
 animal, such as the guinea-pig, is the only way that we 
 can differentiate between those capable of producing 
 diseases from those that are harmless. Still another 
 use of animals is to differentiate between two virulent 
 organisms, which, though entirely different in their 
 specific disease-poisons, are yet so closely allied mor- 
 phologically and in culture characteristics that they 
 cannot always be separated except by studying their 
 
THE INOCULATION OF ANIMALS. 237 
 
 action in the animal body both without and under the 
 influence of specific serums upon them. In this way 
 the typhoid and colon bacilli may be separated, or the 
 pneumococcus and streptococcus. Still, a different use 
 of animals is to measure the protective effect of anti- 
 toxic and bactericidal serums; thus, diphtheria antitoxin 
 is added to diphtheria toxin and injected into guinea- 
 pigs, and streptococcus immunizing serum is mixed 
 with living streptococci and injected into the vein of 
 a rabbit. The use of animals to develop through bac- 
 terial injections protective serums will be dealt with 
 under the special bacteria by whose products they are 
 produced. 
 
 THE INOCULATION OF ANIMALS. 
 
 The inoculation of animals may be made either 
 through natural channels or through artificial ones: 
 
 1. Cutaneous. Cultures are rubbed into the abraded 
 skin. 
 
 2. Subcutaneous. The bacteria are injected by 
 means of a hypodermatic needle under the skin, or 
 are introduced by a platinum loop into a pocket made 
 by an incision. 
 
 3. Intravenous. The bacteria are injected by means 
 of a hypodermatic needle into the vein. This is usually 
 carried out in the ear vein of the rabbit. If rabbits 
 are placed in a holder, so that the rabbit remains quiet 
 and only the head projects, it is usually easy to pass a 
 small needle directly into one of the ear veins, espe- 
 cially those running along their edges. If the ear is 
 first moistened with a 3 per cent, carbolic acid solution, 
 and then supported between the finger inside and the 
 
238 BACTERIOLOGY. 
 
 thumb outside, the vein is usually clearly seen and en- 
 tered with ease, if a small sharp needle is held almost 
 parallel with the ear surface and gently pushed into it. 
 When no holder is present, the rabbit can be held by an 
 assistant seizing the forelegs in one hand and the hind 
 in another and holding the rabbit head downward. 
 
 4. Into the anterior chamber of the eye. 
 
 5. Into the body cavities. The peritoneal and less 
 often the pleural cavities are used for bacterial injec- 
 tion. The hypodermatic needle is usually employed, 
 less often a glass tube drawn out to a fine point. The 
 needle or the pointed glass tube are gently pushed 
 through the abdominal wall, moved about to insure its 
 freedom from the intestines, and the fluid injected. 
 
 6. By inhalation. This method is carried out by 
 forcing the animal to inhale an infected spray or dust. 
 
 7. By the trachea. This method is carried out by 
 making an incision in the trachea and then inoculating 
 the mucous membrane or injected substances into the 
 trachea and bronchi. 
 
 8. Through the intestinal tract by swallowing. 
 
 ' In these injections guinea-pigs are held, as a rule, 
 by an assistant grasping in one hand the forelegs and 
 in the other the hindlegs. 
 
 Rabbits can be held in the same manner, or better 
 placed in some holder. 
 
 Mice, which are usually inoculated subcutaneously at 
 the root of the tail, are best placed in a mouse holder, 
 but can be inoculated by grasping the tail in a pair of 
 forceps, and then, while allowing the mouse to hang 
 head downward in a jar, a glass plate is pushed across 
 the top until only space for its tail is left. 
 
 All these methods must be carried out with the 
 
THE INOCULATION OF ANIMALS. 239 
 
 greatest care as to cleanliness, the hair being clipped 
 and the skin partially, at least, disinfected. After the 
 inoculations the animals should be given the best of 
 care, unless, for special purposes, we want to study 
 them under unusual conditions. For food, rabbits 
 and guinea-pigs require only carrots and hay. 
 
 If animals die, autopsy should be made at the earliest 
 moment possible, for soon after death some of the spe- 
 cies of the bacteria in the intestines are able to penetrate 
 through the intestinal walls and infect the body tissues. 
 If delay is unavoidable, the animals should be placed 
 immediately in a cold place. In making cultures from 
 the dead bodies the greatest care should be taken to 
 avoid contamination. The skin should be disinfected, 
 and any dust prevented by means of a 5 per cent, solu- 
 tion of carbolic acid. All instruments are sterilized 
 by boiling in 3 per cent, soda solution for five minutes. 
 Changes of knives should be made as frequently as the 
 old ones become infected. When organs are examined 
 the portion of the surface through which an incision is 
 to be made must be sterilized, if there is danger that 
 the surrounding cavity is infected, by searing with the 
 flat blade of an iron spatula which has been heated to 
 a dull red heat. 
 
 When it is necessary to transport tissues some dis- 
 tance they should be wrapped in bichloride cloths and 
 sent to the point of destination as soon as possible. In 
 warm weather they may be kept cool by surrounding 
 the vessel which contains them with ice. 
 
 Animals rarely show the same gross lesions as man 
 when both suffer from the same infection. The cell 
 changes are similar, and, also, so far as we can test them, 
 the curative or immunizing effects of protective serums. 
 
CHAPTER XV. 
 
 THE PROCURING OF MATERIAL FOR BACTERIOLOGICAL 
 EXAMINATION FROM THOSE SUFFERING FROM 
 DISEASE. 
 
 A LONG experience has taught me that physicians 
 very frequently take a large amount of trouble, and 
 yet, on account of not carrying out certain simple but 
 necessary precautions, make worthless cultures or send 
 material almost useless for bacteriological study. 
 
 In making cultures from diseased tissues various pro- 
 cedures may be carried out, according to the facilities 
 which the physician has and the kind of information 
 that he desires to obtain. From the dead body culture 
 material should be removed at the first moment possible 
 after death. Every hour's delay makes the results less 
 reliable. From both dead and living tissues the less 
 the alteration that occurs in any substance between its 
 removal from the body and its inoculation upon or in 
 culture media or animals the more exact the informa- 
 tion which will be obtained from its examination. If 
 the material is allowed to dry many bacteria will be 
 destroyed in the process, and certain forms which were 
 present will be obliterated, or, at least, entirely altered 
 in the proportion which they bear to others. If pos- 
 sible, therefore, culture media should be inoculated 
 in the neighborhood of the patient or dead body. For 
 that purpose a bacteriologist should take the most suit- 
 
MATERIAL FOR EXAMINATION IN DISEASE. 241 
 
 able of the culture media to the bedside or autopsy table. 
 Such a list of media, if fairly complete, would comprise 
 nutrient bouillon alone and mixed with one-third its 
 quantity of ascitic fluid, slanted nutrient agar, and 
 firmly solidified slanted blood -serum. If only one 
 variety of media is to be used the solidified blood- 
 serum is most useful for parasitic bacteria, and this can 
 be easily carried by the physician and inoculated by 
 him, even if he is not very familiar with bacterio- 
 logical technique. The material must be obtained in 
 different ways, according to the nature of the infection. 
 For the detection of the bacteria causing septicsernia 
 we are met with the difficulty that there are apt to be 
 very few or no organisms present in the blood until 
 shortly before death. It will, therefore, be useless to 
 take only a drop of blood for cultures, as even when 
 present there may not be more than eight or ten organ- 
 isms in a cubic centimetre. If cultures are to be made 
 at all, it is, therefore, best to make them correctly by 
 taking from 3 to 5 c.c. of blood by means of a sterile 
 hypodermatic needle, or a suitable glass tube armed 
 with a hypodermatic needle, from the vein of the arm, 
 after proper cleansing of the skin and a tiny incision. 
 Into each of five different tubes containing bouillon we 
 add one-fifth of the quantity of blood withdrawn. We 
 have made by this mixture of blood and bouillon a most 
 suitable medium for the growth of all bacteria which 
 produce septicaemia, and at the same time have added a 
 sufficient quantity of blood to insure us the best possible 
 chance of having added some of the bacteria producing 
 the disease. We also streak several nutrient agar plates 
 with blood, so as to indicate roughly the number of 
 organisms present, if they happen to be in abundance, 
 
 16 
 
242 BACTERIOLOGY. 
 
 From wounds, abscesses, cellulitis, etc., the substance 
 for bacteriological examination can, as, a rule, best be 
 obtained by means of small rods armed with a little 
 absorbent cotton. A number of these can be carried 
 in a test-tube. Both rods and tubes must be sterile. 
 The swab is inserted in the wound, then streaked 
 gently over the oblique surface of the nutrient agar in 
 one tube, over the blood-serum in another, and then 
 inserted in the bouillon. Finally, either at the bed- 
 side or in the laboratory, material is thinly streaked 
 over the surface of nutrient agar contained in several 
 Petri dishes. We inoculate several varieties of media, 
 with the hope that one at least will prove a suitable 
 soil for the growth of the organisms present. From 
 surface infections of mucous membranes, as in the 
 nose, throat, vagina, etc., the swab, again, is probably 
 the most useful instrument for obtaining the mate- 
 rial for examination. The greatest care, of course, 
 must be used in all cases to remove the material for 
 study without contaminating it in any way by other 
 material which does not belong to it. Thus, for in- 
 stance, if we wish to obtain material from an abscess 
 of the liver, where the organ lies in a peritoneal cavity 
 iufected with bacteria, here one must first absolutely 
 sterilize the surface of the liver by pressing on it the 
 blade of a hot iron spatula before cutting into the ab- 
 scess, so that we may not attribute the infection which 
 caused the abscess to the germs which we obtained from 
 the infected surface of the liver. From such an organ 
 as the uterus it is only with the greatest care that we 
 can avoid outside contamination, and only an expert 
 bacteriologist familiar with such material will be able 
 to eliminate the vaginal from the uterine bacteria. 
 
MATERIAL FOR EXAMINA TION IN DISEASE. 243 
 
 A statement of the conditions under which materials 
 are obtained should always accompany them when sent 
 to the laboratory for examination, even if the exami- 
 nation is to be made by the one who made the cul- 
 tures. These facts should be noted, or otherwise at 
 some future date they may be forgotten and mislead- 
 ing information sent out. The work of obtaining 
 material for examination without contamination is at 
 times one of extreme difficulty. It simply must be 
 remembered that if contamination does take place our 
 results may become entirely vitiated, and if the diffi- 
 culties are so great that we cannot avoid it, it may 
 simply mean that under such conditions no suitable 
 examination can be made. Where the substance to 
 be studied cannot be immediately subjected to cultures 
 or animal inoculations, it should be transferred in a 
 sterile bottle as soon as possible to a location where the 
 cultures can be made. If for any reason delay must 
 take place, the material should at least be put in a 
 refrigerator, where cold will both prevent any further 
 growth of some varieties of bacteria and lessen the 
 danger of the death of others. After having made 
 the cultures, some of the infected material should 
 always be smeared on a couple of clean slides or cover- 
 glasses and allowed to dry. These can be stained and 
 examined later, and may give much valuable informa- 
 tion. 
 
 In obtaining samples of fluid, such as urine, feces, 
 etc., the bottles in which they are placed should always 
 be sterile, and, of course, no antiseptic should be added. 
 It is necessary to clearly explain this to the nurse, for 
 she has probably been instructed to add disinfectants 
 to all discharges. Disinfected material is, of course, 
 
244 BACTERIOLOGY. 
 
 entirely useless for bacteriological investigations. It 
 cannot be too much emphasized that materials which 
 are not immediately used should be sent to the labora- 
 tory as quickly as possible, for in such substances as 
 feces, where enormous numbers of various kinds of 
 bacteria are present, those which we seek most, such as 
 the typhoid bacilli, frequently succumb to the delete- 
 rious products of the other bacteria present. Even 
 when abundantly present living typhoid bacilli may 
 entirely disappear from the feces in the course of even 
 twelve hours, while at other times they may remain 
 present for weeks. These differences depend on the 
 associated organisms present, the chemical constitution 
 of the feces or urine, and the conditions under which 
 the material is obtained. 
 
CHAPTER XVI. 
 
 BACTERIOLOGICAL EXAMINATION OF WATER AND 
 AIR THE CONTAMINATION AND PURIFICATION 
 OF DRINKING WATERS. 
 
 Bacteriological Examination of Water. The bacterio- 
 logical examination of water is undertaken with two 
 purposes : First, to discover the number of living bac- 
 teria present in the water, and, second, the varieties 
 that may be present. In order to roughly determine 
 the number of living bacteria in water, we thoroughly 
 mingle certain definitely measured quantities of water 
 with suitable quantities of melted but sufficiently cooled 
 nutrient agar or gelatin, the mixtures being immedi- 
 ately poured into Petri dishes, or retained in Esmarch 
 tubes and allowed to quickly harden. The bacteria in 
 the water are thus scattered throughout the solidified 
 media. If nutrient gelatin is employed care must be 
 taken to keep it cool during transportation. The agar 
 is usually allowed to remain at the body-temperature, 
 while the gelatin is necessarily kept at the usual room- 
 temperature (about 70 F.). If nutrient agar alone 
 is used two sets of plates are made, one being kept at 
 body-heat the other at the usual room-temperature. 
 After a suitable length of time has been allowed for 
 the development of the colonies we examine the plate 
 cultures, and by counting the number of colonies de- 
 veloped by means of a low power lens and Wolffhiigel's 
 apparatus (Fig. 29) or some equivalent, we are enabled 
 to find approximately the number of living bacteria 
 
246 BACTERIOLOGY. 
 
 which were present in the water at the moment the 
 water was examined. Any bacteria, however, which, 
 though living in the water, were unable to grow in the 
 media or at the temperatures employed would, naturally, 
 not reveal themselves by the growth of colonies, nor 
 would bacteria clinging together in bunches count as 
 more than a single member. As to the value of learn- 
 ing the number of bacteria in water, we must admit that 
 a single determination of the number of living bacteria 
 in any sample is now known to be of little avail unless 
 the conditions under which the water exists are well 
 known or the number of bacteria is enormous. Thus, 
 for instance, the water in an Adirondack lake might 
 contain in a cubic centimetre far more bacteria than 
 that of a well which was slightly contaminated with 
 typhoid bacilli from human sources. If, however, we 
 knew the usual condition of the well water and the 
 usual number of bacteria present in it, any sudden 
 increase would, of course, give us a strong suspicion, 
 but nothing more than a suspicion, of dangerous con- 
 tamination. In the same way in a stream into which 
 a sewer empties, if we find a great many more bacteria 
 in the stream some distance below the point of entrance 
 of the sewer than there were above, we would have 
 every reason to believe that the increase of bacteria 
 found in the stream below was due to the bacteria added 
 to the stream by the sewer; if we drank that water we 
 would know from the examination we were drinking 
 not only a portion of the sewage but of the bacteria 
 contained therein. It is true, that with our present 
 knowledge, derived from previous bacteriological studies, 
 we would be almost as certain of these facts before as 
 after the bacteriological examination. The determina- 
 
EXAMINATION OF WATER AND AIR. 247 
 
 tion, therefore, of the number of bacteria should only 
 be considered of value, except in the extreme instances 
 where enormous numbers are found, when we know 
 fairly well the conditions, chemical and physical, con- 
 cerning the supply. The examination of water, to de- 
 termine whether or not any forms of parasitic bacteria 
 or other micro-organisms are present, would be more 
 often of practical value than it is if the difficulties 
 were not so great. As a matter of fact, water exami- 
 nations for this purpose are usually negative. The 
 varieties of bacteria most sought, except in the presence 
 of a cholera epidemic, are the typhoid and colon bacilli. 
 If it were possible to readily obtain the typhoid bacilli 
 from water, when they were present in small numbers, 
 its examination for that purpose would be of much 
 greater value than it is now; but we have to remem- 
 ber that we can only examine at one time a few cubic 
 centimetres of water by bacteriological methods, and 
 that although the typhoid bacilli may be sufficiently 
 abundant in the water to give, in the quantity that we 
 ordinarily drink, a few bacilli, yet it must be a very 
 lucky chance if they happen to be in the small amount 
 which we examine. Still, further, although it is very 
 easy to isolate typhoid bacilli from water when they 
 are in considerable numbers, yet when they are a 
 very minute proportion of all the bacteria present it 
 is almost impossible not to overlook them. Many 
 attempts have been made to devise some method by 
 which the relative number of the typhoid and other 
 parasitic bacteria present in water could be increased 
 at the expense of the saprophytic bacteria. Thus to 
 100 c.c. of water 25 c.c. of a 4 per cent, peptone 
 nutrient bouillon is added, and the whole put in the 
 
248 BACTERIOLOGY. 
 
 incubator at 37 C. for twenty-four hours. From 
 this plate cultures are made. In our experience this 
 and other methods have not enabled us to detect the 
 typhoid bacillus where we have failed to find it by 
 making direct plate cultures. As a matter of fact, the 
 typhoid bacillus is found in such a small number of the 
 specimens where we actually know that it is or has 
 been present in the water from which they were ob- 
 tained, because of cases of typhoid fever which have 
 developed from drinking the water, that we must con- 
 sider our lack of finding the bacillus in any given 
 case as absolutely no reason for considering the water 
 to be free from danger. Another serious drawback 
 to the value of the examinations is that they are fre- 
 quently made at a time when the water is really free 
 from contamination, though both earlier and later the 
 bacillus was present; it is hardly worth while, there- 
 fore, except in careful experimental researches, to ex- 
 amine the water for the typhoid bacillus, but rather 
 study the location of the surrounding privies and sources 
 of contamination. The colon bacilli are far more easy 
 to detect, because they are apt to be more abundant, 
 and, also, because they grow more readily in artificial 
 culture media. A method suggested by Theobald 
 Smith is of value in both finding and excluding the 
 presence of bacilli of the colon group. He adds a few 
 drops of the suspected water to glucose nutrient bouillon 
 in fermentation tubes, and keeps it at 37 C. for from 
 thirty-six to forty-eight hours. If no fermentation 
 occurs no colon bacilli are present. If it does occur 
 plates are made and the bacteria isolated and tested. 
 In the bouillon the colon bacilli when present usually 
 increase in numbers and are then readily detected. The 
 
EXAMINATION OF WATER AND A IE. 249 
 
 presence of the colon bacilli in water is, except possibly 
 in rare cases, only of importance as an indication of fecal 
 contamination, and, therefore, of the possibility of dan- 
 gerous infection with other bacteria. Formerly it was 
 considered that its presence indicated human fecal con- 
 tamination, but we now know that many of the animals 
 contain in their intestines colon bacilli so similar to those 
 found in human beings that we cannot differentiate 
 the one variety from the other; therefore, the finding of 
 colon bacilli in water must always be judged according 
 to the conditions surrounding the water-supply. Thus, 
 it may indicate cattle contamination from the barn or 
 surface water, human contamination, or, in certain 
 conditions, simply the accidental contamination of the 
 stream by wandering cattle or animals. Properly 
 judged, however, the examination for the colon bacilli 
 may yield results of considerable practical impor- 
 tance. Whenever, in water examinations, any special 
 variety of bacteria is found in unusual abundance, the 
 fact should be noted, for sometimes it maybe the cause 
 of some prevailing infectious disease ; thus the bacillus 
 pyocyaneus has been found in water producing diar- 
 rhoea with greenish discharges. 
 
 The Obtaining of Water for Examination. Whenever 
 possible the inoculation of the gelatin or agar tubes 
 and the pouring of their contents into the Petri dishes 
 should be done immediately after gathering the samples, 
 otherwise the actual and relative numbers of the differ- 
 ent organisms will change. As a rule, the pathogenic 
 bacteria will decrease and the harmless water species 
 will increase. When the plates cannot be made imme- 
 diately the water held in the sterile vials should be 
 kept nearly at the freezing-point. Even at very low 
 
250 BACTERIOLOGY. 
 
 temperatures some forms of bacteria increase rapidly. 
 Unless the nutrient gelatin or agar inoculations are 
 made within an hour or two the count of the number 
 of colonies is practically useless. Considerable care 
 is necessary in taking the samples of water, so as not 
 to get extraneous organisms in the water from sur- 
 rounding sources. Three slightly different methods 
 will suffice to indicate how it should be done. A 
 simple and accurate method of collecting the water is 
 to have several graduated sterile glass pipettes plugged 
 
 FIG. 35. FIG. 36. FIG. 37. 
 
 Bulb pipette. Graduated pipette. Sternberg bulb. 
 
 at the bottom by a cork and above by cotton. This is 
 inserted the required depth, to avoid the surface water 
 with its particles of dirt, and the cork pushed off by 
 a second pipette or rod and the water allowed to flow 
 or be sucked in. The upper end is now stopped by 
 the finger and the pipette removed and a definite 
 amount of water tested (Figs. 35 and 36). A simple 
 glass tube, sterilized by passing it through a flame and 
 corked below, will answer the same purpose, or, again, 
 a tube, one end of which, after sealing, is blown into a 
 sphere and the other end drawn out into a capillary stem 
 
EXAMINATION OF WATER AND AIR. 251 
 
 (Fig. 37). The stem must be sealed while the bulb is 
 still hot, or while a little water is being boiled in it, so 
 that a partial vacuum may exist in the bulb, in order 
 that the water may be sucked up into it when the 
 stem is broken. The inoculation of the media is now 
 made directly, or water from the tube is emptied into a 
 sterile bottle or test-tube, or the end of the Sternberg 
 bulb is sealed by heat. When water is to be obtained 
 from greater depths or from beneath the surface of 
 wells, more complicated forms of apparatus are neces- 
 sary. A good example is the one devised by Abbott, 
 
 FIG. 38. 
 
 Flask for counting colonies of bacteria. 
 
 and made for him by Charles Leotz & Sons, of Phila- 
 delphia. It consists of a metal framework in which is 
 encased a bottle provided with a ground-glass stopper. 
 To the stopper a spring clamp is attached, and this in 
 turn is operated by a string, so that when the weighted 
 apparatus is allowed to sink into the stream the stopper 
 may be removed at any depth desired by simply pulling 
 on the string. When the bottle is full the stopper is 
 allowed to spring back into position by releasing the 
 spring. Before removing the water the neck of the 
 bottle should be sterilized by pouring a little of a 5 
 
252 BACTERIOLOGY. 
 
 per cent, solution of carbolic acid upon it and drying 
 with a sterile cloth. 
 
 The technique of making plate cultures, of counting 
 the number of colonies, and of isolating and identi- 
 fying pathogenic species are described under the special 
 chapters devoted to these subjects. A point to be re- 
 membered is that about double the number of colonies 
 usually develop at 20 C. as at blood heat (37 C.), 
 many water bacteria not growing at body-temperature. 
 A convenient flat flask with ruled surface (Fig. 38) 
 has been devised to take the place of the Petri dish 
 when the number of bacteria only and not the varieties 
 are wanted. In these there is no danger of contami- 
 nating air-organisms entering during transportation. 
 The stopper can be graduated to hold one c.c. 
 
 The Bacteriological Examination of Air. Saprophytic 
 bacteria are always present in considerable numbers in 
 the air except far out at sea or on high mountains. 
 They are more abundant where organic matter abounds 
 and in dry and windy weather. Pathogenic bacteria, 
 on the other hand, are only occasionally present in 
 the air. The practical results obtained from the ex- 
 amination of air for pathogenic bacteria have been 
 slight. We know that at times they must be in the 
 air, but unless we purposely increase their numbers 
 they are so few in the comparatively small amount of 
 air which it is practicable to examine that we rarely find 
 them. Examination of dust, however, in hospital 
 wards and sick-rooms, in places where only air infec- 
 tion was possible, have revealed tubercle bacilli and 
 other pathogenic bacteria. 
 
 The simplest method of searching for the varieties 
 of bacteria in the air and their number in any place is 
 
PURIFICATION OF WATER. 253 
 
 to expose to the air for longer or shorter periods nutrient 
 agar spread upon the surface of the Petri dish. After 
 exposure the plates are put either in the incubator at 
 37 C. or kept at room-temperature. The more careful 
 examination is made by drawing a given quantity of 
 air through tubes containing sterile sand, which is kept 
 in by pieces of metal gauze. When the operation is 
 completed the sand is poured into a tube containing 
 melted nutrient gelatin or nutrient agar, and after 
 thoroughly shaking the mixture is poured into a Petri 
 dish and the bacteria allowed to develop, either at 
 37 or 20 C., according as a growth of the parasitic 
 or saprophytic varieties is desired. 
 
 THE CONTAMINATION AND PURIFICATION OF 
 DRINKING WATERS. 
 
 Brook and river water is contaminated in two ways: 
 through chemicals, the waste products of manufactur- 
 ing establishments, and through harmful bacteria by 
 the contents of drains, sewers, etc., the latter method 
 being by far the more dangerous. 
 
 When water, which has been soiled by waste products 
 of manufactories only, becomes so diluted or purified 
 that the contamination is not noticeable to the senses 
 and shows no dangerous products on chemical analysis 
 it is probably safe to drink. When sewage is the con- 
 tamination this rule no longer holds, and there may be 
 no chemical impurities and no pathogenic bacteria 
 found and yet disease be produced. That river water 
 which has been fouled by sewage will, in the course of a 
 few miles, through the dilution of additional supplies, 
 through sedimentation, and through oxidation, become 
 
254 BACTERIOLOGY. 
 
 greatly purified is an indisputable fact. The increase in 
 bacteria which occurs from contamination is also largely 
 or entirely lost after ten to twenty miles of river flow. 
 Nevertheless, the history of many epidemics seems to 
 show that a badly contaminated river is never a safe 
 water to drink, although with the lapse of time it be- 
 comes less and less dangerous, nor will sand filter-beds 
 absolutely remove all danger. These statements are 
 founded upon the results of numerous investigations; 
 thus the marked disappearance of bacteria is illustrated 
 by the following : Kummel found below the town of 
 Rosbock 48,000 bacteria to the cubic centimetre ; 
 twenty-five kilometres further down the stream only 
 200 were present about the same number as before 
 the sewage of Rosbock entered. On the other hand, 
 the doubtful security of depending on a river purifi- 
 cation is proved by such experiences as the following: 
 In the city of Lowell, Massachusetts, an alarming 
 epidemic followed the pollution of the Merrimac River 
 three miles above by typhoid feces, and six weeks later 
 an alarming epidemic attacked Lawrence, nine miles 
 below Lowell. It was estimated that the water took 
 ten days to pass from Lowell to Lawrence and through 
 the reservoirs. As typhoid bacilli may live for twenty- 
 five days in water, the Lawrence epidemic is easily 
 explained. Newark-on-Treut, England, averaged 
 seventy-five cases a year from filtered water and only 
 ten when it was changed to deep-well supply. 
 
 The Purification of Water on a Large Scale. Surface 
 waters, if collected and held in sufficiently large lakes 
 or reservoirs, usually become so clarified by sedimen- 
 tation as to require no further treatment so far as its 
 appearance goes. The collection of water in large 
 
PURIFICATION OF WATER. 255 
 
 reservoirs allows not only the living and dead matter 
 to subside, but allows time also for the pathogenic germs 
 to perish through light and antagonistic bacteria and 
 other deleterious influences. Filtration of water ex- 
 erts a very marked purification, taking out 99 per cent, 
 of the organisms in those best constructed and at least 
 90 per cent, in those commonly used in cities. The 
 construction of filters is too large a subject to enter on 
 minutely here; they consist, as a rule, of several layers, 
 beginning with fine sand, and then smaller and larger 
 gravel, and finally rough stones. A certain time elapses 
 before the best results are obtained; this seems to wait 
 for the formation of a film of organic material on the 
 sand, which is full of nitrifying bacteria. Even the 
 best filters only greatly diminish the dangers of pol- 
 luted water. Spring and well waters are, in fact, 
 filtered waters. 
 
 Domestic Purification. Water which requires private 
 filtering should not be supplied for drinking purposes. 
 Unhappily, however, it often is. Filters may be divided, 
 roughly, into those for low and high pressure. The 
 former are directly connected with the water main, while 
 the others simply have the slight pressure of the column 
 of water standing in the filter. Many high-pressure 
 filters contain animal charcoal, silicated carbon, etc., 
 either in a pressed condition or in one porous mass. 
 These filters remove much of the deleterious matter 
 from the suspected waters, but the majority cannot be 
 depended upon to remove all bacteria. Even those 
 which are equipped for self -clean sing become in a little 
 while foul, and, if not cleaned, unfit for use. The best 
 of the class are the Berkefeld and Pasteur filters. 
 These yield a water, if too great pressure is not used, 
 
256 BACTERIOLOGY. 
 
 almost absolutely free from bacteria, and if they are 
 frequently cleansed they are reliable. A large Berke- 
 feld filter will allow sixty gallons of water to pass per 
 hour. The Pasteur filter is more compact and slower. 
 From the best Pasteur filters sterile water may be 
 passed for two to three weeks; from the Berkefeld 
 usually only a few days. A simple typical low- 
 pressure filter is that of Bailey Denton. The upper 
 compartment contains the filtering material, which 
 may be sand or charcoal, and is fed from a cistern or 
 hydrant. After a certain quantity of water has passed 
 in the supply is automatically cut off until the whole 
 amount has filtered. A filter easily made is the follow- 
 ing : Take a large-sized earthenware pot and plug the 
 hole in the bottom with a cork, through which pass a 
 short glass tube. Upon the bottom place an inch of 
 small pieces of broken flower-pot; upon this a couple 
 of inches of well-washed small gravel, and upon this 
 six to twelve inches of well- washed fine, sharp sand. 
 Cover the sand with a piece of filter-paper and hold 
 this down with a few small stones. Mount the pot on 
 a tripod, and it is ready for use. The paper prevents 
 the sand being disturbed when water is added, and as 
 it also holds most of the sediment, this can be readily 
 removed. Every few months the sand can be washed 
 and replaced. Animal charcoal is not a good substance 
 for permanent filters, as bacteria grow well in it. 
 Whenever water is suspected, and there is any doubt 
 as to the filters, it should be boiled for ten minutes; 
 this will destroy all bacteria. This precaution should 
 always be taken in the presence of typhoid fever and 
 cholera epidemics, 
 
CHAPTER XVII. 
 
 THE CLASSIFICATION OF BACTERIA. 
 
 THE PERMANENCE OF VARIETIES. 
 
 BACTERIA have been classified in many different 
 ways by many different observers. As a rule, the genera 
 are based upon morphological characters and the species 
 upon biochemical, physiological, or pathogenic prop- 
 erties. While the form, size, and method of division 
 are the most permanent characteristics of bacteria, and 
 so are naturally utilized for classification, nevertheless, 
 in this basis of division there are decided difficulties. 
 Thus while the form and size of bacteria are fairly con- 
 stant under the same conditions, they are in many quite 
 different under diverse conditions. Another serious 
 drawback is that these morphological characteristics 
 give no indication whatever of the relations of the bac- 
 teria to disease and fermentation the very characteris- 
 tics for which as physicians we study them. Other 
 properties of bacteria which are fairly constant under 
 uniform conditions are those of spore and capsule for- 
 mation, motility, reaction to staining reagents, relation 
 to temperature, to oxygen and other food material, and, 
 finally, their relation to fermentation and disease. 
 
 Taking any one of these properties of bacteria as a 
 basis, we can classify them; but even here there will 
 be groups which under certain conditions would be 
 
 17 
 
258 BACTERIOLOGY. 
 
 placed in one class and under others in another. Thus 
 the power to produce spores may be totally lost or held 
 in abeyance for a time. 
 
 The relations to oxygen may be gradually altered, 
 so that an anaerobic species grows in the presence of 
 oxygen. Parasitic bacteria may be so cultivated as to 
 become saprophytic varieties, and those which have 
 no power to grow in the living body given pathogenic 
 properties. 
 
 The possibility of making any thoroughly satisfac- 
 tory classification is rendered still more difficult by the 
 fact that many necessarily imperfect attempts have 
 already been made, so that there is a great deal of con- 
 fusion, which is steadily increased as new varieties are 
 found or old ones reinvestigated and classified differ- 
 ently in the different systems. 
 
 As one of the more successful attempts to classify 
 bacteria, the system devised by Migula is here given, 
 simply as an example. The morphology of bacteria is 
 used as the basis of the divisions : 
 
 FAMILIES. 
 
 I. Cells globose in a free state, not elongat- 
 ing in any direction before division 
 into 1, 2, or 3 planes . . . 1. Coccacese. 
 II. Cells cylindrical, longer or shorter, 
 and only dividing in one plane, and 
 elongating to twice the normal length 
 before the division. 
 
 (1) Cells straight, rod-shaped, without 
 sheath, non-motile, or motile by 
 
 means of flagella . . . .2. Bacteriaceae. 
 
 ( 2 ) Cells crooked, without sheath . 3. Spirillacese. 
 
 (3) Cells enclosed in a sheath . . 4. Chlamydobacteriace.'e. 
 
 (4) Cells destitute of a sheath, united 
 into threads, motile by means of an 
 
 undulating membrane . . .5. Beggiatoaceae. 
 
THE CLASSIFICATION OF BACTERIA. 259 
 
 GENERA. 
 
 1. Coccacece. 
 Cells without organs of motion. 
 
 a. Division in one plane . . . 1. Streptococcus. 
 
 b. Division in two planes . . .2. Micrococcus. 
 
 c. Division in three planes . . .3. Sarcina. 
 Cells with organs of motion. 
 
 a. Division in two planes . . .4. Planococcus. 
 6. Division in three planes . . . 5. Planosarcina. 
 
 2. Hacleriacece. 
 
 Cells without organs of motion . . 1. Bacterium. 
 Cells with organs of motion (flagella). 
 
 a. Flagella distributed over the whole 
 
 body 2. Bacillus. 
 
 6. Flagella polar . . . . .3. Pseudomonas. 
 
 3. Spirillacece. 
 Cells rigid, not snake-like or flexuous. 
 
 a. Cells without organs of motion . 1. Spirosoma. 
 6. Cells with organs of motion (flagella). 
 
 1. Cells with 1, very rarely 2 to 3 
 
 polar flagella . . . .2. Microspira. 
 
 2. Cells with polar flagella-tufts . 3. Spirillum. 
 Cells flexuous 4. Spirochseta. 
 
 4. Chlamydobacteriacece. 
 Cell contents without granules of sulphur. 
 
 a. Cell threads unbranched. 
 
 I. Cell division always only in one plane 1. Streptothrix. 
 II. Cell division in three planes previous 
 to the formation of conidia. 
 
 1. Cells surrounded by a very delicate, 
 
 scarcely visible sheath (marine) . 2. Phragmidiothrix. 
 
 2. Sheath clearly visible (in fresh water) 3. Crenothrix. 
 
 6. Cell threads branched . . 4. Cladothrix. 
 Cell contents containing sulphur granules 5. Thiothrix. 
 
 5. Seggiatoacea. 
 
 Only one species known (Beggiatoa, Trev.), which is scarcely 
 separable from Oscillana. 
 
260 BACTERIOLOGY. 
 
 A study of the above table will show that it makes 
 changes in the genus of some of the most common 
 bacteria, as in the restoration of the old genus bacte- 
 rium and the assigning to it of all non-motile, rod- 
 shaped organisms, thus altering the genus of some of 
 the most common pathogenic bacteria from bacillus to 
 bacterium. Other changes are seen in the spirilla. 
 Any such scheme is at times very arbitrary in placing 
 some varieties under one generic division and others 
 closely allied in another. It has also the objection, 
 already noted, that it is only one of several classifi- 
 cations already in use, and until some authoritative 
 body agrees on some one it is almost useless in such 
 a volume as this to change the usually employed names 
 for others which are, perhaps, intrinsically somewhat 
 better. Another important reason for waiting is that 
 with the increase of our knowledge we are constantly 
 changing the position of different bacteria. Thus such a 
 well-known germ as the tubercle bacillus is now found 
 to produce, under certain conditions, long thread-like 
 branching forms, so that it ceases to be under the clas- 
 sification of Migula a bacterium. We will, therefore, 
 simply use the usual nomenclature, and consider to- 
 gether, in so far as is practicable, certain groups of 
 bacteria whose members are closely allied to each other 
 in some one or more important directions. 
 
 The Permanence of Bacterial Species. When we come 
 to study special varieties or groups of bacteria, such as 
 the bacilli which produce typhoid fever, diphtheria, and 
 tuberculosis, it is of great importance for us to deter- 
 mine, if possible, to what extent the peculiar character- 
 istics which each of these groups of bacteria possess are 
 permanent in the generations which develop from them. 
 
THE CLASSIFICATION OF BACTERIA. 261 
 
 We can hardly imagine that the multitude of bacte- 
 rial varieties which now exist have always existed. 
 The probability is very strong, that with succeeding 
 generations and changing conditions new bacterial 
 varieties have developed with new characteristics. 
 
 From time to time the changing conditions under 
 which life progressed probably exposed certain animals 
 to the invasion of varieties which never before had 
 gained access to them. If the bacteria found the soil 
 suitable, and also some means of transmission to other 
 animals equally susceptible, a pathogenic species became 
 established which at first, perhaps, found conditions 
 only occasionally favorable to it, but later became more 
 parasitic in its characteristics. Thus in some such way 
 a multitude of bacterial groups arose, some of which 
 accustomed themselves to the conditions present in the 
 soil, others to those in fishes, others to those in birds, 
 and others still to those in man. 
 
 These are, however, theories what has been actually 
 observed in the few years during which bacteria have 
 been studied ? In this short time the pathogenic spe- 
 cies as observed in disease have kept practically unal- 
 tered. The diphtheria bacilli are the same to-day as 
 when Loffler discovered them in 1884, and the dis- 
 ease itself is evidently the same as history shows it to 
 have been before the time of Christ. The same is true 
 for tuberculosis, smallpox, hydrophobia, leprosy, etc. 
 Under practically unchanged conditions, therefore, as 
 exist in the bodies of men, bacteria which have once 
 become established as parasites continue so long as they 
 remain to retain their peculiar (specific) characteristics. 
 Whether new disease varieties, such as the influenza 
 bacillus, are coming into existence from time to time, is, 
 
262 BACTERIOLOGY. 
 
 of course, a possibility, but not a certainty. The one 
 thing we can probably safely assert is that there is no 
 probability that any saprophytic variety now existing 
 can, under any possibility, develop into the now recog- 
 nized varieties of pathogenic bacteria. It is almost 
 impossible to conceive that any such variety should 
 start with the same characteristics and then develop 
 parasitic tendencies under exactly the same circum- 
 stances as those varieties which now produce disease. 
 
 Attenuation. It is now a well-established fact that 
 the great majority of parasitic bacteria can be so altered 
 by change of conditions, and especially by being sub- 
 jected to unfavorable conditions, that they, while mor- 
 phologically the same, lose their power of developing 
 in the body and of producing specific poisons. When 
 either or both these properties are partially destroyed 
 they can usually be redeveloped; but when power to 
 produce specific toxins is absolutely lost, it is, so far 
 as we now know, lost forever. 
 
 The recovery of toxin production is brought about 
 by developing the micro-organism for a considerable 
 length of time under the conditions best suited for it. 
 The recovery of the ability to grow in the body of any 
 animal species is brought about by causing the germ 
 to develop in a series of such animals whose resistance 
 has been overcome by reducing their vitality through 
 poisons, heat, cold, etc. Another method is to ac- 
 custom the micro-organism to the animal's body by 
 letting it remain surrounded by the animal fluids as it 
 rests in a pervious capsule in the peritoneal cavity. 
 
CHAPTER XVIII. 
 
 BACILLUS OF TUBERCULOSIS (KOCH'S TUBERCLE 
 BACILLUS). 
 
 IT was a common belief many years ago in some 
 countries (kingdom of Naples, 1782) that tuberculosis 
 was an infectious disease; but it is only within com- 
 paratively recent times that the infectiousness of tuber- 
 culosis has become an established fact in scientific 
 medicine. Villemin (1868) was the first to show ex- 
 perimentally that tuberculosis might be induced in 
 healthy animals and man by inoculations of tubercu- 
 lous material. Others attempted to microscopically 
 demonstrate the origin of the disease (Ziirn, Buhl, 
 Klebs, Toussaint, etc.); but these investigations, 
 though paving the way to the discovery, which it 
 remained for Robert Koch to make, proved to be un- 
 satisfactory and incomplete. The announcement of 
 the discovery of the tubercle bacillus was made by 
 Koch, in March, 1882, at a meeting of the Physiolog- 
 ical Society of Berlin. At the same time satisfactory 
 experimental evidence was presented as to its etiolog- 
 ical relation to tuberculosis in man and in susceptible 
 animals, and its principal biological characters were 
 given. An innumerable number of investigators now 
 followed Koch into this field, but their observations 
 served only to confirm his original discovery. 
 
264 BACTERIOLOGY. 
 
 The bacilli are found in the sputum of persons suf- 
 fering from pulmonary or laryngeal tuberculosis, either 
 free or in the interior of pus-cells; in miliary tubercles 
 and fresh caseous masses in the lungs and elsewhere; 
 in recent tuberculous cavities in the lungs; in tuber- 
 culous glands, joints, bones, mucous membranes, and 
 skin affections; in the lungs of cattle suffering from 
 pulmonary tuberculosis, and in tubercular nodules, 
 generally in animals which are infected naturally or 
 by experimental inoculations. 
 
 Morphological Characters. The tubercle bacilli are 
 slender, non-motile rods of about 0.2/2 in diameter by 
 1.5 to 4/* in length. (Plate L, Figs. 1, 2, and 3.) 
 Commonly they occur singly or in pairs, and are then 
 usually slightly curved; frequently they are observed 
 in smaller or larger bunches. Under exceptional con- 
 ditions branching forms are observed. In stained 
 preparations there are often seen unstained portions, 
 which have been improperly thought to be spores. 
 From two to six of these unstained spaces may some- 
 times be noticed in a single rod, and under moderate 
 magnification may give to the bacilli the appearance 
 of short chains of streptococci. In old cultures irregu- 
 lar forms may be obtained, the rods being occasionally 
 swollen at one end or presenting lateral projections. 
 
 The staining peculiarities of this bacillus are very 
 important, for by them its differentiation and recogni- 
 tion in microscopical preparations of sputum, etc., are 
 rendered possible. It does not readily take up the 
 ordinary aniline colors, but when once stained it is 
 very difficult to decolorize, even by the use of strong 
 acids. Koch first recognized it in a staining prepara- 
 tion to which an alkali had been added a solution of 
 
PLATE I. 
 
 FIG. i. 
 
 FIG. 2. 
 
 Tubercle bacilli, in red. 
 Strepto-bacilli, in blue. 
 
 X i ioo diameters. 
 
 Tubercle bacilli, in red. 
 Tissue, in blue. 
 
 X i ioo diameters. 
 
 FIG. 3. 
 
 FIG. 4. 
 
 Very large tubercle bacilli. 
 
 Cells in specimen are in blue, 
 
 while bacilli are red. 
 
 X i ioo diameters. 
 
 Short smegma bacilli. 
 Bacilli in specimen are red, 
 rest of material in blue. 
 
 X i ioo diameters. 
 
BACILLUS OF TUBERCULOSIS. 265 
 
 methylene-blue with caustic potash. More recently 
 Ehrlich devised a method of staining which proved 
 to be better, viz., the use of a solution of an aniline 
 color fuchsin or methyl-violet in a saturated aqueous 
 solution of aniline oil and decolorization of other bac- 
 teria with a solution of a mineral acid, to be followed 
 by a contrast stain, such as methylene-blue. (Plate 
 I., Figs. 1 and 2.) Various modifications of Ehrlich's 
 method are now commonly used. The carbol-fuchsin 
 solution of Ziehl is largely employed ; it has the advan- 
 tage of acting quickly and keeping well. The tubercle 
 bacilli can be demonstrated also by Gram's method of 
 staining, but this is not recommended for general use. 
 
 Biological Characters. The bacillus tuberculosis is a 
 parasitic, aerobic, non-motile bacillus, and .grows only 
 at a temperature of about 37 C. It has been assumed 
 that this bacillus is capable of forming spores. The 
 refractile spaces, however, are not found to possess the 
 regular shape and brilliancy of ordinary spores, nor have 
 they any greater resisting power to heat, desiccation, 
 etc., than the homogeneous bacilli. Exposure to 60 C. 
 in water destroys them in fifteen minutes. The bacilli 
 have, however, a somewhat greater resisting power than 
 most other pathogenic bacteria, since frequently the 
 bacilli resist desiccation at the ordinary temperatures 
 for months; many bacilli die, however, soon after dry- 
 ing. Portions of the lung from a tuberculous cow, dried 
 and pulverized, produced tuberculosis in guinea-pigs at 
 the end of 102 days (Cad&ic and Malet). They retain 
 their vitality for a considerable time in putrefying ma- 
 terial. Cold has no effect upon them. When dry the 
 more resistant organisms stand dry heat at 100 C. for 
 hours; but when moist, as in milk, they are more quickly 
 
266 BACTERIOLOGY. 
 
 killed viz., at 55 C. in one hour, at 60 C. in fifteen 
 minutes, at 65 C. in fifteen minutes, at 70 C. in ten 
 minutes, at 80 C. in five minutes, and at 95 C. in one 
 minute. One reason why they appear to withstand in 
 milk high temperatures for a longer time than given in 
 the above figures is, as pointed out by Theobald Smith, 
 that when heated in a test-tube the cream which rises 
 on heating is exposed on its surface to a lower tem- 
 perature than the rest of the milk, and as this contains 
 many bacteria some of them are exposed to less heat 
 than those in the rest of the fluid receive. 
 
 The resisting power of this bacillus against chemi- 
 cal disinfectants is considerable, but not as great as it 
 is apt to appear, for, as in sputum, the bacillus is 
 usually protected by mucus or cell protoplasm from 
 penetration by the germicidal agent. It is not always 
 destroyed by the gastric juice in the stomach, as is 
 shown by successful infection experiments in susceptible 
 animals by feeding them with tubercle bacilli (Baumgar- 
 ten and others). They are destroyed in sputum in six 
 hours or less by the addition of an equal quantity of a 
 3 per cent, solution of carbolic acid, and in about one 
 hour by an equal amount of a 5 per cent, solution. 
 Bichloride of mercury is unsuitable for the disinfection 
 of sputum unless used in very strong solutions (1 : 500). 
 From recent experiments by Yersin upon pure cultures 
 of the bacillus it appears that tubercle bacilli were killed 
 by a 5 per cent, solution of carbolic acid in thirty sec- 
 onds; by 1 per cent, in one minute; absolute alcohol, 
 five minutes; iodoform-ether, 1 per cent., five minutes; 
 mercuric chloride, 1 : 1000 solution, ten minutes. Salt- 
 ing and smoking are said not to destroy the virulence 
 of tuberculous meat (Forster). 
 
BACILLUS OF TUBERCULOSIS. 267 
 
 The tubercle bacillus when exposed to direct sunlight 
 is killed in from a few minutes to several hours, accord- 
 ing to the thickness of the layer and the season of the 
 year; it is also usually destroyed by diffuse daylight in 
 from five to seven days when placed near a window. 
 This fact is worthy of note, as it has an important 
 hygienic bearing. Thus, tuberculous sputum expector- 
 ated upon sidewalks, etc., being exposed to the action 
 of direct sunlight, will in many cases, especially in 
 summer, be disinfected by the time it is in a condi- 
 tion to be carried into the air as dust. For the same 
 reason, consumptive patients should occupy light, sunny 
 rooms and live as much as possible in the open air and 
 exposed to the action of direct sunlight. 
 
 The tubercle bacillus is a strict parasite that is to 
 say, its biological characters are such that it could 
 scarcely find natural conditions outside of the bodies 
 of living animals favorable for its multiplication. But 
 it has been noted that when it is cultivated for a time 
 in artificial media containing glycerin it may grow on 
 the surface of plain veal or chicken bouillon, in which 
 media it fails to develop when introduced directly from 
 a culture originating from the body of an infected ani- 
 mal. This would indicate the possibility of its acquir- 
 ing the ability to grow as a saprophyte. The experi- 
 ments of Nutall also show that the bacillus may multi- 
 ply, under favorable conditions, in tuberculous sputum 
 outside of the body. Notwithstanding these facts, 
 it is probable that the growth of tubercle bacillus out- 
 side of the living bodies of man and animals is so 
 slight as to have no practical importance in causing 
 infection. 
 
 On account of their slow growth and the special con- 
 
268 BACTERIOLOGY. 
 
 ditions which they require, tubercle bacilli cannot be 
 grown in pure culture by the plate method on the 
 ordinary culture media. Koch first succeeded in culti- 
 vating and isolating this bacillus on coagulated blood- 
 serum, which he inoculated by carefully rubbing the 
 surface with sections of tuberculous tissue and then 
 leaving the culture, protected from evaporation, for 
 several weeks in the incubator. Roux and Nocard 
 afterward showed that the bacilli from man and ani- 
 mals occasionally grow on nutrient agar to which 
 glycerin has been added in the proportion of 5 per 
 cent. 
 
 Growth on Coagulated Blood-serum. On this medium, 
 which is regularly used to obtain the first culture, the 
 growth first becomes visible at the end of ten to four- 
 teen days at 37 C., and at the end of three to four 
 weeks a distinct and characteristic development has 
 occurred. Small, grayish-white points and scales first 
 appear on the surface of the medium. As development 
 progresses there is formed an irregular, membranous- 
 looking layer. When a tiny piece of this is removed, 
 placed on a cover-glass without rubbing, stained, and 
 then observed under the microscope the surface growth 
 presents a characteristic appearance, the bacilli being 
 arranged in parallel rows of variously curved figures. 
 
 Owing to the greater facility of preparing and steril- 
 izing glycerin-cigar, and the more rapid and abundant 
 growth of the bacilli, which have become accustomed 
 to growth outside the body on this medium, it is now 
 usually employed in preference to blood-serum for 
 preserving cultures. The development at the end of 
 fourteen to twenty-one days is more abundant than 
 upon blood-serum after several weeks. When numer- 
 
BACILLUS OF TUBERCULOSIS. 269 
 
 ous bacilli have been distributed over the surface of 
 the culture medium, a rather uniform, thick, white 
 layer, which subsequently requires a slightly yellowish 
 tint, is developed; when the bacilli sown are few in 
 number, or are associated in scattered groups, separate 
 colonies are developed, which acquire considerable 
 thickness and have more or less irregular outlines. 
 
 Growth on Peptonized Veal or Beef Broth Containing 
 5 per cent, of Glycerin. On these media the tubercle 
 bacillus also grows readily if a very fresh thin film of 
 growth from the glycerin agar is floated on the surface. 
 The latter of these media is used for the development of 
 tuberculin. The small piece of pellicle removed from 
 the previous culture continues to enlarge while it floats 
 on the surface of the liquid, and in the course of three 
 to six weeks covers it wholly as a single film, which on 
 agitation is easily broken up and then settles on the 
 bottom of the flask, where it ceases to develop further. 
 The liquid remains clear, containing in solution the 
 products formed by the growth of the bacillus, and is 
 really a dilute crude tuberculin. A practical point of 
 importance, if a quick growth is desired, is to remove 
 for the new cultures a portion of the pellicle of a grow- 
 ing bouillon culture, which is very thin and actively 
 increasing. 
 
 The Obtaining of Cultures of the Tubercle Bacillus from 
 Sputa and Infected Materials for Diagnostic Purposes. 
 As this is a matter of great and increasing importance, 
 we will consider in detail the methods which have 
 been successfully employed. Pure cultures can be ob- 
 tained directly from tuberculous material ; but as 
 it is so difficult to get rid of the other bacteria 
 which are almost always present, and which grow 
 
270 BACTERIOLOGY. 
 
 much more rapidly and take possession of the medium 
 before the tubercle bacillus has had time to form 
 visible colonies, it is best, unless human tissues can 
 be obtained free from other infection, first to inocu- 
 late a guinea-pig, both subcutaneously and intraperito- 
 ueally, with the * sputum, and then obtain cultures 
 from the animal as soon as the tubercle infection has 
 fully developed. From acute tuberculosis in man in 
 other regions than the lungs, where mixed infection 
 usually exists, direct cultures on blood-serum may be 
 made. 
 
 The animals thus inoculated usually die at the end 
 of three weeks to four months. It is better, however, 
 to kill a guinea-pig which by its enlarged glands shows 
 evidence of tuberculosis, and to remove, with the greatest 
 care as to cleanliness, one or more nodules from the 
 lungs, spleen, or lymphatic glands. Animals which 
 develop tuberculosis acutely are apt to have abundant 
 tubercle bacilli and give successful cultures, while the 
 chronic cases usually have few bacilli and give unsuc- 
 cessful cultures. The animals after being killed are 
 placed in trays, and after washing with a 5 per cent, 
 solution of carbolic acid, immediately autopsied. The 
 skin over the anterior portion of the body having been 
 carefully turned back, an opening is cut with a fresh 
 set of sterile instruments into the thoracic or abdominal 
 cavity; then with a sterile forceps the lymph-gland 
 portion of spleen or other part which it is desired to 
 examine is removed to a sterile covered beaker. This 
 tissue if suitable may be sliced in thin sections and con- 
 veyed directly to the surface of the solid culture medium 
 and gently rubbed over the surface, and then left on it, 
 or a part of it may first be crushed between two sterilized 
 
BACILLUS OF TUBERCULOSIS. 271 
 
 glass slides and then transferred to the serum and 
 rubbed gently over its surface. Owing to the liability 
 of the blood -serum to become too dry for the develop- 
 ment of the bacillus, it is necessary to keep the cul- 
 ture moist by sealing the end in some way, as by 
 applying a rubber cap over the open end of the test- 
 tube, which prevents evaporation. This cap should be 
 sterilized in a solution of mercuric bichloride (1 : 1000) 
 and the end of the cotton plug burned off just before 
 applying it, to destroy any spores of mould fungi 
 present. Theobald Smith, who has had a very large 
 experience in growing the tubercle bacillus, gives the 
 following details as to his method : 
 
 "Throughout the work solidified dog's serum was 
 used. The dog was bJed under chloroform and the 
 blood drawn from a femoral artery, under aseptic 
 conditions, through sterile tubes directly into sterile 
 flasks. The serum was drawn from the clot with 
 sterile pipettes, and either distributed at once into 
 tubes or else stored with 0.25 to 0.3 per cent, chloro- 
 form added. The temperature required to produce a 
 sufficiently firm and yet not too hard and dry serum is, 
 for the dog, 75 to 76 C.; for horse and beef serum 
 it is from 4 to 5 lower. The tubes containing the 
 serum were set in a thermostat, into which a dish of 
 water was placed, to forestall any abstraction of moist- 
 ure from the serum. About three hours suffice for the 
 coagulation. This procedure dispenses with all sterili- 
 zation excepting that going on during the coagulation 
 of the serum. It prevents the gradual formation of 
 membranes of salts, which, remaining on the surface 
 during coagulation, form a film unsuited for bacteria. 
 Tubes of coagulated serum should be kept in a cold, 
 
272 BACTERIOLOGY. 
 
 closed space, where the opportunities for evaporation 
 are slight. They should always be kept inclined. 
 
 <( The ordinary cotton-plugged test-tubes I do not 
 use, because of the rapid drying out permitted by 
 them as well as the opportunities for infection with 
 fungi. Instead, a tube is used which has a ground- 
 glass cap fitted over it. This cap contracts into a 
 narrow tube plugged with glass-wool; this plug is not 
 disturbed. The tube is cleaned, filled, and inoculated 
 by removing the cap. With sufficient opportunity for 
 the interchange of air very little evaporation takes place, 
 and contamination of the culture is a very rare occur- 
 rence. In inoculating these tubes bits of tissue which 
 include tuberculous foci, especially the most recent, are 
 torn from the organs and transferred to the serum. 
 Very little crushing, if any, is desirable or necessary. 
 I think many failures are due to the often futile 
 attempts to break up firm tubercles. Nor should the 
 bits of tissue be rubbed into the surface, as is some- 
 times recommended. After a stay of several weeks in 
 the thermostat I usually remove the tubes and stir 
 about the bits of tissue. This frequently is the occa- 
 sion for a prompt appearance of growth within a week, 
 as it seems to put certain still microscopical colonies in 
 or around the tissues into better condition for further 
 development. The thermostat should be fairly constant, 
 as urged by Koch in his classic monograph; but I look 
 upon moisture as of more importance. If possible a 
 thermostat should be used which is opened only occa- 
 sionally. Into this a large dish of water is placed, 
 which keeps the space saturated. Ventilation should 
 be restricted to a minimum. As a consequence, moulds 
 grow luxuriantly, and even the gummed labels must be 
 
BACILLUS OF TUBERCULOSIS. 273 
 
 replaced by pieces of stiff manila paper fastened to the 
 tube with a rubber band. By keeping the tubes in- 
 clined no undue amount of condensation of water can 
 collect in the bottom, and the upper portion of the 
 serum remains moist. The only precaution to be 
 applied to prevent infection with moulds is to thor- 
 oughly flame the joint between the tube and cap, as 
 well as the plugged end, before opening the tube." 
 
 At the Saranac Laboratory beef blood-serum is used 
 in ordinary test-tubes, which are sealed by rubber caps. 
 The tubercles are crushed between sterile glass slides 
 and rubbed gently upon the serum surface. The serum 
 itself must not be too firm. It should just be solid 
 enough to stand upright. The results thus obtained 
 by Trudeau and Baldwin have been as good as those 
 reported by Smith. In our experience all methods fre- 
 quently fail with those unfamiliar with them, especially 
 when, as shown by microscopical examination, the 
 tubercular tissue used contains very few bacilli. 
 
 Pathogenesis. The tubercle bacillus is pathogenic not 
 only to man, but to a large number of animals, such as 
 the monkey, pig, cow, etc. Guinea-pigs are extremely 
 susceptible, and are much used for the detection of 
 tubercle bacilli in suspected material. When inoculated 
 with the minutest doses of the living bacilli they 
 usually succumb to the disease. Infection is most 
 rapidly produced by intraperitoneal injection. If a 
 large dose is given death follows in from ten to 
 twenty days. The omentum is found to be clumped 
 together in sausage-like masses and converted into hard 
 knots, which contain many bacilli. There is no serous 
 fluid in the peritoneal cavity, but generally in both 
 pleural sacs. The spleen is enlarged, and it, as well 
 
 18 
 
274 BACTERIOLOGY. 
 
 as the liver and peritoneum, contains large numbers 
 of tubercle bacilli. If smaller doses are given the 
 disease is prolonged. The peritoneum and interior or- 
 gans spleen, liver, etc. are then filled with tubercles. 
 On subcutaneous injection, for instance, into the ab- 
 dominal wall, there is a thickening of the tissues about 
 the point of inoculation, which breaks down in about a 
 week and leaves a sluggish ulcer covered with cheesy 
 material. The neighboring lymph-glands are swollen, 
 and at the end of two or three weeks may attain the 
 size of hazel-nuts. Soon an irregular fever is set up, 
 and the animal becomes emaciated, usually dying within 
 four to eight weeks. If the injected material contained 
 only a small number of bacilli the wound at the point 
 of inoculation may heal up and death be postponed for 
 a long time. On autopsy the lymphatic glands are 
 found to have undergone cheesy degeneration ; the 
 spleen is very much enlarged, and throughout its sub- 
 stance, which is colored dark red, are distributed masses 
 of nodules. The liver is also enormously increased in 
 size, streaked brown and yellow, and the lungs are filled 
 with grayish-white tubercles; but, as a rule, the kidneys 
 contain no nodules. Tubercle bacilli are always found 
 in the affected tissues, but the more chronic the process 
 the fewer the bacilli that are apt to be present. 
 
 Rabbits are also quite susceptible to tuberculosis, but 
 considerably less so than guinea-pigs. In rabbits death 
 almost invariably follows inoculations of tuberculous 
 material into the anterior chamber of the eye. The 
 local effects are iris-tuberculosis and cheesy degeneration 
 of the pupil. The bacilli then penetrate to the neigh- 
 boring lymph-glands, producing softening of these, then 
 pulmonary tuberculosis, general tuberculosis, and finally 
 
BACILLUS OF TUBERCULOSIS. 275 
 
 death at the end of several weeks or months. Subcuta- 
 neous inoculations are less effective, and in small doses 
 do not always kill. Intravenous and intraperitoneal 
 iujections usually produce general tuberculosis and 
 death at the end of a few weeks. The tubercles in 
 rabbits are smaller, as a rule, and the spleen and liver 
 not so much enlarged as in guinea-pigs, but the kidneys 
 not infrequently contain nodules of the size of a pea. 
 
 Of other susceptible animals, field-mice and cats 
 are readily infected by artificial inoculations of tuber- 
 culous material ; rats, white mice, and dogs only 
 when very large doses are given. All these animals 
 present the anatomical lesions of miliary tuberculosis. 
 Bollinger has produced intestinal tuberculosis in calves 
 by inoculating them with material taken from a tuber- 
 culous man. Canaries are also susceptible to inocula- 
 tions of the tubercle bacillus; but not sparrows. Cold- 
 blooded animals of various kinds, according to the 
 experiments of Koch, are immune, unless, as recently 
 demonstrated, the bacilli are first slowly accustomed 
 to growth at low temperatures. Fowls and pigeons 
 are only slightly susceptible to the bacillus derived 
 from man. Among the larger birds, parrots alone 
 would seem to be clearly susceptible. 
 
 Beside the artificial modes of infection already re- 
 ferred to, tuberculosis may be caused in animals by 
 feeding them with tuberculous material. In this case 
 evidence of infection is usually shown in the mesenteric 
 glands before the intestinal walls are affected. Zagari 
 records some experiments in which tubercle bacilli fed 
 to dogs (one of the less susceptible animals) were ab- 
 sorbed by the mucous membranes of the intestines, and 
 thus reached the internal organs without producing any 
 
276 BACTERIOLOGY. 
 
 local lesions whatever. It would seem to be possible, 
 therefore, that tubercular infection may be caused, under 
 certain conditions, by absorption through serous or 
 mucous membranes without the evidence of any local 
 lesion. 
 
 The experimental production of tuberculosis by in- 
 halation of bacilli has been demonstrated by Koch in 
 guinea-pigs, rabbits, rats, and mice, and his results have 
 since been confirmed by many others; but in these ex- 
 periments the bacilli were usually inhaled in the form 
 of a very thin spray in which they were suspended. 
 The experimental inhalation of dry tubercular dust has 
 seldom proved successful. 
 
 Various other tubercular affections which are natural 
 in man have been produced experimentally in animals, 
 as, for instance, tuberculosis of the joints (Pawlowsky), 
 tubercular abscess (Courmont), etc. 
 
 It need hardly be said that the discovery of the 
 tubercle bacillus has elucidated the etiology of many 
 diseases the origin of which was formerly doubtful. 
 Among these may be mentioned the various forms 
 of tuberculosis of the lungs and other organs, lupus, 
 scrofula, fungoid inflammations of the bones and joints, 
 tuberculosis in cattle, monkeys, horses, swine, sheep, 
 goats, and the so-called spontaneous tuberculosis in 
 guinea-pigs and rabbits in cages in which healthy and 
 artificially infected animals have been kept together. 
 
 Of domestic animals cattle are by far the most fre- 
 quently attacked by this disease. It is also not uncom- 
 mon in young swine. Monkeys, when they are kept in 
 confinement, die almost invariably from tuberculosis. 
 Among other domestic and wild animals it is a com- 
 paratively rare disease. Birds, with the exception of 
 
BACILLUS OF TUBERCULOSIS. 277 
 
 parrots, are not subject to tuberculosis, and cold-blooded 
 animals are altogether immune. 
 
 Beside the affections already referred to in man the 
 following diseases have been traced to tubercular origin: 
 Among skin diseases, so-called inoculation-lupus, tuber- 
 culosis- verrucosa cntis, and scrofuloderma; choroidal 
 tuberculosis, idiopathic serous pleurisy and lymphatic 
 enlargements simulating pseudoleuksemia. 
 
 The Action upon the Tissues of the Poisons Produced 
 by the Tubercle Bacillus. Soon after the introduction 
 into the tissues of tubercle bacilli, either living or dead, 
 the cells surrounding them begin to show that some 
 irritant is acting upon them. The connective-tissue 
 cells become swollen and undergo mitotic division, the 
 resultant cells being distinguished by their large size 
 and pale nuclei. A small focus of proliferated epithe- 
 lioid cells is thus formed about the bacilli, and accord- 
 ing to the intensity of the inflammation these cells are 
 surrounded by a larger or smaller number of the lym- 
 phoid cells. When living bacilli are present and multi- 
 plying, the lesions progress, the central cells degenerate 
 and die, and a cheesy mass results, which later may lead 
 to the formation of cavities. Dead bacilli, on the other 
 hand, give off sufficient poison to cause the less marked 
 changes only, and never produce cavities (Prudden and 
 Hodenpyl). Of the gross pathological lesions produced 
 in man by the tubercle bacilli the most characteristic 
 are small nodules, called miliary tubercles. When 
 young, and before they have undergone degeneration, 
 these tubercles are gray and translucent in color, some- 
 what smaller than a millet-seed in size, and hard in 
 consistence. 
 
 But miliary tubercles are not the sole tuberculous 
 
278 BACTERIOLOGY. 
 
 products. The tubercle bacilli may cause the diffuse 
 growth of a tissue identical in structure with that of 
 miliary tubercles that is, composed of a basement 
 substance containing epithelioid, giant, and lymphoid 
 cells. This diffuse tubercle-tissue also tends to undergo 
 cheesy degeneration. 
 
 Distribution of Tubercle Bacilli in the Tissues. In 
 acute tuberculosis, especially when caseation is rapidly 
 spreading, the bacilli are usually abundant. They are 
 generally scattered irregularly through the tissues or 
 in small groups. They are occasionally found in the 
 leucocytes and in the giant and epithelioid cells. In 
 subacute and chronic lesions they are usually few in 
 number. Sometimes in old caseous materials numerous 
 stained granular points are seen ; these are supposed 
 by some to be a resting stage similar to spores. 
 
 Infection. Infection by the tubercle bacillus takes 
 place usually through the respiratory tract or the diges- 
 tive tract, more rarely through wounds of the skin. 
 
 In the majority of cases the mode of infection is 
 evident. Pulmonary tuberculosis as a primary dis- 
 ease, and not occurring in young children, may be con- 
 sidered to be caused chiefly by the direct transmission 
 of tubercle bacilli through kissing, soiled hands, hand- 
 kerchiefs, etc., or by the inhalation of tuberculous dust. 
 Intestinal and mesenteric tuberculosis, which is rare 
 among adults and common with children, is probably 
 due not only to swallowing the bacilli received in the 
 above ways, but also to the ingestion of tuberculous 
 milk. Lupus is probably always produced by the 
 inoculation of tubercle bacilli on the skin or mucous 
 membranes, which is indicated by the fact that the 
 original seat of the disease is so often on a wounded 
 
BACILLUS OF TUBERCULOSIS. 279 
 
 surface. Localized skin tuberculosis is sometimes pro- 
 duced by inoculation at autopsies. 
 
 Infection by Inhalation of Tuberculous Dust. Cer- 
 tainly one of the common modes of infection is by 
 means of tuberculous sputum, which, being coughed 
 up by consumptives and carelessly expectorated, dries 
 and distributes numerous virulent bacilli in the dust. 
 As long as the sputum remains moist there is no 
 danger of dust infection, but only of direct contact; 
 it is only when it becomes dry, as on handkerchiefs, 
 bedclothes, and the floor, etc., that the dust is a 
 source of danger for infection. A great number of 
 the expectorated and dried tubercle bacilli undoubtedly 
 die, especially when exposed to the action of direct 
 sunlight; but when it is considered that from one-half 
 to three billion virulent tubercle bacilli (according to 
 the experiments of Nutall) may be expectorated by a 
 single tuberculous individual in twenty-four hours, it 
 is evident that even a much smaller proportion than are 
 known to stay alive will suffice in the immediate 
 vicinity of consumptives to produce infection unless 
 precautions are taken to prevent it. The danger 
 of infection is greatest, of course, in the close neigh- 
 borhood of tuberculous patients who expectorate pro- 
 fusely and indiscriminately that is, without taking 
 the necessary means for preventing infection. There 
 is comparatively little danger of infection at a distance, 
 as in the streets, for instance, where the tubercle bacilli, 
 even if present in the dust, have become so diluted that 
 they are not much to be feared. Exhaustive experi- 
 ments made by many observers have shown that parti- 
 cles of dust collected from the immediate neighborhood 
 of consumptives, when inoculated into guinea-pigs, 
 
280 BACTERIOLOGY. 
 
 produced tuberculosis in a considerable percentage of 
 them; whereas the dust from rooms inhabited by healthy 
 persons or the dust of the streets did so only in an ex- 
 tremely small percentage. Fliigge is probably right 
 in thinking that the dust which is fine enough to 
 remain for a long time in suspension in the air is prac- 
 tically free from living pathogenic bacteria. It is the 
 coarser particles in which the bacilli are protected 
 by an envelope of mucus that resist drying for consider- 
 able periods. These are carried only short distances 
 by air currents. Such results as those obtained by 
 Straus, who, examining the nasal secretions of twenty- 
 nine healthy persons living in a hospital with con- 
 sumptive patients, found tubercle bacilli in nine of 
 them, must be accepted with some reserve, since we 
 know that in the air there are bacilli derived from 
 grasses which look and stain like tubercle bacilli and 
 yet are totally different. It has been argued by some, 
 from the fact that about one-seventh of all men die from 
 tuberculosis, that the tubercle bacilli must be ubiquitous, 
 and that precautions are useless; but, as Cornet has 
 pointed out, this does not mean that one-seventh of all 
 men living are tuberculous, for no man is tuberculous 
 during the entire course of his life, but only for a lim- 
 ited period (variously estimated at from three to eight 
 years). It may, therefore, be said that the danger of 
 infection from tuberculosis in general is not so great 
 after all, but that on this account it is all the more to 
 be feared and guarded against in the immediate neigh- 
 borhood of consumptives. Those who are most liable 
 to infection from this source are the families, the nurses, 
 the fellow-workmen, and fellow-prisoners of persons 
 suffering from the disease. In this connection, also, 
 
BACILLUS OF TUBERCULOSIS. 281 
 
 attention may be drawn to the fact that rooms which 
 have been recently occupied by consumptives are not 
 infrequently the means of producing infection (as has 
 been clinically and experimentally demonstrated) from 
 the deposition of tuberculous dust on furniture, walls, 
 floors, etc. Fliigge has recently drawn attention to the 
 fact that in coughing, sneezing, etc., very fine parti- 
 cles of throat secretion are thrown out and carried by 
 air currents many feet from the patient and remain 
 suspended in the air for a considerable time. This 
 is another means of infection, but probably an in- 
 frequent one. We have now to encourage us a mass 
 of facts which go to show that when the sputa is care- 
 fully looked after there is very little danger of the 
 infection of others except by close personal contact. 
 
 Individual Susceptibility. It is believed by many 
 that in demonstrating the possibility of infection in 
 pulmonary tuberculosis its occurrence is sufficiently 
 explained; but they leave out another and most impor- 
 tant factor in the production of an infectious disease 
 individual susceptibility. That this susceptibility, 
 or " predisposition/ ' as it is improperly called, may 
 be either inherited or acquired is now an accepted 
 fact in medicine. It is even thought that the phys- 
 ical signs and characters the phthisical habit which 
 indicate this susceptibility can be externally recog- 
 nized. Whatever may be the opinion with regard to 
 these outward signs, there is no doubt that personal 
 susceptibility is of the greatest importance in the pro- 
 duction of this disease. Unquestionably, vast differ- 
 ences exist in different individuals in the intensity 
 of the tubercular process in the lung. That this does 
 riot depend chiefly upon a difference in virulence of 
 
282 BACTERIOLOGY. 
 
 the infection is evident, from the fact that individuals 
 contracting tuberculosis from the same source are at- 
 tacked with different severity, and that there is, as a 
 rule, no great difference in degrees of virulence in the 
 tubercle bacilli obtained from different sources. As is 
 seen from the results of post-mortem examinations in 
 which the remains of old tubercular processes have been 
 found in the lungs of about one-third of all the bodies 
 examined, many cases of pulmonary phthisis must occur 
 without showing any visible evidences of disease, and 
 heal of their own accord. The possibility of favorably 
 influencing in many an existing tuberculosis by treat- 
 ment also proves that, under natural conditions, there is 
 a varying susceptibility to the disease. Clinical experi- 
 ence teaches, likewise, that poor hygienic conditions, 
 depressing surroundings (as in asylums and prisons), 
 obstinate bronchial affections, diabetes, and other ex- 
 hausting diseases increase the susceptibility to phthisis. 
 Animal experiments have shown that not only are there 
 differences of susceptibility in various animal species, 
 but also an individual susceptibility in the same species. 
 This is not so evident among guinea-pigs, which are 
 so susceptible that they succumb to an inoculation of 
 the minutest dose of virulent bacilli; but rabbits are 
 not always killed by subcutaneous inoculations, though 
 some individuals die from very small doses. Dogs, 
 rats, and other more resistant animals show this still 
 more plainly. Man cannot be placed on the same plane 
 of susceptibility to tuberculosis with guinea-pigs, for 
 with him the disease often remains local or is entirely 
 cured. The doctrine of individual susceptibility, there- 
 fore, is seen to be founded on fact, although the reasons 
 for it are only partially understood. 
 
BACILLUS OF TUBERCULOSIS. 283 
 
 Infection by Ingestion of Milk and Meat. Phthisi- 
 cal sputum, however, is not held responsible for the 
 occurrence of all human tuberculosis. Milk also 
 serves as a conveyer of infection, whether it be the milk 
 of nursing mothers suffering from consumption or the 
 milk of tuberculous cows. The transmission of tubercle 
 bacilli in the milk of tuberculous individuals has only 
 been indirectly established in human beings, but in 
 cow's milk it has been abundantly proved. Formerly 
 it was thought that in order to produce infection by 
 milk there must be local tubercular affection of the 
 udder; but it is known now that tubercle bacilli may 
 be found in the milk when an internal organ is infected 
 and when careful search fails to detect any udder dis- 
 ease. So that the milk of every cow which has any 
 internal tubercular infection must be considered as pos- 
 sibly containing tubercle bacilli. Rabinowitsch and 
 Kempner proved beyond all question that not only the 
 milk of tubercular cattle which showed no appreciable 
 udder disease, but also those in which tuberculosis was 
 only detected through tuberculin, frequently contained 
 tubercle bacilli. Different observers have found tubercle 
 bacilli in the milk of from 20 to 60 per cent, of tuber- 
 culous cows. When we consider the prevalence of tuber- 
 culosis among cattle we can readily realize, if the 
 bovine bacillus readily infects human beings, the 
 danger to which children are exposed from this source 
 of infection. Thus, taking the abattoir statistics of 
 various countries, we find that in Prussia 8.3 per cent, 
 of the cattle slaughtered were tuberculous; in Dresden, 
 14.4 per cent.; in London, 25 per cent.; in Berlin, 12 
 per cent.; in New York, about 7 per cent. Another 
 possible source of infection in intestinal tuberculosis is 
 
284 BACTERIOLOGY. 
 
 the flesh of tuberculous cattle. Here the same condi- 
 tions hold good as in the infection by milk, only the 
 danger is considerably less, from the fact that meat is 
 usually cooked, and also because the muscular tissues 
 are seldom attacked. In view of the great mortality 
 from tubercular diseases among mankind, the legisla- 
 tive control and inspection of cattle and milk would 
 seem to be an absolute necessity. As a practical and 
 simple method of preventing infection, especially 
 among children, the sterilization (by heat) of the milk 
 used as food must commend itself to all. It is only right 
 to state, however, that the actual proof that human tuber- 
 culosis has come from milk or food infected with bovine 
 tuberculosis is very small, and that it is perfectly pos- 
 sible that the bovine bacilli may not be as virulent for 
 man as for animals, still we know that human tubercu- 
 losis produces bovine tuberculosis in young and suscep- 
 tible animals, and the reverse is in all probability true. 
 The relation of bovine to human tubercle bacilli will 
 be discussed later in this chapter. 
 
 Auto-infection by Swallowing Sputum. The secondary 
 forms of tuberculosis which often succeed a primary 
 infection of the lungs may be explained as an auto- 
 infection from the swallowing of sputum containing 
 bacilli, these passing through the gastric juice unaf- 
 fected. It is a wonder, indeed, that intestinal tuber- 
 culosis is not more common than it is in consumption; 
 but this is probably due to the fact that in adults the 
 intestines are comparatively insusceptible. Tubercu- 
 losis may also begin as a local infection in the lungs or 
 intestines, and theiice extend to other parts of the body, 
 until, passing into the circulation, a general miliary 
 tuberculosis results. 
 
BACILLUS OF TUBERCULOSIS. 285 
 
 Hypothesis of Transmissibility of Tubercle Bacilli to 
 the Foetus. Baumgarten and others have advanced a 
 hypothesis to account for certain obscure cases of tuber- 
 culosis namely, that of the transmissibility of tubercle 
 bacilli from the mother to the unborn babe. There 
 seems to be some evidence of the possible transmission 
 of tubercular poison from the mother to the foetus in 
 animals. The first authentic case recorded is that 
 reported by Johne of an eight-months-old calf foetus; 
 other cases have since been reported. With regard to 
 tuberculosis in the human foetus the evidence is not so 
 clear, though several cases have been reported of tuber- 
 culosis in very young babies only a few weeks old, and 
 two cases are recorded of placental tuberculosis. The 
 fact that statistics show a greater frequency of tuber- 
 cular diseases in children during the first than in the 
 following years of life does not strengthen the hypo- 
 thesis of infection in utero ; for nursing babies would 
 naturally be more exposed to infection through the 
 mother's milk and through personal contact than others, 
 and, beside, the more tender the life of the infant the more 
 susceptible it would be ordinarily to indirect infection 
 from a tuberculous mother. Experimental proof, how- 
 ever, of the actual transmission of tubercle bacilli from 
 the mother to the foetus in animals has recently been 
 furnished. De Blenzi found that in five out of eighteen 
 cases in guinea-pigs such transmission of bacilli did 
 take place, and Gartner confirmed the same in numer- 
 ous experiments on rabbits, mice, and canaries. The 
 infection resulted not only from animals affected with 
 general miliary tuberculosis, but also from local dis- 
 ease of the lungs; but in the majority of cases very 
 few bacilli were transmitted to the foetus so few, 
 
286 BACTERIOLOGY. 
 
 indeed, that it required the inoculation of the entire 
 contents of the body to cause tuberculosis in guinea- 
 pigs; moreover, only one or two of a litter were affected 
 at one time. According to these experiments one would 
 expect to find in man foetal or placental tubercular in- 
 fection more common than it is, whereas it is extremely 
 rare, even if the few cases reported be accepted as proven. 
 Possibly the few bacilli which may be transmitted to 
 the foetus do not find conditions favorable for their 
 development, and, being so few in number, die; or 
 they may remain latent, as has been suggested, for 
 certain lengths of time without producing visible effects, 
 and only show symptoms of infection later; but we have 
 no experimental confirmation of any such latency ex- 
 isting with regard to the tubercle bacillus, and it is not 
 to be assumed that it does exist. As to the infection of 
 the foetus from the paternal side, where the father has 
 tuberculosis of the scrotum or seminal vessels (which 
 have been found to be tuberculous in exceptional cases), 
 we have no reason to suppose that such can occur. 
 There are, however, some grounds for belief that infec- 
 tion in this way may take place from husband to wife. 
 Thus, Gartner found, as a result of his experiments in 
 animals, that a large majority of the guinea-pigs and 
 rabbits which were brought together with males whose 
 semen contained tubercle bacilli died of primary gen- 
 ital tuberculosis; but from the rarity of this affection 
 in women and cows it may be assumed that tubercle 
 bacilli occur very much less frequently in semen of men 
 and cattle than in that of the smaller animals. 
 
 Length of Time Tubercle Bacilli Remain Virulent in 
 Sputum. Of considerable importance in studying the 
 subject of tubercular infection is the question of the 
 
BACILLUS OF TUBERCULOSIS. 287 
 
 length of time daring which the tubercle bacillus re- 
 tains its virulence, and whether there are any naturally 
 attenuated varieties. According to experimental in- 
 vestigations, the virulence of dried tubercular sputum 
 is not suddenly but gradually lost, a certain pro- 
 portion of it retaining its specific infective power 
 under ordinary conditions, as in a dwelling-room, for 
 at least two or three months. An instance is reported 
 by Ducor (Paris, 1890) of a healthy family having 
 been infected with tuberculosis from living in a room 
 which had been occupied by a consumptive two years 
 before, and on examining the sputum-stained wall-paper 
 not only were tubercle bacilli found in it, but upon being 
 inoculated into guinea-pigs they died of tuberculosis. 
 
 Attenuation. Metschnikoff states that when kept 
 at a temperature of 42 C. for some time the tubercle 
 bacillus undergoes a notable diminution in its patho- 
 genic power, and that when kept at a temperature of 
 43 to 44 C. it after a time only induces a local abscess 
 when injected subcutaneously into guinea-pigs. The 
 experiments of Lote also indicate that an attenuation 
 of virulence has occurred in the cultures preserved in 
 Koch's laboratory, originating in 1882 from the lungs 
 of a tuberculous ape. A culture of ours which we ob- 
 tained from Trudeau, and which has grown now either 
 at Saranac or in our laboratory for six years, is no 
 longer capable of causing tuberculosis in guinea-pigs, 
 although originally virulent. 
 
 Mixed Infection. Some time ago attention was drawn 
 to the fact that tuberculosis, whether of the lungs, 
 lymphatics, or cold abscesses, was often a mixed in- 
 fection. The other micro-organisms with which the 
 tubercle bacillus is most commonly associated are the 
 
288 BACTERIOLOGY. 
 
 streptococcus, pneumococcus, and influenza bacillus. 
 Besides these many other varieties are met with occa- 
 sionally in individual cases. What the influence of 
 this secondary or mixed infection is, under all circum- 
 stances, is not exactly known; but generally the effect 
 is an unfavorable one, and not infrequently on their 
 invasion the disease takes on a septicsemic character. 
 For the technique employed in examining sputa for 
 mixed infection, see later in this chapter. 
 
 Immunization. As in other infectious diseases, many 
 attempts have been made to produce an artificial immu- 
 nity against tuberculosis, but so far the results have 
 been unsatisfactory. Among the numerous medicinal 
 agents that have been tried to protect animals against 
 the action of the tubercle bacillus may be mentioned 
 tannin, menthol, sulphuretted hydrogen, mercuric chlo- 
 ride, creosote, creolin, phenol, arsenic, eucalyptol, etc. 
 Various inoculation experiments with cultures of the 
 tubercle bacilli and their products have been made, 
 and though the results reported in some cases have 
 been temporarily favorable, immunization has never 
 been satisfactorily produced. 
 
 Koch's Tuberculin. The discovery by Koch of toxins 
 in cultures of the tubercle bacillus which possess prop- 
 erties which explain its pathogenic power must rank 
 as one of the first importance in scientific medicine, 
 on account of what it has led up to, even if as ap 
 pears probable the final verdict may be that its thera- 
 peutic value in the treatment of tubercular diseases in 
 man is very slight. 
 
 Tuberculin contains all the products of the growth 
 of the tubercle bacilli in the nutrient bouillon as well 
 as some substances extracted from the bodies of the 
 
BACILLUS OF TUBERCULOSIS. 289 
 
 bacilli themselves. It also contains all the albuminoid 
 and other materials originally contained in the bouillon 
 which have remained unaffected by the activities of the 
 bacilli. There are two preparations known respec- 
 tively as the old and the new tuberculin. 
 
 Old tuberculin is prepared as follows : The tubercle 
 bacillus is cultivated in an infusion of calf s flesh, or of 
 beef flesh, or extract to which 1 per cent, of peptone 
 and 4 to 5 per cent, of glycerin have been added, the 
 culture liquid being slightly alkaline. The inoculation 
 is made upon the surface from a piece of very thin 
 pellicle from a young bouillon culture, or, if the bou- 
 illon culture is unobtainable, with small masses from a 
 culture on glycerin-agar. These masses, floating on 
 the surface, give rise in from three to six weeks, accord- 
 ing to the rapidity with which the culture grows, to an 
 abundant development and to the formation of ,a toler- 
 ably thick and dry, white crumpled layer, which finally 
 covers the entire surface. At the end of four to eight 
 weeks development ceases, and the layer after a time 
 sinks to the bottom. Fully developed cultures, after 
 having been tested for purity by a microscopical exami- 
 nation, are passed into a suitable vessel and evaporated 
 to one-tenth of their original bulk over a water-bath 
 at a temperature of 70 to 80 C. The liquid is then 
 filtered through chemically pure sterilized filter-paper. 
 The crude tuberculin thus obtained contains 40 to 50 per 
 cent, of glycerin and keeps well, retaining its activity 
 indefinitely. 
 
 The method of treatment and the results obtained 
 from the old tuberculin have been described recently 
 by Koch briefly as follows : After each injection, which 
 should be large enough to cause a slight but not a great 
 
 19 
 
290 BACTERIOLOGY. 
 
 rise of temperature, a noticeable improvement in the 
 tuberculous process results. The amount of tuberculin 
 injected is continually increased, so as to continue the 
 moderate reactions. After several months all reactions 
 cease, the patients having become temporarily immune 
 to the toxin, but not to the growth of the bacillus. 
 Further injections are now useless until this immunity 
 has passed. During the treatment the bacilli themselves 
 have not been directly affected, and when the treatment 
 is interrupted the tuberculous process is apt to progress. 
 Many cases, however, of pure tuberculosis become cured 
 or greatly benefited by several periods of treatment. 
 
 The substances produced in the body by the old tuber- 
 culin neutralized the tubercular toxins, according to 
 Koch, but were not bactericidal. After a series of ex- 
 periments, he considered the difficulty to be due to the 
 nature of the envelope of the tubercle bacillus, which 
 made it difficult to obtain the substance of the bacilli 
 in soluble form without so altering it by heat or chem- 
 icals that it was useless to produce immunizing sub- 
 stances. He conceived that immunity was not produced 
 in man for somewhat similar reasons possibly, the 
 bacilli never giving out sufficient toxin to cause cura- 
 tive substances to be produced. He therefore decided 
 to grind up the dried bacilli and soak them in water, 
 and thus obtain, if possible, without the addition of 
 heat, a soluble extract of the body-substance of the 
 bacilli, which he hoped would be immunizing. He 
 also tried to eliminate as much as possible of the toxic 
 products which produce fever. Biichner by a different 
 method, through crushing under a great pressure 
 tubercle bacilli mixed with sand, and thus squeezing 
 out their protoplasm, obtained a very similar substance. 
 
BACILLUS OF TUBERCULOSIS. 291 
 
 The new tuberculin formed by either of these methods 
 is a watery extract of the soluble portions of the un- 
 altered tubercle bacilli. As can be readily seen, in a 
 preparation thus made contamination is difficult to 
 avoid, freedom from intact bacilli is uncertain, and the 
 strength of the solution prepared at different times is 
 variable. Twenty per cent, of glycerin is added to 
 preserve the tuberculin from contamination. After 
 three years of trial the results obtained with the new 
 tuberculin preparations cannot be considered to have 
 exerted either very different or very superior effects to 
 the older product. 
 
 As to the results obtained in general the reports are 
 as yet conflicting. Lupus seems to be decidedly bene- 
 fited for a time both by the old and the new tuberculin. 
 Relapses are, however, common. On advanced phthisis, 
 laryngeal tuberculosis, arid other tubercular processes 
 no effects have been noted, and nearly every one dis- 
 approves of their use in these cases as well as in those 
 where mixed infection is suspected ; even in cases of 
 beginning infection, opinions, as a whole, are not very 
 enthusiastic. The new tuberculin is, except when 
 prepared with the utmost care, .a dangerous substance, 
 for Trudeau, Baldwin and others found that guinea-pigs 
 injected with it not only did not become immunized, 
 but actually became infected from the living bacilli in 
 the fluid. 
 
 The chief use to which tuberculin has been put is as 
 an aid to the diagnosis of tuberculosis in cattle and 
 human beings, and for this purpose it has proved to be 
 of inestimable value. Numerous experiments made by 
 veterinary surgeons show that the injection of tuber- 
 culin in tuberculous cows in doses of 25 to 50 centi- 
 
292 BACTERIOLOGY. 
 
 grammes produces in at least 95 per cent, a rise of tem- 
 perature of from 1 to 3 C. The febrile reaction occurs 
 in from twelve to fifteen hours after the injection. Its 
 intensity and duration do not depend upon the extent 
 of the tuberculous lesions, but is even more marked 
 when these are slight than in advanced cases. In non- 
 tuberculous animals no reaction occurs, or one much 
 less than in tuberculous animals, and the results ob- 
 tained on autopsy justify the suspicion that tubercu- 
 losis exists if an elevation of temperature of a degree 
 or more occurs from the subcutaneous injection of the 
 dose mentioned. For these injections the crude old 
 tuberculin is used, which for the convenience of admin- 
 istration is diluted with water. The following are the 
 directions for inspecting herds for tuberculosis : 
 
 (t Inspections should be carried on while the herd is 
 stabled. If it is necessary to stable animals under 
 unusual conditions or among unusual surroundings that 
 make them uneasy and excited the tuberculin test should 
 be postponed until the cattle have become accustomed 
 to the conditions they are subjected to, and then begin 
 with a careful physical examination of each animal. 
 This is essential, because in some severe cases of tuber- 
 culosis, on account of saturation with toxins, no reaction 
 follows the injection of tuberculin, but experience has 
 shown that these cases can be discovered by physical 
 examination. This should include a careful examina- 
 tion of the udder and of the superficial lymphatic glands 
 and auscultation of the lungs. 
 
 " Each animal should be numbered or described in 
 such a way that it can be recognized without difficulty. 
 It is well to number the stalls with chalk and transfer 
 these numbers to the temperature-sheet, so that the 
 
BACILLUS OF TUBERCULOSIS. 293 
 
 temperature of each animal can be recorded in its 
 appropriate place without danger of confusion. The 
 following procedure has been used extensively and has 
 given excellent results : 
 
 " (a) Take the temperature of each animal to be tested 
 at least twice, at intervals of three hours, before tuber- 
 culin is injected. 
 
 " (b) Inject the tuberculin in the evening, prefer- 
 ably between the hours of six and nine. The injection 
 should be made with a carefully sterilized hypoder- 
 matic syringe. The most convenient point for injec- 
 tion is back of the left scapula. Prior to the injection 
 the skin should be washed carefully with a 5 per cent, 
 solution of carbolic acid or other antiseptic. 
 
 " (c) The temperature should be taken nine hours 
 after the injection, and temperature measurements 
 repeated at regular intervals of two or three hours 
 until the sixteenth hour after the injection. 
 
 " (d) When there is no elevation of temperature at 
 this time (sixteen hours after the injection) the exam- 
 ination may be discontinued; but if the temperature 
 shows an upward tendency, measurements must be con- 
 tinued until a distinct reaction is recognized or until 
 the temperature begins to fall. 
 
 " (e) If a reaction is detected prior to the sixteenth 
 hour, the measurements of temperature should be con- 
 tinued until the expiration of this period. 
 
 " (/) If there is an unusual change of temperature 
 of the stable, or a sudden change in the weather, this 
 fact should be recorded on the report-blank. 
 
 " (g) If a cow is in a febrile condition tuberculin 
 should not be used, because it would be impossible to 
 determine whether, if a rise of temperature occurred, 
 it was due to the tuberculin or to some transitory illness. 
 
294 BACTERIOLOGY. 
 
 " (Ji) Cows should not be tested within a few days 
 before or after calving, for experience has shown that 
 the result at these times may be misleading. 
 
 " (i) The tuberculin test is not recommended for 
 calves under three months old. 
 
 " (j) In old, emaciated animals and in re-tests use 
 twice the usual dose of tuberculin, for these animals 
 are less sensitive. 
 
 " (k) Condemned cattle must be removed from the 
 herd and kept away from those that are healthy. 
 
 " (/) In making post-mortems the carcasses should 
 be thoroughly inspected, and all of the organs should 
 be examined." 
 
 Tuberculin injections are also made in man to reveal 
 a suspected tuberculosis. At first some believed that the 
 irritation aroused in the tuberculous foci by the tuber- 
 culin sometimes caused a dissemination of the bacilli and 
 an increase in the disease. When carefully used, how- 
 ever, in suitable cases there is probably no danger. A 
 drawback to its usefulness is that it does not reveal at 
 all the extent of the disease, nor whether the tuber- 
 culosis is active or dormant. It is, however, of great 
 value in selected cases, both surgical and medical, 
 where slight tuberculosis is suspected, and yet no de- 
 cision can be reached. I quote here Dr. Trudeau upon 
 the use of the test. 
 
 "In the absence of any well-defined rules founded 
 upon the experience of others at the time I began to 
 use the test, the method I adopted has been a purely 
 arbitrary one, and I make no claim for its being the 
 best or the most reliable, although, as far as my own 
 personal experience goes, I have as yet seen no objec- 
 tion to it or any reason to modify it. 
 
 
BACILLUS OF TUBERCULOSIS. 295 
 
 " The range of the patient's temperature is ascer- 
 tained by taking it at 8 A.M., 3 P.M., and 8 P.M. for 
 three or four days before making the test. The first 
 injection should not exceed 0.5 mg., and if any fever 
 is habitually present should be even less, and is best 
 given early in the morning or late at night, as the 
 typical reaction usually begins, in my experience, 
 within six or twelve hours. Such a small dose, while 
 it will often be sufficient to produce the looked-for rise 
 of temperature, has, under my observation, never pro- 
 duced unpleasant or violent symptoms. An interval of 
 two or three days should be allowed between each of the 
 two or three subsequent injections it may be necessary 
 to give, as reaction in very rare cases may be delayed 
 for twenty-four or even thirty-six hours. On the third 
 day a second dose of 1 mg. is given, and if no effect is 
 produced a third, of 2 mg., three days later. In the 
 great majority of cases of latent tuberculosis an appre- 
 ciable reaction will be produced by the time a dose of 
 2 mg. has been reached. If no effect has been caused 
 by the tests applied as above I have usually gone no 
 further, and concluded that no tuberculous process was 
 present, or at least not to a degree which need be taken 
 into account in advising the patient or which would 
 warrant insisting on a radical change in his surround- 
 ings and mode of life. If some slight symptoms, how- 
 ever, have been produced by a dose of 2 mg., it may 
 be necessary to give a fourth injection of 3 mg. in order 
 to reach a positive conclusion. Nevertheless, it should 
 be borne in mind that in a few cases the exhibition 
 of even larger doses may cause reaction and indicate 
 the existence of some slight latent tuberculous lesion, 
 and the test should not, when applied within the 
 
296 BACTERIOLOGY. 
 
 moderate doses described, be considered absolutely 
 infallible. 
 
 " No evidence in connection with the tuberculin test 
 as applied to man and animals has been forthcoming 
 thus far from those who have made use of it, which 
 would tend to sustain the general impression that this 
 method is necessarily dangerous and tends invariably 
 to aggravate the disease, and my own experience has 
 developed nothing which would seem to confirm this 
 impression. It is evident that the size of the doses 
 given has much to do with the limitations of this method 
 for usefulness and the correctness of the conclusions 
 reached by its application. The tuberculin used is also 
 a matter of some importance in determining the dosage, 
 as different samples vary considerably in their efficiency. 
 The minute amounts adopted by Grasset and Vedel 
 i. e., from 0.0002 to 0.0005 while they have the advan- 
 tage of absolute safety, may lead into error, as they 
 are insufficient, on the evidence of these observers them- 
 selves, to cause reaction in cases proven to be tuberculous 
 by the presence of the bacillus in the expectoration. If, 
 on the other hand, the test be pushed to the injection of 
 such large amounts as 10 mg. or more, as advocated by 
 Maragliano, such doses are by no means free from the 
 objection of occasionally causing unpleasant and some- 
 times dangerous symptoms; and even if the amount 
 given be not carried to the dose of 10 mg., which is 
 known to produce fever in healthy subjects, it is likely 
 that on account of individual susceptibility or the pres- 
 ence of some other morbid process in the body, reaction 
 will be found to occur with the larger doses when no 
 tuberculous process exists. The adoption of an initial 
 dose so small as to guard against the absolute possibility 
 
BACILLUS OF TUBERCULOSIS. 297 
 
 of producing violent reactionary symptoms, and the 
 graded increase of the subsequent doses within such 
 quantities as are known never to produce reaction in 
 healthy individuals, would seem to afford the best 
 protection against unpleasant results and misleading 
 evidence. " 
 
 Antituberculous Serum. Whether serum-therapy is 
 destined to solve the problem of the treatment of tuber- 
 culosis remains for the future to decide, but up to the 
 present the results obtained with antituberculous serum 
 do not warrant our forming such an opinion. The at- 
 tempts to obtain from animals chiefly horses a serum 
 which would be protective have been carried out along 
 very much the same lines as Koch's experiments upon 
 man. The methods adopted have been as follows: Old 
 cultures of tubercle bacilli grown in 5 per cent, glycerin 
 bouillon have been filtered either with or without pre- 
 vious boiling, and then injected into animals, this 
 process being similar to Koch's with his first tuberculin. 
 Others have injected living virulent or non-virulent 
 tubercle bacilli, either alone or with their culture fluids; 
 others still (Biichner) have injected the bacterial proto- 
 plasm obtained by crushing tubercle bacilli together 
 with sand and squeezing them ; this, like Koch with 
 his new tuberculin, being an attempt to get from the 
 unaltered products and cell-contents of the bacilli the 
 formation in the body of bactericidal or immunizing 
 substances. 
 
 Among the many claiming good results in man or 
 animals thus treated may be mentioned Hericourt, 
 Bichet, Bernheim, Maragliano, Yiquerat, Paquin, 
 de Schweinitz and Dorset, McFarland, and others. 
 The majority claim for their serum the power to neu- 
 
298 BACTERIOLOGY. 
 
 tralize the effect of tuberculin when injected into tuber- 
 culous guinea-pigs; but this test is insufficient and prob- 
 ably valueless, since tuberculin is not the same as the 
 unaltered products of the tubercle bacillus. Moreover, 
 it has been shown by Trudeau and Baldwin that other 
 substances which have no specific properties whatever 
 will have much the same effect as the serum under 
 certain conditions. Some make the further claim that 
 guinea-pigs injected with serum acquire an immunity 
 to the virulent tubercle bacilli, and that those already 
 infected live longer than the controls which receive no 
 serum; and some even claim to be able to cure animals 
 eighteen days after inoculation with, a culture of tu- 
 bercle bacilli. Very few observers, however, have suc- 
 ceeded in obtaining appreciable results with the serums 
 prepared by other experimenters. In spite of such con- 
 flicting testimony, it is probably safe to assert that no 
 serums now obtainable have any great value. Nor as 
 we look at the progressive nature of tuberculosis can 
 we see much ground to hope for the abundant de- 
 velopment of curative substances in the blood of ani- 
 mals. 
 
 Prophylaxis. Meanwhile all energies should be 
 directed to the prevention of tuberculosis, not only 
 by the enforcement of proper sanitary regulations as 
 regards the care of sputum, milk, meat, disinfection, 
 etc., but also by continued experimental work and by 
 the establishment of free consumptive hospitals, and 
 by efforts to improve the character of the food, dwell- 
 ings, and condition of the people in general, we should 
 endeavor to build up the individual resistance to the 
 disease. It may be years yet before the public are 
 sufficiently educated to co-operate with the sanitary 
 
BACILLUS OF TUBERCULOSIS. 299 
 
 authorities in adopting the necessary hygienic measures 
 to stamp out tuberculosis entirely; but, judging from 
 the results which have already been obtained in reducing 
 the mortality from this dread disease, we have reason to 
 believe that in time it can be completely controlled. 
 
 The Tubercle Bacillus of Cattle and its Relation to 
 Human Tuberculosis. Among the domestic animals 
 tuberculosis is most common in cattle. On account 
 of the milk which they provide for our use, and which 
 is liable to contain bacilli, the relation of these to human 
 tuberculosis is a matter of extreme importance. 
 
 The chief seat of the lesions is apt to be the lungs 
 and with them the pleura; less often the abdominal 
 organs and the udder are affected. In pigs and horses 
 the abdominal organs are most often involved, then the 
 lungs and lymphatic glands. In sheep and goats tuber- 
 culosis is rare. The bovine bacillus, as the most im- 
 portant of the group, will be alone considered here. 
 
 The bacilli derived from cattle are on the average a 
 little shorter and straighter than the average human 
 bacillus; but there are many derived from cattle exactly 
 similar to those derived from man in size, shape, and 
 staining. In guinea-pigs, and especially in rabbits, the 
 bovine bacilli are more virulent than those from human 
 sources. Animals infected with the bacilli from cattle, 
 as well as those from the other domestic animals, react 
 to the tuberculin test. All these bacilli are, therefore, 
 undoubtedly from the same original stock, and at 
 first glance we might consider it unnecessary to prove 
 that those derived from cattle were capable of causing 
 human tuberculosis. There are facts, however, which 
 tend to make us doubtful of the extent to which this 
 infection takes place. As we investigate we find that 
 
300 BACTERIOLOGY. 
 
 all facts tend to show that the great majority of cases, 
 in adults at least, come from human infection. The 
 cases where fairly strong proof of bovine infection has 
 been obtained are certainly rare. 
 
 Further, we have the undoubted fact that constant 
 sojourn in one species of animal tends to increase the 
 virulence of the germ for that animal and to lessen it 
 for others. 
 
 Theobald Smith has made the interesting discovery 
 that there is a wide difference between the culture 
 growth of the average bovine bacillus and the average 
 one from human sources, the bovine bacilli being shorter 
 and straighter, and growing less luxuriently than those 
 from man ; and, further, that the bovine bacilli are 
 much more virulent for rabbits. He has found these 
 differences persist for long periods, and believes that 
 the simple passage through a single person in a case of 
 human tnberculosis would not be sufficient to change 
 these characteristics. He has not yet had a chance to 
 examine the bacilli of any case in young children where 
 milk infection was strongly suspected, but in adults 
 not one of some half a dozen cultures showed the 
 bovine characteristics. 
 
 At present it seems fair to assume that bovine bacilli 
 are capable of infecting only those that are very sus- 
 ceptible, such as young children. This question is in 
 great need of further study, and unless proof is 
 brought to show that bovine bacilli never infect human 
 beings, no cattle which are shown to be tubercular 
 should be allowed to furnish milk, or at least none un- 
 sterilized should be used for drinking purposes. The flesh 
 is less harmful, as muscular tissue is seldom infected. 
 
 Bird (Avian) Tuberculosis. Tuberculosis is very com- 
 
BACILLUS OF TUBERCULOSIS. 301 
 
 mon and infectious among fowls. The bacilli them- 
 selves grow more readily on artificial culture media 
 and produce a more even and moist growth. The 
 bacilli are more apt to show branching forms than the 
 human. In rabbits they produce very similar lesions. 
 They are probably from the same stock as the mamma- 
 lian varieties; but it is not believed that they are any, 
 and certainly not any great, factor in the production of 
 human tuberculosis. 
 
 Diagnosis. One of the most important results of the 
 discovery of the tubercle bacillus relates to the practical 
 diagnosis of tuberculosis. The staining peculiarities 
 of this bacillus render it possible by the bacteriological 
 examination of microscopical preparations to make an 
 almost absolutely positive diagnosis in the majority of 
 cases. A still more certain test in doubtful cases is 
 the subcutaneous or intraperitoneal injection of guinea- 
 pigs, which permits of the determination of the presence 
 of numbers of bacilli so small as to escape detection by 
 microscopical examination. For the animal test, how- 
 ever, time is required at least three weeks, and, when 
 the result is negative, several months before any posi- 
 tive conclusion can be reached, for when only a few ba- 
 cilli are present tuberculosis develops slowly in animals. 
 
 LABORATORY TECHNIQUE IN THE EXAMINATION 
 FOR TUBERCLE BACILLI AND OTHER ASSO- 
 CIATED BACTERIA. 
 
 I. Microscopical Examination of Sputum for the Presence 
 of Tubercle Bacilli. 
 
 1. Collection of Material. The sputum should be col- 
 lected in a clean bottle (two-ounce) with a wide mouth 
 and a water-tight stopper, and the bottle labelled with 
 
302 BACTERIOLOGY. 
 
 the name of the patient or other distinguishing mark. 
 The expectoration discharged in the morning is to be 
 preferred, especially in recent cases, and the material 
 should be coughed up from the lungs. Care should be 
 taken that the contents of the stomach, nasopharyngeal 
 mucus, etc., are not discharged during the act of expec- 
 toration and collected instead of pulmonary sputum. If 
 the expectoration be scanty the entire amount discharged 
 in twenty-four hoars should be collected. In pul- 
 monary tuberculosis the purulent, cheesy, and muco- 
 purulent sputum usually contains bacilli; while pure 
 mucus, blood, and saliva, as a rule, do not. When 
 hemorrhage has occurred, if possible some purulent, 
 cheesy, or mucopurulent sputum should be collected 
 for examination. The sputum should not be kept any 
 longer than necessary before examination, for, though 
 a slight delay or even till putrefaction begins, does not 
 entirely vitiate the result, it is best to examine it in as 
 fresh a condition as possible. 
 
 2. Methods of Examination, (a) EXAMINATION FOR 
 TUBERCLE BACILLI. Pour the specimen into a clean, 
 shallow vessel having a blackened bottom a Petr 
 dish placed upon a sheet of dull black paper answers 
 the purpose and select from the sputum one of the 
 small, white or yellowish-white, cheesy masses or 
 11 balls " which it is seen to contain. From this make 
 a cover-glass " smear " in the usual way. Immerse 
 this in a solution of Ehrlich's aniline-water fuchsin (see 
 page 198), contained in a thin watch-glass or porcelain 
 dish, and steam over a small flame for two minutes. 
 Then remove the cover-glass from this and wash with 
 water. Now decolorize by immersing the stained prep- 
 aration in a 3 per cent, hydrochloric acid solution in 
 
BACILLUS OF TUBERCULOSIS. 303 
 
 alcohol for from a few seconds up to one minute, re- 
 moving at the time when all color is just about gone 
 from the cover-glass smear. Wash thoroughly with 
 water and make a contrast stain by applying a cold 
 solution of Loffler's alkaline methylene-blue 
 
 Concentrated alcoholic solution of methyl blue 30 c. c. 
 Caustic potash (1:1 0,000 solution) . .100" 
 
 for from fifteen to thirty seconds. Wash with water; 
 press between folds of filter-paper; dry in the air; 
 mount and examine. 
 
 The tubercle bacilli are distinguished by the fact that 
 they retain the red color imparted to them in the 
 fuchsin solution, while the other bacteria present, 
 having been decolorized in the acid solution, take the 
 contrast stain and appear blue. (See plate II., Figs. 
 1 and 2.) 
 
 Various methods have been suggested for the staining 
 of tubercle bacilli, but the original method as employed 
 by Koch, or some slight modification of it, is so satis- 
 factory in its results that it seems unnecessary to substi- 
 tute others for it. The above is a slight modification of 
 the Koch-Ehrlich method, differing from it chiefly in 
 the use of a weak for a strong acid decolorizer. It 
 has been found that the strong acid solution originally 
 employed (5 per cent, sulphuric acid solution in alcohol) 
 often decolorizes some of the bacilli entirely by its too 
 energetic action, and that a weaker decolorizer, such 
 as the above, gives more uniform results. 
 
 Instead of the Koch-Ehrlich aniline-water solution, 
 ZiehVs carbol-fuchsin solution may be used, and is by 
 many preferred (see page 198). Instead of floating 
 the cover-glass smears on the staining fluid they can be 
 
304 BACTERIOLOGY. 
 
 held in the Cornet forceps, covered and kept covered 
 completely with fluid while steamed for two minutes 
 over the flame. 
 
 The Koch-Ehrlich solution decomposes after having 
 been made for a time, so that it must be freshly pre- 
 pared as needed. Solutions older than fourteen days 
 should not be used. The advantages in using ZiehPs 
 carbol-fuchsin solution are that it keeps well and is 
 more convenient for use in small quantities. 
 
 Another method, which is often of value on account 
 of its simplicity and rapidity of performance, is that 
 of Frankel as modified by Gabbett. This consists in 
 staining the cover-glass " smear " with steaming ZiehPs 
 carbol-fuchsin solution for from one to two minutes, 
 and then after washing in water placing it from one- 
 half to one minute directly in a second solution which 
 contains both the acid for decolorizing and the contrast 
 stain. This second solution consists of 
 
 Sulphuric acid . . . . .25 c.c. 
 Methylene-blue in substance . . 2 grammes. 
 Water . . . . . . 75 c.c. 
 
 It is then washed with water and is ready for examina- 
 tion. The tubercle bacilli will remain red as stained by 
 the fuchsin, while all other bacteria will be tinted blue. 
 When the number of tubercle bacilli in sputum is 
 very small they may easily escape detection. Methods 
 have, therefore, been suggested for finding them under 
 these circumstances. Ribbert proposed the addition to 
 the sputum of a 2 per cent, solution of caustic potash 
 and boiling the mixture. The mucus is dissolved, and 
 when the mixture is placed in a conical glass vessel any 
 bacilli present are deposited at the bottom, and may be 
 found in the sediment after removing the supernatant 
 
BACILLUS OF TUBERCULOSIS. 3Q5 
 
 fluid. The sedimentation may be obtained more quickly 
 by the centrifugal machine. 
 
 II. Examination for Other Bacteria (Mixed Infection). 
 
 With regard to the bacteriological diagnosis of pul- 
 monary phthisis, many consider that it is not enough 
 to show only the presence of tubercle bacilli; it is held 
 to be of equal importance, both for purposes of prognosis 
 and treatment, that the presence of other micro-organ- 
 isms which may be associated with the tubercle bacillus 
 should also be determined. It is now usual to dis- 
 tinguish pure tuberculosis of the lungs from a mixed 
 infection. Phthisis due to the tubercle bacillus alone, 
 which constitutes but a small percentage of all cases, 
 may occur without febrile reaction; or when fever occurs 
 the prognosis is unfavorable, thus indicating that the 
 disease is already advanced. It is in the uncomplicated 
 forms of phthisis, moreover, where one must expect if 
 anywhere the best results from treatment with tuber- 
 culin or antituberculous serum. The majority of cases, 
 however, of pulmonary tuberculosis show a mixed in- 
 fection, especially with varieties of the streptococcus 
 and pneumococcus. These cases may be active, with 
 fever, or passive, without fever, according, perhaps, 
 as the parenchyma of the lung is invaded by the bac- 
 teria; or they are only superficially located in cavities, 
 bronchi, etc. Mixed infection with the staphylococcus 
 and with the influenza and pneumonia bacilli have 
 also been frequently met with by us. The tetragenus 
 has not been detected by us in thoroughly washed 
 fresh sputum, but has been found by others. At present 
 the facts seem to prove that the tubercle bacilli have 
 in the great majority of cases at least until shortly 
 
 20 
 
306 BACTERIOLOGY. 
 
 before death, a more important role than the associated 
 bacteria. 
 
 The great majority of stained smears from specimens 
 of sputa show not only the tubercle bacilli stained in 
 red, but many other bacteria stained blue. Some of 
 these associated bacteria have come from the diseased 
 areas of the lungs, while others were merely added 
 to the sputa as it passed through the mouth, or have 
 developed after gathering. To separate the one from 
 the other we wash the sputa. 
 
 Sputum Washing. The first essential is that the 
 material be washed within a few minutes, and certainly 
 within an hour, of being expectorated. If a longer 
 time is allowed to intervene, the bacteria from the 
 mouth will penetrate into the interior of the mucus, and 
 thus appear as if they came from the lungs. Sputum 
 treated twenty-four hours after its expectoration is use- 
 less for examining for anything except the tubercle 
 bacillus. A rough method is to pour some of the speci- 
 men of sputum to be examined into a convenient re- 
 ceptacle containing sterile water, and withdraw, by 
 means of a sterilized platinum wire, one of the cheesy 
 masses or thick " balls' 7 of mucus. Pass this loop five 
 times through sterile water in a dish; repeat the oper- 
 ation in freshwater in a second and third dish. Spread 
 what remains of the mass on cover -glasses and make 
 smear preparations; stain and examine. With another 
 mass inoculate ascitic bouillon in tubes and agar in 
 plates. 
 
 If it is desired to examine the specimens for capsule 
 bacteria, pneumococci, etc., they may be stained by 
 Welch's acetic-acid method (page 203) or by Gram's 
 method (page 203). 
 

 BACILLUS OF TUBERCULOSIS. 307 
 
 When we wish to thoroughly exclude mouth bacteria 
 a lump of the sputum raised by a natural cough is seized 
 by the forceps and transferred to a bottle of sterile 
 water and thoroughly shaken ; it is then removed to a 
 second bottle of bouillon and again thoroughly shaken. 
 From this it is passed in the same way through four 
 other bottles of bouillon. A portion of the mass is 
 now smeared over cover-glasses, and the rest inoculated 
 in suitable media, such as agar in Petrie dishes, and 
 ascitic fluid bouillon in tubes. If desired the bacteria 
 washed . off in the different washings are allowed to 
 develop. 
 
 Practical Notes on the Examination for Mixed Infec- 
 tion. 1. The difficulties to be overcome, in order to 
 obtain sputum consisting presumably of exudate from 
 the deeper portions of the lungs, are so great that the 
 collection of the specimens should be supervised by the 
 bacteriologist in charge of the work of examination. 
 
 2. Specimens of sputum collected even with the 
 greatest precaution may give evidence of decided 
 mouth infection unless immediately washed. 
 
 3. The sputum must be examined very soon after 
 collection. 
 
 4. The culture medium used for the final cultures 
 must be suitable for the growth of the micro-organisms. 
 
 5. At least two successive examinations of sputum 
 should be made in each case. 
 
 6. The results, especially as to the number of colo- 
 nies, vary according to the size and tenacity of the ball 
 of sputum washed e.g., a small ball of sputum which 
 becomes more or less broken up upon thorough shaking 
 may contain very few or no bacteria. 
 
 Williams, in the examination of the sputum in some 
 
308 BACTERIOLOGY. 
 
 forty cases, came to the following conclusions : 1. The 
 presence of a large number of bacteria in a satisfactory 
 and thoroughly washed specimen of sputum indicates 
 that these bacteria probably play an active part in the 
 
 2. The presence of a small number of bacteria in 
 such sputum does not necessarily indicate that they are 
 not active in that case, for they may penetrate more or 
 less deeply into the lung tissue, and produce patho- 
 logical changes without being thrown off in large num- 
 bers with the exudate. It is probable, however, that, 
 as a rule, the smaller the number found the less the 
 degree of mixed infection. 
 
 3. Cases of clinically secondary infection frequently 
 give pure cultures of some one organism which appeared 
 to be capable of causing the symptoms. 
 
 4. In the majority of severe cases of clinically 
 mixed infection many organisms have been found which 
 usually have belonged to several different species or 
 varieties. 
 
 5. In the majority of cases of clinically non-mixed 
 infection very few organisms have been found. 
 
 6. Only bacteria which might cause pathological 
 changes were present. 
 
 7. Very few of the organisms found were virulent 
 in rabbits, even though coming from severe cases of 
 mixed infection. 
 
 The virulence for laboratory animals of bacteria ob- 
 tained from the sputum is, therefore, no indication of 
 their virulence for man, because of the impossibility of 
 reproducing in such animals the exact condition of sus- 
 ceptibility present in human infection. 
 
 General Rules in Microscopical Examination of Sputum. 
 
BACILLUS OF TUBERCULOSIS. 309 
 
 Always make two cover-glass preparations from each 
 specimen. Report no result as negative until at least 
 two preparations have been subjected to a thorough 
 search with a 1/12 oil-immersion or 2 mm. apochromatic 
 lens by means of a mechanical stage. From a very 
 large experience in the examination of sputum for 
 tubercle bacilli, the New York Health Department 
 bacteriologists have concluded that the examination 
 of two preparations of each specimen in the careful 
 manner described above is usually sufficient to demon- 
 strate the presence of the bacilli when they are pres- 
 ent in the sputa, and they are usually found to be 
 present to this extent in fairly well-developed cases 
 of pulmonary tuberculosis, and in many cases which 
 are in the incipient stage. There are, however, un- 
 doubted cases of incipient pulmonary tuberculosis which 
 require the examination of many preparations before 
 the tubercle bacillus can be found; and that cases also 
 occur in which the sputum for a time does not contain the 
 bacilli, which were, nevertheless, present at an earlier 
 period, and which again later appear. Therefore, if cases 
 occur which may be still regarded as possibly tubercu- 
 losis, further examinations of the sputum should be 
 made. It should also be constantly borne in mind 
 that the demonstration of the presence of tubercle 
 bacilli in the sputum proves about as conclusively as 
 anything can the existence of some degree of tubercu- 
 losis; but that the absence of tubercle bacilli or the 
 failure to find them microscopically does not positively 
 exclude the existence of the disease. Here injections 
 of tuberculin can be made use of. 
 
310 BACTERIOLOGY. 
 
 III. Staining of Tubercle Bacilli in Tissues. 
 
 Thin sections of tuberculous tissues may be stained 
 by the same methods recommended for cover-glass 
 preparations, except that it is best not to employ heat 
 to any extent. 
 
 The Ehrlich's Method. Place the paraffin sections in 
 aniline fuchsin and leave for from one to twelve hours; 
 then decolorize by placing them for about half a minute 
 in dilute nitric acid (10 per cent.); wash in 60 per cent, 
 alcohol until no more color is given off; counter stain 
 for two or three minutes in a saturated aqueous solu- 
 tion of methylene-blue, wash in water; dehydrate with 
 absolute alcohol; clear up in oil of cedar or xylol and 
 mount in xylol balsam. 
 
 Method of Ziehl-Neelson. Stain the section in warmed 
 carbol-fuchsin solution for one hour, the temperature to 
 be not over 45 to 50 C. Decolorize for a fe\y seconds 
 in 5 per cent, sulphuric acid, then in 70 per cent, alcohol, 
 and from this on as in the Ehrlich method. 
 
CHAPTER XIX. 
 
 BACILLI SHOWING SIMILAR STAINING REACTIONS TO 
 THOSE OF THE TUBERCLE BACILLI SYPHILIS 
 BACILLUS SMEGMA BACILLUS LEPROSY BACIL- 
 LUS GRASS BACILLI. 
 
 SYPHILIS BACILLUS. 
 
 DISCOVERED by Lustgarten in syphilitic lesions and 
 secretions of syphilitic ulcers (1884), and believed by 
 him to be the specific .cause of this disease. It has 
 since been shown that in normal smegma from the 
 prepuce or the vulva bacilli are found in great abun- 
 dance, similar in their morphology to the bacillus of 
 Lustgarten, but differing, as a rule, slightly in certain 
 staining peculiarities. (See Fig. 39, page 313.) 
 
 Morphology. Straight or curved bacilli, which bear 
 considerable resemblance to tubercle bacilli, but differ 
 from them in staining reactions. They are from 3 to 
 5// long and from 0.2 to 0.3/* broad, usually curved 
 or bent at a sharp angle, or S-shaped, often thickened at 
 one end and irregularly notched. With a high-power 
 lens bright, shining spaces in the deeply stained rods 
 may be observed; these, from two to four in a single rod, 
 are believed by Lrustgarten to be spores. The bacilli 
 are not usually found free in the tissues, but commonly 
 lie singly or sometimes in groups within the interior of 
 cells having a round, oval, or polygonal form, and 
 apparently somewhat swollen. 
 
312 BACTERIOLOGY. 
 
 The bacillus of Lustgarten stains with equal diffi- 
 culty as the tubercle bacillus, but is much less resistant 
 to the action of certain decolorizing agents, such as 
 mineral acids, particularly sulphuric acid. It is, as a 
 rule, more resistant to the decolorizing action of alcohol 
 than the smegma bacillus. 
 
 Biological and Pathogenic Properties. Numerous at- 
 tempts have been made to cultivate the bacillus of 
 Lustgarten on artificial media, but without success. 
 The inoculation of animals with syphilitic tissues and 
 secretions has also given only negative results, though 
 in man, as is well known, such inoculation has often 
 taken place, the tertiary products only being non- 
 infectious; but as the bacillus has never been obtained 
 in pure culture, we have no ppsitive information as to 
 its biological characters or pathogenesis. 
 
 Lustgarten' s bacillus has been found in various syph- 
 ilitic tissues and lesions, in beginning sclerosis, in the 
 papules, in condylomata and gummata, and not only 
 in the vicinity of the genitals, but also in the mouth, 
 throat, heart, and brain. No satisfactory experimental 
 evidence has been given of its causative relation to 
 syphilis, but the failure to find other micro-organisms, 
 and the occurrence of these characteristic bacilli in vari- 
 ous parts of the body, would seem to point to their etio- 
 logical importance; while, on the other hand, the long 
 immunity in syphilis, so different from that in any 
 known bacterial disease, casts doubt not only on the 
 status of this bacillus, but also upon the bacterial 
 nature of the micro-organism. The fact that the ba- 
 cilli have been found occasionally in tertiary lesions 
 which, however, are known to possess no infectious 
 property may possibly be explained by the some- 
 
SYPHILIS BACILL US. 313 
 
 what improbable assumption that the bacilli here 
 present have become attenuated or have died. The 
 finding of saprophytic bacilli the so-called smegma 
 bacilli (Fig. 39 and Plate I., Fig. 4), almost identical 
 
 FIG. 39. 
 
 S 
 
 Smegma bacilli, similar in appearance to syphilis bacilli. X 1000 diam. 
 
 morphologically with the bacillus of Lustgarten, under 
 the prepuce of healthy persons, does not prove the 
 identity of the two bacilli, though in the absence of 
 cultures and inoculation experiments we have not the 
 means of establishing their relationship to one another. 
 The smegma bacilli have never been identified in other 
 parts of the body except in the neighborhood of the 
 genitals. While the bacillus of Lustgarten cannot 
 resist the prolonged decolorizing action of acids, but 
 is resistant to the action of alcohol, the smegma ba- 
 cillus, when stained, is quickly decolorized by alcohol, 
 but quite resistant to 5 per cent, sulphuric acid solution. 
 Beside, the syphilis bacillus has been found in papules, 
 in gummata, and other syphilomata where there seems 
 no probability whatever of the smegma bacillus having 
 emigrated. Baumgarten, who has searched in vain for 
 Lustgarten' s bacillus in uncomplicated visceral syphilo- 
 
314 BACTERIOLOGY. 
 
 mata, suggests that the bacilli found in such lesions were, 
 perhaps, tubercle bacilli, and represented a mixed in- 
 fection. This may have been true of some cases, no 
 doubt, as the differentiation of new-growths of tertiary 
 syphilis and tuberculosis is often difficult; but the differ- 
 entiation of the two bacilli can usually be made by their 
 different powers of resistance to the decolorizing action 
 of acids. Finally, other micro-organisms have been 
 described and claimed to be the specific cause of syph- 
 ilis, but none of these discoveries have been confirmed. 
 From this it appears that though there is no conclusive 
 proof of the fact, there is some possibility, but hardly 
 a probability, that Lustgarten's bacillus is the true cause 
 of syphilis. Its position at present is too doubtful to 
 make its detection of any diagnostic value. 
 
 Syphilitic Infection. Infection of those not immune 
 can take place at any time when an abrasion, however 
 small, is brought in contact with the blood or secretions 
 from the primary or secondary lesions of syphilitics. 
 
 The differential diagnosis of Lustgarten's bacillus 
 must be made from the tubercle bacillus, the smegma 
 bacillus, and the leprosy bacillus. According to Hueppe, 
 the differential diagnosis between these four organisms 
 depends upon the following reactions : When stained 
 by the carbol-fuchsin method commonly employed in 
 staining the tubercle bacillus, the syphilis bacillus be- 
 comes almost instantly decolorized by treatment with 
 mineral acids, particularly sulphuric acid; whereas the 
 smegma bacillus resists such treatment for a much 
 longer time, and the lepra and tubercle bacillus for a 
 still longer time. On the other hand, if decolorization 
 is practised with alcohol instead of acids the smegma 
 bacillus is the first to lose its color. The bacillus tuber- 
 
LEPROSY BA GILL US. 315 
 
 culosis and the bacillus of leprosy are both very retentive 
 of their color, even after treatment with acids and 
 alcohol. If, then, we treat the preparation, stained with 
 carbolfuchsin, with sulphuric acid, the syphilis bacillus 
 becomes almost at once decolorized. If it is not imme- 
 diately decolorized, treat with alcohol; if it is then 
 decolorized, it is the smegma bacillus. If it is still 
 not decolorized, it is either the leprosy or the tubercle 
 bacillus. 
 
 By these methods the differential diagnosis can usually 
 be made. In all investigations of importance, however, 
 animal inoculations should also be made, as by this 
 means alone can a positive diagnosis from tuberculosis 
 be established. Especial care should be observed in 
 the examination of syphilitic ulcers of the genital re- 
 gion, as in this situation the smegma bacilli are almost 
 always present. 
 
 LEPROSY BACILLUS. 
 
 The bacillus of leprosy was discovered by Hansen 
 and Neisser (1879) in the leprous tubercles of persons 
 afflicted with the disease. This discovery was confirmed 
 by many subsequent observers. 
 
 Morphology. Small, slender rods resembling the 
 tubercle bacilli in form, but somewhat shorter and 
 not so frequently curved. The rods have pointed ends, 
 and in stained preparations unstained spaces, similar to 
 those observed in the tubercle bacillus, are seen. They 
 stain readily with the aniline colors and also by Gram's 
 method. Although differing from the tubercle bacillus 
 in the ease with which they take up the ordinary aniline 
 dyes, they behave like tubercle bacilli in retaining their 
 
316 BACTERIOLOGY. 
 
 color when subsequently treated with strong solutions 
 of the mineral acids and alcohol. Thus double-stained 
 preparations may be made by first staining sections or 
 cover-glass preparations in ZiehPs carbol-fuchsin solu- 
 tion or in an aqueous solution of methyl-violet, de- 
 colorizing in acid, washing in alcohol, and counter- 
 staining with methylene-blue or fuchsin. 
 
 Biological Characters. Attempts to cultivate the bacil- 
 lus leprse have been frequently made, but so far with 
 only questionable results. None of the cultures ob- 
 tained have given positive results when inoculated 
 into animals. 
 
 Pathogenesis. Numerous inoculation experiments 
 have been made on animals with portions of leprous 
 tubercles, excised for the purpose from lepers, but 
 although a few positive results have been reported, 
 there is no conclusive evidence that leprosy can be 
 transmitted to the lower animals by inoculation. The 
 inference that this bacillus bears an etiological relation 
 to the disease with which it is associated is based 
 entirely upon the demonstration of its constant pres- 
 ence in leprous tissues. 
 
 The bacilli are found in all the diseased parts and 
 usually in large numbers, especially in tubercles on the 
 skin, in the conjunctiva and cornea, and the mucous 
 membranes of the mouth, gums, and larynx, and in 
 the interstitial processes of the nerves, the testicles, 
 spleen, liver, and kidneys. The rods lie almost exclu- 
 sively within the peculiar round or oval cells of the 
 granulation tissue which composes the leprous tubercles, 
 either irregularly scattered or arranged parallel to one 
 another. In old centres of infection the leprosy cells 
 containing the bacilli are larger and often polynuclear. 
 
LEPROSY BACILLUS. 317 
 
 Giant-cells, such as are found in tuberculosis, are claimed 
 to have been observed by a few investigators (Boinet 
 and Borrel). In the interior of the skin tubercles the 
 hair follicles, sebaceous and sweat-glands are often 
 attacked, and bacilli have sometimes been found in 
 these (Unna, etc.). Quite young eruptions often con- 
 tain a few bacilli. A true caseation of the tubercles 
 does not occur, but ulceration results. 
 
 In the anaesthetic forms of leprosy the bacilli are 
 found most commonly in the nerves and less frequently 
 in the skin. They have been demonstrated in the sym- 
 pathetic nervous system, in the spinal cord, and in the 
 brain. The bacillus leprae occurs also in the blood, 
 partly free and partly within the leucocytes, especially 
 during the febrile stage which precedes the breaking 
 out of fresh tubercles (Walters and Doutrelepont). The 
 bacilli have also been found in the intestines, in the 
 lungs, and in the sputum, but not in the urine. 
 
 With regard to the question of the direct inheri- 
 tance of the disease from the mother to the unborn 
 child there is considerable difference of opinion. Some 
 cases have been reported, however, in which a direct 
 transmission of the bacillus during intra-uterine life 
 seems to have been the only or most plausible expla- 
 nation of the infection. At the same time, we have no 
 positive experimental evidence to prove that such an 
 infection does take place. Although many attempts 
 have been made to infect healthy individuals with 
 material containing the bacilli of leprosy, the results 
 are not conclusive. Even the experiments made by 
 Arning, who inoculated a condemned criminal in the 
 Sandwich Islands with fresh leprous tubercles, and 
 which has been generally regarded as positive evidence 
 
318 BACTERIOLOGY. 
 
 of the transmissibility of the disease in this way, is by 
 no means conclusive; for, according to Swift, the man 
 had other opportunities for becoming infected. These 
 negative results, together with the fact that infection 
 does not more frequently occur in persons exposed to 
 the disease, may possibly be explained by the assump- 
 tion that the bacilli contained in the tubercular tissue 
 are mostly dead, or much more probably that an indi- 
 vidual susceptibility to the disease is requisite for its 
 production. 
 
 The wide-spread idea, before the discovery of the 
 leprosy bacillus, that the disease was associated with the 
 constant eating of dried fish or a certain kind of food 
 has now been entirely abandoned. 
 
 The relation of leprosy to tuberculosis is sufficiently 
 evident from their great similarity in many respects. 
 This is rendered still more remarkable if the ob- 
 servation recently made is true, that leprosy reacts, 
 both locally and generally, to an injection of tuberculin 
 in the same manner as tuberculosis (Babes and Kalin- 
 dero). 
 
 Differential Diagnosis. The differential diagnosis be- 
 tween leprosy and tuberculosis is not difficult in typical 
 cases. The large numbers of bacilli found in the inte- 
 rior of the cells would point with great probability to 
 leprosy. Too much importance should not be placed 
 upon the staining peculiarities, as these are not con- 
 stant. Moreover, the two diseases not infrequently 
 occur together in the same individual. In making the 
 diagnosis, therefore, all the signs, histological and 
 pathogenic, must be considered and animal inoculations 
 made. 
 
TIMOTHY AND OTHER GRASS BACILLI. 319 
 
 TIMOTHY AND OTHER GRASS BACILLI. 
 
 On various grasses, in cow's manure, in butter, and in 
 milk, there have been discovered a number of varieties 
 of bacteria which have more or less of the characteristics 
 of the tubercle bacillus. Some of them are as difficult to 
 stain and as resistant to decolorizing action of mineral 
 acids and alcohol as the tubercle bacillus found in man. 
 Many of them are of the same general size and shape as 
 the tubercle bacillus,, and, strangely enough, produce 
 in animals small diseased areas, which not only macro- 
 scopically but also microscopically resemble miliary 
 tubercles due to the tubercle bacillus. They, however, 
 are entirely different in their culture characteristics, 
 producing in twenty-four to forty-eight hours on ordi- 
 nary culture media moist, round colonies of an eighth 
 to a quarter of an inch in diameter, and of a more or 
 less intense pink color. In animals they produce only 
 localized lesions, causing death only when injected in 
 large numbers. The injected animals are unaffected 
 by tuberculin injections. The chief interest which 
 these bacilli have for us is the possibility of confusing 
 them with the tubercle bacilli. This danger is always 
 present in milk, for the grass bacilli find so many 
 means of gaining entrance to it. In the examination 
 of dust, healthy throat and nose secretions, etc., the 
 simple microscopical examination might lead to error. 
 
 They can be separated from tubercle bacilli by in- 
 oculating animals, and then if they show any infection, 
 injecting tuberculin, when if infected with tuberculosis 
 they will die, but if by grass bacilli they will show 
 no reaction. Cultures from the lesions will also show 
 on ordinary media pink colonies if grass bacilli are 
 present, and no growth if only tubercle bacilli. 
 
CHAPTEE XX. 
 
 INFLUENZA BACILLUS. 
 
 AFTER numerous unsuccessful attempts during the 
 epidemic of 1889 and succeeding years to discover the 
 specific cause of influenza, Pfeiffer succeeded in isolating 
 a bacillus (1892) from the purulent bronchial secretion 
 of patients suffering from epidemic influenza which he 
 
 FIG. 40. 
 
 Influenza bacilli. X 1100 diameters. 
 
 showed was the probable cause of the disease. This 
 discovery has since been confirmed by many observers, 
 the results of whose researches give us reason to believe 
 that this bacillus is the chief etiological factor in the 
 production of influenza. 
 
INFLUENZA BACILLUS. 321 
 
 Morphology. Very small, moderately thick bacilli, 
 usually occurring singly or united in pairs, but threads 
 or chains of three, four, or more elements, are occa- 
 sionally found. 
 
 The bacillus stains with difficulty with the ordinary 
 aniline colors best with dilute ZiehPs solution or 
 Loffler's methylene-blue solution, with heat. When 
 faintly stained the two ends of the bacilli are some- 
 times more deeply stained than the middle portion. 
 Those we have examined, all obtained from cases in 
 New York, were not stained by Gram's method, but 
 some report instances in which they were. 
 
 Biological Characters. An aerobic, non-motile bacil- 
 lus; does not form spores; no growth occurs below 
 26 C., or above 43 C., or in the entire absence of oxy- 
 gen. This bacillus is best cultivated at 37 C., and on 
 the surface of the ordinary nutrient culture media con- 
 taining haemoglobin or purulent material. Plain or 
 glycerin-agar, or blood-serum streaked with sputum, 
 pus, or blood, make a good soil for their growth. At 
 the end of eighteen to twenty-four hours in the incu- 
 bator very small, drop-like colonies are developed, 
 which, under a low magnification (100 diameters), ap- 
 pear as shining, transparent, homogeneous masses, and 
 even under a No. 7 lens scarcely show at all the indi- 
 vidual organisms. Older colonies are sometimes col- 
 ored yellowish-brown in the centre. A characteristic 
 feature of the influenza bacillus is that the colonies tend 
 to remain separate from each other, although when they 
 are thickly sown in a film of moist blood upon nutri- 
 ent agar they may become confluent. Transplantation 
 of the original culture to ordinary agar or serum can- 
 not, as a rule, be successfully performed, owing to the 
 
 21 
 
322 BACTERIOLOGY. 
 
 want of sufficient haemoglobin; but if sterile rabbit, 
 pigeon, or human blood be added to these media trans- 
 plantation may be indefinitely performed, provided it is 
 done every three or four days. Cultures may remain 
 alive up to seventeen days in the ice-chest. 
 
 The Detection of the Influenza Bacillus in Sputum. 
 When it is desired to obtain cultures of the bacillus 
 of influenza for diagnostic purposes from material sus- 
 pected to contain this organism, it is advisable from 
 the start to make use of plate cultures, the best medium 
 being nutrient agar freshly smeared with rabbit's blood. 
 The sputum, blood, or other substance to be examined 
 is streaked across several plates of blood-smeared agar, 
 so as to leave on some considerable of the material and 
 on others merely the slightest trace. An easy way to 
 get blood when a large number of plates are to be made 
 is to kill a rabbit and autopsy it immediately. The 
 skin is turned back from the chest and the thorax 
 opened aseptically. The heart is cut off at its base 
 and dragged over some twenty to forty plates, as de- 
 sired. The blood collecting in the thorax is used to 
 smear the agar in a number of tubes, which can be kept 
 in the ice-chest until needed. With a little skill blood 
 .can be withdrawn aseptically from the ear vein of a 
 rabbit by means of a glass tube armed with a hypo- 
 dermic needle. 
 
 When cultures are made from sputa the endeavor 
 should be made to collect the expectoration which comes 
 up naturally, so as not to get any more than necessary 
 of the mouth bacteria. If the mouth is at all foul, it 
 should be cleansed before gathering the sputum. Cul- 
 tures should be made as soon as possible after obtaining 
 the material. The plates are put in the incubator for 
 
INFLUENZA BACILLUS. 323 
 
 eighteen hours and then examined under a magnifica- 
 tion of about 100 diameters. The influenza colonies, 
 when present, will be found in the neighborhood of 
 the blood-cells, much lighter in hue, somewhat smaller, 
 and more finely granular than those of the pneumococci. 
 They appear scarcely more noticeable than the groups of 
 blood-cells that have lost their color and largely disin- 
 tegrated. With higher magnification the colonies do not 
 show the individual bacteria distinctly, and thus con- 
 trast with the pneumococci. The suspicious colonies are 
 fished out, inoculated upon blood and simple nutrient 
 agar, and examined microscopically. When the detec- 
 tion of the bacilli is important, and there are any puru- 
 lent masses in the sputum, as in influenza complicating 
 phthisis, these are washed, as under directions for ex- 
 amination of sputa for mixed infection (page 306). 
 
 On 1.5 per cent, sugar-agar growth also occurs, the 
 colonies appearing as extremely small droplets, clear 
 as water, often only recognizable with a lens (Pfeiffer). 
 In bouillon a very scanty development takes place, un- 
 less blood is added. At the end of twenty-four hours 
 small, white particles are seen on the surface, which 
 subsequently sink to the bottom, forming a white, 
 woolly deposit, while the bouillon remains clear. 
 
 EESISTANCE AND LENGTH OF LIFE. The influenza 
 bacillus is very sensitive to desiccation; a pure culture 
 diluted with water and dried is destroyed with certainty 
 in twenty-four hours; in dried sputum the vitality, 
 according to the completeness of drying, is retained 
 from twelve to forty-eight hours. It does not grow, 
 but soon dies in water. The thermal death-point is 
 60 C. with five minutes' exposure (Pfeiffer and Beck). 
 In bouillon cultures and in sputum at 20 C. they retain 
 
324 BACTERIOLOGY. 
 
 their vitality for from a few days to two or three 
 weeks. 
 
 Pathogenesis. The bacillus of influenza, in so far as 
 experiments show, produces the disease only in monkeys 
 and to a less extent in rabbits. From numerous ex- 
 periments made by Pfeiffer on guinea-pigs, rats, mice, 
 and pigeons these animals seem to be more or less in- 
 susceptible to influenza. When a small quantity of 
 culture on blood-agar, twenty-four hours old, sus- 
 pended in 1 c.c. of bouillon, was injected intrave- 
 nously into rabbits, Pfeiffer found that a characteristic 
 pathogenic effect was produced. The first symptoms 
 were developed in one and a half to two hours after 
 the injection. The animals became extremely feeble, 
 lying flat upon the floor, with their limbs extended, 
 and suffered from extreme dyspnoea. The temperature 
 rose to 41 C. or above. At the end of five or six 
 hours they were able to sit up on their haunches again, 
 and in twenty-four hours had recovered. Larger doses 
 caused the death of the animals inoculated. These 
 results are attributed by Pfeiffer to toxic products 
 present in the cultures, and in none of his experiments 
 was he ever able to obtain effects resembling septicsemic 
 infection. In some of the experiments on monkeys, 
 these animals, when cultures were rubbed into the nasal 
 mucous membrane, showed a febrile condition, lasting 
 for a few days, and in one case an abscess was produced 
 from an injection into the subcutaneous intercellular 
 tissues: but in no instance has Pfeiffer observed a mul- 
 tiplication of the bacilli introduced. Recently Cantani 
 has shown that it is possible to produce an infection of 
 influenza in rabbits when inoculated with small doses 
 ( to J c.c.) of living bacilli, provided the point of least 
 
INFLUENZA BACILLUS. 325 
 
 resistance is chosen for the inoculation viz., the brain, 
 upon which the toxic products of the bacillus influenzae 
 acts most powerfully. 
 
 The cell bodies of the bacilli seem to possess consid- 
 erable pyogenic action. 
 
 Immunity. Possibly an immunity for a short period 
 against the influenza poison may be established after an 
 attack. At least in three experiments made by Pfeiffer 
 on monkeys, these animals, after recovering from an 
 inoculation with bacilli, seemed to be much less suscep- 
 tible to a second injection. 
 
 In patients suffering from influenza the bacilli are 
 found chiefly in the nasal and bronchial secretions. 
 In acute uncomplicated cases they may be observed 
 microscopically in large masses and often in absolutely 
 pure culture; the green, purulent sputum derived from 
 the bronchial tubes is especially suitable for examina- 
 tion. The older the process is the fewer bacilli will be 
 found, and the more frequently will they be seen lying 
 within the pus -cells instead of being embedded free in 
 the secretion as at first. At the same time they stain 
 less readily and present more irregular and swollen 
 forms. Very frequently, perhaps almost invariably 
 (Finkler), the influenza process invades portions of the 
 lung tissue. In severe cases a form of pneumonia is 
 the result, which is lobular and purulent in character, 
 and accompanied by symptoms almost identical with 
 bronchopneumonia due to the pneumococcus. The walls 
 of the bronchioles and alveolar septa become densely 
 infiltrated with leucocytes, and the lumina of the bron- 
 chial tubes and alveoli are similarly filled. The pus- 
 cells are found to contain more or less influenza bacilli. 
 There may be partial softening of the tissues, or even 
 
326 BACTERIOLOGY. 
 
 abscess formation. Bacilli are found in fatal cases 
 to have penetrated from the bronchial tubes not only 
 into the peribronchitic tissue, but even to the sur- 
 face of the pleura, and rarely they have been obtained 
 in pure cultures in the purulent exudation. The pleu- 
 risy which follows influenza, however, is usually a 
 secondary infection, due to the streptococcus or pneu- 
 mococcus. Ordinarily influenza runs an acute or sub- 
 acute course, and not infrequently it is accompanied by 
 mixed infections, with the pneumococcns and the strep- 
 tococcus. Pfeiffer was the first to draw attention to 
 certain chronic conditions depending upon the influenza 
 bacillus. According to this observer, these bacilli may 
 be retained in the lung tissue for months at a time, 
 remaining latent awhile, and then becoming active 
 again, with a resulting exacerbation of the disease. 
 Consumptives are particularly susceptible to attacks of 
 influenza. Williams, in the examination of washed 
 sputa in cases of pulmonary tuberculosis, has on numer- 
 ous occasions found abundant influenza bacilli, and this 
 in the summer, when no influenza was known to be 
 present in New York. Taken together with Pfeiffer's 
 results in Berlin, this indicates that at all times of the 
 year many consumptives carry about with them influ- 
 enza bacilli, and that very likely many healthy persons 
 also harbor a few. Given proper climatic conditions, 
 we have at all times the seed to start an epidemic. 
 
 The influenza bacillus does not occur, as a rule, in 
 the blood. According to Pfuhl and Nauwerck, the 
 influenza bacilli have been found in the interior organs 
 and the brain, but these observations require further 
 confirmation. So far as positive results have shown, 
 influenza would seem to be a local infection confined to 
 
INFLUENZA BACILLUS. 327 
 
 the air-passages; the general symptoms produced are 
 due probably to the absorption of the toxic products of 
 the specific organism, these poisons being particularly 
 active in their effects on the central nervous system. 
 
 The discovery of this bacillus enables us to explain 
 many things, previously unaccountable, in the cause of 
 epidemic influenza. We now know, from the prop- 
 erty of the influenza bacillus not being able to exist for 
 long periods in dust, that the disease is not transmis- 
 sible to great distances through the air. We also 
 know that the infective material is contained only in 
 the catarrhal secretions. Sporadic cases, or the sudden 
 eruption of epidemics in any localities from which the 
 disease has been absent for a long time, or where there 
 has been no new importation of 'infection, may possibly 
 be explained by the fact that the bacilli, as already 
 mentioned, often remain latent in the lungs or bronchial 
 secretions of the body for many months, and perhaps 
 years, and then become active again, when under favor- 
 able circumstances they may be communicated to others. 
 The bacteriological diagnosis of influenza is of consider- 
 able importance for the identification of clinically doubt- 
 ful cases, which, from their clinical symptoms, may be 
 mistaken for grippe, or vice versa, such as bronchitis, 
 pneumonia, or tuberculosis. Up to the present time, 
 however, the diagnosis gives us little help in prognosis 
 or treatment. 
 
 In acute uncomplicated cases the probable diagnosis 
 can be frequently made by microscopical examinations 
 of stained preparations of the sputum, there being pres- 
 ent enormous numbers of small bacilli. In chronic cases 
 or those of mixed infection the culture method usually 
 gives a positive result. The bacillus of influenza is so 
 
328 BACTERIOLOGY. 
 
 well characterized by its morphological, staining, and 
 cultural peculiarities that it may be distinguished with 
 sufficient certainty for practical purposes from all other 
 bacteria by an expert bacteriologist who is familiar 
 with it. The only bacillus which resembles it at all 
 closely is the pseudo-influenza bacillus found by Pfeiffer 
 in three cases of bronchopneumonia. This bacillus is 
 culturally very similar to the true bacillus influenzse, 
 but may be distinguished from it by its larger size and 
 tendency to grow out into long threads. It is not cer- 
 tain but that it is a form of the influenza bacillus. 
 There is no doubt that other infections are also included 
 under the clinical forms of influenza, and during an 
 epidemic bronchopneumonias, irregular types of lobar 
 pneumonias, and cases of bronchitis frequently have 
 symptoms so closely alike that the nature of the bac- 
 teria active in the case is very frequently different from 
 that supposed by the clinician. Thus in four consecu- 
 tive autopsies examined by the writer the influenza 
 bacillus was found almost in pure culture in one case 
 believed to be due to the pneumococcus, and entirely 
 absent in two of the three believed to be due to it. 
 Except for these examinations the clinician would be 
 of the opinion that he had clearly diagnosticated bacte- 
 riologically the cases, while in fact he had been wrong 
 in three of the four. 
 
 The striking symptoms in acute respiratory diseases 
 are frequently more due to the location and amount of 
 the poisons than to the special variety of organisms pro- 
 ducing them. 
 
CHAPTER XXI. 
 
 DIPHTHERIA BACILLUS. 
 
 History. The specific contagious disease which we 
 now call diphtheria, and, therefore, according to our 
 present belief, the bacilli which cause it, can be traced 
 back to almost the Homeric period of Grecian history. 
 The Greeks believed that it had been communicated to 
 their country from Egypt. The description of the 
 pharyngeal and laryngeal manifestations of this dis- 
 ease left by Aretseus leaves no doubt that it was of 
 diphtheria that he wrote. Galen, in his remarks on 
 the Chironion ulcer, tells us that the pseudomembrane 
 was gotten rid of by coughing in the laryngeal form 
 of the disease, and by hawking in the pharyngeal type. 
 From time to time during the next one thousand years 
 we hear of epidemics both in Italy and in other por- 
 tions of the civilized world which indicate that the 
 specific bacteria continued to be handed down from 
 case to case. In 1517 we read of a malignant form 
 of the disease raging in Switzerland, along the Rhine, 
 and in the Netherlands. In 1557 we read of further 
 epidemics in France, Germany, Holland, and Spain. 
 The disease now crossed to America, and in the New 
 England States we get clear accounts of its ravages. 
 Thus, Samuel Danforth, in 1659, Jost four of his eleven 
 children within a fortnight by a ( ' malady of the blad- 
 ders in the windpipe/' In 1765, Home, a Scotchman, 
 
330 BACTERIOLOGY. 
 
 tried to show that " croup " and pharyngeal diphthe- 
 ria were different diseases, or, in bacteriological terms, 
 due to different micro-organisms, and this subject re- 
 mained under controversy until it was recently settled 
 that while most cases were undoubtedly due, at least 
 to a great extent, to diphtheria bacilli, a few were not. 
 
 Bard, an American, supported, in 1771, the opposite 
 theory from Home, considering the process the same 
 wherever located. In this ground he was much nearer 
 to the facts than Home. His observations upon diph- 
 theria were very important and accurate. 
 
 In 1821, Bretoaneau published his first essay on diph- 
 theria in Paris and gave to the disease its present name. 
 His observations were so extensive and so correct that 
 little advance in knowledge took place until the causal 
 relations of the diphtheria bacilli and their associated 
 micro-organisms to the disease began to be recognized. 
 Since then the combined clinical, bacteriological, and 
 pathological studies have sufficed to make diphtheria 
 one of the best understood of diseases. 
 
 The Bacillus. In the year 1883 bacilli which were 
 very peculiar and striking in appearance were shown 
 by Klebs to be of constant occurrence in the pseudo- 
 membranes from the throats of those dying of true 
 epidemic diphtheria. One year later Loftier published 
 the results of a very thorough and extensive series of 
 investigations on this subject. He found the bacillus 
 described by Klebs in many cases of throat inflam- 
 mations which had been diagnosticated as diphthe- 
 ria. He separated these bacilli from the other bac- 
 teria present and obtained them in pure culture. When 
 he inoculated the bacilli upon the abraded mucous mem- 
 brane of susceptible animals, more or less characteristic 
 
DIPHTHERIA BACILLUS. 331 
 
 pseudomembranes were produced, and frequently death 
 or paralysis followed, with characteristic lesions. 
 
 In 1887 and 1888 further studies by Loffler, Eoux, 
 and Yersin added to the proof of the dependence of 
 diphtheria on this bacillus. It was found that while 
 no other forms of bacteria were constantly met with, 
 the diphtheria bacilli were present in all characteristic 
 cases of diphtheria, and that these bacilli possessed the 
 morphological, cultural, and pathogenic qualities of 
 those described by Klebs and Loffler. The results of 
 these investigations have since been confirmed by a 
 great number of combined clinical and bacteriological 
 observations both in animals and human beings. A 
 very instructive accidental experiment was carried out 
 under my observation some years ago. One of the 
 laboratory workers unintentionally sucked through a 
 defective pipette a few drops into the mouth of a 
 bouillon culture of a virulent diphtheria bacillus, and 
 two days later characteristic diphtheria of a serious type 
 developed. All the conditions have been fulfilled for 
 diphtheria which are necessary to the most rigid proof 
 of the dependence of an infective disease upon a given 
 micro-organism viz., the constant presence of this 
 organism in the lesions of the disease, the isolation of 
 the organism in pure culture, the reproduction of the 
 essential lesions of the disease in animals and in man 
 by inoculation with pure cultures, the failure to produce 
 all the characteristic lesions of this disease by any other 
 bacteria, and the additional proof of the immunizing 
 value of the specific substances developed in animals 
 subjected to injections of diphtheria toxin. In view of 
 these facts we are now justified in saying that the name 
 diphtheria, or at least primary diphtheria, should be 
 
332 
 
 BACTERIOLOGY. 
 
 applied, and exclusively applied, to that acute infectious 
 disease usually associated with pseudomembranous affec- 
 tion of the mucous membranes which is primarily 
 caused by the bacillus diphtherise of Loffler. Other 
 bacteria do, indeed, occasionally produce lesions which 
 simulate in one way or another those caused by the 
 diphtheria bacillus, but none of them ever produce 
 lesions similar in their totality to those of a charac- 
 teristic case of diphtheria. 
 
 Morphology. When cover-glass preparations made 
 from the cultures grown on blood-serum are examined 
 the diphtheria bacilli are found to possess the following 
 morphological characteristics : The diameter of the 
 bacilli varies from 0.2^ to 0.8/j. and the length usually 
 
 FIG. 41. 
 
 FIG. 42. 
 
 One of very characteristic forms ot 
 diphtheria bacilli from blood-serum 
 cultures, showing clubbed ends and 
 irregular stain. X 1100 diameters 
 Stain, methylene-blue. 
 
 Extremely long form of diphtheria 
 bacillus. This culture has grown on 
 artificial media for four years and 
 produces strong toxin. X 1100 diam- 
 eters. 
 
 from I/jt to 6//, but exceptionally even longer (see 
 Fig. 42). They occur singly and in pairs (see Figs. 41 
 to 44), and very infrequently in chains of three or four. 
 At times, especially in the tissues, branching forms are 
 
DIPHTHERIA BACILLUS. 
 
 333 
 
 observed. The rods are straight or slightly curved, and 
 usually are not uniformly cylindrical throughout their 
 entire length, but are swollen at the end or pointed at 
 
 FIG. 43. 
 
 FIG. 44. 
 
 Diphtheria bacilli characteristic in 
 shapes but showing even staining. In 
 appearance similar to the xerosis ba- 
 cillus. X 1100 diameters. Stain, me- 
 thylene-blue. 
 
 Non-virulent diphtheria bacilli, 
 showing stain with Neisser's solu- 
 tions, supposed to be characteristic 
 of virulent bacilli. Bodies of bacilli 
 in smear, faint brown ; points, dark 
 blue. 
 
 FIG. 45. 
 
 Small type of pseudodiphtheria bacilli. X 1000 diameters. 
 
 the ends and swollen in the middle portion. The 
 average length of the bacilli in cultures from different 
 sources frequently varies greatly, and even from the 
 
334 BACTERIOLOGY. 
 
 same culture individual bacilli differ much in their 
 size and shape. The two bacilli of a pair may lie 
 with their long diameter in the same axis or at an 
 obtuse or an acute angle, or the pairs of bacilli may 
 lie side by side or irregularly across each other. The 
 bacilli possess no spores, but have in them highly 
 refractile bodies at certain stages in their life. 
 
 The Klebs-Loffler bacilli stain readily with ordinary 
 aniline dyes, and retain fairly well their color after 
 staining by Gram's method. When Loffler's alkaline 
 solution of methylene-blue is applied cold for five min- 
 utes or warm for one minute the bacilli from blood- 
 serum cultures especially, and from other media less 
 constantly, stain in an irregular and extremely charac- 
 teristic way (see Fig. 41). Many of the bacilli do not 
 stain uniformly. In many cultures round or oval 
 bodies situated at the ends or in the central portions 
 stain much more intensely than the rest of the bacillus. 
 Sometimes these highly stained bodies are thicker than 
 the rest of the bacillus; again, they are thinner and 
 surrounded by a more slightly stained portion. The 
 bacilli seem to stain in this peculiar manner at a cer- 
 tain period of their growth, and more when grown on 
 some media than on others, so that only a portion of 
 the organisms taken from a culture at any one time 
 will show the characteristic staining. In old cultures 
 the bacilli stain poorly and not at all in a characteristic 
 way. The same round or oval bodies which take the 
 methylene-blue more intensely than the remainder of 
 the bacillus are brought out still more distinctly by the 
 Neisser stain. 
 
 The Neisser stain is carried out by placing the cover- 
 slip smear of diphtheria or other bacilli in solution No. 
 
DIPHTHERIA BACILLUS. 335 
 
 1 for from two to three seconds, and then, after washing, 
 in No. 2 for from three to five seconds. The bacilli 
 will then appear either entirely brown or will show at 
 one or both ends a dark-blue round body. With char- 
 acteristic diphtheria bacilli taken from a twelve to 
 eighteen hours' growth on serum nearly all will show 
 the blue bodies (Fig. 44), while with the pseudotype 
 (Fig. 45), to be described hereafter, few, if any, will 
 be seen. 
 
 The solutions are as follows : 
 
 No 1. 
 
 Alcohol (96 per cent. ) . . . . 20 parts 
 
 Methylene blue (Griibler) . . .- 1 part 
 
 Distilled water . *. . . . 950 parts. 
 
 Acetic acid (glacial) ... . 50 " 
 
 No. 2. 
 
 Bismark brown . . . . . 1 part. 
 Boiling distilled water ... . 500 parts. 
 
 The Neisser stain has been advocated in order to 
 separate the virulent from the non-virulent bacilli 
 without the delay of inoculating animals; but in our 
 hands, with a very large experience, neither the Neisser 
 stain nor other stains, such as the modifications of the 
 Roux stain, have given any more information as to the 
 virulence of the bacilli than the usual methylene-blue 
 solution of Loffler. A small percentage of virulent 
 bacilli fail to take the Neisser stain, and quite a few 
 non-virulent psetidodiphtheria bacilli show the dark 
 bodies. In New York there are also a large number 
 of bacilli which seem to have all the staining and cul- 
 tural characteristics of the virulent bacilli, and yet are 
 non-virulent in the sense that they produce no specific 
 
336 BACTERIOLOGY. 
 
 toxin. To one who is accustomed to the Loffler stain 
 it gives as much information as any other as to the 
 specific virulence of the bacilli. The Neisser stain will 
 undoubtedly cause the examiner to suspect more strongly 
 some bacilli of being virulent than the Loffler stain, 
 but with the varieties met with in New York this sus- 
 picion is as apt to be wrong as right. As will be stated 
 more fully later, nothing but the animal inoculations 
 with control injections of antitoxin will separate spe- 
 cifically virulent from non-virulent bacilli. 
 
 The morphology of the diphtheria bacillus varies con- 
 siderably with the different culture media employed. 
 On glycerin agar or simple nutrient agar it is smaller, 
 and, as a rule, more regular in form than when grown 
 on other usual culture media (Fig. 46). Short, spindle, 
 
 PIG. 46. 
 
 Diphtheria bacilli from agar culture. X 1000 diameters. 
 
 lancet, or club-shaped forms, staining uniformly, are 
 here commonly observed. The bacilli which have de- 
 veloped in the pseudomembranes or exudate in cases of 
 diphtheria resemble in shape those grown on blood- 
 serum, but stain more evenly. 
 
DIPHTHERIA BACILLUS. 337 
 
 But though the morphology of the diphtheria bacillus 
 is more regular under some circumstances than others, 
 its chief morphological characteristic is its irregularity 
 of form and size. 
 
 Biology. The Klebs-Loffler bacillus is non-motile 
 and non-liquefying. It is aerobic. It grows most 
 readily in the presence of oxygen, but also without 
 it; it is thus facultative anaerobic. It does not form 
 spores. Its thermal death-point with ten minutes' 
 exposure is about 58 C., and with longer exposure a 
 lower temperature; it is more easily destroyed by dis- 
 infectants than many other bacteria. In the dry state 
 and exposed to diffuse light diphtheria bacilli usually 
 die in a few days or may live for weeks or months; 
 when in the dark, or protected by a film of mucus or 
 albumin, they may live for even longer periods. Thus 
 I found scrapings from a dry bit of membrane to 
 contain vigorous and virulent living bacilli for a 
 period of four months after removal from the throat, 
 and if the membrane had not been at that time com- 
 pletely used, living bacilli could probably have been 
 obtained for a much longer period; in culture media 
 when kept at the blood heat they usually die after a 
 few weeks, but under certain conditions, as when sealed 
 in tubes and protected from heat and light, they retain 
 their virulence for years. The bacillus is not sensitive 
 to cold, for I found it to retain its virulence after ex- 
 posure for two hours to several hundred degrees below 
 zero. It begins to develop, but grows slowly, at a 
 temperature of 20 C., or even less. It grows more 
 rapidly as the temperature rises, and attains its maximum 
 development at 37 C. It may grow at a temperature 
 as high as 41 C. and retain its virulence for months. 
 
 22 
 
338 BACTERIOLOGY. 
 
 Growth on Blood-serum. Blood-serum, especially in 
 the form of L6 frier's mixture, is the most favorable 
 medium for the growth of the diphtheria bacillus, 
 and is used particularly for diagnostic purposes in 
 examining cultures from the throats of persons sus- 
 pected of having diphtheria. For its preparation, 
 see p. 377. If we examine the growth of the diph- 
 theria bacillus in pure culture on blood-serum we shall 
 find at the end of from eight to twelve hours small colo 
 nies of bacilli, which appear as pearl-gray, whitish-gray, 
 or, more rarely, yellowish-gray, slightly raised points. 
 The colonies when separated from each other may in- 
 crease in forty-eight hours so that the diameter may 
 be one-eighth of an inch. The borders are usually 
 somewhat uneven. The colonies lying together be- 
 come confluent and fuse into one mass, when the 
 serum is moist. During the first twelve hours the 
 colonies of the diphtheria bacilli are about equal in size 
 to those of the other pathogenic bacteria which are often 
 present in the throat; but after this time the diphtheria 
 colonies become larger than those of the streptococci and 
 smaller than those of the staphylococci. The diph- 
 theria bacilli in their growth never liquefy the blood- 
 serum. 
 
 Growth on Agar. On 1 per cent, slightly alkaline, 
 plain nutrient or glycerin-agar the growth of the 
 diphtheria bacillus is less certain and luxuriant than 
 upon blood-serum; but the appearance of the colonies 
 when examined under a low-power lens, though very 
 variable, is often far more characteristic. (See Fig. 30, 
 page 229, and Fig. 47, page 339.) The diphtheria 
 bacillus obtained from cultures which have developed 
 for some time on culture media grows well, as a rule, 
 
DIPHTHERIA BACILLUS. 339 
 
 on suitable nutrient or glycerin-agar, but when fresh 
 from pseudomembranes it frequently grows on these 
 media with great difficulty, and the colonies develop 
 so slowly as to be covered up by the more luxuriant 
 growth of other bacteria, or fail to develop at all. 
 
 FIG. 47. 
 
 Colonies of diphtheria bacilli. X 200 diameters. 
 
 If the colonies develop deep in the substance of the 
 agar they are usually round or oval, and, as a rule, pre- 
 sent no extensions; but if near the surface, commonly 
 from one, but sometimes from both sides, they spread 
 out an apron-like extension which exceeds in surface 
 area the rest of the colony. When the colonies develop 
 entirely on the surface they are more or less coarsely 
 granular, and usually have a dark centre and vary 
 very much in their thickness. Some are almost trans- 
 lucent; others are thick and almost as luxuriant as the 
 staphylococcus. The edges are sometimes jagged and 
 frequently shade off into a delicate lace like fringe; at 
 other times the margins are more even and the colonies 
 
340 BACTERIOLOGY. 
 
 are nearly circular. With a high-power lens the edges 
 show sprouting bacilli. The colonies are gray or 
 grayish-white by reflected light and pure gray with 
 an olive tint by transmitted light. 
 
 The growth of the diphtheria bacillus upon agar 
 presents certain peculiarities which are of practical 
 importance. If a large number of the bacilli from a 
 recent culture are implanted upon a properly prepared 
 agar plate a certain and fairly vigorous growth will 
 always take place. If, however, the agar is inoculated 
 with an exudate from the throat which contains but 
 few bacilli, no growth whatever may occur, while the 
 tubes of coagulated blood-serum inoculated with the 
 same exudate contain the bacilli abundantly. Again, 
 agar prepared from broth made from different speci- 
 mens of beef or to which different peptones have been 
 added, varies as to its suitability for the growth of the 
 bacilli. Because of the uncertainty, therefore, of ob- 
 taining a growth by the inoculation of agar with bacilli 
 unaccustomed to this medium, agar is a far less reliable 
 medium than blood-serum for use in primary cultures 
 for diagnostic purposes. If used the agar should at 
 least be tested by means of a culture before being em- 
 ployed. A mixture composed of two parts of a 1J 
 per cent, nutrient agar and one part of sterile ascitic 
 fluid makes a medium upon which the bacillus grows 
 much more luxuriantly but not so characteristically. 
 The mixture is made by adding the warmed ascitic 
 fluid to the tubes containing the melted agar cooled to 
 60. After shaking the Petri plates are filled. 
 
 The Isolation of the Diphtheria Bacillus from Plate 
 Cultures. Nutrient plain or glycerin-agar, with or 
 without the addition of ascitic fluid, is, however, the 
 
DIPHTHERIA BACILLUS. 341 
 
 medium employed to get by plating methods a pure 
 culture from the original serum tube. The agar should 
 be freshly melted and poured in the Petri dish for this 
 purpose. After it has hardened the layers in a number 
 of plates are streaked across with bacteria from colonies 
 on the serum culture, which appear in size and color 
 like the diphtheria bacilli. Other plates are made from 
 a general mixture of all the bacteria, selected, as a rule, 
 from the drier portion of the serum. The plates are left 
 in the incubator for twelve hours at 37 C. In the 
 examination of the plates one should first seek for 
 typical colonies and then later for any that look nearest 
 the characteristic picture. Diphtheria colonies are very 
 apt to be found at the edges of the streaks of bacterial 
 growth. 
 
 Growth in Bouillon. The diphtheria bacillus usu- 
 ally grows readily in broth slightly alkaline to litmus. 
 The characteristic growth in neutral bouillon is one 
 showing fine grains. These deposit along the slles and 
 bottom of the tube, leaving the broth nearly clear. A 
 few cultures in neutral bouillon and many in alkaline 
 bouillon produce for twenty-four or forty-eight hours 
 a more or less diffuse cloudiness, and frequently a film 
 forms over the surface of the broth. On shaking the 
 tube this film breaks up and slowly sinks to the bottom. 
 This film is more apt to develop during the growth of 
 cultures which have long been cultivated in bouillon, and 
 indeed after a time the entire development may appear 
 on the surface in the form of a friable pedicle. The 
 diphtheria bacillus in its growth causes a fermentation 
 of the meat sugars and the glucose, and thus changes 
 the reaction of the bouillon, rendering it distinctly less 
 alkaline within forty-eight hours, and then, after a vari- 
 
342 BACTERIOLOGY. 
 
 able time, when all the fermentable sugars have been 
 decomposed, more alkaline again through the progress- 
 ing fermentation of other substances. Among the 
 products formed by its growth is the diphtheria toxin. 
 
 Growth in Ascitic Bouillon. Many diphtheria bacilli 
 grow but feebly in nutrient bouillon when first re- 
 moved from the throat. These develop more luxuri- 
 antly when to the bouillon 25 per cent, aseitic fluid or 
 blood-serum is added. 
 
 Growth on Gelatin. The growth on this medium 
 is much slower, more scanty, and less characteristic than 
 that on the other media mentioned, on account of the 
 lower temperature at which it is used. 
 
 Growth in Milk. The diphtheria bacillus grows 
 readily in milk, beginning to develop at a compara- 
 tively low temperature (20 C.). Thus milk having 
 become inoculated with the bacillus from some cases of 
 diphtheria may under certain conditions be the means 
 of conveying infection to previously healthy persons. 
 Though this growth takes place, the milk remains un- 
 changed in appearance. 
 
 Pathogenesis. The diphtheria bacillus is pathogenic 
 for guinea-pigs, rabbits, chickens, pigeons, small birds, 
 and cats; also in a lesser degree for dogs, goats, cattle, 
 and horses, but hardly at all for rats and mice. In 
 spite of its pathogenic qualities for these animals true 
 diphtheria occurs in them with extreme rarity. As a 
 rule, supposed diphtheritic inflammations in them are 
 due to other bacteria which cannot produce the disease 
 in man. 
 
 The virulence of diphtheria bacilli from different 
 sources, as measured by their toxin production, varies 
 enormously. Thus 0.002 c.c. of a forty-hour bouillon 
 
DIPHTHERIA BACILLUS. 343 
 
 culture of one bacillus will kill a guinea-pig, while it 
 would require 1 c.c. of the culture of another bacillus 
 to kill. The same marked variation occurs in the 
 amount of toxin produced by different bacilli in their 
 growth in media outside of the body. There are also 
 bacilli which produce no specific toxin whatever and 
 yet appear to have all the other characteristics of viru- 
 lent bacilli. Moreover, the diphtheria bacilli differ 
 greatly in the tenacity with which they retain their 
 virulence when grown outside the body. The bacillus 
 that we have used in the laboratory of the health de- 
 partment has retained its virulence unaltered for four 
 years in frequently renewed bouillon cultures. Other 
 bacilli have lost 50 per cent, of their virulence after 
 being kept for only a few months. The passage of 
 diphtheria bacilli through the bodies of susceptible 
 animals does not increase their virulence to any con- 
 siderable extent, this being probably due to the fact 
 that the bacilli multiply but little in the tissues. 
 
 At the autopsy of animals dying from the poisons 
 produced by the bacilli the characteristic lesions de- 
 scribed by Loftier are found. At the seat of inocula- 
 tion there is a grayish focus surrounded by an area of 
 congestion; the subcutaneous tissues for some distance 
 around are oedematous; the adjacent lymph-nodes are 
 swollen; and the serous cavities, especially the pleural 
 and the pericardial, frequently contain an excess of 
 fluid, usually clear, but at times turbid; the lungs are 
 generally congested. In the organs are found numer- 
 ous smaller and larger masses of necrotic cells, which 
 are permeated by leucocytes. The heart and voluntary 
 muscular fibres usually show degenerative changes. 
 Occasionally there is fatty degeneration of the liver and 
 
344 BACTERIOLOGY. 
 
 kidneys. The number of leucocytes in the blood is in- 
 creased. From the area surrounding the point of inocu- 
 lation virulent bacilli may be obtained, but in the in- 
 ternal organs they are only occasionally found, unless an 
 enormous number of bacilli have been injected. Paral- 
 ysis, commencing usually in the posterior extremities 
 and then gradually extending to other portions of the 
 body and causing death by paralysis of the heart or 
 respiratory organs, is also produced in many cases in 
 which ihe inoculated animals do not succumb to a too 
 rapid intoxication. In rare instances the muscles of 
 the neck or of the larynx are first paralyzed, and thus 
 characteristic symptoms are caused. In a number of 
 animals I have seen recovery take place three to six 
 weeks after the onset of the paralysis. 
 
 Diphtheria Toxin. It is evident that a micro organ- 
 ism which, when injected subcutaneonsly, destroys the 
 life of susceptible animals and produces such marked 
 anatomical changes in the internal organs, while it is 
 found only at or near the point of inoculation, must 
 owe its pathogenic power to the formation of a poison 
 which, being absorbed, gives rise to toxaemia and death. 
 This poison or toxin has been partially isolated by Roux 
 and Yersin, and others, by filtration through porous 
 porcelain from cultures of the living bacilli. It has not 
 yet been successfully analyzed, so that its chemical 
 composition is unknown, but it has many of the prop- 
 erties of proteid substances, and can well be designated 
 by the term active proteid (see page 72). Diphtheria 
 toxin is totally destroyed by boiling for five minutes, 
 and loses some 95 per cent, of its strength when ex- 
 posed to 75 C. for the same time ; 73 C. destroys 
 only about 85 per cent, and 60 very little. Lower 
 
DIPHTHERIA BACILLUS. 345 
 
 temperatures only alter it very gradually. Kept from 
 light and air and in cold storage it keeps almost un- 
 altered for years. 
 
 The Production of Toxin in Culture Media. The arti- 
 ficial production of toxin in cultures of the diphtheria 
 bacillus has been found to depend upon definite condi- 
 tions, which are of practical importance in obtaining 
 toxin for the inoculation of horses, and also of theoret- 
 ical interest in explaining why cases of apparently equal 
 local severity have such different degrees of toxic ab- 
 sorption. The researches of Roux and Yersin laid the 
 foundation of our knowledge. Their investigations have 
 been continued by Theobald Smith, Spronck, ourselves, 
 and others. After an extensive series of investigations 
 we (Park and Williams) came to the following conclu- 
 sions : Toxin is produced by fully virulent diphtheria 
 bacilli at all times during their life when the conditions 
 are favorable. Under less favorable conditions some 
 bacilli are able to produce toxin while others are not; 
 or it may be that some conditions favor some bacilli 
 while they are deleterious to others. Diphtheria ba- 
 cilli may find conditions suitable for luxuriant growth, 
 but unsuitable for the production of toxin. The re- 
 quisite conditions for a good development of toxin, as 
 judged by the behavior of a number of cultures, are a 
 temperature from about 35 to 37.5 C., a suitable cul- 
 ture medium, such as a 2 per cent, peptone nutrient 
 bouillon of an alkalinity which should be about 8 c.c. 
 of normal soda solution per litre above the neutral 
 point to litmus, and prepared from a suitable peptone 
 and meat. The culture fluid should be in compara- 
 tively thin layers and in large-necked Erlenmeyer 
 flasks, so as to allow of a free access of air. The 
 
346 BACTERIOLOGY. 
 
 greatest accumulation of toxin in bouillon is after a 
 duration of growth of the culture of from five to ten 
 days, according to the peculiarities of the culture em- 
 ployed. At a too early period toxin has not sufficiently 
 accumulated, at a too late period it has begun to de- 
 generate. In our experience the amount of muscle 
 sugar present in the meat makes no appreciable differ- 
 ence in the toxin produced, so long as the bouillon has 
 been made sufficiently alkaline to prevent the acid 
 produced by the fermentation of the sugar from pro- 
 ducing in the bouillon an acidity sufficient to inhibit the 
 growth of the bacilli. In neutral bouillon, as pointed 
 out by Smith and Spronck, the sugar does produce suffi- 
 cient acid to interfere with the growth of the bacilli 
 and the development of toxin. This can be prevented 
 by the previous destruction of the sugar through the 
 fermentation caused by the growth of the colon bacilli. 
 After the fermentation 0.1 per cent, of glucose should 
 be added. Beside the sugar and allied bodies in the 
 meat there are other substances, whose nature is un- 
 known, which hinder or aid a full growth of the bacilli 
 or production of toxin. This is true of bouillon made 
 directly from fresh meat, fermented meat, or meat ex- 
 tracts. With the meat as we obtain it in New York we 
 get better results with uufermented meat than with fer- 
 mented. In Boston, with the same bacillus, Smith 
 gets more toxin from the fermented bouillon. Con- 
 tradictory results have been obtained by others, and 
 must be attributed to the difference in the materials used. 
 Under the best conditions we can devise, toxin begins 
 to be produced by bacilli from some cultures when 
 freshly sown in bouillon some time during the first 
 twenty-four hours; from other cultures, for reasons not 
 
DIPHTHERIA BACILLUS. 347 
 
 well understood, not for from two to four days. In 
 neutral bouillon the culture fluid frequently becomes 
 slightly acid and toxin production may be delayed for 
 from one to three weeks. The greatest accumulation 
 of toxin is on the fourth day, on the average, after the 
 rapid production of toxin has commenced. After that 
 time the number of living bacilli rapidly diminishes 
 in the culture, and the conditions for those remaining 
 alive are not suitable for the rapid production of toxin. 
 As the toxin is not stable, the deterioration taking place 
 in the toxin already produced is greater than the amount 
 of new toxin still forming. 
 
 Bacilli, when repeatedly transplanted from bouillon 
 to bouillon, gradually come to grow on the surface 
 only. This characteristic seems to aid in the develop- 
 ment of toxin. 
 
 The relations of toxin to antitoxin will be described 
 after the subject of antitoxin has been considered. 
 
 Non-virulent Diphtheria Bacilli. Xerosis Bacilli. In 
 the very large number of tests for virulence of the 
 bacilli obtained from hundreds of cases of suspected 
 diphtheria which have been carried out during the 
 past six years in the laboratories of the Health De- 
 partment of New York City, in over 95 per cent, of 
 cases the bacilli derived from exudates or pseudomem- 
 branes and possessing the characteristics of the Loffler 
 bacilli have been found to be virulent, that is producers 
 of diphtheria toxin. But there are, however, in inflamed 
 throats as well as in healthy throats, either alone or 
 associated with the virulent bacilli, occasionally bacilli, 
 which though morphologically and in their behavior on 
 culture media identical with the Klebs-Loffler bacillus, 
 yet producers, at least in artificial culture media and 
 
348 BACTERIOLOGY. 
 
 the usual test animals, of no appreciable diphtheria 
 toxin. Between bacilli which produce a great deal 
 of toxin and those which apparently produce none 
 we find all grades of virulence. We believe, there- 
 fore, that in accordance with Roiix and Yersiri these 
 bacilli should be considered as attenuated varieties 
 of the diphtheria bacillus which have lost their power 
 to produce diphtheria toxin. These observers, and 
 others following them, have shown that the virulent 
 bacilli can be artificially attenuated by cultivating them 
 at a temperature of 39.5 to 40 C. in a current of air. 
 So far as we know, bacilli which produce no specific 
 toxin have never later been found to develop it. In our 
 experience some cultures hold their virulence even when 
 grown at 41 C. for a number of months, while others 
 lose it more quickly. Bacilli are also found which 
 resemble diphtheria bacilli very closely except in toxin 
 production, but differ in one or more particulars. Both 
 these and the characteristic non virulent bacilli are found 
 occasionally upon all the mucous membranes, both when 
 inflamed and when apparently normal. From varieties 
 of this sort having been found in a number of .cases 
 of the condition known as xerosis conjunctivas by 
 Kuschbert and Neisser, these bacilli are often called 
 xerosis bacilli. Under this name different observers 
 have placed bacilli identical with the diphtheria bacilli 
 and others differing quite markedly from them. Fig. 
 43, though taken from virulent bacilli, gives an exact 
 picture of many of the xerosis variety. These bacilli 
 may be almost non-pathogenic in guinea-pigs, or they 
 may kill, as we have found in a number of instances, 
 in doses of 2 to 5 c.c. hypodermatically injected. Ani- 
 mals are not protected by diphtheria antitoxin from 
 
DIPHTHERIA BACILLUS. 349 
 
 the action of these bacilli. At autopsy the bacilli are 
 usually found more or less abundantly in the blood and 
 internal organs. In this very same location, however, 
 diphtheria bacilli are found of very low toxic power, 
 so that here, again, we cannot assert that these xerosic 
 bacilli have not come from true diphtheria stock. 
 
 Location of Diphtheritic Inflammations and Viru- 
 lence of Bacilli. Virulent bacilli produce and are 
 found not only in pseudomembranous inflammations 
 of the fauces, larynx, and nasal cavities, but also occa- 
 sionally in membranous affections of the skin, vagina, 
 rectum, conjunctiva, nose, and ear (simple mem- 
 branous rhinitis and otitis media). From the severity 
 of an isolated case the virulence of the bacilli cannot be 
 accurately determined. The most virulent bacillus I 
 have ever found was obtained from a mild case of diph- 
 theria simulating tonsillitis. Another case, however, 
 infected by this bacillus proved to be very severe. In 
 localized epidemics the average severity of the cases 
 probably indicates roughly the virulence of the bacillus 
 causing the infection, as here the individual susceptibil- 
 ity of the different persons infected would, in all likeli- 
 hood, when taken together, be similar to that of other 
 groups; but even in this instance special conditions of 
 climate, food, or race may influence certain localities. 
 Moreover, the bacteria associated with the diphtheria 
 bacilli, and which are liable to be transmitted with them, 
 may influence the severity of and the complications 
 arising in the cases. 
 
 Virulent Bacilli in Healthy Throats. Fully virulent 
 bacilli have frequently been found in healthy throats 
 of persons who have been brought in direct contact 
 with diphtheria patients or infected clothing without 
 
350 BACTERIOLOGY. 
 
 contracting the disease. It is, therefore, apparent that 
 infection in diphtheria, as in other infectious diseases, 
 requires not only the presence of virulent bacilli, but 
 also a susceptib lity to the disease, which may be 
 inherited or acquired. Among the predisposing in- 
 fluences which contribute to the production of diph- 
 theritic infection may be mentioned the breathing of 
 foul air and living in overcrowded and ill-ventilated 
 rooms, poor food, certain diseases, more particularly 
 catarrhal inflammations of the mucous membranes, and 
 depressing conditions generally. Under these condi- 
 tions an infected mucous membrane may become sus- 
 ceptible to disease. In connection with Beebe (1894) 
 
 1 made an examination of the throats of 330 healthy 
 persons who had not come in contact, so far as known, 
 with diphtheria, and we found virulent bacilli in 8 only, 
 
 2 of whom later developed the disease. In 24 of the 330 
 healthy throats non-virulent bacilli or attenuated forms 
 of the diphtheria bacillus were found. Very similar 
 observations have been made by others in many widely 
 separated countries. 
 
 The Persistence of Diphtheria Bacilli in the Throat. 
 The continued presence of virulent diphtheria bacilli 
 in the throats of patients who have recovered from the 
 disease, and after the disappearance of the exndate, has 
 been repeatedly demonstrated. Beebe and I found that 
 in 304 of 605 consecutive cases the bacilli disappeared 
 within three days after the disappearance of the pseudo- 
 membrane; in 176 cases they persisted for seven days, 
 in 64 cases for twelve days, in 36 cases for fifteen days, 
 in 12 cases for three weeks, in 4 cases for four weeks, 
 and in 2 cases for nine weeks. Since then 1 have met 
 with a case in which they persisted for six months. 
 
DIPHTHERIA BACILLUS. 351 
 
 Pseudodiphtheria Bacilli. Beside the typical baciili 
 which produce diphtheria toxin and those which do not, 
 but which, so far as we can determine, are otherwise iden- 
 tical with the Loffler bacillus, there are other bacilli 
 found in positions similar to those in which diphtheria 
 bacilli abound, which, though resembling these organ- 
 isms in many particulars, yet differ from them as a class 
 in others equally important. The variety most preva- 
 lent is rather short, plump, and more uniform in size 
 and shape than the true Loffler bacillus (Fig. 45). On 
 blood-serum their colony growth is very similar to that 
 of the diphtheria bacilli. The great majority of them 
 in any culture show no polar granules when stained 
 by the Neisser method, and stain evenly throughout 
 with the alkaline rnethylene-blue solution. They do 
 not produce acid by the fermentation of glucose, as 
 do all known virulent and many non-virulent diph- 
 theria bacilli; therefore, there is no increase in acidity 
 in the bouillon in which they are grown during the 
 first twenty-four hours from the fermentation of the 
 meat sugar regularly present. They are found in vary- 
 ing abundance in different localities in about 1 per 
 cent, of the normal throat and nasal secretions, in New 
 York City, and seem to have now at least no con- 
 nection with diphtheria; whether they were originally 
 derived from diphtheria bacillus is doubtful; they cer- 
 tainly seem to have no connection with it now. They 
 never produce diphtheria toxin, and to them properly 
 has been applied the name pseudodiphtheria bacilli. In 
 bouillon they grow, as a rule, less luxuriantly than the 
 diphtheria bacilli. Some of the varieties of the psewdo- 
 diphtheria bacilli are as long as the shorter forms of 
 the virulent bacilli. When these are found in cultures 
 
352 BACTERIOLOGY. 
 
 from cases of suspected diphtheria they may lead to an 
 incorrect diagnosis. The Neisser staining method is of 
 value here, but, unfortunately, the absence of the stained 
 bodies is not a sufficient ground to exclude the possi- 
 bility of their being true diphtheria bacilli. There are 
 also some varieties which resemble the short pseudo- 
 bacilli in form and staining, but which produce acid in 
 glucose bouillon. These bacilli are found occasionally 
 in all countries where search has been made for them. 
 It may be added here that no facts have come to light 
 which indicate that bacilli which do not produce diph- 
 theria toxin in animals ever produce it in man. It 
 must also be borne in mind, however, that such proof 
 is necessarily very difficult to obtain. 
 
 Mixed Infection in Diphtheria. Virulent diphtheria 
 bacilli, however, are not the only bacteria present in 
 human diphtheria. Various cocci, more particularly 
 streptococci, staphylococci, and pneumococci, are almost 
 always found associated with Loffler bacilli in diph- 
 theria, playing an important part in the disease and 
 leading often to serious complications (sepsis and bron- 
 chopneumonia). Indeed, the prognosis in a case of 
 diphtheria is now judged to be graver, other things 
 being equal, according to the degree in which other 
 pathogenic bacteria influence the course of the disease. 
 These cases of so-called mixed infection in diphtheria 
 have within recent years attracted considerable atten- 
 tion, and have been the subject of a number of animal 
 experiments. Though the results of these investiga- 
 tions so far have been somewhat indefinite, they would 
 seem to iudicate that when other bacteria are associated 
 with the diphtheria bacilli they mutually assist one 
 another in their attacks upon the mucous membrane, 
 
DIPHTHERIA BACILLUS. 353 
 
 the streptococcus being particularly active in this 
 respect, often opening the way for the invasion of the 
 Loffler bacillus into the deeper tissues or supplying 
 needed conditions for the development of its toxin. 
 Thus diphtheria is not always a primary, but often a 
 secondary disease, following some other infection, as 
 measles or scarlet fever. In most fatal cases of bron- 
 chopneumonia following laryngeal diphtheria we find 
 not only abundant pneumococci or streptococci in the 
 inflamed lung areas, but also in the blood and tissues 
 of the organs. As these septic infections due to the 
 pyogenic cocci are in no way influenced by the diph- 
 theria antitoxin, they frequently are the cause of the 
 fatal termination. Other bacteria cause putrefactive 
 changes in the exudate, producing alterations in color 
 and offensive odors. 
 
 Pseudomembranous Exudative Inflammations Due to 
 Bacteria other than the Diphtheria Bacilli. The diph- 
 theria bacillus, though the most usual, is not the only 
 micro-organism that is capable of producing pseudo- 
 membranous inflammations. There are numerous bac- 
 teria present almost constantly in the throat secretions, 
 which, under certain conditions, can (ause local lesions 
 very similar to those in the less marked cases of true 
 diphtheria. The streptococcus and pneumococcus are 
 the two forms most frequently found in these cases, 
 but there are also others which, under suitable condi- 
 tions, take an active part in producing this form of 
 inflammation. Some of these bacteria do not develop 
 on artificial media, so that we know little of their 
 characteristics. Among these is a long slender bacillus 
 which is occasionally found in great abundance in the 
 middle layers of pseudomembranes when the diphtheria 
 
 23 
 
354 BACTERIOLOGY. 
 
 bacillus is absent. This, or one similar to it, has been 
 described by Vincent. 1 It does not grow on artificial 
 media and is not pathogenic in animals. From its 
 presence in the false membrane of a number of cases, 
 it is believed to have some causal relation to them. 
 
 These cases show most of the local appearances of 
 true diphtheria, the superficial necrosis of the epithe- 
 lium, the membrane of the glandular swellings. The 
 pseudomembranes may persist for from one to two 
 weeks, or even, in exceptional cases, longer. This 
 bacillus is apparently frequently present in the normal 
 throat, and is probably only able under certain favor- 
 ing conditions, such as syphilis, to produce lesions. 
 Nerve degeneration and paralysis do not follow an 
 attack. 
 
 The pseudomembranous angina accompanying scarlet 
 fever, and to a less extent other diseases, may not 
 show the presence of diphtheria bacilli, but only the 
 pyogenic cocci, especially streptococci, or, more rarely, 
 some varieties of little known bacilli. The deposit 
 covering the inflamed tissues in these non-specific cases 
 is, it is true, usually but not always, rather an exudate 
 than a true pseudomembrane. The majority of these 
 cases, however, are mild affections, being only of im- 
 portance in adding to the severity of the disease which 
 they complicate. An exception should be made when 
 the larynx is affected, as here the lungs are often sec- 
 ondarily involved. The bacteria which occur in false 
 diphtheria are streptococci, staphylococci, diplococci, 
 and sometimes pseudodiphtheria bacilli or bacilli which 
 are morphologically and culturally distinct from the 
 
 i Annales de 1'Institut Pasteur, August, 1899. 
 
DIPHTHERIA BACILLUS. 355 
 
 Loffler bacilli. These will be referred to further under 
 their respective organisms. 
 
 The Transmission of Diphtheria. The possibility of 
 the transmission of diphtheria from animals to man 
 cannot be disputed, for cats and many animals can be 
 infected, but there are no authentic cases of such 
 transmission on record. So-called diphtheritic disease 
 in animals and birds is usually due to other micro- 
 organisms than the diphtheria bacilli. Diphtheritic 
 infection, however, can generally be traced, directly or 
 indirectly, to its source; though there are undoubtedly 
 some cases of diphtheria in which we cannot determine 
 the source of the infection, for we have no reason to 
 believe that diphtheria is ever spontaneous. 
 
 Let us consider some of the means by which the dis- 
 ease may be communicated. In actual experiment the 
 bacilli have been observed to remain virulent in bits 
 of dried membrane by Loffler for fourteen weeks, 
 by us for seventeen weeks, and by Roux and Yersin 
 for twenty weeks. Dried on silk threads Abel reports 
 that they may sometimes live one hundred and seventy- 
 two days, and upon a child's plaything which had been 
 kept in a dark place they lived for five months. The 
 virulent bacilli have been found on soiled bedding or 
 clothing of a diphtheria patient, on drin king-cups, 
 shoes, hair, slate-pencils, etc. Beside these sources 
 of infection by which the disease may be indirectly 
 transmitted, virulent bacilli may be directly received 
 from the pseudo membrane, exudate, or discharges of 
 diphtheria patients; from the secretions of the nose 
 and throat of convalescent cases of diphtheria in which 
 the virulent bacilli persist; and from the healthy 
 throats of individuals who acquired the bacilli from 
 
356 BACTERIOLOGY. 
 
 being in contact with others having virulent germs on 
 their persons or clothing. In such cases the bacilli 
 may sometimes live and develop for days or weeks in 
 the throat without causing any lesion. When we con- 
 sider that it is only the severe types of diphtheria that 
 remain isolated during their actual illness, the wonder 
 is not that so many, but that so few, persons contract 
 the disease. It indicates that very frequently virulent 
 bacilli are received into the mouth, and then either find 
 no conditions there suitable for their growth or are 
 swept away by food or drink before they could effect 
 a lodgement. 
 
 Susceptibility to and Immunity against Diphtheria. 
 An individual susceptibility, both general and local, to 
 diphtheria, as in all infectious diseases, is necessary to 
 contract this disease. Moreover, the diphtheria poison 
 does not produce the same effect on the mucous mem- 
 branes of all persons. Age has long been recognized 
 to be an important factor in diphtheria. Children 
 within the first six months of life. are but little sus- 
 ceptible, the greatest degree of susceptibility being 
 between the third and the tenth year, while adults 
 are almost immune. An inherited susceptibility or 
 "family predisposition" to the disease has also been 
 observed. 
 
 Long before the discovery of the Klebs-Loffler 
 bacillus it was a well known fact that two attacks of 
 diphtheria seldom occurred in the same individual within 
 short periods of time, and none of us would fear to 
 leave a convalescent case in the same room with one still 
 suffering from the disease. To what this natural suscep- 
 tibility or immunity is due is still only partially under- 
 stood; when we remember, however, that simply a slight 
 
DIPHTHERIA BACILLUS. 357 
 
 increase in the acidity or alkalinity of the bouillon in 
 which the diphtheria bacilli are producing their toxin 
 will prevent further production, it is easy to imagine that 
 many changes in the throat secretion or in its mucous 
 membrane may prevent the development of the bacil- 
 lus or of the production by the bacillus of its toxin, 
 and, therefore, of its disease-producing power. But, 
 as the result of animal experiments, it is now known 
 that an artificial immunity against diphtheria can be 
 produced, at least for a considerable length of time, 
 by the development of substances directly antidotal to 
 the diphtheria toxin. By the inoculation of virulent or 
 somewhat attenuated cultures or of diphtheria toxin, 
 Fraenkel, Behring, Wernicke, Aronson, Roux, and 
 since then many others, have succeeded in immunizing 
 animals; but the most important and valuable results 
 are those which have been obtained by Behring, in 
 conjunction with others, who showed that the blood 
 of immune animals contains a substance which neutial- 
 izes the diphtheria toxin. The blood-serum of persons 
 who have recovered from diphtheria has been found 
 also to possess this protective property, which it acquires 
 about a week after the beginning of the disease, and loses 
 again in a few weeks or months. Moreover, the blood- 
 serum of many individuals, usually adults, who have 
 never had diphtheria often has a slight general anti- 
 toxic property. 
 
 Antitoxic Serum. The knowledge derived from these 
 remarkable investigations into the protective powers of 
 the blood-serum of immunized animals has been em- 
 ployed with the most brilliant results for the prevention 
 and early treatment of diphtheria in man. The dis- 
 covery of the method of the production of antitoxic 
 
358 BACTERIOLOGY. 
 
 serum or antitoxin in animals, and its practical appli- 
 cation to the treatment and cure of diphtheria, has 
 been shared by many experimenters, at first chiefly 
 in Germany and France, and later in this country. 
 Among those whose labors in this direction have ren- 
 dered them most worthy of mention are Behring, 
 Ehrlich, Boer, Kossel, and Aronson in Germany; 
 Roux, Martin, and Chaillou in France. 
 
 Results of the Antitoxin Treatment of Diphtheria. 
 Though the results of the antitoxin treatment of 
 diphtheria belong properly to the province of serum- 
 therapy rather than to bacteriology, in view of the 
 great practical importance of the subject it may not be 
 amiss to quote here the conclusions arrived at by Biggs 
 and Guerard after a review of all the statistics and 
 opinions published since the beginning of the antitoxin 
 treatment in 1892 : 
 
 " It matters not from what point of view the sub- 
 ject is regarded, if the evidence now at hand is properly 
 weighed, but one conclusion is or can be reached 
 whether we consider the percentage of mortality from 
 diphtheria and croup in cities as a whole, or in hospi- 
 tals, or in private practice; or whether we take the 
 absolute mortality for all the cities of Germany whose 
 population is over 15,000, and all the cities of France 
 whose population is over 20,000; or the absolute mor- 
 tality for New York City, or for the great hospitals in 
 France, Germany, and Austria; or whether we con- 
 sider only the most fatal cases of diphtheria, the laryn- 
 geal and operative cases; or whether we study the ques- 
 tion with relation to the day of the disease on which 
 treatment is commenced, or the age of the patient 
 treated; it matters not how the subject is regarded or 
 
DIPHTHERIA BACILLUS. 359 
 
 how it is turned for the purpose of comparison with 
 previous results, the conclusion reached is always the 
 same namely, there has been an average reduction of 
 mortality from the use of antitoxin in the treatment of 
 diphtheria of not less than 50 per cent., and under the 
 most favorable conditions a reduction to one-quarter, 
 or even less, of the previous death-rate. This has 
 occurred not in one city at one particular time, but in 
 many cities, in different countries, at different seasons 
 of the year, and always in conjunction with the intro- 
 duction of antitoxin serum and proportionate to the 
 extent of its use. 7 ' 
 
 The Production of Diphtheria Antitoxin for Therapeutic 
 Purposes. As a result of the work of years in the 
 laboratories of the Health Department of New York 
 City, the following may be laid down as a practical 
 method : 
 
 The strongest diphtheria toxin possible should be 
 obtained by taking a very virulent culture and grow- 
 ing it under the conditions described on page 345. 
 The culture, after a week's growth, is removed, and 
 having been tested for purity by microscopical and 
 culture tests is rendered sterile by the addition of 10 
 per cent, of a 5 per cent, solution of carbolic acid. 
 On the following day the sterile culture is filtered 
 through ordinary sterile, filter-paper and stored in 
 full bottles in a cold place until needed. Its strength 
 is then tested by giving a series of guinea-pigs care- 
 fully measured amounts. Less than 0.01 c.c., when 
 injected hypodermatically, should kill a 250-gramme 
 guinea-pig. 
 
 The horses used should be young, vigorous, of fair 
 size, and absolutely healthy. Vicious habits, such as 
 
360 BACTERIOLOGY. 
 
 kicking, etc., make no difference, of course, except to 
 those who handle the animals. A number of such 
 horses are severally injected with an amount of toxin 
 sufficient to kill five thousand guinea-pigs of 250 
 grammes' weight (about 20 c.c. of strong toxin). 
 After from three to five days, so soon as the fever 
 reaction has subsided, a second subcutaneous injection 
 of a slightly larger dose is given. With the first three 
 injections of toxin 10,000 units of antitoxin are given. 
 If antitoxin is not mixed with the first doses of toxin 
 only one-tenth of the doses advised is to be given. 
 At intervals of from five to eight days increasing injec- 
 tions of pure toxin are made, until at the end of two 
 months from ten to twenty times the original amount 
 is given. There is absolutely no way of judging which 
 horses will produce the highest grades of antitoxin. 
 Very roughly, those horses which are extremely sensi- 
 tive and those which react hardly at all are the poorest, 
 but even here there are exceptions. The only way, 
 therefore, is at the end of six weeks or two months to 
 bleed the horses and test their serum. If only high- 
 grade serum is wanted all horses that give less than 
 150 units per c.c. are discarded. If moderate grades 
 only are desired, all that yield 100 units may be 
 retained. The retained horses receive steadily in- 
 creasing doses, the rapidity of the increase and the 
 interval of time between the doses (three days to one 
 week) depending somewhat on the reaction following 
 the injection, an elevation of temperature of more than 
 3 F. being undesirable. At the end of three months 
 the antitoxic serum of all the horses should contain 
 over 300 units, and in about 10 per cent, as much as 
 800 units in each cubic centimetre. Very few horses 
 
DIPHTHERIA BACILLUS. 361 
 
 ever give above 1000 units, and none so far has given 
 as much as 2000 units per c.c. The very best horses 
 continue to furnish blood containing the maximum 
 amount of antitoxin for several months, and then, in 
 spite of increasing injections of toxin, begin to furnish 
 blood of gradually decreasing strength. If every nine 
 months an interval of three months' freedom from 
 inoculations is given, the best horses furnish high- 
 grade serum during their periods of treatment for from 
 two to four years. 
 
 In order to obtain the serum the blood is withdrawn 
 from the jugular vein by means of a sharp- pointed 
 canula, which is plunged through the vein wall, a slit 
 having been made in the skin. The blood is carried 
 by a sterile rubber tube into large Erlenmeyer flasks 
 and allowed to clot, the flasks, however, being placed 
 in a slanting position before clotting has commenced. 
 The serum is drawn off after four days by means of sterile 
 glass and rubber tubing, and is stored in large flasks. 
 From this, as needed, small phials are filled. The 
 phials and their stoppers, as indeed all the utensils used 
 for holding the serum, must be absolutely sterile, and 
 every possible precaution must be taken to avoid 
 contamination of the serum. An antiseptic may be 
 added to the serum as a preservative, but it is not 
 necessary and probably inadvisable, except when the 
 serum is to be sent to great distances, where it cannot 
 be kept under supervision. 
 
 Kept from access of air and light and in a cold place 
 it is fairly stable, deteriorating not more than 40 per 
 cent., and often much less, within a year. Diphtheria 
 antitoxin, when stored in phials and kept under the 
 above conditions, contains within 10 per cent, of its 
 
362 BACTEEIOLOOY. 
 
 original strength for at least two months; after that it 
 can be used by allowing for a maximum deterioration 
 of 10 per cent, for each month. The antitoxin in old 
 serum is just the same as in that freshly-bottled, only 
 there is less of it. 
 
 The nature of diphtheria antitoxin has until recently 
 been known almost wholly from its physiological prop- 
 erties. Recently experiments have seemed to show that 
 it was either closely bound to the globulins or was itself 
 a globulin. Mr. J. P. Atkinson, assistant chemist in 
 the laboratory, has kindly permitted me to state the 
 results of his investigations, which will soon appear in 
 the Journal of Experimental Medicine. He found that 
 antitoxic and normal horse-serum react similarly toward 
 M'gSO 4 , in that the globulin is precipitated completely 
 from the other constituents of the serum. In the case 
 of antitoxic serum the globulin precipitate carries with 
 it all of the antitoxic power of the serum, leaving the 
 filtrate without any neutralizing power against the diph- 
 theria toxin. When watery solutions of this globulin 
 are saturated with NaCl a precipitate occurs. When 
 the solution is heated a series of further precipitates take 
 place, as follows : Cloudiness appears at 40, 49, 57, 
 and 67 C. ; complete precipitate occurs at 45, 54, 
 62, and 72 C. Each of these precipitates has anti- 
 toxic properties, and the total quantities contain all the 
 original antitoxin except some 5 per cent., which is evi- 
 dently destroyed by the higher temperatures required 
 for the last two precipitates. After the last precipitate 
 the solution is free of globulin and also of all antitoxic 
 properties. 
 
 A further fact developed by Atkinson is that the 
 globulins increase markedly in the serum of horses as 
 
DIPHTHERIA BACILLUS. 363 
 
 the antitoxin strength increases. It seems, therefore, 
 from the above facts that diphtheria antitoxin has the 
 characteristics of the globulins. Whether it is a union 
 of diphtheria toxin and globulin, or an increase of cer- 
 tain globulin-like substances through the stimulation of 
 the toxin, we have as yet no facts to tell us. Antitoxin 
 is destroyed by prolonged moderate heat (60 C.) and 
 by short exposure to higher temperatures (95 to 100 
 C.). It is much less sensitive than diphtheria toxin. 
 
 Diphtheria antitoxin has the power of neutralizing 
 diphtheria toxin, so that when a certain amount is in- 
 jected into an animal before or together with the toxin 
 it overcomes its poisonous action. As already stated, 
 there is a great difference of opinion as to whether anti- 
 toxin acts by direct chemical neutralization of the toxin 
 or indirectly on the cells. The facts in favor of a direct 
 action of antitoxins upon their corresponding toxins 
 have recently been briefly summarized by Cobbett as 
 follows : 
 
 1. Certain reactions have been observed to take 
 place between these substances outside the animal body 
 (venom, ricln, crotin, tetanus toxin, diphtheria toxin, 
 and their corresponding antitoxins). 
 
 2. Various attempts to separate the toxins and anti- 
 toxins from neutral mixtures have been failures. Par- 
 tial successes have, at least in some instances, been 
 shown to depend upon the fact that insufficient time for 
 their complete union was allowed, separation being no 
 longer possible if this were granted. 
 
 3. The accuracy of the titration of toxins and anti- 
 toxins to within 1 per cent, of error. 
 
 4. The fact that to save an animal from 1000 fatal 
 doses of diphtheria toxin requires little more than a 
 
364 BACTERIOLOGY. 
 
 hundred times as much antitoxin as is required for ten 
 fatal doses, the resistance of the animal it-elf account- 
 ing for the difference. 
 
 5. The fact that the potency of antitoxin is greatly 
 increased if it is allowed to come in contact with the 
 toxin outside the animal body; and is increased still 
 further if allowed to remain for sufficient time in con- 
 tact with the toxin at a suitable temperature. 
 
 On the other hand, the conclusions which Buchner 
 and Roux drew from their experiments have been shown 
 to have been based on a misconception, for they ignored 
 the capacity of an animal to deal with a certain minimal 
 quantity of poison, and, consequently, made no distinc- 
 tion between a physiologically neutral and a completely 
 neutral mixture. 
 
 The facts now known, therefore, indicate rather 
 strongly that the antitoxins of tetanus and diphtheria, 
 of snake-poison, of ricin, etc., enter into direct chem- 
 ical combination with their respective toxins a com- 
 bination which is, perhaps, not exactly comparable to 
 that of an acid with an alkali; for, as we have seen, 
 it is a much slower one, but one which possibly as 
 Ehrlich has suggested more closely resembles the for- 
 mation of a double salt. Some facts seem to indicate 
 that the antitoxin has a stronger affinity for toxin than 
 the toxin has for the cells. Many points, however, are 
 still far from clear as to the manner in which both 
 toxins and antitoxins act. 
 
 The Testing of Antitoxin. This power, possessed 
 by a definite quantity of antitoxin to neutralize a cer- 
 tain amount of toxin, is utilized in testing antitoxin. 
 Guinea-pigs of about 250 grammes' weight are sulicu- 
 taneously injected with one hundred or with ten fatal 
 
DIPHTHERIA BACILLUS. 365 
 
 doses of toxin which have been previously mixed with 
 an amount of antitoxin believed to be sufficient to pro- 
 tect from the toxin. If the guinea-pig lives four days, 
 but dies soon after, the amount of antitoxin added to 
 the toxin was just 1 or 0.1 unit, according as one hun- 
 dred or ten fatal doses were employed. If the guinea- 
 pig dies earlier, less than 1 unit was added. 
 
 The Use of Antitoxin in Treatment and Immuniza- 
 tion. The antitoxin in the higher grades is iden- 
 tical with that in the lower grades ; there is simply 
 more of it in each drop of the serum. In treatment, 
 however, for the same amount of antitoxin we have to 
 inject less blood-serum with the higher grades, and, 
 therefore, have somewhat less danger of rashes and 
 other deleterious results. With concentrated globulin 
 solutions we may hope still further to avoid all dis- 
 agreeable effects (see page 362). The amount of anti- 
 toxin required for immunization is 200 units for an 
 infant, 500 for an adult, and proportionately for those 
 between these extremes. After the observation of the 
 use of antitoxin in the immunization of several thousand 
 cases, I have absolute belief in its power to prevent 
 an outbreak of diphtheria for at least two weeks, and 
 also of its harmlessness in the small doses required. If 
 it is desired to prolong the immunity the antitoxin in- 
 jection is repeated every two weeks. For treatment, 
 mild cases should be given 1500 units, moderate cases 
 2000 units, and severe cases 3000 units. Where no 
 improvement follows in twelve hours the dose should 
 be repeated. Antitoxin is useless when given by the 
 mouth, as very little of it is absorbed. 
 
 No deleterious effects are to be feared except a rash, 
 with some rise of temperature, in about 20 per cent, of 
 
366 BACTERIOLOGY. 
 
 the cases. With the serum from some horses the rashes 
 are very infrequent, while with that from others they 
 occur more often. The same horse will at one time 
 furnish a serum which produces no rashes and at an- 
 other one which gives a great number. No way has 
 yet been found to eliminate them entirely. Filtering 
 and moderate heating produce little effect. Standing 
 for some months causes a precipitate to occur, and the 
 clear serum seems somewhat less liable to produce 
 rashes than when it was fresh. 
 
 The Persistence of Antitoxin in the Blood. When in- 
 jections of toxin are stopped in a horse the antitoxin is 
 slowly eliminated, so that there is a loss of about 20 
 per cent, a week. In from three to five months all 
 appreciable antitoxin has been eliminated. Immunity 
 in human beings lasts from two to six weeks after an 
 injection of 500 units of antitoxin. 
 
 Technical Points upon the Testing of Diphtheria Anti- 
 toxin and the Relations between the Toxicity and Neu- 
 tralizing Value of Diphtheria Toxin. Until within a 
 fairly recent time the filtered or sterilized bouillon in 
 which the diphtheria bacillus had grown and produced 
 its " toxin" was supposed to require for its neutraliza- 
 tion an amount of antitoxin directly proportional to its 
 toxicity as tested in guinea-pigs. Thus, if from one 
 bouillon culture ten fatal doses of " toxin" were re- 
 quired to neutralize a certain quantity of antitoxin, it 
 was believed that ten fatal doses from every culture, 
 without regard to the way in which it had been pro- 
 duced or preserved, would also neutralize the same 
 amount of antitoxin. Upon this belief was founded 
 the Behring-Ehrlich definition of an antitoxin unit. 
 
 The results of tests by different experimenters with 
 
DIPHTHERIA BACILLUS. 
 
 367 
 
 the same antitoxic serum, but with different diphtheria 
 toxins, proved this opinion to be incorrect. Ehrlich 1 
 deserves the credit for first clearly perceiving and 
 publishing this. He obtained from various sources 
 twelve toxins and compared their neutralizing value 
 upon antitoxin ; these tests gave most interesting and 
 important information. The results in six toxins, 
 which are representative of the twelve, are as shown 
 in the following table : 
 
 o> 
 
 a 
 
 i 
 
 '*""' '^ O L^ ^ j /^ 
 
 JfJI*. 
 
 
 
 
 
 a 
 
 || 
 
 2^2|g? 
 
 l" ?1 
 
 
 a 
 
 .2 ^ 
 
 laSf^ + ^"a^lsSj L,-Ln 
 
 
 'oJ3 
 
 si 
 
 z; 
 
 *sl| 
 
 -'0 cS 
 
 9(-1^^T3*-^ S^^.^ 3 >>.5 o3 o 
 
 ^Sobcg:, isS^^Ss? = fatal 
 a ^^-a-as 'gS'S-g sc-a doses - 
 
 ^Ou^gP^3 f OO^^ J r^U3&^ 
 
 Data upon " toxin" 
 specimen given by 
 Ehrlich. 
 
 _. J3 1 > O> 
 SS 9-3 fl 
 
 ^w-sSSfl : _S5'S5'3^ 
 
 ^-tDPCfl>-^H *3 R T5 B 3 o ; 
 
 
 p^J .SJ-2 
 
 1 JB &3 S y ^ 8 2 S 3_! 
 
 
 
 E-i 
 
 ^=2^ 
 
 |-2a^s 
 
 ^=^^oa. 
 
 
 
 2 
 
 0.03 
 
 42 
 
 32 
 
 10 
 
 Preserved two years. 
 
 4 
 
 0.009 
 
 39.4 
 
 33.4 
 
 6 
 
 Old, deteriorated from 
 
 
 
 
 
 
 0.003 to 0.009. 
 
 7 
 
 0.0165 
 
 76.3 
 
 54.4 
 
 22 
 
 Fresh toxin, preserved 
 
 
 
 
 
 
 with tricresol. 
 
 9 
 
 0.039 
 
 123 
 
 108 
 
 15 
 
 A number of fresh cul- 
 
 
 
 
 
 
 tures grown at 37 C. 
 four and eight days. 
 
 10 
 
 0.001 
 
 29.2 
 
 27.5 
 
 1.7 
 
 Precipitated from 
 greatly deteriorated 
 
 
 
 
 
 
 "toxin." 
 
 12 
 
 0.0025 
 
 100 
 
 50 
 
 50 
 
 Tested immediately 
 after its withdrawal. 
 
 From the facts set forth in the tables, Ehrlich has 
 derived interesting theories, which, if true, would add 
 greatly to our knowledge of toxins, and would also have 
 a very direct influence upon the present methods of 
 standardizing antitoxin. He believes that the diphtheria 
 bacilli in their growth produce a toxin which, so long as 
 
 1 " Die Wertbemessung des Diphtherieheilserums und deren theoretische 
 Grundlagen," Klinisches Jahrbuch, 1897. 
 
368 BA CTERIOL OGY. 
 
 it remains chemically unaltered, has a definite poisonous 
 strength with a definite value in neutralizing antitoxin. 
 This neutralization he believes to be a chemical union, 
 in which two hundred fatal doses of toxin for a 250 
 grammes' weight guinea-pig combine with one unit of 
 antitoxin. The toxin is, however, an unstable com- 
 pound, and begins to change almost immediately into 
 substances which are not, at least acutely, poisonous, 
 but which retain their full power to neutralize anti- 
 toxin. These substances, according to Ehrlich, fall 
 into three groups. The first has more affinity for 
 combining with the antitoxin than the toxin itself 
 (protoxoids). The second has the same affinity (syn- 
 toxoids). The third has less affinity (epitoxoids). 
 
 According to him, if a mixture of toxoids and toxin 
 is added to antitoxin, the protoxoids first combine with 
 the antitoxin, then the syntoxoids and the toxin com- 
 bine in equal proportions, so long as the supply lasts, 
 with the amount of antitoxin remaining, or, if there 
 is a surplus, with enough to satisfy them; finally, if 
 any antitoxin remains, the epitoxoids unite with it. 
 
 If to a mixture in which all three toxoids, as well as 
 toxin, have united with antitoxin, some additional toxic 
 culture bouillon be added, the new protoxoids displace 
 first the epitoxoids, and then, if free protoxoids remain, 
 the toxin and the syntoxoids from their antitoxin, and 
 thus liberate as well as add free toxin to the solution. 
 
 Ehrlich gives an interesting theory to explain the pro- 
 duction of antitoxin in the blood. This he does upon 
 the supposition that, when absorbed, the toxin combines 
 with a portion of certain selected cells, and that this 
 portion, by its union with toxin, becomes at least 
 physiologically dead. The cell replaces this dead 
 
DIPHTHERIA BACILLUS. 369 
 
 matter with new and similar substance; after the stimuli 
 following several repeated losses and replacements of 
 this substance the cells produce it in excess. This sub- 
 stance, whether originally in the normal cell or repro- 
 duced there, and whether remaining in the cell or 
 thrown out into the circulation, is antitoxin. 
 
 The above summary merely gives an outline of 
 some of the points in Ehrlich' s most interesting article. 
 To become fully acquainted with the reason for his 
 theories the article itself must be carefully read. 
 
 Interest in both his theoretical reasoning and in his 
 practical conclusions led us to subject both to a series 
 of tests which have, I believe, added some interesting 
 facts to those already published by Ehrlich as well as 
 cast doubts on some of his conclusions. 
 
 The results of these experiments of Atkinson and 
 myself * were fully in accord with those published by 
 Ehrlich as to the varying neutralizing value of a 
 minimal fatal dose of "toxin"; they, however, also 
 indicate roughly a general law in accordance with which 
 these changes occur. 
 
 The neutralizing value of a fatal dose of toxin is at 
 its lowest in the culture fluid when the first consider- 
 able amounts of toxin have been produced. After a 
 short period, during which the quantity of toxin in the 
 fluid is increasing, the neutralizing value of the fatal dose 
 begins to increase, at first rapidly, then more slowly. 
 
 While the culture is still in vigorous growth and new 
 toxin is being produced, the neutralizing value of the 
 fatal dose fluctuates somewhat, but with a generally 
 upward tendency. After the cessation of toxin pro- 
 
 1 Journal of Experimental Medicine, vol. Hi., No. 4. 
 24 
 
370 BACTERIOLOGY. 
 
 duction the neutralizing value of the fatal dose increases 
 steadily until it becomes five to ten times its original 
 amount. 
 
 In our experiments the greatest value for L+ was 
 126, the least 27. As at six hours L+ was only 72 
 and at twenty-eight hours only 91, we doubt whether 
 L^ ever reaches above 150. 1 When we seek to analyze 
 the above-described process we find certain facts which 
 seem partly to explain it. Experiments have shown 
 that filtered toxin, preserved for any length of time in 
 conditions under which access of air occurs, gradually 
 loses in both its toxicity and neutralizing power, and 
 that it loses more rapidly in the former property than 
 in the latter. Thus, while the fatal dose of a toxin pre- 
 served for one year rose from 0.01 c.c, to 0.55 c.c., it 
 lost only half as much in neutralizing value, 1 unit 
 neutralizing at first 1 c.c., at the end of the year 0.25 
 c.c. These processes take place more rapidly at room- 
 temperature than in the ice-chest, and in the incubator 
 than in the room. 
 
 In the fluid holding the living bacilli we have, there- 
 fore, after the first few hours of toxin formation, a 
 double process going on one of deterioration in the 
 toxin already accumulated, which tends to increase the 
 neutralizing value of the fatal dose; the other of new 
 toxin formation, which probably tends to diminish the 
 neutralizing value. The chemical changes produced 
 by the growth of the bacilli in the bouillon tend to aid 
 one or the other of these processes, and so to make, from 
 hour to hour, slight changes in the value of the fatal 
 
 1 Li = fatal doses of toxin required to kill a guinea-pig in four days after 
 having been mixed with one unit of antitoxin. 
 L = fatal doses of toxin required to fully neutralize one unit of antitoxin- 
 
DIPHTHERIA BACILLUS. 371 
 
 dose. Later, with the period of cessation of toxin pro- 
 duction, the gradual deterioration of the toxicity alone 
 continues, and the fatal dose gradually and steadily in- 
 creases in its neutralizing value. 
 
 Ehrlich's theories as to the splitting up of " toxin" 
 into toxoids having little or no toxicity, but on the 
 average full neutralizing power for antitoxin have 
 not, in our opinion, been substantiated by the results 
 of these experiments. The difference between the 
 amount of toxin mixed with a unit of antitoxin which 
 causes the first symptoms and that causing death upon 
 the fourth day would be, it is true, explained by his 
 theory; but the failure of this difference to be greater 
 where, by his theories, epitoxoids should be in great 
 abundance, prevents our acceptance of his views. The 
 fact of the greater neutralization value of a fatal dose 
 of a deteriorated toxin would be accounted for on his 
 protoxoid theory. This, however, is not proof of its 
 correctness, as other theories, such as the production by 
 the diphtheria bacillus of two or more closely allied 
 toxins, similar to the allied alkaloids produced by 
 plants, would equally account for it if we supposed 
 the one which had the greater neutralization value was 
 more resistant to destruction than the other. 1 We only 
 advance this theory to call attention to the fact that 
 many theories can on paper explain a process without 
 necessarily being thereby established. 
 
 While we do not believe, therefore, that he has 
 changed the principles of testing antitoxin, yet we 
 believe he has contributed greatly to uniformity in 
 results by calling attention to the necessity of selecting 
 
 1 The incomplete precipitation of the diphtheria toxin by MgSO 4 makes it 
 probable that more than one poison exists. 
 
372 BACTERIOLOGY. 
 
 a suitable toxin and by employing and distributing an 
 antitoxin as a standard to test toxins by. In this way 
 smaller testing stations can make their results corre- 
 spond with those of the central station. 
 
 In spite of the great variations in the neutralizing 
 value of a fatal dose in different toxins we do not be- 
 lieve there has been any such great difference in the 
 toxins used by the different stations for testing pur- 
 poses. Most laboratories have taken the culture fluid 
 at about the time of its greatest toxicity, and the 
 neutralizing value of a fatal dose of this toxin would 
 seldom vary more than 10 per cent, above or below the 
 standard now adopted in Germany by the government 
 testing station, this latter being presumably as close as 
 possible to that used to establish the original Behring- 
 Ehrlich unit. 
 
 Where error has been made it has usually been by 
 taking too old culture fluids, which would cause the 
 antitoxin strength of samples tested to be estimated 
 below and not above its real value. Culture 8, which is 
 used not only by the New York Board of Health Labor- 
 atory but by many other laboratories in the United 
 States and Europe, fortunately produces on the sixth 
 to eighth day the time at which the culture is usually 
 removed a toxin which grades Ehrlich's antitoxin 
 within 5 per cent, of the strength given by him. 
 
 We believe that by using such a bacillus we can, 
 after gaining a fuller knowledge of its characteristics, 
 obtain a toxin of a known and suitable neutralizing 
 value, and thus always correctly standardize an anti- 
 toxic serum. This is certainly true for the bacillus 
 which we have used for the past four years. Mean- 
 while, a fairly permanent preparation of a carefully 
 
DIPHTHERIA BACILLUS. 373 
 
 tested antitoxin is of immense value in insuring a 
 uniform, though not necessarily correct, standard among 
 the different testing stations and in allowing of com- 
 parison between them. 
 
 The old definition of Behring and Ehrlich, that an 
 antitoxin unit contains the amount of antitoxin which 
 will protect the life of a guinea-pig from one hundred 
 fatal doses of toxin, must be modified so as to be de- 
 fined as that amount of antitoxin which will neutral- 
 ize one hundred fatal doses of a toxin similar to that 
 adopted as the standard namely, one having the char- 
 acteristics of toxins in cultures at the height of their 
 toxicity. 
 
 The actual test of an antitoxin serum is, therefore, 
 carried out as follows: Six guinea-pigs are injected 
 with mixtures of toxin and antitoxin. In each of the 
 mixtures there is 100 times the amount of a toxin such 
 as just described, which will kill 250 grammes of 
 guinea-pig on an average in 96 hours. In each of the 
 mixtures the amount of antitoxin varies; for instance, 
 No. 1 would contain 0.002 c.c. serum, No. 2, 0.003 
 c.c., No. 3, 0.004 c.c., No. 4, 0.005 c.c., etc. If, at 
 the end of the fourth day, Nos. 1, 2, and 3 were dead, 
 and Nos. 4, 5, and 6 were alive, we would consider 
 the serum to contain 200 units of antitoxin for each c.c. 
 When we mix only ten fatal doses of toxin with one- 
 tenth of the amount of antitoxin used with 100 fatal 
 doses we usually consider that the guinea-pig must not 
 only live but remain well. 
 
 The Relation of Bacteriology to Diagnosis. I believe 
 that all experienced clinicians will agree that, when 
 left to judge solely by the appearance and symptoms 
 of a case, there are certain mild exudative inflamma- 
 
374 BACTERIOLOGY. 
 
 tions of the throat belonging to both diphtheritic and 
 to non-diphtheritic inflammations which appear exactly 
 alike, having apparently similar symptoms and similar 
 duration; that it is even possible to examine two cases, 
 knowing that one is surely diphtheria, or at least that 
 diphtheria bacilli are present in the exudate, and the 
 other surely is not, and yet be unable to determine 
 clinically which is true diphtheria and which is pseudo- 
 diphtheria. It is not meant to imply that a case is 
 one of true diphtheria simply because the diphtheria 
 bacilli are present, but rather that the doubtful cases 
 not only have the diphtheria bacilli in the exudate, 
 but are capable of giving true characteristic diphtheria 
 to others, or later develop it characteristically them- 
 selves; and that those in whose throats no diphtheria 
 bacilli exist can under no condition give true character- 
 istic diphtheria to others or develop it themselves unless 
 they receive a new infection. It is, indeed, true, as a 
 rule, that cases presenting the appearance of ordinary 
 follicular tonsillitis in adults are not due to the diph- 
 theria bacillus. It is also true that now and then a case 
 having this appearance is one of diphtheria, and almost 
 every physician has seen such cases from time to time 
 in households infected with diphtheria. On the other 
 hand, in small children mild diphtheria very frequently 
 occurs with the semblance of rather severe ordinary 
 follicular tonsillitis, due to the pyogenic cocci, and in 
 large cities where diphtheria is prevalent all such cases 
 must be watched as being more or less suspicious. 
 As showing our doubt in our own judgment, I think 
 most would feel that if in any case exposure to diph- 
 theria is known to have occurred, even a slightly sus- 
 picious sore-throat would be regarded as probably due to 
 
DIPHTHERIA BACILLUS. 375 
 
 the diphtheria bacilli. If, on the other hand, no cases 
 of diphtheria have been known to exist in the neigh- 
 borhood, even cases of a more suspicious nature would 
 probably not be regarded as diphtheria. 
 
 Appearances Characteristic of Diphtheria. The pres- 
 ence of irregular-shaped patches of adherent grayish or 
 yellowish-gray pseudomembrane on some other por- 
 tions than the tonsils is, as a rule, an indication of the 
 activity of the diphtheria bacilli. Kestricted to the 
 tonsils alone their presence is less certain. 
 
 Occasionally, in scarlatinal angina or in severe phleg- 
 monous sore-throats, patches of exudate may appear on 
 the uvula or borders of the faucial pillars, and still the 
 case may not be due to the diphtheria bacilli; these 
 are, however, exceptional. Thick, grayish pseudo- 
 membranes which cover large portions of the tonsils, 
 soft palate, and nostrils are almost invariably the 
 lesions produced by diphtheria bacilli. 
 
 The very great majority of cases of pseudomem- 
 branous or exudative laryngitis, in the coast cities at 
 least, whether an exudate is present in the pharynx or 
 not, are due to the diphtheria bacilli. Cases in which 
 no exudate is apparent and those in which the laryngeal 
 obstruction is sudden and the spasmodic element is 
 marked, are, however, frequently due to the activity of 
 other bacteria. Nearly all membranous affections of 
 the nose are true diphtheria. When the membrane 
 is limited to the nose the symptoms are, as a rule, very 
 slight; but when the nasopharynx is involved the 
 symptoms are usually grave. Usually a small area of 
 inflammation indicates a slight or moderate severity, 
 and an extensive area a severe infection. 
 
 Most cases of pseudomembranes and exudates entirely 
 
376 BACTERIOLOGY. 
 
 confined to portions of the tonsils in adults are not due 
 to the diphtheria bacilli, although a few cases presenting 
 these symptoms are. The more complete the involve- 
 ment of the tonsils the more apt the case is to be due 
 to them. Cases presenting the appearances found in 
 scarlet fever, in which a thin, grayish membrane lines 
 the borders of the uvula and faucial pillars, are rarely 
 diphtheritic. As a rule, pseudomembranous inflam- 
 mations complicating scarlet fever, syphilis, and other 
 infectious diseases are due to the activity of the patho- 
 genic cocci and other bacteria induced by the inflamed 
 condition of the mucous membranes due to the scarla- 
 tinal or other poison. But from time to time such 
 cases, if they have been exposed to diphtheria, may be 
 complicated by it, and in some epidemics mixed in- 
 fection is common. 
 
 The Exudate Due to the Diphtheria Bacilli Contrasted 
 with That Due to Other Bacteria. As a rule, the exudate 
 in diphtheria is firmly incorporated with the underlying 
 mucous membrane, and cannot be removed without 
 leaving a bleeding surface, at least until convalescence. 
 The tissues surrounding the exudate are more or less 
 inflamed and swollen. Where other bacteria produce 
 the irritant the exudate, except in the cases due to the 
 bacillus described by Vincent, is usually loosely at- 
 tached, collected in small masses, and easily removable. 
 Exceptions, however, occur in both these diseases, so 
 that in true diphtheria the exudate may be easily re- 
 moved, and in lesions due to other bacteria the exudate 
 may be firmly adherent. 
 
 Paralysis following a pseudomembranous inflamma- 
 tion is an almost positive indication that the case was 
 one of diphtheria, although slight paralysis has followed 
 
DIPHTHERIA BACILLUS. 377 
 
 in a very few cases in which careful cultures revealed 
 no diphtheria bacilli. These, if not true diphtheria, 
 must be considered very exceptional cases. 
 
 Bacteriological Diagnosis. From the above it is appar- 
 ent that fully developed characteristic cases of diph- 
 theria are readily diagnosticated, but that many of the 
 less marked, or at an early period undeveloped, cases 
 are difficult to differentiate the one from the other. 
 In these cases cultures are of the utmost value, since 
 they enable us to isolate those in which the bacilli are 
 found, and to give preventive injections of antitoxin 
 to both the sick and those in contact with them, if this 
 has not already been done. As a rule, cultures do not 
 give us as much information as to the gravity of the 
 case as the clinical appearances, for by the end of 
 twenty-four to forty-eight hours the extent of the dis- 
 ease is usually easy of determination. The reported 
 absence of bacilli in a culture must be given weight in 
 proportion to the skill with which the culture was made, 
 the suitableness of the media, and the knowledge and 
 experience of the one who examined it. 
 
 Diphtheria does not occur without the presence of 
 the diphtheria bacilli; but there have been many cases 
 of diphtheria in which for one or another reason no 
 bacilli were found in the cultures by the examiner. 
 In many of these cases later cultures revealed them. 
 In a convalescent case the absence of bacilli in any one 
 culture indicates that there are certainly not many 
 bacilli left in the throat. Only repeated cultures can 
 prove their total absence. 
 
 TECHNIQUE OF THE BACTERIOLOGICAL DIAGNOSIS. 
 Collection of the Blood-serum and its Preparation for Use 
 in Cultures. A covered glass jar which has been thor- 
 
378 BACTERIOLOGY. 
 
 oughly cleansed with hot water is taken to the slaughter- 
 house and filled with freshly-shed blood from a calf or 
 sheep. The blood is received directly in the jar as it 
 spurts from the cut in the throat of the animal. After 
 the edge of the jar has been wiped it is covered with 
 the lid and set aside, where it may stand quietly until 
 the blood has thoroughly clotted. The jar is then car- 
 ried to the laboratory and placed in an ice-chest. If 
 the jar containing the blood is carried about before the 
 latter has clotted, very imperfect separation of the serum 
 will take place. It is well to inspect the blood in the 
 jar after it has been standing a few hours, and if the 
 clot is found adhering to the sides, to separate it by a 
 rod. The blood is allowed to remain twenty -four hours 
 on the ice, and then the serum which surrounds the clot 
 is siphoned off by a rubber tube and mixed with one- 
 third its quantity of nutrient beef broth, to which 1 
 per cent, glucose has been added. This constitutes the 
 Loffler blood-serum mixture. This is poured into tubes, 
 which should be about four inches in length and two- 
 thirds of an inch in diameter, having been previously 
 plugged witli cotton and sterilized by dry heat at 
 150 C. for one hour. Care should be taken in filling 
 the tubes to avoid the formation of air-bubbles, as they 
 leave a permanently uneven surface when the strum 
 has been coagulated by heat. To prevent this the end 
 of the pipette or funnel which contains the serum should 
 be inserted well into the test-tube. About 2 c.c. are 
 sufficient for each tube. The tubes, having been filled 
 to the required height, are now to be coagulated and 
 sterilized. They are placed slanted at the proper angle 
 and then kept for two hours at a temperature just below 
 95 C. For this purpose a Koch serum coagulator 
 
DIPHTHERIA BACILLUS. 379 
 
 (Fig. 22) or a double boiler serves best, though a steam 
 sterilizer will suffice. If the latter is used a wire 
 frame must be arranged to hold the tubes at the proper 
 inclination, and the degree of heat must be carefully 
 watched, as otherwise the temperature may go too 
 high, and if the serum is actually boiled the culture 
 medium will be spoiled. After sterilization by this 
 process the tubes containing the sterile, solidified blood- 
 serum can be placed in covered tin boxes or stopped 
 with sterile corks and kept for months. The serum thus 
 prepared is quite opaque and firm. A mixture of blood- 
 cells renders the serum darker, but it is not less useful. 
 
 The Swab for Inoculating Culture Tubes. The swab 
 to inoculate the serum is made as follows : A stiff, thin 
 iron rod, six inches in length, is roughened at one end 
 by a few blows of a hammer, and about this end a little 
 absorbent cotton is firmly wound. Each swab is then 
 placed in a separate glass tube, and the mouths of the 
 tubes are plugged with cotton. The tubes and rods are 
 then sterilizt d by dry heat at about 150 C. for one 
 hour, and stored for future use. Th<se cotton swabs 
 have proved much more serviceable for making inocu- 
 lations than platinum wire needles, especially in young 
 children and in laryngeal cases. It is easier to use the 
 cotton swab in such cases, and it gathers up so much 
 more material for the inoculation that it has seemed 
 more reliable. 
 
 For convenience and safety in transportation a " cul- 
 ture outfit' 7 has been devised, which consists of a small 
 wooden box containing a tube of blood-serum, a tube 
 holding a swab, and a record blank. These " culture 
 outfits " may be carried or sent by messenger or ex- 
 press to any place desired. 
 
380 BACTERIOLOGY. 
 
 Directions for Inoculating Culture Tubes with the Ex- 
 udate. The patient is placed in a good light, and, if a 
 child, properly held. The swab is removed from its 
 tube, and, while the tongue is depressed with a spoon, 
 is passed into the pharynx (if possible, without touch- 
 ing the tongue or other parts of the mouth) and is 
 rubbed gently but firmly against any visible membrane 
 on the tonsils or in the pharynx, and then, without 
 being laid down, the swab is immediately inserted in 
 the blood-serum tube, and the portion which has pre- 
 viously been in contact with the exudate is rubbed a 
 number of times back and forth over the whole sur- 
 face of the serum. This should be done thoroughly, 
 but it is to be gently done, so as not to break the sur- 
 face of the serum. The swab should then be placed 
 in its tube, and both tubes, thin cotton plugs having 
 been inserted, are reserved for examination or sent to 
 the laboratory or collecting station (as in New York 
 City). If sent to the health department laboratories 
 for examination the blank forms of report which usu- 
 ally accompany each "outfit" should be filled out and 
 forwarded with the tubes. 
 
 Where there is no visible membrane (it may be 
 present in the nose or larynx) the swab should be 
 thoroughly rubbed over the mucous membrane of the 
 pharynx and tonsils, and in the nasal cavities, and a 
 culture made from these. In very young children care 
 should be taken not to use the swab when the throat 
 contains food or vomited matter, as then the bacterio- 
 logical examination is rendered more difficult. Under 
 no conditions should any attempt be made to collect 
 the material shortly after the application of strong 
 
DIPHTHERIA BACILLUS. 381 
 
 disinfectants (especially solutions of corrosive subli- 
 mate) to the throat. 
 
 Examination of Cultures. The culture tubes which 
 have been inoculated, as described above, are kept in 
 an incubator at 37 C. for twelve hours, and are then 
 ready for examination. When great haste is required, 
 even five hours will often suffice for a sufficient growth 
 of bacteria for a skilled examiner to decide as to the 
 presence or absence of the bacilli. On inspection it 
 will be seen that the surface of the blood-serum is 
 dotted with numerous colonies, which are just visible. 
 No diagnosis can be made from simple inspection ; if, 
 however, the serum is found to be liquefied or shows 
 other evidences of contamination the examination will 
 probably be unsatisfactory. 
 
 In order to make a microscopical preparation a clean 
 platinum needle is inserted in the tube and quite a 
 large number of colonies are swept with it from the 
 surface of the culture medium, a part being selected 
 where small colonies only are found. A sufficient 
 amount of the bacteria adherent to the needle are 
 washed off in the drop of water previously placed on 
 the cover-glass and smeared over its surface. The 
 bacteria on the glass are then allowed to dry in the 
 air. The cover-glass is then passed quickly through 
 the flame of a Bunsen burner or alcohol lamp, three 
 times in the usual way, covered with a few drops of 
 Loffler's solution of alkaline methylene-blue, and left 
 without heating for ten minutes. It is then rinsed off 
 in clear water, dried, and mounted in balsam. When 
 other methods of staining are desired they are carried 
 out in the proper way. 
 
 In the great majority of cases one of two pictures 
 
382 BACTERIOLOGY. 
 
 will be seen with the 1/12 oil immersion lens either 
 an enormous number of characteristic Loffler-ba( illi, 
 with a moderate number of cocci, or a pure culture of 
 cocci, mostly in pairs or short chains (see Streptococ- 
 cus). In a few cases there will be an approximately 
 even mixture of Loffler bacilli and cocci, and in others 
 a great excess of cocci. Beside these, there will be 
 occasionally met preparations in which, with the cocci, 
 there are mingled bacilli more or less resembling the 
 Loffler bacilli. These bacilli, which are usually of 
 the pseudodiphtheria type of bacilli (see Fig. 46), are 
 especially frequent in cultures from the nose. 
 
 In not more than one case in twenty will there be 
 any serious difficulty in making the diagnosis, if the 
 serum in the tube was moist and had been properly 
 inoculated. In such a case another culture must be 
 made or the bacilli plated out and tested in pure 
 culture. 
 
 Direct Microscopical Examination of the Exudate. 
 An immediate diagnosis without the use of cultures 
 is often possible from a microscopical examination of 
 the exudate. This is made by smearing a slide or 
 cover-^lass with a little of the exudate from the swab, 
 drying, heating, staining, and examining it microscop- 
 ically. This examination, however, is much more diffi- 
 cult, and the results are more uncertain than when the 
 covers are prepared from cultures. The bacilli from 
 the membrane are usually less typical in appearance 
 than those found in cultures, and they are mixed with 
 fibrin, pus, and epithelial cells. They may also be 
 very few in number in the parts reached by the swab, 
 or bacilli may be met with which closely resemble the 
 Loffier bacilli in appearance, but which differ greatly 
 
DIPHTHERIA BACILLUS. 383 
 
 in growth and in other characteristics, and have abso- 
 lutely no connection with them. When in a smear 
 containing mostly cocci a few of these doubtful bacilli 
 are present, it is impossible either to exclude or to make 
 the diagnosis of diphtheria with certainty. Although 
 in some cases this immediate examination may be of 
 the greatest value, it is not a method suitable for gen- 
 eral use, and should always be controlled by cultures. 
 
 Animal Inoculation as a Test of Virulence. If the 
 determination of the virulence of the bacilli found is 
 of importance, animal inoculations must be made. Ex- 
 periments on animals form the only method of deter- 
 mining with certainty the virulence of the diphtheria 
 bacillus. For this purpose, alkaline broth cultures of 
 forty-eight hours 7 growth should be used for the sub- 
 cutaneous inoculation of guinea-pigs. The amount 
 injected should not be more than one-fifth per cent, of 
 the body-weight of the animal inoculated unless con- 
 trols with antitoxin are made. In the large majority 
 of cases, when the bacilli are virulent, this amount 
 causes death within seventy-two hours. At the autopsy 
 the characteristic lesions already described are found. 
 Bacilli which in cultures and in animal experiments 
 have shown themselves to be characteristic may be 
 regarded for practical purposes as certainly true diph- 
 theria bacilli, and as capable of producing diphtheria 
 in man under favorable conditions. 
 
 For an absolute test of specific virulence antitoxin 
 must be used. A guinea-pig is injected with antitoxin, 
 and then this and a control animal, with double the 
 fatal dose of a broth culture of the bacilli to be tested; 
 if the guinea-pig which received the antitoxin lives, 
 while the control dies, it was surely a diphtheria bacil- 
 
384 BACTERIOLOGY. 
 
 lus which killed by means of diphtheria toxin or, in 
 other words, not simply a virulent bacillus, but a viru- 
 lent diphtheria bacillus. When the bacilli to be tested 
 grow poorly in the simple nutrient bouillon they should 
 be grown in bouillon to which one-third its quantity of 
 ascitic fluid has been added. Quite a number of bacilli 
 have been met with which killed 250 gramme guinea- 
 pigs in doses of 2 to 15 c.c., and yet were unaffected 
 by antitoxin. These bacilli, though slightly virulent 
 to guinea-pigs, produce no diphtheria toxin, and so 
 cannot, to the best of our belief, produce diphtheria in 
 man. 
 
CHAPTER XXII. 
 
 THE BACILLUS OF TETANUS. 
 
 IN 1884, Nicolaier, a student in Fliigge's Institute, 
 produced tetanus in mice and rabbits by the subcutaneous 
 inoculation of particles of garden earth, and showed that 
 the disease was transmissible by inoculation from these 
 animals to others. Carle and Rattoiie, in 1884, demon- 
 strated the infectious nature of tetanus as it occurs in 
 man. Finally, Kitasato, in 1889, obtained the bacillus 
 of tetanus in pure culture and described his method of 
 obtaining it and its biological characters. 
 
 The tetanus bacillus occurs in nature as a common 
 inhabitant of the soil, at least in places where manure 
 has been thrown, being abundant in many localities, 
 not only in the superficial layers, but also at the depth 
 of several feet. It has been found in many different 
 substances and places in hay-dust, in horse and cow 
 manure, in the mortar of old masonry, in the dust from 
 horses' hair, in the dust in rooms of houses, barracks, 
 and hospitals, in the air, and in the arrow poison of 
 certain savages in the New Hebrides, who obtained it 
 by smearing the arrow-heads with dirt from crab holes 
 in the swamps. 
 
 Morphology. Motile, slender rods, with rounded ends, 
 0.3// to 0.5/jt. in diameter by 2/j. to 4fj. in length, usually 
 occurring singly, but, especially in old cultures-, often 
 growing in long threads. They form round spores, 
 
 25 
 
386 BACTERIOLOGY. 
 
 thicker than the cell (from I ft to 1.5// in diameter), 
 occupying one of its extremities and giving to the rods 
 the appearance of small pins (Fig. 48). It is stained 
 with the ordinary aniline dyes, and is not decolorized 
 by Gram's method. The spores may be demonstrated 
 by double-staining with ZiehPs method. 
 
 FIG. 48. 
 
 Tetanus bacilli with spores in distended ends. X 1100 diameters. 
 
 Biology. An anaerobic, liquefying, motile (though not 
 very actively motile) bacillus. Forms spores, and in 
 the spore stage it is not motile. It does not grow at 
 temperatures below 14 C., but grows slowly at tem- 
 peratures from 20 to 24 C., and best at 37 C., when 
 it rapidly forms spores. It will not grow in the pres- 
 ence of oxygen or carbon dioxide gas, but grows well in 
 an atmosphere of pure hydrogen. 
 
 The bacillus of tetanus grows in ordinary nutrient 
 gelatin and agar of a slightly alkaline reaction. The 
 addition to the media of 1.5 per cent, of glucose causes 
 the development to be more rapid and abundant. It 
 also grows abundantly in alkaline bouillon under an 
 atmosphere of hydrogen. 
 
THE BACILLUS OF TETANUS. 387 
 
 Its growth in the animal organism is comparatively 
 scanty, and is usually associated with other bacteria; 
 hence, it is difficult to obtain it in pure culture. The 
 method of procedure proposed by Kitasato, which, how- 
 ever, is not always successful, consists in inoculating an 
 agar tube with the tetanus-bearing material (pus from 
 the inoculation wound), keeping this for twenty-four to 
 forty-eight hours at a temperature of 37 C., and, after 
 the tetanus spores have formed, heating it for about an 
 hour at 80 C., to destroy the associated bacteria. The 
 spores of the tetanus bacillus being able to survive this 
 exposure, anaerobic cultures are then made in the usual 
 way, and the tetanus colonies thus isolated. The fur- 
 ther development is unattended with difficulty. On 
 gelatin plates the colonies develop slowly; they resemble 
 somewhat the colonies of the bacillus subtilis, and have 
 a dense, opaque centre surrounded by fine, diverging 
 rays. Liquefaction takes place more slowly, however, 
 than with the bacillus subtilis, and the resemblance to 
 these colonies is soon lost. In old cultures the entire 
 mass is made iip of a number of fine threads, and the 
 colonies are not unlike those of the common mould. 
 
 The colonies on agar are quite characteristic (San- 
 felice). To the naked eye they present the appearance 
 of light, fleecy clouds; under the microscope, a tangle 
 of fine threads. The extreme fineness of the threads 
 enables them to be distinguished from the colonies of 
 -other anaerobes. 
 
 The stab cultures in gelatin exhibit the appearance of 
 a cloudy, linear mass, with prolongations radiating into 
 the gelatin from all sides. Liquefaction takes place 
 slowly, generally with the production of gas. In stab 
 cultures in agar a growth occurs not unlike in structure 
 
388 BACTERIOLOGY. 
 
 that of a miniature pine-tree. Alkaline bouillon is ren- 
 dered somewhat turbid by the growth of the tetanus 
 bacillus. In all cases a production of gas results, accom- 
 panied by a characteristic and very disagreeable empy- 
 reumatic odor. It also grows in acid culture media, 
 but of itself produces no acid. It develops in milk 
 without coagulating it, and starch is not hydrated by 
 it in its growth (Sanfelice). 
 
 The spores of the tetanus bacillus are very resistant 
 to outside influences; they retain their vitality for 
 months and years in a desiccated condition, and are 
 not destroyed in two and a half months when present in 
 putrefying material (Turco). They withstand an ex- 
 posure of one hour to 80 C., but are killed by an ex- 
 posure of five minutes to 100 C. in the steam sterilizer. 
 They resist the action of 5 per cent, carbolic acid for 
 ten hours, but succumb when exposed to it for fifteen 
 hours. A 5 per cent, solution of carbolic acid, how- 
 ever, to which 0.5 per cent, of hydrochloric acid has 
 been added, destroys them in two hours. When acted 
 upon for three hours by bichloride of mercury (1 : 1000) 
 they are killed, and in thirty minutes when 0.5 per cent. 
 HC1 is added to the solution. If the solution contains 
 1 : 1000 bichloride, with 5 per cent, carbolic and a 0.5 
 per cent. HC1, the spores are killed in ten minutes. 
 Silver nitrate solutions destroy the spores in one minute 
 in 1 per cent, solution and in five minutes in 1 : 1000 
 solution. 
 
 Pathogenesis. In mice, guinea-pigs, rabbits, rats, 
 horses, goats, and a number of other animals inocula- 
 tions of pure cultures of the tetanus bacillus cause typi- 
 cal .tetanus after an incubation of from one to three 
 days. A mere trace only as much as remains cling- 
 
THE BACILLUS OF TETANUS. 389 
 
 ing to a platinum needle of an old culture is often 
 sufficient to kill very susceptible animals like mice and 
 guinea-pigs. Other animals require a larger amount. 
 Birds are but little susceptible, and fowls scarcely at 
 all. It is a remarkable fact that an amount of toxin 
 sufficient to kill a hen would suffice to kill 500 horses. 
 On the inoculation of less than a fatal dose in test- 
 animals a local tetanus may be produced, which lasts 
 for days and weeks and then ends in recovery. On 
 killing the animal there is found at autopsy, just at the 
 point of inoculation, a hemorrhagic spot, and no changes 
 here or in the interior organs other than these. A few 
 tetanus bacilli may be detected locally with great diffi- 
 culty, often none at all; possibly a few may be found 
 in the region of the lymphatic glands. From this 
 scanty occurrence of bacilli the conclusion has been 
 reached that the bacilli of tetanus, when inoculated in 
 pure culture, do not multiply in the living body, but 
 only produce lesions through the absorption of the 
 poison which they produce at the point of infec- 
 tion. These authors also found that pure cultures of 
 tetanus, after the germs had sporulated and the toxins 
 had been destroyed by heat, could be injected into 
 animals without producing tetanus. Even one or two 
 millions of spores, if deprived of the toxins, proved 
 harmless to guinea-pigs, and from 15 to 30 c.c. of 
 broth cultures were harmless to rabbits. But if a cul- 
 ture of non-pathogenic organisms was injected simul- 
 taneously with the spores, or if there was an effu- 
 sion of blood at the point of injection, or if there was 
 a previous bruising of the tissues, the animals surely 
 died of tetanus. Even irritating foreign bodies were 
 introduced along with the spores deprived of their 
 
390 BACTERIOLOGY. 
 
 toxins, and tetanus did not develop; bat if the wounds 
 containing the foreign bodies became infected with 
 other bacteria, tetanus developed and the animals died. 
 From these experiments it seems that a mixed infection 
 is necessary to the development of tetanus when the 
 infection is produced by spores. 
 
 This fact i's of the greatest importance in natural 
 tetanus. Here the infection may be considered as 
 prubably invariably produced by the bacilli in their 
 spore state, and the conditions favoring infection are 
 almost always present. A wound of some kind has 
 occurred, penetrating at least through the skin, though 
 perhaps of a most trivial character, such as might be 
 caused by a dirty splinter of wood, and the bacilli or 
 their spores are thus introduced from the soil in which 
 they are so widely distributed. If in any given case, 
 the tissues being healthy, the ordinary saprophytic 
 germs are killed by proper disinfection at once, a 
 mixed infection does not take place, and tetanus will 
 not develop. If, however, the tissues infected be 
 badly bruised or lacerated, the spores may develop 
 and produce the disease. With regard to the persist- 
 ence of tetanus spores upon objects where they have 
 found a resting-place, Henrijean reports that by means 
 of a splinter of wood which had once caused tetanus 
 he was able after eleven years to again cause the dis- 
 ease by inoculating an animal with the same splinter. 
 The bacilli of tetanus are apparently more numerous 
 in certain localities than in others for example, some 
 parts of Long Island and New Jersey, which have 
 become notorious for the number of cases of tetanus 
 caused by small wounds but they are very generally 
 distributed, as the experiments on animals inoculated 
 
THE BACILLUS OF TETANUS. 391 
 
 with garden earth have shown, and are fairly common 
 in New York City. 
 
 Man and almost all domestic animals are subject to 
 tetanus. On examination of an infected individual 
 very little local evidence of the disease can be discov- 
 ered. Generally at the point of infection, if there is 
 an external wound, some pus is to be seen, in which, 
 along with numerous other bacteria, tetanus bacilli or 
 their spores may be found. By successive inoculation 
 of this pus in susceptible animals the disease can often be 
 reproduced for from four to five generations; but some- 
 times there is a break in the chain, which proves that 
 in such cases the infection has been brought about less 
 by the bacilli than by the toxin which was transmitted 
 with them. 
 
 Not only traumatic tetanus, but also all the various 
 forms of tetanus, are now conceded to be produced by 
 the tetanus bacillus puerperal tetanus, tetanus neona- 
 torum, and idiopathic and rheumatic tetanus. In 
 tetanus neonatorum and puerperal tetanus the infection 
 is introduced through the navel and the inner surface 
 of the uterus. It should be borne in mind, however, 
 that when there is no external and visible wound there 
 may be an internal one. Carbone and Perrero report 
 a case of so-called rheumatic tetanus in which attenu- 
 ated forms of tetanus bacilli were found in the bronchial 
 secretions. These bacilli possessed the morphological 
 and cultural peculiarities of the tetanus bacilli, but 
 they did not produce toxin. Similar anaerobes have 
 been found in meat-juices and in the soil. The bacilli 
 found in the bronchial secretions, therefore, may have 
 been tetanus bacilli which, owing to certain conditions, 
 had lost their virulence, just as we know it to happen 
 
392 BACTERIOLOGY. 
 
 in diphtheria. It may well be supposed that the mucous 
 membranes of the bronchi, and other similar mem- 
 branes, in a condition of catarrhal inflammation, may 
 be more susceptible to tetanus infection than they 
 normally are. 
 
 Tetanus Toxin. It is evident from the localization of 
 the tetanus bacilli at the point of inoculation and their 
 slight multiplication at this point that they owe their 
 action to the production of a powerful toxin. While 
 there are a few cases on record in which the bacilli 
 have been found in the tissues of the animal body 
 other than the point of infection, the fact remains that 
 in the vast majority of cases the tetanus bacillus is 
 localized. This toxin can be readily separated from 
 cultures by filtration. One-hundredth of a milligramme 
 of an eight-day filtered bouillon culture is sufficient, as 
 a rule, to kill a mouse. From this filtrate, however, 
 the active toxic substance has been obtained in a much 
 more concentrated form. The purified and dried tetanus 
 toxin prepared by Brieger and Cohn was surely fatal to 
 a 15 gramme mouse in a dose of 0.00000005 gramme. 
 Reckoning according to the body-weight of 75 kilo- 
 grammes, or 175 pounds, it would require but 0.00023 
 gramme, or 0.23 milligramme of this toxin, to prove 
 fatal to a man. By comparing this with snake- 
 poison, Calmette has found that dried cobra venom 
 requires 0.25 milligramme to kill a rabbit of 4 kilo- 
 grammes' weight, and according to body-weight, it 
 would require 4.375 milligrammes to kill a man of 70 
 kilogrammes. As the fatal dose of atropine for an 
 adult is 130 milligrammes, of strychnine from 30 to 
 100 milligrammes, and of anhydrous prussic acid 54 
 milligrammes, the appalling strength of the tetanus toxin 
 
THE BACILLUS OF TETANUS. 393 
 
 can readily be appreciated (Lambert). What the true 
 composition and constitution of the tetanus poisons are 
 is unknown. It has been shown, however, that it pos- 
 sesses neither the characteristics of an alkaloid (ptomain) 
 nor of an albuminous body (toxalbumin); it is largely 
 precipitated from fluids saturated with ammonium sul- 
 phate. 
 
 The quantity of the toxin produced varies, even 
 when derived from one and the same culture, according 
 to the age of the culture, its composition, reaction, etc. ; 
 and partly it is due to the extreme sensitiveness of the 
 toxin, which cannot bear keeping any length of time or 
 exposure to light, being sensibly affected by most chem- 
 ical reagents and destroyed by heating to 55 to 60 C. 
 for any length of time. It retains its strength best in 
 the dry state. 
 
 Some authors (Kitasato and Sanfelice) have main- 
 tained that the tetanus cultures retain their viru- 
 lence unaltered; others, again, have observed consider- 
 able alteration in toxicity. Righi, for instance, has 
 observed that the tetanus bacillus cultivated under 
 aerobic conditions may entirely lose its virulence. 
 Certain chemical agents also produce on cultures of the 
 tetanus bacillus an attenuation of virulence, if only a 
 temporary one. 
 
 The Action of Tetanus Toxin in the Body. The parts 
 first to be affected with tetanus are in about one- third of 
 the cases in man, and usually in animals the muscles 
 lying in the vicinity of the inoculation for instance, 
 the hind foot of a mouse inoculated on that leg is first 
 affected, then the tail, the other foot, the back and 
 chest muscles on both sides, and the forelegs, until 
 finally there is a general tetanus of the entire body. 
 
394 BACTERIOLOGY. 
 
 In mild cases, or when a dose too small to be fatal 
 has been received, the tetanic spasm may remain con- 
 fined to the muscles adjacent to the point of inocula- 
 tion or infection. According to Gumprecht, the action 
 of tetanus depends upon an increased reflex excita- 
 bility, as in strychnine-poisoning; but it is different 
 from strychnine in its mode of distribution, and prob- 
 ably takes place chiefly through the nervous system, as 
 in rabies. This view is supported by Brunner, Brusch- 
 ettini, and others. Beck has described a peculiar degen- 
 eration in the motor cells of the cord in animals killed 
 by tetanus. This degeneration does not seem to attack 
 the entire cells, but only a peripheral part, and seems 
 to be confined chiefly to the body of the cell, usually 
 leaving the nucleus intact. Only very late do the 
 nucleus and the nucleolus take part in the changes. 
 The changes consist in a swelling of the cell and a 
 homogeneous or finely granular degeneration with a 
 swelling, and, finally, coarse lumping together of the 
 chromatin This is especially evident at the tiny emi- 
 nence from which the axis-cylinder arises and in the 
 axis-cylinder itself. Beck considers this as proving 
 that the poison travels along the axis-cylinder, and 
 that, as the nucleus is the last portion affected, the 
 change is not a necrosis but only a modification of 
 cell function. 
 
 But there is also, in addition, undoubtedly a diffusion 
 of the poison by means of the blood and lymph. The 
 blood usually contains the poison, as has been proved 
 experimentally on animals. Neisser showed that the 
 blood of a tetanic patient was capable of inducing 
 tetanus in animals when injected subcutaneously. 
 Kitasato also found the serous exudates of the pleural 
 
THE BACILLUS OF TETANUS. 395 
 
 and pericardial cavities as well as the blood of tetanic 
 animals would cause tetanus when transferred to other 
 animals. Kahlmeyer, Bruschettini, and others have 
 obtained similar results. The toxin has also been 
 demonstrated in the urine when large amounts have 
 been inoculated. 
 
 Courmont and Doyon believe that the so-called toxin 
 elaborated by the tetanus bacillus is not the true poison, 
 but is a ferment which forms from the poison in the 
 body at the expense of the organism, and is found 
 in the blood, sometimes in the urine, and in, especial 
 abundance in tetanized muscles. The action of tetanus 
 toxin is never suddenly produced, though when once 
 formed its absorption is rapid, but always requires a 
 certain period of incubation. These authors hold that 
 the substances produced by the tetanus bacillus must 
 undergo a chemical change in the body, because after 
 it is formed in the tissues it can be extracted from 
 them by boiling, and when injected into other animals 
 causes immediate tetanic symptoms without any period 
 of incubation. But other observers repeating these 
 experiments have failed to confirm Courmont and 
 Doyon' s results, and appear to have proved their 
 theory to be untenable. 
 
 Tetanus Antitoxin. Behring and Kitasato were the 
 first to show the possibility of immunizing animals 
 against tetanus infection. Here the question of immu- 
 nity against infection does not consist in producing an 
 increased power of resistance against the development 
 of the infecting agent, as is the case in most infectious 
 diseases, but similar to diphtheria, in bringing about 
 an immunity to the effects of the tetanus toxin. The 
 bacillus of tetanus, as we have seen, does not belong 
 
396 BACTERIOLOGY. 
 
 to the septicsemic class of organisms which spreads 
 through the body, and by their growth and increase 
 produce their effects, but, on the contrary, remains 
 localized at the original point of infection. It pro- 
 duces, however, in its growth a most powerful toxin. 
 The treatment of tetanus is, therefore, directed against 
 the production of toxin and its neutralization in the 
 body. The methods originally proposed by Behring and 
 by Roux for producing a curative serum consisted 
 chiefly in weakening the tetanus toxin by means of 
 chemical, disinfectants (iodine trichloride, Gram's and 
 LugoPs solutions), so that when inoculated into the 
 test-animals they produced comparatively little reac- 
 tion. At the present time we inject the pure unaltered 
 toxin either alone in small doses or along with anti- 
 toxin. After the first dose of toxin the animals acquire 
 a certain tolerance which enables them to stand a dose 
 of a less attenuated toxin or of a greater amount of un- 
 changed toxin. Thus by gradually increasing the doses 
 or the strength of the toxin administered, the animals 
 are finally able to bear injections of large quantities of 
 the strongest toxin. 
 
 These immunizing experiments in tetanus have borne 
 practical fruit, for it was through them that the prin- 
 ciple of serum-therapeutics first became known the 
 protective and curative effects of the blood -serum of 
 immunized animals. It was thus shown that animals 
 could be protected from tetanus infection by the pre- 
 vious or simultaneous injection of tetanus antitoxin, 
 provided that such antitoxic serum was obtained from 
 a thoroughly immunized animal; and from this it was 
 assumed that the same result could be produced in natu- 
 ral tetanus in man; but, unfortunately, the conditions 
 
THE BACILLUS OF TETANUS. 397 
 
 in the natural disease are very much less favorable, in- 
 asmuch as treatment is usually commenced not shortly 
 after the infection has taken place, but often only on 
 the appearance of tetanic symptoms, when the poison 
 has already diffused itself through the body. 
 
 Tetanus Antitoxin. The tetanus antitoxin is developed 
 in the same manner as the diphtheria antitoxin by 
 inoculating the tetanus toxin in increasing doses into 
 horses. The toxin is produced in bouillon cultures 
 grown anaerobically. After ten or fifteen days the 
 culture fluid is filtered through porcelain, and the germ- 
 free filtrate is used for the inoculations. The horses 
 receive half a c.c. as the initial dose of a toxin of which 
 1 c.c. kills 250,000 grammes of guinea-pig, and along 
 with this a sufficient amount of antitoxin to neutralize 
 it. In five days this dose is doubled, and then every 
 five to seven days larger amounts are given. The dose 
 is increased, as rapidly as the horse s can stand it, until 
 they support 700 to 800 c.c. or more at a single injec- 
 tion. After some months of this treatment the blood 
 of the horse contains the antitoxin in sufficient amount 
 for therapeutic use. When the animals 7 temperatures 
 are normal and they have recovered from the dose of 
 toxin last given, they are bled into sterile flasks and 
 the serum collected. 
 
 Technique of Testing Antitoxin Serum for Value in 
 Antitoxin. Tetanus antitoxin is tested exactly in the 
 same manner as diphtheria antitoxin, except that the 
 standard unit is different. The test toxin used in the 
 German method is one of which 1 gramme destroys 
 150,000,000 grammes of mouse. This is dissolved in 
 33 J c.c. of 10 per cent. NaCl solution. Ten times the 
 amount of antitoxic serum which neutralizes 1 c.c. of 
 
398 BACTERIOLOGY. 
 
 this dilution of the test toxin contains one unit of anti- 
 toxin. In the French method the amount of antitoxin 
 which is required to protect a mouse from a dose of 
 toxin sufficient to kill in four days is determined, and 
 the strength of the antitoxin is stated by determining 
 the amount of serum required to protect one gramme 
 of animal. If 0.001 c.c. protected a 10 gramme mouse 
 the strength of that serum would be 1 : 10,000. Guinea- 
 pigs are sometimes used in place of mice. Knorr's toxin 
 is preserved by precipitating it with saturated ammo- 
 nium sulphate and drying and preserving the precipi- 
 tate in sealed tubes. As required, it is dissolved in 10 
 per cent, salt solution, as above stated. For small 
 testing stations the best way is to obtain some freshly 
 standardized antitoxin and compare serums with this. 
 
 The Persistence of Antitoxin in the Blood. Ransom 
 has recently shown that the tetanus antitoxin is elimi- 
 nated just about as rapidly from the blood of an animal 
 when produced by toxin injections as when injected 
 with antitoxin, so long as the serum was from an ani- 
 mal of the same race. When from a different race, it 
 is much more quickly eliminated. From this we see 
 a possible explanation of the fact that immunity in 
 man, due to an injection of the antitoxic serum of the 
 horse, is less persistent than immunity conferred by an 
 attack of the disease. 
 
 He found some interesting facts in testing the anti- 
 toxic values of the serum of an immunized mare, of its 
 foal, and of the milk. The foal's serum was one-third 
 the strength of the mare's, and one hundred and fifty 
 times that of the mare's milk. In two months the 
 mare's serum lost two-thirds in antitoxic strength, the 
 foal's five-sixths, and the milk one-half. Injections of 
 
THE BACILLUS OF TETANUS. 399 
 
 toxin were then given the mare, so that it doubled its 
 original strength in one month. The milk increased 
 eightfold, but the foal's continued to lose in antitoxin, 
 although it was feeding on the antitoxic milk. 
 
 Eesults of the Antitoxin Treatment in Tetanus. Tetanus 
 is a comparatively rare disease both in man and animals, 
 though in some localities it is more common than in 
 others. In New York city there are usually fifteen 
 to thirty cases following every fourth of July. Most of 
 them are caused by infection through blank cartridge 
 wounds. Recovery sometimes follows from the ordi- 
 nary symptomatic treatment or without treatment at 
 all, so that the statistics of cures of the disease by the 
 injection of antitoxic serum must be very carefully 
 sifted before they can be accepted as reliable. Lambert, 
 however, who has recently made an exhaustive study 
 of tetanus, states that in a total of 114 cases of this 
 disease treated with antitoxin, according to published 
 and unpublished reports, there was a mortality of 40.35 
 per cent. Of these, 47 were acute cases that is, cases 
 with an incubation period of eight days or less and with 
 rapid onset, or cases with a longer period of incubation, 
 but intensely rapid onset of symptoms; of these the 
 mortality was 74.46 per cent. Of the chronic type 
 those with an incubation period of nine days or more, 
 or those with shorter incubation with slow onset there 
 were 61 cases, with a mortality of 1 6.39 per cent. With 
 a still larger number of cases the results indicate that 
 with tetanus antitoxin about 20 per cent, better results 
 are obtained than without. The new method of inject- 
 ing from 3-15 c.c. of antitoxic serum into the lateral 
 ventricles has not, in the writer's opinion, shown itself 
 to be superior to the intravenous or subcutaneous 
 
400 -BA CTERIOL OGY. 
 
 methods. Some speak well of it. No bad results have 
 followed the injections when the serum was sterile and 
 the operation was performed aseptically. 
 
 The Dosage of Tetanus Antitoxin. For immunization 
 10 c.c. of a serum of a strength of 1:1,000,000,000 
 will suffice unless the danger seems great, when the 
 injection is repeated at the end of a week. For treat- 
 ment, it is well to begin with 50 c.c., and then, accord- 
 ing to the severity of the case, give from 20 to 50 c.c. 
 each day until the symptoms abate. In the gravest 
 cases no curative effect will be noticed from the serum. 
 
 Though these few cases are not sufficient to form a 
 final judgment of any treatment, Lambert concludes 
 that by means of the antitoxin treatment, combined 
 with other rational methods, the prognosis, even in 
 acute cases of tetanus, has been improved; but that 
 it still remains exceedingly grave so much so that 
 the preventive inoculation of serum in all cases where 
 dirt has been ground into serious contusions de- 
 serves a much more extensive consideration than has 
 heretofore been given it. The striking results which 
 have been obtained, particularly in veterinary practice, 
 with the prophylactic injection of tetanus antitoxin, 
 would seem to warrant the treating of patients with 
 immunizing doses of serum at least in neighborhoods 
 where tetanus is not uncommon when the lacerated 
 and dirty condition of their wounds may indicate the 
 possibility of a tetanus infection. 
 
 Differential Diagnosis. The differential diagnosis of 
 the bacillus of tetanus is, generally speaking, not diffi- 
 cult, inasmuch as animal inoculation affords a sure test 
 of the specific organism. No other micro-organism 
 known produces similar effects to the tetanus bacillus, 
 
THE BACILLUS OF TETANUS. 4Q1 
 
 nor is any other neutralized by tetanus antitoxin. The 
 other characteristics also of this bacillus are usually dis- 
 tinctive, though microscopical examination alone cannot 
 be depended on to make a differential diagnosis. Diffi- 
 culty arises when other anaerobic or aerobic bacilli, 
 almost morphologically identical with the tetanus 
 bacillus, are encountered which are non-pathogenic, 
 such as the bacillus pseudotetanicus anaerobius, already 
 mentioned, and the bacillus pseudotetanicus aerobius. 
 It is possible, however, that both these bacilli, when 
 characteristic in cultures, are only varieties of the 
 tetanus bacillus, which, under unfavorable conditions of 
 growth, have lost, their virulence. These non-virulent 
 types do not, as a rule, have spores absolutely at their 
 ends, and the spores themselves are usually more ovoid 
 than those in the true tetanus bacilli. 
 
 26 
 
CHAPTER XXIII. 
 
 BACILLUS TYPHOSUS (EBERTH-GAFFKY ? S BACILLUS OF 
 TYPHOID FEVER ; BACILLUS TYPHI ABDOMINALIS). 
 
 THIS organism was first observed by Eberth, and inde- 
 pendently by Koch, in 1880, in the spleen and diseased 
 organs of the intestine in typhoid cadavers, but was 
 not obtained in pure culture and its principal biologi- 
 cal cultures described until the researches of Gaffky, 
 in 1884. Its etiological relationship to typhoid fever 
 has been particularly difficult of demonstration, for 
 although pathogenic for many animals when subcuta- 
 neously or intravenously inoculated, it has been almost 
 impossible to produce infection or in any way give rise 
 to lesions corresponding to those occurring generally in 
 man. It has been recently shown, however, that 
 animals under certain conditions, when their power 
 of resistance has been reduced, as by exposure to the 
 influence of noxious gases, may be rendered susceptible 
 to infection, with the production of more or less char- 
 acteristic lesions. These results, together with the 
 specific reactions of the blood-serum of typhoid patients, 
 as first pointed out by Pfeiffer, Gruber, Widal, and 
 others, and the constant presence of the bacillus 
 typhosus in the intestines and in some of the organs 
 of the typhoid cadavers, as shown by its frequent 
 isolation from the spleen, blood, and excretions of the 
 sick during life and its absence in healthy persons, 
 
BACILLUS TYPHOSUS. 
 
 403 
 
 unless they are convalescent from typhoid infection, 
 have demonstrated, on a scientific basis, that this bacil- 
 lus is the chief etiological factor in the production of 
 typhoid fever. 
 
 Morphological Characters. The typhoid bacilli are 
 rods of about l/i to 3/2 in length by 0.5// to Sfjt in 
 diameter, with rounded ends, often growing into long 
 threads. They are usually longer and somewhat more 
 slender in form than the bacilli coli communis under 
 similar conditions. The typhoid bacilli vary, however, 
 in shape when grown in different culture media. (See 
 Figs. 49, 50, and Fig. 6, page 39.) 
 
 FIG. 49. 
 
 FIG. 50. 
 
 Typhoid bacilli from nutrient agar. 
 X 1100 diameters. 
 
 Typhoid bacilli from nutrient gelatin. 
 X 1100 diameters. 
 
 The typhoid bacilli stain with the ordinary aniline 
 colors, but a little less readily than do most other bac- 
 teria, though there is no constant difference in staining 
 characteristics between these and other bacilli of this 
 group the colon bacilli. They are decolorized by 
 Gram's iodine solution. Not infrequently, particu- 
 larly when grown on potato, refractive granules may 
 
404 BACTERIOLOGY. 
 
 be seen at the ends of the rods, which stain more in- 
 tensely, and either at the extremities or along the body 
 " vacuoles" are observed, which remain unstained; but 
 as these show even less resistant power than the homo- 
 geneous bacilli found in other cultures, they are cer- 
 tainly not spores, but probably are evidences only of 
 retrograde changes and effects of the drying prepara- 
 tory to staining. 
 
 FIG. 51. 
 
 
 Flagella, heavily stained, attached to bacilli. 
 
 The bacilli, when existing under favorable conditions, 
 are, although in various cultures to a different degree, 
 very actively motile, the smaller ones having often an 
 undulating motion, while the larger rods dart about 
 rapidly, with a snake-like movement. This movement 
 is produced by a number of delicate locomotive organs 
 in the form of fine, hair-like flagella, which are arranged 
 around the bodies of the bacilli. (Fig. 10, page 43, 
 and Fig. 51.) The flagella are usually from eighteen 
 to twenty in number, but many short rods have but a 
 single terminal flagellum. They are not seen in un- 
 stained preparations, nor are they rendered visible by 
 
BACILLUS TYPHOSUS. 405 
 
 the ordinary methods of staining. (See Staining of 
 Flagella, page 205.) 
 
 Biological Characters. The typhoid bacillus is a 
 motile, aerobic, non-liquefying bacillus, developing 
 best at 37 C.; over 40 and below 30 its growth is 
 retarded; below 10 it ceases. It grows most abun- 
 dantly in the presence of oxygen, but oxygen is not 
 essential to its development. 
 
 Its growth on most culture media is similar to that 
 of the bacillus coli conimunis, but it is somewhat 
 slower and not quite so luxuriant. 
 
 FIG. 52. 
 
 A superficial and a deep colony of typhoid bacilli in gelatin. 
 X 50 diameters. 
 
 Growth on Gelatin Plates. (Fig. 52.) The colonies 
 growing deep down in this plate medium have nothing 
 in their appearance to distinguish them; they appear 
 as round points with a -sharp margin, of a yellowish- 
 
406 BACTERIOLOGY. 
 
 brown color, and finely granular. The superficial 
 colonies, however, particularly when young, are often 
 quite characteristic; they are transparent, bluish-white 
 in color, with an irregular outline, not unlike a grape- 
 leaf in shape. Slightly magnified they appear homo- 
 geneous in structure, but marked by a delicate network 
 of furrows. 
 
 In stick cultures in gelatin the growth is mostly on the 
 surface, appearing as a thin, scalloped extension, which 
 gradually reaches out to the sides of the tube. In the 
 track of the needle there is but a limited growth, which 
 may be streaked, granular, or uniform in structure, and 
 of a yellowish-brown color. There is no liquefaction. 
 
 Growth in Bouillon. This medium is uniformly 
 clouded by the typhoid bacillus, but the clouding is not 
 so intense as by the colon bacillus. A film is frequently 
 formed on the surface after eighteen to twenty-four 
 hours' growth. A very slight amount of acid is pro- 
 duced. 
 
 Growth on Agar. The streak cultures on agar are 
 not distinctive; a transparent, grayish streak is formed. 
 
 Growth on Potato. The growth on this medium 
 has been held by some to be very important, but it 
 varies considerably. When characteristic the growth 
 is invisible, but luxuriant, usually covering the surface 
 of the medium, and when scraped with the needle offers 
 a certain resistance. In some cases, however, the 
 growth is restricted to the immediate vicinity of the 
 point of inoculation, not very luxuriant, and of the 
 same color as the potato. Again, the growth may be 
 quite heavy and colojred yellowish-brown, and with a 
 greenish halo, when it is very similar to the growth of 
 the colon bacillus. These differences of growth on this 
 
BACILLUS TYPHOSUS. 407 
 
 medium appear to be chiefly due to variations in the 
 substance, especially in the reaction, of the potato. 
 
 Milk. The typhoid bacillus does not cause coagu- 
 lation when grown in sterilized milk. 
 
 Fermentation. It does not produce fermentation in 
 either glucose, lactose, saccharose, or glycerin bouillon, 
 and evolves no gas as the result of fermentation. 
 
 Lactose-litmus Agar. It grows usually as pale blue 
 colonies on lactose-litmus agar, but occasionally causes 
 slight reddening of the surrounding medium. 
 
 Indol Reaction. It does not produce indol. This 
 test was proposed by Kitasato for differentiating the 
 typhoid bacillus from other similar bacilli, such as 
 those of the colon group, which, as a rule, give the 
 indol reaction. 
 
 The reaction, being a very delicate one, requires 
 great care in its performance to arrive at accurate con- 
 clusions. (For test of indol, see page 77.) Instead of 
 bouillon, the simple peptone-water (which consists of 
 dried peptone, 1 part; sodium chloride, 0.5 part, and 
 distilled water, 100 parts) is to be preferred for this 
 purpose, because its pale color does not mask the reac- 
 tion*. 
 
 Pathogenic Properties. It has been extremely diffi- 
 cult to show experimentally that the bacillus typhosus 
 is specifically pathogenic for animals. A great many 
 experiments have been made, with the view of repro- 
 ducing in the tissues of lower animals the pathological 
 lesions of typhoid fever as seen in man, but the results 
 have not been completely satisfactory ; nor is this sur- 
 prising when one considers that this disease does not 
 occur naturally, so far as is known, among animals. 
 Sickness or fatal results without the appearance of the 
 
408 BACTERIOLOGY. 
 
 typical pathological changes have regularly followed 
 animal inoculations, but in most cases they could easily 
 be traced to the toxaemia produced by the substances in 
 the bodies of the bacilli injected, not necessarily accom- 
 panied by the growth of the organism, rather than to 
 infection due to the development of the typhoid bacillus 
 in the tissues. 
 
 In a certain number of cases subcutaneous and intra- 
 peritoneal inoculations in animals have been productive 
 of more or less typical typhoid lesions. Among the 
 most successful efforts in this direction are the experi- 
 ments of Cygnaeus and Seitz, who, by the inoculation 
 of the typhoid bacillus into dogs, rabbits, and mice, 
 produced in the small intestines conditions that were 
 histologically and to the naked eye analogous to those 
 found in the human subject, but their results were not 
 constant. Of a number of experiments made by Ab- 
 bott, with the same object in view, only one positive 
 result followed the introduction of typhoid bacilli into 
 the circulation of rabbits. In this case the ulcer in 
 the ileum was macroscopically and microscopically iden- 
 tical with those found at autopsy in the small intestines 
 of the human subject dead of this disease. The bacilli 
 were found in the spleen. 
 
 Experiments indicate that the presence of other bac- 
 teria in the body, and of exposure to the effect of nox- 
 ious gases in lowering the natural resistance of the 
 individual, render him more susceptible to infection 
 from typhoid fever and, indeed, from other infectious 
 diseases. 
 
 But whatever conclusions may be drawn from these 
 results, with regard to the typhoid process in animals, 
 typhoid fever in the human subject is now recognized 
 
BACILLUS TYPHOSUS. 409 
 
 as a true infection, caused by the introduction and 
 growth of typhoid bacilli. It belongs to that class of 
 infectious diseases which are known as metastatic that 
 is to say, diseases in which the specific bacilli do not 
 abound in the entire circulation, as in septicaemia, nor 
 remain localized in one place, but are distributed in 
 groups throughout the body. The characteristic lesions 
 of typhoid fever are seated in the lymphatic structures of 
 the intestine namely, the solitary follicles and patches 
 of Peyer, the mesenteric glands, and the spleen. The 
 liver and kidneys are less commonly attacked. Wher- 
 ever found the typhoid bacilli are observed to be arranged 
 in groups or foci; only occasionally, as in the walls of 
 the intestine, are they singly or loosely aggregated to- 
 gether. These foci are formed, most probably, during 
 life, as is proved by the degenerative changes often 
 seen about them; but it is possible that the bacilli may 
 also multiply somewhat after death. 
 
 The production of the lymph-nodules so often found 
 in typhoid fever in the internal organs is due to the 
 effects of the toxic substances eliminated by the typhoid 
 bacilli. This hyperplasia is particularly evident in the 
 lymphatic structures of the intestine, these being more 
 directly under the influence of the concentrated products 
 of the bacilli. To these, however, other inflammatory 
 processes are added, until finally necrosis or sloughing 
 of the tissues takes place. Possibly all these series of 
 changes may be at times caused solely by the products 
 of the typhoid bacilli which are gathered at certain 
 points. There is no question, however, that usually 
 other organisms take part in the production of these 
 processes in the intestines, but it remains to be deter- 
 mined when they begin to do so. In typhoid fever 
 
410 BACTERIOLOGY. 
 
 necrosis of the tissues of the internal organs is of 
 comparatively rare occurrence. Caseation of the mes- 
 enteric glands, which is commonly observed, is due 
 probably to mixed infection. There are, however, a 
 number of cases now on record in which the typhoid 
 bacillus has played the part of pus producer. Cases 
 of sacculated and general peritonitis, subphrenic ab- 
 scess, osteomyelitis, periostitis, and inflammatory pro- 
 cesses of other kinds have been reported as being 
 due to the typhoid bacillus. Kruse also reports an 
 abscess of the spleen which contained only bacillus 
 typhosus, and typhoid abscess of the liver has been 
 recorded by many. In certain cases of typhoid pneu- 
 monia, serous pleurisy, empyema, and meningitis, 
 typhoid bacilli exclusively have occurred. The in- 
 flammation produced may or may not be accompanied 
 by the formation of pus. As argument against the 
 observations above cited there has been brought forward 
 the supposition that probably the real cause of the 
 disease had been destroyed before the entrance of the 
 typhoid bacillus. Though this may be true of some 
 cases, as in pneumonia, which is caused usually by the 
 short-lived pneumococcus, there is no reason to doubt 
 the causal relation of the typhoid bacillus to the other 
 diseases, inasmuch as it has been proved by numerous 
 investigations. 
 
 Such cases, however, are of comparatively rare occur- 
 rence, because only exceptionally do the bacilli suffi- 
 ciently mass together in such numbers as to become 
 pus producers. As a rule, when complications occur 
 in typhoid fever they are due to secondary or mixed in- 
 fection with the staphylococcus, pneumococcus, strepto- 
 coccus, pyocyaneus, and colon bacillus. Frequently 
 
BACILLUS TYPHOSUS. 411 
 
 these bacteria are found side by side with the typhoid 
 bacilli; in such cases it is difficult to say which was the 
 primary and which was the secondary infection. 
 
 The peculiar arrangement of the typhoid bacilli in the 
 body can only be explained by their passage through 
 the circulation; and this is proved by the bacilli being 
 found in the spleen almost constantly and in smaller 
 numbers in the blood itself. Thus, Neuhauss has had 
 nine positive results out of fifteen in cultures from vein 
 blood. 
 
 The typhoid bacillus can be transmitted also from 
 the blood of the mother to the foetus (E berth, Fraenkel, 
 etc.). In one case reported by Ernst a living child, 
 four days after birth, showed evidences of general 
 typhoid infection, icterus and rose-spots. Frascani 
 reports that in animal experiments he has frequently 
 found typhoid bacilli in the foetus. 
 
 Not infrequently typhoid bacilli are found in the 
 secretions. They are present in the urine in about 20 
 per cent, of the cases in the third and fourth week of 
 typhoid fever. Slight pathological lesions in the kid- 
 neys almost always occur in typhoid fever, but severe 
 lesions also sometimes occur. In a case under our ob- 
 servation the urine was distinctly purulent and crowded 
 with typhoid bacilli. The bacillus typhosus is not 
 commonly found in the sweat, but Geisler observed it 
 once. It has also been detected, though rarely, in the 
 sputum and secretions of the throat. 
 
 In cases of pneumonia due to the typhoid bacillus it 
 is abundantly present in the sputa, and care should be 
 taken to disinfect the expectoration of typhoid patients. 
 According to Chiari, in typhoid fever the bacilli are 
 almost always present in the gall-bladder. The bacilli 
 
412 BACTERIOLOGY. 
 
 are frequently eliminated by the feces being derived 
 from the inflamed^ mucous surface of the intestines; 
 their growth within the intestinal canal itself, even if 
 it occurs to a limited extent, is probably not extensive. 
 Methods of Infection. With regard to the mode of 
 invasion of the typhoid bacilli, there is no doubt that 
 it is principally by way of the mouth, through the 
 stomach to the intestines. Mayer reports a particularly 
 convincing illustration of this fact in a case where death 
 ensued on the second day of the disease. On autopsy 
 were found hypersemia of the lungs, spleen, and kid- 
 ney; in the lower portion of the ileum great enlarge- 
 ment of the solitary follicles and patches of Peyer, but 
 nowhere a trace of necrosis or loss of substance; nor 
 were the mesenteric glands enlarged. Microscopically 
 an extraordinary deposit of characteristic bacilli were 
 found in the submucosa and interstitial spaces of the 
 muscles; many hundred bacilli lay in one field. On 
 the other hand, several cases are recorded in which the 
 intestinal changes were entirely wanting, and only a 
 localization of bacilli and lesions in the mesenteric 
 glands and spleen revealed the nature of the infection. 
 Inasmuch as they were present in the lymph-glands 
 which belong to the intestines, it may be assumed, 
 thinks Kruse, who reports one of these cases, that the 
 bacilli were here more rapidly absorbed than usual with- 
 out multiplying to any extent in the intestines. The 
 case mentioned by Guarnieri is also worthy of notice: 
 in this there was apparently a primary infection of the 
 gall-ducts, with no accompanying lesions in the intes- 
 tine. Bacilli were found in the blood twelve days be- 
 fore death, and on autopsy pure cultures were obtained 
 from the liver and spleen. 
 
BACILLUS TYPHOSUS. 413 
 
 Not only do the very great majority of cases exam- 
 ined bacteriologically and pathologically, but the epi- 
 demiological history of the disease, prove that the 
 chief mode of invasion of the typhoid bacillus is by 
 way of the mouth and stomach. The infective mate- 
 rial is discharged principally by means of the excretions 
 and secretions of. the sick namely, by the feces, the 
 urine, and occasionally by the sputum. 
 
 Of considerable practical importance is it to know for 
 what length of time the typhoid bacillus is capable of 
 living outside of the body; but, unfortunately, owing 
 to the great difficulties in proving the presence of this 
 organism in natural conditions, our knowledge on this 
 point is very deficient. In feces the length of life of 
 the typhoid bacilli is very variable; sometimes they 
 live but a few hours, usually a few days, exceptionally 
 for very long periods. Thus, according to Uffelmann, 
 typhoid bacilli may remain alive in feces for five and 
 a half months, and, according to Karlinski, for at least 
 several months. Foote says that they can be found in 
 living oysters for a month at a time. Their life in 
 feces and in water, however, is usually very much 
 shorter. As a rule, they can be detected in water no 
 longer than fourteen days after introduction. The 
 life of the typhoid bacillus varies according to the 
 abundance and varieties of the bacteria associated with 
 it and according to the presence or absence of such in- 
 jurious influences as high temperature, light, desicca- 
 tion, etc., to which it is peculiarly sensitive. That the 
 bacilli do live much longer under favorable circum- 
 stances, as to protection and nourishment, than is gen- 
 erally supposed, is shown by the fact, as reported by 
 Buschke, that they were found in an old bone-centre 
 
414 BACTERIOLOGY. 
 
 seven years after the original infection. There is no 
 reason to deny that such opportunities for a latent ex- 
 istence of the typhoid bacillus may not occur outside 
 of the body. Indeed, many epidemics of typhoid 
 fever can only be accounted for by some such assump- 
 tion of latency in or outside of the body. 
 
 The bacilli may reach the mouth by means of infected 
 fingers or articles of various kinds, or by the ingestion 
 of infected food, milk, water, etc., or by more obscure 
 ways, such as the contamination of food by flies and 
 other insects, or by the inhalation through the mouth 
 of dust containing typhoid bacilli. Of the greatest 
 importance, however, is the production of infection 
 by contaminated drinking-water or through drinking- 
 water or milk, which is the most plausible explana- 
 tion for the majority of epidemics of typhoid fever. 
 In many cases indirect proof of this mode of infec- 
 tion has been found in the known contamination of 
 the water with typhoid feces or urine, and in some few 
 cases it has been confirmed by direct proof in finding 
 the bacilli. Examples of infection from water and 
 milk have come frequently under our direct observa- 
 tion for instance, a large force of workmen obtained 
 their drinking-water from a well very near to their 
 work. Typhoid fever broke out, and continued to 
 spread until the well was filled up. Investigation 
 showed that some of the sick, before their discovery, 
 repeatedly infected the soil surrounding the well with 
 their urine and feces. Another instance of milk in- 
 fection secondary to water infection was the case of a 
 milk dealer whose son came home suffering from 
 typhoid fever. The intestinal movements were thrown 
 into a small stream which ran into a pond from which 
 
BACILLUS TYPHOSUS. 415 
 
 the milk cans were washed. A very alarming epidemic 
 of typhoid developed, which was confined to the houses 
 and asylums supplied with this milk. In our late war, 
 not only water infection but food infection was notice- 
 able, as in the case of a regiment where certain com- 
 panies were badly infected, while others nearly escaped. 
 Each company had its separate kitchen and food-supply, 
 and much of the infection could be traced to the food. 
 
 In this, as in all infectious diseases, individual sus- 
 ceptibility plays an important role in the production of 
 infection. Without a suitable soil upon which to grow 
 the seed cannot thrive. There must in many be some 
 disturbance of the digestion, excesses in drinking, etc., 
 or a general weakening of the power of resistance of 
 the individual, caused by bad food, exposure to heat, 
 overexertion, etc., as with soldiers and prisoners, for 
 example, to bring about the conditions suitable for the 
 production of typhoid fever. 
 
 The supposition that the breathing of noxious gases 
 is conducive to the disease, though possibly true to a 
 certain extent, as some animal experiments already 
 referred to would seem to indicate, has not yet been 
 conclusively proven; nor do Pettenkofer's investiga- 
 tions, into the relation of the frequency of typhoid 
 fever to the ground-water level, satisfactorily explain 
 the occurrence of the disease in most cases, whether 
 sporadically or in epidemics. 
 
 Immunization. Specific immunization against experi- 
 mental typhoid infection has been produced in mice, 
 guinea-pigs, rabbits, dogs and other animals by the 
 usual method of injecting at first small quantities of 
 the living or dead typhoid culture and gradually in- 
 creasing the dose. The blood-serum of animals thus 
 
416 BACTERIOLOGY. 
 
 immunized has been found to acquire protective and 
 curative bactericidal and perhaps feeble antitoxic prop- 
 erties against the typhoid bacillus. These character- 
 istics have also been observed in the blood-serum of 
 persons who are convalescent from typhoid fever 
 (Pfeiffer and Kolle, Widal and Chantemesse). Re- 
 cently the attempt has been made to employ the 
 typhoid-serum for the cure of typhoid fever in man, 
 but no marked results have been obtained. The injec- 
 tion in man of very small amounts (0.3 c.c. of bouillon 
 culture) of dead typhoid bacilli produces for a day or 
 two a slight fever reaction, to be followed in a few days 
 by the development of bactericidal substances in the 
 blood, which apparently are sufficient in amount to 
 give immunity for some weeks. The use of immu- 
 nized serum, or when this cannot be obtained of dead 
 cultures, would seem to be advisable where great 
 danger of typhoid infection exists. 
 
 The Diagnosis of Typhoid Fever, or rather of Typhoid 
 Infection, by Means of the Widal or Serum Reaction. 
 The chief practical application of our knowledge of the 
 specific substances developed in the blood of persons 
 sick with typhoid fever has been in the way of 
 diagnosis. In view of the interest which has been 
 manifested in this test, and of the fact that it is now 
 so largely used, a brief history of the investigations 
 which led up to its discovery may be given. 
 
 In 1894-95, Pfeiffer showed that when cultures con- 
 taining dead or living cholera spirilla or typhoid bacilli 
 are injected subcutaneously into animals or man, specific 
 protective substances are formed in the blood of the in- 
 dividuals thus treated. These substances grant a more 
 or less complete immunity against the invasion of the 
 
BACILLUS TYPHOSUS. 417 
 
 living germs of the respective diseases. He also de- 
 scribed the occurrence of a peculiar phenomenon when 
 a portion of a fresh culture of the typhoid bacillus on 
 agar is added to a small quantity of the serum of an 
 animal immunized against typhoid and the mixture 
 injected into the peritoneal cavity of a non-immunized 
 guinea-pig. After this procedure, if from time to time 
 minute drops of the liquid be withdrawn in a capillary 
 tube and examined microscopically, it is found that the 
 bacteria, which were formerly and in control animals, 
 which remain, actively motile and vigorous, become 
 in a very short time, under the influence of the serum, 
 entirely motionless and later dead. They are first im- 
 mobilized, then they become somewhat swollen and 
 agglomerated into balls or clumps, which gradually 
 become paler and paler, until finally they are dissolved 
 in the peritoneal fluid. This process takes place regu- 
 larly in about twenty minutes, provided a sufficient 
 degree of immunity be present in the animals from 
 which the serum was obtained. The animals injected 
 with the mixture of the serum of immunized animals 
 and typhoid cultures remain unaffected, while control 
 animals treated with a fluid containing only the serum 
 of non-immunized animals mixed with typhoid cultures 
 die. Pfeiffer claimed that the reaction of the serum 
 thus employed is so distinctly specific that it may serve 
 for the differential diagnosis of the cholera vibrion or 
 typhoid bacillus from other vibrions or allied bacilli, 
 such as Finkler's and Prior's or colon groups. 
 
 In March, 1896, Pfeiffer and Kolle published an 
 article entitled " The Differential Diagnosis of Typhoid 
 Fever by Means of the Serum of Animals Immunized 
 Against Typhoid Infection/ ' in which they claimed 
 
 27 
 
418 BACTERIOLOGY. 
 
 that by the aid of the presence or absence of this reac- 
 tion in the serum of convalescents from suspected 
 typhoid fever the nature of the disease could be deter- 
 mined. It was further found if the serum of an animal 
 thoroughly immunized to the typhoid bacillus was 
 diluted with 40 parts of bouillon, and a similar dilu- 
 tion made of the serum of non-immunized animals, 
 and both solutions were then inoculated with a culture 
 of the typhoid bacillus and placed in the incubator at 
 37 C., that after the expiration of one hour macro- 
 scopical differences in the culture could be observed, 
 which increased in distinctness for four hours and then 
 gradually disappeared. The reaction occurring is de- 
 scribed as follows : In the tubes in which the typhoid 
 culture is mixed with typhoid serum the bacilli are 
 agglomerated in fine, whitish flakes, which settle to the 
 bottom of the tube, while the supernatant fluid is clear 
 or only slightly cloudy. On the other hand, the tubes 
 containing mixtures of bouillon with cholera or coli 
 serum, or the serum of non-immunized animals inocu- 
 lated with the typhoid bacilli, became and remained 
 uniformly and intensely cloudy. These serum mix- 
 tures, examined microscopically in a hanging drop, 
 show distinct differences. The typhoid serum mixture 
 inoculated with the typhoid bacilli exhibits the organ- 
 isms entirely motionless, lying clumped together in 
 heaps; in the other mixtures the bacilli are actively 
 motile. 
 
 These observations were made independently by 
 Gruber and Durham, who maintained, however, that 
 the reaction described by Pfeiffer was by no means 
 specific, and that when the reaction is positive the 
 diagnosis still remains in doubt, for the reaction is 
 
BA CILL US TYPHOS US. 419 
 
 quantitative only, and not qualitative, so far as the 
 cholera spirillum and typhoid bacillus, at least, are 
 concerned. They conclude, nevertheless, that these 
 investigations will render valuable assistance in the 
 clinical diagnosis of cholera and typhoid fever. It 
 developed through further research that before the 
 development of the bactericidal substances agglutina- 
 tive substances usually appeared in the blood. 
 
 WIDAI^XES^ The first practical application of the 
 use Trf^serum, however, for the early diagnosis of 
 typhoid fever on a more extensive scale was made by 
 Widal, and reported with great fulness and detail in a 
 communication published in June, 1896. Widal con- 
 firmed the reaction as above described, proved that the 
 agglutinative reaction was one of infection and usually 
 occurred early, elaborated the test, and proposed a 
 method by which it may be practically applied for 
 diagnostic purposes. Since then the serum test for 
 the diagnosis of typhoid fever has come into general 
 use in bacteriological laboratories in all parts of the 
 world, and though the extravagant expectations raised 
 at the time when Widal first announced his method of 
 applying this test have not been entirely fulfilled, it 
 has, nevertheless, proved to be of great assistance in 
 the diagnosis of obscure cases of the disease, and it is 
 now one of the recognized tests for the differentiation 
 of the typhoid bacillus. 
 
 It should also be mentioned that to Wyatt Johnson, 
 of Montreal, belongs the credit of having brought this 
 test more conspicuously before the public by intro- 
 ducing its use into municipal laboratories, suggesting 
 that dried blood should be employed in place of blood- 
 serum (Widal having previously noticed that drying 
 
420 BACTERIOLOGY. 
 
 did not destroy the agglutinating properties of typhoid 
 blood); and that in October, 1896, the serum test was 
 regularly employed in the New York Board of Health 
 Laboratory for the routine examination of the blood- 
 serum of suspected cases of typhod fever. Since then 
 numerous health departments have followed the example 
 set by those of Montreal and New York. 
 
 USE OF DRIED BLOOD. Directions for Preparing 
 Specimens of Blood. The skin covering the tip of the 
 finger or the ear is thoroughly cleansed, and is then 
 pricked with a needle deeply enough to cause several 
 drops of blood to exude. Two fair-sized drops are then 
 placed on a glass slide, one near either end, and allowed 
 to dry. Paper may also be employed, but it is not as 
 good, for the blood soaks more or less into it, and 
 later, when it is dissolved, some of the paper-fibre is 
 apt to be rubbed off with it. The slide is placed in a 
 box for protection. 
 
 Preparation of Specimen of Blood for Examination. 
 In preparing the specimens for examination the dried 
 blood is brought into solution by adding to it and mix- 
 ing it with about five times the quantity of water ; then 
 a minute drop of this decidedly reddish mixture is 
 placed on a cover-glass, and to it is added a similar 
 drop of an eighteen to twenty-four-hour-old bouillon 
 culture of the typhoid bacillus, which, if it has a slight 
 pellicle, should be well shaken. The drops, after being 
 mixed, should have a faint reddish or pink tinge. The 
 cover-glass with the mixture on the surface is inverted 
 over a hollow slide (the edges about the concavity 
 having been smeared with vaseline, so as to make a 
 closed chamber), and the hanging drop then examined 
 under the microscope (preferably by gaslight), a high- 
 
BACILLUS TYPHOSUS. 
 
 421 
 
 power dry lens (about 1/8 inch) being used, or, some- 
 what less serviceably, a 1/12 oil-immersion lens. 
 
 THE REACTION. If the reaction takes place rapidly 
 the first glance through the microscope reveals the com- 
 pleted reaction, all the bacilli being in loose clumps 
 and nearly or altogether motionless (Fig. 53). Be- 
 
 FlG. 53. 
 
 Widal reaction. Bacilli gathered into one large and two small clumps, the 
 few isolated bacteria being motionless or almost so. 
 
 tween the clumps are clear spaces containing few or no 
 isolated bacilli. If the reaction is a little less complete 
 a few bacilli may be found moving slowly between the 
 clumps in an aimless way, while others attached to the 
 clumps by one end are apparently trying to pull away, 
 much as a fly caught on fly-paper struggles for freedom. 
 If the agglutinating substances are still less abundant 
 the reaction may be watched through the whole course 
 of its development. Immediately after mixing the blood 
 and culture together it will be noticed that the bacilli 
 
422 BACTERIOLOGY. 
 
 move more slowly than before the addition of serum. 
 Some of these soon cease all progressive movement, 
 and it will be seen that they are gathering together in 
 small groups of two or more, the individual bacilli 
 being still somewhat separated from each other. Grad- 
 ually they close up the spaces between them, and clumps 
 are formed. According to the completeness of the 
 reaction, either all of the bacilli may finally become 
 clumped and immobilized or only a small portion of 
 them, the rest remaining freely motile, and those 
 clumped may appear to be struggling for freedom. 
 With blood containing a large amount of agglutinating 
 substances all the gradations in the intensity of the re- 
 action may be observed, from those shown in a marked 
 and immediate reaction to those appearing in a late and 
 indefinite one, by simply varying the proportions of 
 blood added to the culture fluid. 
 
 Pseudo-reactions. If too concentrated a solution of 
 dried blood from a healthy person is employed there 
 will be an immobilization of the bacilli, but no true 
 clumping. This is sometimes mistaken for a reaction. 
 Again, dissolved blood always shows a varying amount 
 of detritus, partly in the form of fibrinous clumps; and 
 prolonged microscopical examination of the mixture of 
 dissolved blood with a culture fluid shows that the 
 bacilli, inhibited by substances in the blood, often be- 
 come more or less entangled in these clumps, and in 
 the course of one-half to one hour very few isolated 
 motile bacteria are seen. The fibrinous clumps alone, 
 especially if examined with a poor light by a beginner, 
 may be easily mistaken for clumps of bacilli. Again, 
 the bacilli may become clumped after remaining for 
 one-half to two hours by slight drying of the drop or 
 
BACILLUS TYPHOSUS. 423 
 
 the effect of substances on the cover-glass. The reac- 
 tion in typhoid is chiefly due to specific substances, 
 but clumping and inhibition of movement similar in 
 character may be caused by other substances such as 
 exist in normal horse and other serums. This is a 
 very important fact to keep in mind. 
 
 USE OF SERUM. Mode of Obtaining Serum for Ex- 
 amination. Fluid blood-serum can be easily obtained in 
 two ways: First, the serum may be obtained directly from 
 the blood, thus : The tip of the finger or ear is pricked 
 with a lancet-shaped needle, and the blood as it issues 
 is allowed to fill by gravity a capillary tube having a 
 central bulb. The ends of the tube are then sealed 
 by heat or wax, and as the blood clots a few drops of 
 serum separate. This method of obtaining blood- 
 serum has the advantage of rapidity; but it has also 
 disadvantages namely, that the serum thus separated 
 is apt to contain more or less blood-cells, which some- 
 what obscure the field when the liquid serum is imme- 
 diately mixed with the culture, and the needle stab is 
 often objected to. Second, the serum may be obtained 
 from blisters. This gives more satisfactory results, but 
 causes twelve hours' delay. The method is as follows: 
 A section of cantharides plaster, the size of a 5-cent 
 piece, is applied to the skin at some spot on the chest 
 or abdomen. A blister forms in from six to eighteen 
 hours. This should be protected from injury by a 
 vaccine shield or bunion plaster. The serum from 
 the blister is collected in a capillary tube, the ends 
 of which are then sealed. Several drops of the serum 
 can be easily obtained from a blister so small that it is 
 practically painless and harmless. The serum obtained 
 is clear and admirablv suited for the test. 
 
424 BACTERIOLOGY. 
 
 Advantages and Disadvantage of Serum and Dried 
 Blood for the Serum Test. The dried blood is easily 
 and quickly obtained, and does not deteriorate or be- 
 come contaminated by bacterial growth. It is readily 
 transported, and seems to be of nearly equal strength 
 with the serum in its agglutinating properties. It 
 must in use, however, be diluted with at least five 
 times its bulk of water, otherwise it is too viscid to be 
 properly employed. The amount of dilution can only 
 be determined roughly by the color of the resulting 
 mixture, for it is impossible to estimate accurately the 
 amount of dried blood from the size of the drop, and 
 it is too much trouble to weigh it accurately. Serum, 
 on the other hand, can be used in any dilution desired, 
 varying from a mixture which contains equal parts of 
 serum and broth culture to that containing 1 part of 
 serum to 100 parts of culture, and this can be exactly 
 measured by a graduated pipette, or, roughly, by a 
 measured platinum loop. The disadvantages in the 
 use of serum are entirely due to the slight difficulty in 
 collecting and transporting it and the delay in obtain- 
 ing it when a blister is employed. If the serum is 
 obtained from blood after clotting has occurred a 
 greater quantity of blood must be drawn than is neces- 
 sary when the dried-blood method is used ; if it is 
 obtained from a blister, a delay of six to eighteen 
 hours is required. The transportation of the serum 
 in capillary tubes presents no difficulties if tubes of 
 sufficiently thick and tough glass are employed and 
 placed in tiny wooden boxes. For scientific investiga- 
 tions and for accurate results, particularly in obscure 
 cases, the use of fluid serum is to be preferred to dried 
 blood. Practically, however, the results are nearly as 
 
BACILLUS TYPHOSUS. 425 
 
 good for diagnostic purposes from the dried blood as 
 from the serum. 
 
 The Typhoid Culture Employed. It is important that 
 the culture employed for serum-tests should be a suit- 
 able one, for although in our experience all cultures 
 show the reaction, yet some respond much better than 
 others. A broth culture of the typhoid bacillus de- 
 veloped at 35 C., not over twenty-four hours old, in 
 which the bacilli are isolated and actively motile, has 
 been found to give us the most satisfactory results. 
 Stock cultures of typhoid bacilli can be preserved on 
 nutrient agar in sealed tubes and kept in the ice-box. 
 These remain alive for months or even years. From 
 time to time one of these is taken out and used to 
 start a fresh series of bouillon cultures. 
 
 The Dilution of the Blood-serum to be Employed and 
 the Time Required for the Development of Reaction. The 
 serum test, as has been pointed out, is quantitative and 
 not qualitative. By this it is not meant to assert that the 
 agglutinating and immobilizing substances produced in 
 the blood of a patient suffering from typhoid infection 
 are the same as those present at times in normal blood, 
 or those produced in the blood of persons sick from 
 other infections. It is intended, however, to maintain 
 that the effect upon the bacilli, as seen under the micro- 
 scope, is identical, the difference being that in typhoid 
 fever, as a rule, substances which cause this reaction 
 are usually far in excess of the amount which ever 
 appears in non typhoid blood, so that the reaction 
 occurs after the addition to the culture of far smaller 
 quantities of serum than in other diseases, or when the 
 same dilution is used it occurs far more quickly and 
 completely with the typhoid serum. 
 
426 BACTERIOLOGY. 
 
 The results obtained in the health department labor- 
 atories, as well as elsewhere, have shown that in a 
 certain proportion of cases not typhoid fever there 
 occurs a delayed moderate reaction in a 1 to 10 dilution 
 of serum or blood (the proportion originally proposed 
 by Widal); but very rarely, if ever, excepting in 
 typhoid fever, or at least typhoid infection, does a 
 complete reaction occur in this dilution within jive 
 minutes. When dried blood is used the slight ten- 
 dency of non-typhoid blood in 1 to 10 dilution to 
 produce agglutination is increased by the presence of 
 the fibrinous clumps, and perhaps by other substances 
 derived from the disintegrated blood-cells. From many 
 cases examined by Fraenkel, Stern, Forster, Scholtz, 
 ourselves and others, it has been found that in dilutions 
 of 1 to 20 or more a decided, quick reaction is never 
 produced in any febrile disease other than that due to 
 typhoid infection, while in typhoid fever such a distinct 
 reaction often occurs with dilutions of 1 to 50. 
 
 The mode of procedure, therefore, as now employed 
 is as follows : The test is first made with the typhoid 
 bacillus in a 10 per cent, solution of serum or blood. 
 In the case of serum, one part of serum is added to 
 nine of the bouillon culture. With dried blood, a solu- 
 tion of the blood is first made, and the final dilution 
 guessed from the color of the mixed culture and blood 
 solution. To obtain an idea of the dilution by the 
 color, known amounts of blood are dried and then 
 mixed with definite amounts of water ; the colors re- 
 sulting are fixed in the memory as guides for future 
 tests. If there is no reaction that is to say, if 
 within five minutes no marked change is noted in the 
 motility of the bacilli, and no considerable clumping 
 
BACILLUS TYPHOSUS. 427 
 
 occurs nothing more is needed; the result is negative 
 as far as this specimen is concerned. If marked 
 clumping and immobilization of the bacilli immedi- 
 ately begin and become complete within five minutes, 
 this is denominated a marked immediate typhoid re- 
 action, and no further test is considered necessary, 
 though it is always advisable to confirm the reaction 
 with higher dilutions up to 1 to 20 and 1 to 50. If, 
 however, upon examination of the mixture there is no 
 marked immediate reaction, but the bacilli only show 
 in the first few minutes an inhibition in their motility 
 and a tendency to clump, which becomes more marked 
 but not complete within five minutes, this must be 
 tested with the higher dilution of 1 to 20, so as to 
 measure the exact strength of the reaction. If in the 
 1 to 20 dilution a complete reaction takes place within 
 thirty minutes, the blood is considered to have come 
 from a case of typhoid infection, while if a less com- 
 plete reaction occurs it is considered that a probability 
 only of typhoid infection has been established. The 
 time allowed for the development of the reaction with 
 the high dilutions is by many from one to two hours, 
 but to us thirty minutes seem safer. Positive results 
 obtained in this way may be taken to be conclusive 
 unless there be grounds for suspecting that the reaction 
 may be owing to a previous fairly recent attack. The 
 absence of reaction in one examination is considered 
 by us to in no way exclude typhoid infection. If the 
 case remains clinically doubtful, the examination should 
 be repeated within a few days. 
 
 Proportion of Oases of Typhoid Fever in which a Defi- 
 nite Reaction Occurs and the Time of its Appearance. 
 As the result of a large number of cases examined in 
 
428 BACTERIOLOGY. 
 
 the health department laboratories, it has been found 
 that about 20 per cent, gave positive results in the first 
 week, about 60 per cent, in the second week, about 80 
 per cent, in the third week, about 90 per cent, in the 
 fourth week, and about 75 per cent, in the second 
 month of the disease. In 88 per cent, of the cases in 
 which repeated examinations were made (hospital cases) 
 a definite typhoid reaction was present at some time 
 during the illness. 
 
 Persistence of the Reaction. This peculiar property 
 of the blood-serum may persist in persons who have 
 recovered from typhoid fever for a number of months. 
 Thus a definite typhoid reaction has been reported in 
 serum from three months to a year, and a slight reac- 
 tion, though much less than sufficient to establish a 
 diagnosis of typhoid infection, from one to fifteen 
 years, after convalescence from the disease. 
 
 The Reaction with the Blood-serum of Healthy Persons 
 and of Those III with Diseases other than Typhoid Fever. 
 An immediate marked reaction has not been observed 
 in a 1 to 10 dilution of the blood-serum of over one 
 hundred healthy persons examined in the health depart- 
 ment laboratories. In several hundred cases of diseases, 
 not believed by the physicians in charge to be at the 
 end of the disease typhoid fever, only very rarely did 
 the serum give a marked immediate reaction in a 1 to 
 10 dilution, and here in the light of past experience, I 
 believe a typhoid infection, though not a typical typhoid 
 fever, to have existed. These results have been con- 
 firmed by others, the question of dilution having been 
 recently made the subject of elaborate investigations, 
 with the view of determining, if possible, at what 
 dilution the typhoid serum would react and others 
 
BACILLUS TYPHOSUS. 429 
 
 would not. Thus, Schultz has reported lately that 
 among 100 cases of non-typhoid febrile diseases 
 apparently positive results were obtained in 19 with 
 dilutions of 1 to 5, in 11 of these with 1 to 10, in 
 7 with 1 to 15, in 3 with 1 to 20, and in 1 a very faint 
 reaction with 1 to 25; whereas in as many cases of true 
 typhoid he never failed with dilutions of 1 to 50. In 
 these experiments it must be noted, however, that the 
 time-limit was from one to two hours. A faint re- 
 action with a 1 to 25 dilution with a time-limit of two 
 hours indicates less agglutinating substance than an 
 immediate complete reaction with a 1 to 10 dilution. 
 
 From an experience with the practical application 
 of the serum test for the diagnosis of typhoid fever 
 extending over three years, it may be said that this 
 method of diagnosis is simple and easy of perform- 
 ance in the laboratory by an expert bacteriologist, but 
 it is not to be recommended for routine employment 
 by practising physicians as a clinical test unless they 
 have had experience; that with the modifications as 
 now employed, and due regard to the avoidance of all 
 possible sources of error, it is as reliable a method as 
 any other bacteriological test at present in use; and 
 that as such, though not absolutely infallible, the Widal 
 test is an indispensable aid to the clinical diagnosis of 
 irregular or slightly marked typhoid fever. 
 
 The Isolation of Typhoid Bacilli from Suspected Feces, 
 Urine, Blood, Water, etc. In the bacteriological study 
 of typhoid infection for diagnostic and other purposes, 
 attempts have been made to isolate the specific bacilli 
 from the blood, rose-spots, sweat, urine, feces, and by 
 spleen puncture. Although the results obtained by 
 puncture of the spleen have been encouraging and have 
 
430 BACTERIOLOGY. 
 
 thrown light upon the distribution of the organism in 
 the body daring life, yet as a regular means of diag- 
 nosis it is to be discouraged, on account of the possible 
 danger to the patient. The results of the examination 
 of the blood and rose-spots of typhoid patients have 
 in the main proved unsatisfactory, though the investi- 
 gations of some of the later observers have given a 
 number of positive results from the blood. The exam- 
 ination of the urine and feces of typhoid patients has 
 more often given positive results than the blood, 
 and these positive results have become more fre- 
 quent and satisfactory as the methods for differentiating 
 the bacillus typhosus have grown more exact and re- 
 fined. 
 
 There are at present several recently devised media 
 employed for the isolation and identification of the 
 typhoid bacillus, which are much better than any of 
 those formerly used. These are the Hiss, Capaldi, 
 and Eisner media. In the hands of trained bacteri- 
 ologists they give satisfactory results. 
 
 THE Hiss MEDIA : l T/ieir Composition and Prepa- 
 ration. Two are used: one for the isolation of the 
 typhoid bacillus by plate culture, and one for the 
 differentiation of the typhoid bacillus from all other 
 forms in pure culture in tubes. 
 
 The plating medium is composed of 10 grammes of 
 agar, 25 grammes of gelatin, 5 grammes of sodium 
 chloride, 5 grammes of Liebig's beef extract, 10 
 grammes of glucose, and 1000 c.c. of water. When 
 the agar is thoroughly melted the gelatin is added and 
 
 1 This description is taken Irom an article by Dr. Philip Hanson Hiss, Jr., 
 " On a Method of Isolating and Identifying Bacillus Typhosus and Members 
 of the Colon Group in Semi-solid Culture Media," published in the Journal 
 of Experimental Medicine, 1897, vol. ii. No. 6. 
 
BACILLUS TYPHOSUS. 431 
 
 completely dissolved by a few minutes' boiling. The 
 medium is then titrated, to determine its reaction, 
 phenol phthalein being used as the indicator. The 
 requisite amount of normal hydrochloric acid or sodium 
 hydrate solution is added to bring it to the desired 
 reaction i. e., a reaction indicating 2 per cent, of nor- 
 mal acid. To clear the medium add one or two eggs, 
 well beaten it) 25 c.c. of water, boil for forty-five min- 
 utes, and filter through a thin filter of absorbent cotton. 
 Add the glucose after clearing. The reaction of the 
 medium is most important; it should never contain less 
 than 2 per cent, of normal acid. 
 
 The tube medium contains agar, 5 grammes; gelatin, 
 80 grammes; sodium chloride, 5 grammes; meat ex- 
 
 FIG. 54. 
 
 Hiss' plate media: Small light colony (t) is composed of typhoid bacilli; 
 large colony (c) of colon bacilli. (From Hiss.) 
 
 tract, 5 grammes, and glucose, 10 grammes to the litre 
 of water, and reacts to 1.5 per cent, acid by the indi- 
 cator. The mode of preparation is the same as for the 
 plate medium, care being taken always to add the 
 
432 BACTE&IOLOGY. 
 
 gelatin after the agar is thoroughly melted, so as not 
 to alter this ingredient by prolonged exposure to high 
 temperature. The glucose is added after clearing. 
 The medium must contain 1.5 per cent, of normal 
 acid. 
 
 Growth of the Colonies. The growth of the typhoid 
 bacilli in plates made from the medium as above de- 
 scribed gives rise to small colonies with irregular out- 
 growth and fringing threads (Figs. 54 and 55). The 
 
 FIG. 55. 
 
 Colony of typhoid bacilli more highly magnified. (Hiss.) 
 
 colon colonies, on the other hand, are much larger, 
 and, as a rule, are darker in color and do not form 
 threads. The growth of the typhoid bacilli in tubes 
 produces uniform clouding at 37 C. within eighteen 
 hours. The colon cultures do not give the uniform 
 clouding, and present several appearances, probably 
 dependent upon differences in the degree of their 
 motility and gas-producing properties in media. Some 
 of the varieties of the colon bacillus grow only locally 
 
BACILLUS TTPHOSUS. 433 
 
 where they were inoculated by the platinum needle. 
 Others grow diffusely through the medium, but owing 
 to the production of gas and the passage of gas-bubbles 
 through the medium, clear streaks- ramify through the 
 otherwise diffusely cloudy tube contents. This charac- 
 teristic appearance is not produced when the medium 
 is incorrect in reaction or in consistency. With un- 
 tried media it is always well to insert a platinum wire 
 into the tube contents and stir it about ; if any gas is 
 liberated the culture is not one of the typhoid bacillus 
 and the medium is not correct. 
 
 Method of Making the Test. The usual method of 
 making the test is to take enough of the specimen of 
 feces or urine i. e., from one to several, loops and 
 transfer it to a tube containing broth. From this 
 emulsion in broth five or six plates are generally 
 made by transferring one to five loops of the emulsion 
 to tubes containing the melted plate medium and then 
 pouring the contents of these tubes into Petri dishes. 
 These dishes are placed in the incubator at 37 C. 
 and allowed to remain for eighteen to twenty-four 
 hours, when they may be examined. If typical 
 thread-forming colonies are found the tube medium 
 is inoculated from them and the growth in the tubes 
 allowed to develop for about eighteen hours at 37 C. 
 If these tubes then present the characteristic clouding, 
 experience indicates that the diagnosis of typhoid may 
 be safely made, for the typhoid bacillus alone, of all 
 the organisms investigated, has displayed the power 
 of giving rise both to the thread- forming colonies in 
 the plating medium and the uniform clouding in the 
 tube medium when exposed to a temperature of 37 C. 
 The organisms isolated in this manner have been sub- 
 
 28 
 
434 B A OTERIO LOGY. 
 
 jectecT to the usual tests for the recognition of the 
 bacillus typhosus, and have always corresponded in 
 all their reactions to those given by the typical typhoid 
 bacillus. 
 
 ELSNER'S METHOD. 1 As Eisner himself gives no 
 very definite details as to the steps to be taken in 
 making up his medium, those working with it have 
 developed their own modifications. We found the 
 following method to give satisfactory results : 
 
 1. Grate 0.5 kilogramme of small potatoes to a fine 
 pulp, add 1 litre of water, and allow the mixture to 
 stand in a cool place over night. 
 
 2. Mash thoroughly (meat-press is best) and strain 
 through a fine cloth. This must be done when the 
 mixture is cold or the swelling of the starch-granules 
 will prevent the filtering process. 
 
 3. Boil the filtrate and filter again. 
 
 4. Add 10 per cent, of gelatine and dissolve by 
 boiling. 
 
 5. Test for the acidity. Eisner used litmus as an 
 indicator, and advised that the medium be of such an 
 acidity as to require the addition of 2.5 c.c. of deci- 
 normal hydroxide solution to make it neutral. If 
 more than 2.5 c.c. are required the acidity must be 
 reduced by normal sodic hydrate solution. Abbot 
 advises using phenolphtalein as the indicator, and 
 making the reaction such that 3 c.c. of the decinormal 
 solution will neutralize 10 c.c. of the medium. 
 
 6. Boil and clarify with an egg. 
 
 7. Filter first through cotton and then through 
 paper. 
 
 i Zeitschrift fur Hyg. und Infek., 1896, Bd. xxi. S. 25. 
 
BACILLUS TYPHOSUS. 435 
 
 8. To the filtrate add 1 per cent, of potassium 
 iodide. (Use a solution so made that 1 c.c. is equiva- 
 lent to 1 gramme of the salt.) 
 
 9. Decant into tubes and sterilize. 
 
 One of the most important points in working with 
 this medium is that the incubator must be kept at a con- 
 staut temperature of from 22 to 24 C. If the plates 
 be put away before the gelatin has thoroughly cooled, 
 or if the room becomes a very little too warm at any 
 time during the colony growth, so as to soften the gel- 
 atin, both the typhoid and the colon bacilli will develop 
 threads or become oval, and thus the characteristic dif- 
 ferentiation will be lost. Care must be taken, also, 
 that the room in which the plates are examined be not 
 too warm. This causes great inconvenience during the 
 summer months in most parts of the United States, 
 and requires special methods for keeping a room at the 
 proper temperature and keeping the plates cool during 
 their examination. 
 
 The iodide of potash prevents the great majority of 
 bacteria, especially the liquefiers, from developing; in 
 fact, little but colon and typhoid bacilli appear on the 
 plates. This is one of the chief advantages of the 
 medium in the examination of both water and feces. 
 
 Appearance of the Colonies. The colon is the first to 
 develop; the colonies are rough and granular in appear- 
 ance and greenish-brown in color; for the greater part 
 the colonies are on or near the surface. The typhoid 
 develop later, and their colonies usually show the clas- 
 sical " dew-drop " appearance small, white, gleaming, 
 generally without variation in substance, but occasion- 
 ally slightly granular. This point causes some trouble 
 to one first using the medium, as the young colon colo- 
 
436 BACTERIOLOGY. 
 
 nies sometimes present much the same appearance. 
 However, it was found that the typhoid colonies usu- 
 ally grew near the surface, while the colon colonies, 
 which are small and white, are almost invariably very 
 deep. This is especially true after forty-eight hours; 
 therefore, if one is not sure of a colony from the 
 appearance, an attempt to "fish" it will usually 
 identify it. 
 
 To one familiar with the medium, the characteristic 
 appearance of a plate when typhoid is present is almost 
 unmistakable, and it would seem that one would be 
 almost sure to find the typhoid in some one of a 
 series of plates, even if there were but few in the 
 specimen. 
 
 Eisner says that the typhoids do not develop for 
 forty-eight hours. Although the differentiation is 
 more accurate after that time, still for practical 
 methods of work twenty-four hours was found to be 
 quite sufficient. When the plates are first taken from 
 the incubator the diagnosis is not quite as certain, for 
 the colon colonies will not have developed the charac- 
 teristic color; but if the plates be allowed to stand in 
 the light for a couple of hours the diagnosis will be 
 found quite easy. After forty-eight hours all the large 
 colonies will be colon and most of the small ones 
 typhoid, if there be any typhoid present. Even after 
 several days' standing in the ice-chest the typhoid colo- 
 nies do not develop color. 
 
 Except for the difficulty in obtaining an exact tem- 
 perature for growth and a cool room for examination, 
 this method was found very satisfactory. 
 
 THE CAPALDI PLATE MEDIUM. In his original 
 paper, Capaldi gives the following recipe: 
 
BACILLUS TYPHOSUS. 437 
 
 Aqua dest t . 1000 
 
 Gelatin 20 
 
 Mannite (grape-sugar) . . . . . 10 
 
 Sodium chloride ...... 5 
 
 Potassium chloride ...... 5 
 
 Boil, filter, add 2 per cent, agar and 10 c.c. of normal sodic 
 hydrate solution ; boil, filter, and sterilize. 
 
 In making up the medium for work the only varia- 
 tion was that the agar was added when the gelatin was 
 put in in the original recipe, and the gelatin was 
 added after the first filtration. 
 
 The Capaldi medium is usually employed for surface 
 cultures, but can be inoculated while melted in the 
 tubes. Plates may be made beforehand, so that they 
 are ready for use when the specimen comes in. As 
 these plates are to be kept at 37 C., the difficulties in 
 regard to temperature are avoided; but, unlike the 
 Eisner plates, other organisms beside the colon and 
 typhoid develop and may cause some confusion. In 
 making the plates one or two are inoculated by gently 
 carrying across their surface a platinum loop of feces 
 or urine. Others are then inoculated with a loop of 
 urine or much diluted feces. In this way we are apt 
 to have some plates with just the right amount of 
 colonies. 
 
 Appearance of the Colonies. Capaldi thus describes 
 the differentiation: Typhoid: Small, gleaming, transpa- 
 rent, almost colorless colonies (by reflected light, blue). 
 Colon: Large, milky colonies (reflected light, brown). 
 
 In using the medium it was found that even in a 
 pure plate of typhoid the colonies vary much in size 
 and appearance, while different typhoids show indi- 
 vidual differences in growth. In general, a medium- 
 sized, gray-white colony, with a few refractive granules, 
 
438 BACTERIOLOGY. 
 
 is the typhoid (Fig. 56). However, it is often trans- 
 parent, without the refractive granules; sometimes 
 with a nuclear centre, and sometimes of equal con- 
 sistency throughout. Streptococci simulate typhoid, 
 but a high-power lens will show the coccus. 
 
 FIG. 56. 
 
 Colonies of typhoid bacilli, on Capaldi medium, slightly magnified. 
 
 Colon colonies are usually much larger than the 
 typhoid; a decided brown color, very large, refractive 
 granules, and in general quite different in appearance 
 (Fig. 57). 
 
 The best way to work with the Capaldi medium is to 
 make several plates with different typhoid cultures, 
 observe carefully all the variations in the colonies, and 
 bear these in mind when working with the mixed 
 plates. After these precautions have been taken the 
 
BACILLUS TYPHOSUS. 
 
 439 
 
 medium will be found very satisfactory. The colonies, 
 as a rule, appear characteristically in twelve to eigh- 
 teen hours, and thus give a quick method of diagnosis. 
 The two media together (Capaldi and Eisner) work 
 excellently, as one is an aid to the other. When 
 many colonies of the typhoid bacilli were present the 
 
 FIG. 57. 
 
 Colonies of colon bacilli. Capaldi medium slightly magnified. 
 
 differentiation was usually easily seen upon both media, 
 and the two together made diagnosis almost certain. 
 The bacilli from the suspected typhoid colonies can be 
 quickly tested sufficiently for practical purposes on the 
 Hiss tube medium and by the reaction between the 
 bacilli and the serum from an immunized horse. 
 
 As to the comparative merits of these three media, 
 it is probably safe to say that any one of them will, in 
 the hands of one accustomed to them, reveal the typhoid 
 bacilli, if they are present, except perhaps when they 
 
440 -B4 CTERIOL OGY. 
 
 exist in only the most minute numbers. The Eisner 
 method has the objection that it is very difficult to work 
 with in hot weather. The Hiss plate medium has the 
 objection that it is a difficult medium to prepare. If 
 the acidity is not just right the thread outgrowths do 
 not appear. Indeed, the only sure way is to test a new 
 batch of medium with a pure culture and alter the 
 reaction until the culture grows correctly. A very few 
 varieties of the typhoid bacillus do not produce typical 
 thread outgrowths from the colonies. 
 
 The Capaldi medium has the objection that some of 
 the typhoid and some of the colon colonies frequently 
 look much alike. If one, however, will always pick 
 out the colonies which look most like the typhoid, it 
 will usually turn out that typhoid bacilli have been 
 obtained if any were present. Personally, for general 
 use I prefer the Capaldi medium for the plate cultures 
 and the Hiss tube medium for identifying the bacilli 
 obtained. Through these media we are now in a posi- 
 tion to obtain and identify typhoid bacilli from feces, 
 urine, etc., within forty-eight hours. 
 
 Recently numerous investigations have been carried 
 out to discover how frequently and at what period in 
 typhoid fever cases bacilli were present in the feces 
 and urine. In the laboratory Hiss has recently exam- 
 ined the feces in forty- three consecutive cases, thirty- 
 seven of which were in the febrile stage and six con- 
 valescent. In a number of instances only one stool was 
 examined, but even under these adverse conditions the 
 average of positive results in the febrile stage was 66.6 
 per cent. 
 
 Out of 26 cases of typhoid fever in hospitals exam- 
 ined ; 21 were in the febrile stage and 5 convalescent. 
 
BACILLUS TYPHOSUS. 441 
 
 In the febrile cases in 17 the presence of typhoid bacilli, 
 often in great numbers, was demonstrated. Thus in 
 these carefully followed cases the statistics show over 
 80 per cent, of the febrile cases positive. The bacilli 
 were isolated from these cases as early as the sixth day, 
 and as late as the thirtieth day, and in a case of relapse 
 on the forty-seventh day of the disease. The conval- 
 escent cases gave uniformly negative results, the earliest 
 examination having been made on the third day after 
 the disappearance of the fever. The bacilli seemed to 
 be more numerous in the stools from about the tenth or 
 twelfth day on. These observations, with regard to the 
 appearance of the bacilli in the stools during the febrile 
 stage and their usually quick disappearance after the 
 defervescence, have been confirmed by others. In 
 several cases in which no Widal reaction was demon- 
 strated the bacilli were isolated. From private sources 
 between the seventh and twenty-first day of the disease, 
 experience thus far obtained seems to indicate that the 
 bacilli may be obtained from about 25 per cent, of all 
 cases on the first examination and from about 75 per 
 cent, after repeated examinations. In some samples of 
 feces typhoid bacilli die out within twenty -four hours; 
 in others they remain alive for days or even weeks. 
 This seems to depend on the bacteria present in the 
 feces and upon its chemical formation. Probably the 
 presence of typhoid bacilli in some stools and their ab- 
 sence in others must be explained largely upon the 
 characteristics of the intestinal contents. The short 
 life of the typhoid bacillus in many specimens of feces 
 suggests that stools be examined as quickly as possible. 
 In fact, unless the physician wishes to take the 
 trouble to have the sample of feces sent immediately 
 
442 BACTERIOLOGY. 
 
 to the laboratory, it is hardly worth while for the bac- 
 teriologist to take the trouble to make the test. 
 
 Typhoid Bacilli in the Urine. Of even more interest 
 than the presence of the bacilli in feces is their frequent 
 occurrence in great numbers in the urine. The results 
 of the examinations of others as well as those of our 
 own indicate that the typhoid bacilli are not apt to be 
 found in the urine until the beginning of the third week 
 of the fever, and may not appear until much later. 
 From this on to convalescence they appear in about 25 
 per cent, of the cases, and usually in pure culture and in 
 enormous numbers. Of nine positive cases examined 
 by Richardson 1 two died and seven were discharged. 
 At the time of their discharge their urine was loaded 
 with typhoid bacilli. We have noted similar cases. In 
 one the bacilli persisted for five weeks. Undoubtedly 
 in some cases they persist for months. When we think 
 of the chances such cases have to spread infection as 
 they pass from place to place, we begin to realize 
 how epidemics can start without apparent cause. The 
 more we investigate the persistence of bacteria in con- 
 valescent cases of disease the more difficult the preven- 
 tion of their dissemination is seen to be. The disin- 
 fection of the urine should always be looked after in 
 typhoid fever, and convalescents should not be allowed 
 to go to places where contamination of the water-supply 
 is possible without at least warning them of the neces- 
 sity of great care in disinfecting their urine and feces 
 for some weeks. Richardson made the interesting dis- 
 covery that after washing out the bladder with a very 
 weak solution of bichloride of mercury the typhoid 
 bacilli no longer appeared in the urine. 
 
 1 Journal of Experimental Medicine, May, 1898. 
 
BACILLUS TYPHOSUS. 443 
 
 The Detection of Typhoid Bacilli in Water. This sub- 
 ject is considered on pages 247 and 248. There is 
 absolutely no doubt that the contamination of streams 
 and reservoirs is the frequent cause of the outbreak of 
 epidemics of typhoid fever, but the actual finding and 
 isolation of the bacilli is a very rare occurrence. This 
 is owing to the contamination often having occurred 
 and passed away before the bacteriological examination 
 is undertaken, and also because of the great difficulties 
 met with in detecting a few typhoid bacilli when they 
 are associated with large numbers of other bacteria. 
 The greater the amount of contamination which is 
 thrown into the water, and the shorter the time which 
 elapses between the infection and the drinking of the 
 water, the greater is the danger. 
 
 The typhoid bacillus and the colon bacillus of 
 Escherich resemble each other in many respects. The 
 characteristics of each, which allow us to differentiate 
 the one from the other, will be considered at the end of 
 the description of the colon bacillus. 
 
CHAPTER XXIV. 
 
 BACILLUS COLI COMMTJNIS (OR COLON BACILLUS OF 
 ESCHERICIl). 
 
 THIS organism was first described by Emmerich 
 (1885), who obtained it from the blood, various organs, 
 and intestinal discharges of cholera patients at Naples. 
 It was afterward found by Escherich (1886) in the 
 feces of healthy milk-fed infants and by Weisser in 
 the alvine discharges of healthy men. It has since 
 been demonstrated to be a normal inhabitant of the 
 intestines of man and of many of the lower animals. 
 
 Morphology. The size and shape of the bacillus coli 
 varies considerably in its morphology according to the 
 sources and the culture media from which it is obtained. 
 The typical form is that of short rods with rounded 
 ends, from 0.4/z to 0.7/* in diameter by I/JL to 3// in 
 length; but sometimes the rods are so short as to be 
 almost spherical, resembling micrococci in appearance, 
 and, again, they are somewhat oval in form or are seen 
 as threads of 6^ or more in length. The various 
 forms may often be associated in the same culture (Fig. 
 58). The bacilli may occur as single cells or as 
 pairs joined end-to-end, rarely as short chains. In 
 unfavorable culture media in stained preparations they 
 may present unstained spaces (vacuoles) and more in- 
 tensely stained portions at the extremities, closely 
 resembling spores, but these are due, according to 
 
BACILLUS COLI COMMUNIS. 445 
 
 Escherich, to degenerative changes in the protoplasm. 
 The colon bacillus does not form spores. There is 
 nothing in the morphology of this bacillus which is 
 characteristic or which may aid in its identification, 
 for in this respect it simulates many other organisms. 
 
 FIG. 58. 
 
 Colon bacilli. Twenty-four-hour agar culture. X 1100 diameters. 
 
 The colon bacillus stains readily with the ordinary 
 aniline colors; it is quickly decolorized by Gram's 
 method. 
 
 Biology. It is an aerobic, facultative anaerobic, non- 
 liquefying bacillus. It is motile, but its movements 
 are so sluggish that a positive opinion is often difficult, 
 being exhibited often by one or two individuals, in fresh 
 cultures, and at a high temperature only. These move- 
 ments are produced by flagella, which may be demon- 
 strated by Loffler's method of staining, though not 
 usually in such numbers as are seen to occur on the 
 bacillus typhosus. 
 
446 BACTERIOLOGY. 
 
 Growth on Gelatin. In gelatin plates colonies are 
 developed in from twenty-four to forty-eight hours, 
 which vary considerably in their appearance according 
 to their age, and in different cultures in the same 
 medium. They resemble greatly the colonies of the 
 typhoid bacillus, except that they are somewhat larger 
 for the same period of growth. When located in the 
 depths of the gelatin and examined by a low-power 
 lens they are at first seen to be finely granular, almost 
 homogeneous, in structure round, and of a pale yellow- 
 ish to brownish color; later they become larger, denser, 
 darker, and more coarsely granular. In shape they 
 may be round, oval, or " whetstone-like. " The super- 
 ficial colonies appear as small, dry, irregular, flat, 
 blue- white points that are commonly somewhat dentated 
 at the margin. 
 
 In stab cultures on gelatin the growth usually takes 
 the form of a nail with a flattened head, the surface 
 extension generally reaching out rapidly to the sides 
 of the tube. 
 
 On Nutrient Agar and Blood-serum. On nutrient 
 agar and blood-serum an abundant, soft, white layer is 
 quickly developed in the incubator, but the growth 
 is not characteristic. 
 
 In Bouillon. In bouillon the bacillus coli produces 
 diffuse clouding with sedimentation; in some cultures 
 a tendency to pellicle formation on the surface is seen 
 occasionally. In old cultures, in the absence of sugar, 
 the reaction becomes alkaline, and a decided fecal odor 
 may be noticed. 
 
 The colon bacillus produces indol in bouillon and in 
 peptone solutions, this reaction being most pronounced 
 after a week's development in the incubator. It pos- 
 
BACILLUS COLI COMMUNIS. 447 
 
 sesses also a considerable reducing power, converting 
 nitrates into nitrites, as may be demonstrated by the 
 addition of sulphuric acid in the proper proportion to 
 a bouillon or peptone culture, when a pink coloration 
 results. 
 
 On Potato. On potato the growth is rapid and 
 abundant, appearing after twenty-four to thirty-six 
 hours in the incubator as a yellowish-brown to dark 
 cream-colored deposit covering the greater part of the 
 surface. But there are considerable variations from 
 the typical growth on potato; there may be no growth 
 at all, or it may be scanty and of a white color. These 
 variations are due at times to the bacillus, but more 
 often to variations in the potato. 
 
 Gas-production. The bacillus coli grows rapidly 
 in media containing glycerin and sugar, particularly 
 glucose, causing active fermentation with liberation of 
 carbonic acid and hydrogen gas. Cultivated in solid 
 media, to which glucose has been added, the gas-pro- 
 duction is recognized by the appearance of numerous 
 bubbles along and about the points of growth ; in fluid 
 media it may be demonstrated in the fermentation- tube. 
 Grown on lactose-litmus-agar, the colonies are pink 
 and the color of the surrounding medium is changed 
 from blue to red, showing the production of acid. 
 
 Milk is coagulated by the growth of the bacillus 
 coli after twenty-four to seventy-three hours in the in- 
 cubator, with the production of gas and acid; very 
 rarely acid may be produced and no coagulation occur. 
 The coagulation of the milk is hastened by warming. 
 
 The thermal death-point of the colon bacillus from 
 feces was found by Weisser to be 60 C., the time of 
 exposure being ten minutes. When the bacilli from 
 
448 BACTERIOLOGY. 
 
 a bouillon culture were dried upon thin glass covers 
 they failed to grow after twenty-four hours (Weisser). 
 Waliczek found that when dried upon pieces of sterile 
 filter-paper they failed to grow at the end of eighteen 
 hours. These results give confirmation to the view 
 that the colon bacillus does not form spores. 
 
 Pathogenesis. The colon bacillus is pathogenic in 
 varying degrees for test animals, though the results of 
 the inoculations, as with the typhoid bacillus, cannot 
 always be predicted with certainty. Intraperitoneal 
 injections of from 0.1 to 1 c.c. of fresh, virulent cul- 
 tures usually produce death in mice at the end of from 
 one to eight days, but death does not invariably follow. 
 The more rapidly death ensues the greater the number 
 of bacilli found in the body; they are always more 
 abundant in the abdominal cavity than in the blood; 
 in other words, the result is to be attributed to the 
 toxic rather than to the infective properties of the 
 culture used. But the fact that the bacilli are found 
 in the blood and internal organs when death rapidly 
 follows inoculation proves that they do multiply to 
 some extent in the body. When less virulent cultures, 
 however, are injected and death results, this is due to 
 the poisonous products formed by the bacilli and given 
 up at their death. The lesions produced are those of 
 enteritis : the duodenum and jejunum are found to con- 
 tain fluid, the spleen is somewhat enlarged, and there 
 is marked hypersemia and ecchymosis of the small in- 
 testines, together with swelling of Peyer's patches. 
 
 Intraperitoneal and intravenous inoculation of guinea- 
 pigs and rabbits may also produce death, which, when 
 it follows, usually takes place within the first forty-eight 
 hours, accompanied by a decided fall of temperature, 
 
BACILLUS COLI COMMUNIS. 449 
 
 the symptoms of enteritis, diarrhoea, etc., and finally 
 fibro-purulent peritonitis. 
 
 When subcutaneous inoculations of mice and guinea- 
 pigs are made it requires the introduction of much 
 larger quantities of the culture to produce infection; 
 in rabbits this is followed only by abscess formation at 
 the point of inoculation. Dogs and cats are similarly 
 affected. 
 
 Bazy and Guyon have succeeded in producing infec- 
 tion of the bladder in animals by injection of pure 
 cultures into the blood with simultaneous tying of the 
 ureters; Albaran and Halle have caused cystitis and 
 pyelonephritis by direct injections into the bladder and 
 ureters, the urine being artificially suppressed; Chassin 
 and Roger produced angiocholitis and abscess of the 
 liver in the same way. Loruelle, Fraenkel, and Bar- 
 hacci, by injuring or tying the intestines and intro- 
 ducing dirt into the abdominal cavity, with or without 
 the simultaneous injection of cultures of the colon 
 bacillus, succeeded in causing diffuse peritonitis in 
 animals. Akermann produced osteomyelitis in young 
 rabbits by intravenous injections of cultures. So far 
 all attempts to produce experimental infection of the 
 intestines by the ingestion of cultures of the colon 
 bacillus have failed to give positive results (Emmerich 
 and Korkunoff). 
 
 Certain peculiar effects have been observed by Black- 
 stein and by Gilbert and Lion as the result of intra- 
 venous inoculation of rabbits with pure cultures of the 
 bacillus coli, which are worthy of note. The former 
 of these authors found, from eight to thirty-eight days 
 after injection, that the liver frequently contained 
 opaque, whitish or yellowish-white spots, and streaks 
 
 29 
 
450 BACTERIOLOGY. 
 
 of irregular size and shape, giving a peculiar mottling 
 to the organ when present in large numbers. By micro- 
 scopical examination these were found to represent 
 places where the liver cells had undergone necrosis, 
 accompanied by emigration of leucocytes, and the cells 
 about them were in a condition of fatty degeneration. 
 In sections of the liver, masses of the bacilli were dis- 
 covered in and about the necrotic foci. The bacilli 
 were not found generally distributed through the body, 
 but only in the bile, liver, and occasionally in the 
 spleen. Gilbert and Lion found in addition that hemi- 
 plegia and paraplegia were often produced in conse- 
 quence of atrophy of the cells of the cord. These 
 observations have been confirmed by Thoinot and 
 Massilin, but in their experiments the nerve-lesions 
 were not commonly present. 
 
 From experiments on animals it would, therefore, 
 appear that the true explanation of the palhogensis of 
 the colon bacillus is undoubtedly to be found in the 
 toxic effects of the chemical products of the organism 
 rather than in its mechanical presence in the tissues. 
 
 Variation in Virulence. The virulence of the colon 
 bacillus varies considerably as derived from different 
 sources. An attempt has been made to establish 
 certain rules for this. Thus, Lesage and Macaigne 
 express the opinion that when obtained from a healthy 
 body it is only slightly virulent, while that isolated 
 from a diseased person is much more virulent. The 
 infective power is thought to bear a definite relation 
 to the severity of the disease with which the organism 
 is associated; for instance, to be greatest in cultures 
 taken from cholera patients and least in those obtained 
 from pus. Dreyfus also confirms this view. He found 
 
BACILLUS CO LI COMMUNIS. 451 
 
 by experiment that 1 c.c. of a fresh bouillon culture 
 of the B. coli from normal feces was required to kill 
 guinea-pigs by intraperitoneal and rabbits by intraven- 
 ous injection, whereas less than one-fifth as much of a 
 culture from a fatal case of cholera nostras was suf- 
 ficient to kill the same animal; but this rule has prob- 
 ably many exceptions, even if it be true in some cases. 
 
 All observers, however, agree that the virulence 
 of the B. coli is diminished by continued cultivation 
 through successive generations, and that it is increased 
 by passage through animals. 
 
 Immunization. Immunization against colon infec- 
 tion is comparatively easy to produce in the usual way 
 by the inoculation of gradually increasing doses of 
 cultures of the living bacilli or dead bacilli. 
 
 Occurrence in Man and Animals. The bacillus coli 
 communis is a common inhabitant of the intestinal 
 canal in man and in many animals. According to 
 Fremlin, it is found normally in dogs, mice, and rab- 
 bits, but not in rats, pigeons, or guinea-pigs. Accord- 
 ing to Dyas and Keith, it occurs in goats, rabbits, dogs, 
 cats, swine, and cows, but not in horses. Grimbert 
 claims to have found it in the intestines of almost all 
 domestic animals, and in the mouth as well as the in- 
 testines of man. It is also frequently found in water 
 and food (milk, etc.), so that it is one of the most wide- 
 spread saprophytic bacteria known. Formerly it was 
 thought that the presence of the B. coli in water was 
 sufficient proof of its contamination by feces; but the 
 recent investigations of Weichselbaum, Kruse, Beck- 
 mann, and Refith would seem to show that there are 
 no grounds for this assumption, as the colon bacillus 
 may reach the water from many different sources. 
 
452 BACTERIOLOGY. 
 
 From its common seat in the intestines it may, 
 under favoring conditions, penetrate other organs after 
 death which fact may account for its being found 
 so often at autopsy in the interior of the body; but it 
 may also be absorbed during life, more especially if 
 there is obstruction of the intestines or if the mucosa 
 has been deprived of its epithelium. For this reason, 
 no doubt, the B. coli is so frequently found in cholera, 
 typhoid fever, and dysentery, producing often a second 
 infection. The absorption of the colon bacillus from 
 the intestinal canal plays an important part, probably, 
 in the production of many diseases, such as cystitis 
 and other inflammatory affections. It has been con- 
 sidered to be the cause of epidemic infectious enteritis 
 and cholera nostras, this assumption being based upon 
 the facts that the colon bacillus in these diseases is 
 found in greater abundance than usual in the alvine 
 discharges and often in pure culture ; that it then pos- 
 sesses an increased virulence, and that it often pene- 
 trates the interior organs, as has been shown by autop- 
 sies. But the conclusion drawn from these facts as to 
 the etiology of the diseases above mentioned is not 
 positive, though it cannot be denied that under certain 
 conditions the colon bacillus may be productive of dis- 
 ease. This is brought about, according to the com- 
 monly accepted view, either by an increase of virulence 
 of the B. coli normally present in the intestines or by 
 the introduction of especially virulent bacilli in the 
 food. The colon bacillus has also been assumed to be 
 the cause of cholera infautum; but the investigations 
 of Booker, Baginsky, Escherich, and Fliigge would 
 seem, to indicate that this disease is of a much more 
 complicated origin. The B. coli, moreover, is associ- 
 
BACILLUS COLI COMMUNIS. 453 
 
 ated with dysentery, probably as a secondary affection, 
 as in amoebic or tropical dysentery. It is also found 
 frequently in cases of diffuse and circumscribed peri- 
 tonitis, appendicitis, etc., either alone or together with 
 other bacteria which play a part in the etiology of these 
 diseases along with certain chemical ferments and toxins 
 and foreign bodies in the intestines. The origin of in- 
 fections of the gall-ducts (with at times the production 
 of gallstones) and multiple abscess of the liver is also 
 explained in this way by Dmochowski and Jauowski, 
 though, according to Letienne, the mere presence of 
 the B. coli in the bile, in which it has been found 
 under normal conditions, is not sufficient to account 
 for these affections. Puerperal fever is not infre- 
 quently caused by the colon bacillus by infection of 
 the vagina or uterus. Other diseases to which the 
 colon bacillus seems to stand in a certain relation, 
 though rarely, are : Endocarditis, meningitis, tropical 
 abscess of the liver, bronchopneumoriia and an irregu- 
 lar type of lobar pneumonia, fetid bronchitis, chronic 
 amygdalitis, and abscess of the lachrymal sac. The B. 
 coli has been found in a case of urethritis (pseudo- 
 gonorrhoea) lying inside the cells like gonococci, and it 
 is often associated with the pyogenic cocci in cutaneous 
 and subcutaneous purulent inflammations. 
 
 In the above-mentioned diseases the colon bacillus 
 has been found either alone or associated with other 
 pathogenic bacteria in such numbers as to be con- 
 sidered a factor in the etiology of the disease, and in 
 some cases there is no reason to doubt that it is the 
 primary cause of infection. Though further study and 
 investigation are required to show the specific patho- 
 genic properties of this micro-organism, it is evident 
 
454 BACTERIOLOGY. 
 
 that under certain conditions it may become pathogenic 
 to man. 
 
 According to many authorities there are a great 
 number of varieties of the colon bacillus, some main- 
 taining even that it may become, under suitable con- 
 ditions, identical with the typhoid bacillus; but there 
 has been no proof whatever of this. 
 
 Differential Diagnosis. By comparing what has been 
 said of the bacillus coli and the bacillus typhosus it 
 will be seen that while certain varieties of each simu- 
 late each other in many respects, the characteristic 
 varieties of each still possess individual characteristics 
 by which they may be readily differentiated: 
 
 1. The motility of the B. coli is, as a rule, much less 
 conspicuous than that of the B. typhosus. It is also 
 shorter, thicker, and has fewer flagella. 
 
 2. In gelatin the colonies of the colon bacillus de- 
 velop more rapidly and luxuriantly than those of the 
 typhoid bacillus. 
 
 3. On potato the growth of the colon bacillus is 
 usually rapid, luxuriant, and visible, though not inva- 
 riably so; while that of the typhoid bacillus is ordina- 
 rily invisible. 
 
 4. The colon bacillus coagulates milk in from thirty- 
 six to forty-eight hours in the incubator, with acid 
 reaction. The typhoid bacillus does not cause coagula- 
 tion. 
 
 5. The colon bacillus is conspicuous for its power of 
 causing fermentation, with the production of gas in 
 media containing glucose. The typhoid bacillus never 
 does this. 
 
 6. In nutrient agar or gelatin containing lactose and 
 litmus tincture, and of a slightly alkaline reaction, the 
 
BACILLUS CO LI COM MUNIS. 455 
 
 color of the colonies of the colon bacillus is pink and 
 the surrounding medium becomes red; while the colonies 
 of the typhoid bacillus are blue, and there is little or no 
 reddening of the surrounding medium. 
 
 7. The colon bacillus possesses the property of pro- 
 ducing indol in cultures of bouillon or peptone; the 
 typhoid bacillus in these solutions does not produce 
 indol, except in a few rare exceptions. 
 
 8. The colon bacillus rarely produces thread out- 
 growths in the Hiss plate medium. The typhoid bacil- 
 lus produces thread outgrowths and smaller colonies in 
 this medium. In the Hiss tube medium the colon 
 bacillus produces either a growth limited to the area 
 inoculated or a diffuse growth streaked with clear lines 
 and spaces. The typhoid bacillus produces a diffuse 
 growth evenly clouding the entire medium. 
 
 9. On the Capaldi medium the colon colonies are 
 more granular and darker than those of the typhoid 
 bacilli. 
 
 10. On the Eisner medium the colon colonies appear 
 earlier and become larger and more opaque than the 
 average typhoid colonies. 
 
 11. Finally, we have the test of placing the bacilli 
 in animals and in the hanging drop, together with the 
 serum of animals immunized to either the colon or the 
 typhoid bacillus. 
 
 None of these tests alone can be depended upon for 
 making a differential diagnosis of the colon bacillus 
 from the typhoid bacillus or other similar bacilli. 
 
 Unfortunately in most, at least, of these characters 
 certain degrees of variation may often be observed in 
 different cultures of the typhoid and colon bacillus 
 which may lead to confusion. For instance, the mor- 
 
456 BACTERIOLOGY. 
 
 phology may vary considerably, even at times when 
 grown on the same culture media, and the motility is 
 not always equally active; the flagella formation may 
 vary; the rapidity of growth may differ, especially 
 between freshly made and old cultures; the grape-leaf 
 appearance of the surface colonies on gelatin, which is 
 usually characteristic, may vary with the composition 
 of the gelatin, at times no typical colonies at all being 
 presented; the threads in the Hiss media may be lack- 
 ing; the indol test requires great care in its perform- 
 ance, and in rare instances the typhoid bacillus produces 
 it; the growth on potato is not to be depended on, being 
 often visible and not characteristic; the virulence of 
 both the bacilli is so little characteristic that it can 
 hardly be used for diagnostic purposes; and, finally, the 
 serum test is not absolutely infallible in all cases, for 
 once we met with a bacillus in feces which grew in a 
 manner utterly at variance with the typhoid bacillus, 
 yet still gave the Widal reaction perfectly with the 
 serum of an immunized horse. It is also stated by 
 Abbott that all typhoid bacilli do not give the Widal 
 reaction with the serum derived from a typhoid in- 
 fection with a single variety of a typhoid bacillus. 
 This is an experience that as yet we have not met 
 with. The Pfeiffer reaction in guinea-pigs is a matter 
 of extreme delicacy, aud varying results are sometimes 
 obtained. 
 
 In spite, however, of these difficulties it is very easy 
 to sufficiently identify the typhoid bacillus for all prac- 
 tical purposes. A bacillus which grows typically in 
 the Hiss tube media and shows the Widal reaction with 
 a high dilution of the serum of an animal immunized 
 
BACILLUS COLI COMMUNIS. 457 
 
 to the typhoid bacillus, is in all probability the typhoid 
 bacillus. The same could probably be stated of a 
 bacillus which grew characteristically in glucose bouil- 
 lon and nutrient gelatin, and also showed the specific 
 serum reaction. Probably not one time in ten thousand 
 would such bacilli prove on further investigation not 
 to be typhoid bacilli. A still further test is to inocu- 
 late animals with several doses of the dead bacilli, 
 whose identification is sought, and note whether there 
 is produced a serum which agglutinates typhoid bacilli. 
 
CHAPTER XXV. 
 
 PNEUMOBACILLUS ; FRIEDLANDER' S BACILLUS. 
 
 DISCOVERED by Friedlander (1883), and declared by 
 him to be the cause of fibrinous pneumonia. Subse- 
 quent researches have shown that it is present in only 
 a small proportion of the cases of this disease. It is 
 found also not infrequently in the mucous membranes 
 of the mouth and air-passages of healthy individuals, 
 and in the air. 
 
 Morphology. Short bacilli with rounded ends, often 
 resembling micrococci, especially in recent cultures; 
 commonly united in pairs or in chains of four, and, 
 under certain circumstances, surrounded by a trans- 
 parent capsule. This capsule is not seen in prepara- 
 tions made from artificial culture media, but is visible 
 in well-stained preparations from the blood of an in- 
 oculated animal. 
 
 Friedlander' s bacillus stains readily with the aniline 
 colors, but is not stained by Gram's method. 
 
 Biological Characters. An aerobic, non-motile, non- 
 liquefying bacillus; also facultative anaerobic; does not 
 form spores. In gelatin stick cultures it presents the 
 " nail-shaped " growth first described by Friedlander, 
 which is not, however, peculiar to this bacillus. Gas- 
 bubbles occasionally develop in gelatin, and in old 
 cultures the gelatin acquires a distinct brownish colora- 
 tion. This latter characteristic distinguishes the growth 
 
PNE UMOBA GILL US. 459 
 
 of this bacillus from that of the bacillus aerogenes, 
 which is otherwise very similar to it morphologically 
 and culturally. On gelatin plates colonies appear at the 
 end of twenty-four hours as small white spheres, which 
 rapidly increase in size. These colonies, when ex- 
 amined by a low-power lens, present a somewhat 
 irregular outline and a slightly granular appearance. 
 The growth on agar is in quite large and moist grayish 
 colonies. On blood-serum abundant, grayish-white, 
 viscid masses are developed. The growth on potato 
 is luxuriant a thick, yellowish-white, glistening layer 
 rapidly covering the entire surface. Milk is not coagu- 
 lated. Indol is produced in bouillon or peptone solu- 
 tions. Fermentation of rnilk-sugar and glucose is 
 caused. Growth occurs at 16 to 20 C., but is more 
 rapid at 37 C. 
 
 Pathogenesis. Friedlander's bacillus is pathogenic 
 for mice and guinea-pigs, less so for dogs, and rabbits 
 are apparently immune. In Friedlander's experiments 
 mice proved to be particularly susceptible. These 
 animals, when pure cultures of the bacillus are in- 
 jected through the thoracic wall into the tissue of the 
 lung, invariably succumb to the disease. On autopsy 
 the pleural cavities are found to contain a sero-puru- 
 lent fluid, the lungs are intensely congested, and in 
 places show limited areas of red hepatization; the 
 spleen is considerably enlarged, and bacilli are present 
 in the lungs, the pleuritic fluid, and the blood. In 
 guinea-pigs which are killed by the inoculation similar 
 appearances are observed. 
 
 Friedlander's bacillus has been found in man, not 
 only in patients suffering from croupous pneumonia 
 and other respiratory diseases, but also in healthy indi- 
 
460 BACTERIOLOGY. 
 
 viduals, and in the outside world. Thus, Pawlowsky 
 found it in the atmosphere and Mori in canal water; 
 Netter observed it in 4.5 per cent, of the cases ex- 
 amined by him in the saliva of healthy individuals, 
 and Pansini in cases of pulmonary tuberculosis in 
 the sputum. Friedlander believed that the bacillus 
 described by him was the specific cause of croupous 
 pneumonia; but in 129 cases examined by Weichsel- 
 baum this bacillus was found in only 9; of 70 cases 
 examined by Wolf only 3 showed the presence of Fried- 
 lander's pneumobacillus. It is evident, therefore, that 
 though this micro-organism may be concerned in the 
 production of certain forms of the disease, it is not the 
 specific cause of croupous pneumonia. The cases which 
 are due primarily to the pneumobacillus are distin- 
 guished, according to Weichselbaum and Netter, by 
 their peculiarly malignant type and by the viscidity of 
 the exudate produced. This bacillus is also probably 
 concerned, primarily or secondarily, under certain cir- 
 cumstances, in the production of pleurisy, abscess of 
 the lungs, pericarditis, endocarditis, otitis media, and 
 meningitis, in all of which diseases it has been found 
 at times to be present. 
 
CHAPTER XXVI. 
 
 THE PRODUCERS OF ABSCESS, CELLULITIS, SEPTI- 
 CAEMIA, ETC. 
 
 THE STAPHYLOCOCCI. THE MICROCOCCUS 
 TETRAGENUS. 
 
 The Staphylococcus Pyogenes Aureus. (The Golden 
 Staphylococcus.) 
 
 THE Staphylococcus pyogenes aureus is one of the 
 commonest pathogenic bacteria, being almost every- 
 where present, and is the organism most frequently 
 concerned in the production of acute, circumscribed, 
 suppurative inflammations. It was first observed by 
 Ogston (1881) in the pus of acute abscesses, but was 
 not obtained by him in pure culture. It was isolated 
 from the pus of acute abscesses and accurately described 
 by Rosenbach (1884). 
 
 Morphology. Small, spherical cells, having a diameter 
 of 0.87/y. (Passet), occurring solitary, in pairs as diplo- 
 cocci, in short chains of three or four elements, or in 
 groups of four, but most commonly in irregular masses, 
 simulating clusters of grapes ; hence the name staphylo- 
 COOGUS. (See Fig. 59.) 
 
 It stains quickly in aqueous solutions of the basic 
 aniline colors. When previously stained with methyl- 
 violet it is not decolorized by Gram's method. 
 
 Biology. The Staphylococcus pyogenes aureus is an 
 aerobic, facultative anaerobic, liquefying micrococcus, 
 
462 BACTERIOLOGY. 
 
 growing readily at a temperature from 18 to 20 C., 
 but best at 37, in milk, bouillon, and other liquid 
 media, and in nutrient gelatin or agar, accompanied by 
 liquefaction of the gelatin. 
 
 Growth on Gelatin. Grown on gelatin plates it 
 develops, at room-temperature within forty-eight hours, 
 punctiform colonies, which, when examined under a 
 low-power lens, appear as circular disks of a pale brown 
 color, somewhat darker in the centre, and surrounded 
 by a smooth border. The colonies grow rapidly. The 
 
 FIG. 59. 
 
 Staphylococcus. X 1100 diameters. 
 
 appearance of the growth is most characteristic. Im- 
 mediately surrounding the colonies, which are of a pale 
 yellow color, there is a deepening of the surface of the 
 gelatin, due to its liquefaction. By suitable light a 
 number of these shallow depressions with sharply de- 
 fined outlines may be seen on the gelatin plate, having 
 a diameter of from 5 to 10 mm., in the centres of which 
 lie the yellow colonies. Later, the liquefaction becomes 
 general, the colonies running together. In stick cul- 
 tures in gelatin a white confluent growth at first appears 
 
PRODUCERS OF ABSCESS, CELLULITIS, ETC. 463 
 
 along the line of puncture, followed by liquefaction of 
 the medium, which rapidly extends to the sides of the 
 test-tube. At the end of two days the yellow pigmen- 
 tation begins to form, and this increases in intensity 
 for eight days. Finally, the gelatin is completely 
 liquefied, and the " golden staphylococci " form a 
 golden-yellow or orange-colored deposit at the bottom 
 of the tube. Under unfavorable conditions the staphyl- 
 ococcus aureus gradually loses its ability to make pig- 
 ment and to liquefy gelatin. 
 
 Growth on Agar. In streak and stick cultures on 
 agar a whitish growth is at first produced, and this 
 at the end of a few days becomes golden-yellow on the 
 surface. The yellow pigmentation is produced only in 
 the presence of oxygen; colonies found at the bottom 
 of a stab culture or under a layer of oil remain white. 
 
 Milk inoculated with this inicrococcus at the end of 
 from one to eight days is coagulated; bouillon and 
 peptone solutions are densely clouded by the lux- 
 uriant growth produced. 
 
 In the three last-named culture media, as the result 
 of the growth of the staphylococcus aureus, there is a 
 production of acid in considerable quantities, these con- 
 sisting chiefly of lactic, butyric, a.nd valerianic acids. 
 These acids have been supposed to play a part in the 
 production of pus, in which, according to some ob- 
 servers, they are often present. 
 
 The staphylococcus is distinguished from most other 
 pathogenic bacteria by its comparatively greater power 
 of resistance to outside influences, desiccation, etc., as 
 well as to chemical disinfectants. Cultures of the 
 staphylococcus pyogenes in gelatin or agar retain their 
 vitality for a year or more. Its thermal death-point is 
 
464 BACTERIOLOGY. 
 
 between 56 and 60 0., the time of exposure being 
 ten minutes (Sternberg). Bolton found that a 1 per 
 cent, solution of carbolic acid destroyed the vitality 
 after two hours' exposure. Mercuric chloride, 1 : 1000, 
 destroys it in from five to ten minutes, according to 
 most authorities, though Abbott found that in the 
 same culture there may be a considerable difference in 
 the resisting power of the cocci, all being frequently 
 destroyed in five minutes, while, again, some may sur- 
 vive after an exposure to a solution of 1 : 1000 for ten, 
 twenty, and even thirty minutes. 
 
 Pathogenesis. The pathogenic effect of the staphylo- 
 coccus pyogenes aureus on test animals varies consider- 
 ably according to the mode of application and the 
 virulence of the special culture employed. In the exper- 
 iments so far made this micrococcus, as found in sup- 
 purative processes in the human subject, has not proved 
 to be as infectious for animals as it is for man. In 
 man a simple rubbing of the surface of the unbroken skin 
 with pus from an acute abscess is, as a rule, sufficient 
 to produce purulent inflammation, and the introduction 
 of a few germs from a septic case into a wound may lead 
 to a fatal pysemia. These conditions can only be repro- 
 duced in lower animajs with difficulty and by the inocu- 
 lation of large quantities of the culture. Subcutaneous 
 injections, or the inoculation of open wounds in mice, 
 guinea-pigs, and rabbits, are commonly without result; 
 occasionally abscess formation may follow at the point 
 of inoculation, which usually ends in recovery. The 
 pus-producing property of the organism is exhibited 
 in proportion to the virulence of the culture employed. 
 Slightly virulent cultures, which constitute the majority 
 of those obtained from pus taken from the human sub- 
 
PRODUCERS OF ABSCESS, CELLULITIS, ETC. 465 
 
 ject, when injected subcutaneously in large quantities 
 (several c.c. of a fresh bouillon culture) in rabbits or 
 guinea-pigs, give rise to local pathological lesions acute 
 abscesses. When virulent cultures are used which are 
 rarely obtainable 0.5 c.c. of a fresh bouillon culture 
 is sufficient to produce similar results. The abscesses 
 heal generally without treatment; sometimes the ani- 
 mals die from marasmus in consequence of the sup- 
 purative process. In intraperitoneal inoculations the 
 degree of virulence of the culture employed is still 
 more conspicuous in the effects produced. The ani- 
 mals usually die in from two to nine days. The most 
 characteristic pathological lesions are found in the kid- 
 neys, which contain numerous small collections of pus, 
 and under the microscope present the appearances 
 resulting from embolic nephritis. Punctiform, whitish- 
 yellow masses of the size of a pea are found permeating 
 the pyramids. Many of the capillaries and some of the 
 smaller arteries of the cortex are plugged up with 
 thrombi consisting of micrococci. Metastatic abscesses 
 may also be observed in the joints and muscles. The 
 micrococci may be recovered in pure cultures from the 
 blood and the various organs; but they are not numer- 
 ous in the blood and are often difficult to demonstrate 
 microscopically. Intravenous inoculations of animals 
 are followed by similar pathological changes Orth 
 and Wyssokowitsch first pointed out that injection of 
 staphylococci into the circulation, after injuring the 
 cardiac valves in rabbits, produced ulcerative endo- 
 carditis. Subsequently, Weichselbaum, Prudden, and 
 Fraenkel and Sanger obtained confirmatory results, 
 thus establishing the fact that when the valves are first 
 injured, mechanically or chemically, the injection into a 
 
 30 
 
466 BACTERIOLOGY. 
 
 vein of a pure culture of staphylococcus aureus gives 
 rise to a genuine ulcerative endocarditis. It has been 
 further shown by Kibbert that the same result may be 
 obtained without previous injury to the valves by in- 
 jecting into a vein the staphylococcus from a potato 
 culture suspended in water. In his experiments not 
 only the micrococci from the surface, but the super- 
 ficial layer of the potato was scraped off with a steril- 
 ized knife and mixed with distilled water, and the 
 successful result is ascribed to the fact that the little 
 agglomerations of micrococci and infected fragments of 
 potato attach themselves to the margins of the valves 
 more readily than isolated cocci would do. Not infre- 
 quently, also, in intravenous inoculations of young ani- 
 mals there occurs a localization of the injected material 
 in the marrow of the small bones. This may take place 
 in full-grown animals when the bones have been injured 
 or fractured. The experimental osteomyelitis thus pro- 
 duced has been demonstrated to be anatomically analo- 
 gous to this disease in man. With regard to the lesions 
 found in the kidneys after intraperitoneal or intravenous 
 inoculation of cultures of the staphylococcus, it has been 
 found that when injected in considerable quantities the 
 organism may be obtained in cultures from the urine, 
 but not sooner than six or eight hours after the injec- 
 tion, and not until the formation of purulent foci in 
 the kidneys has already occurred. 
 
 The Production of Toxic Substances. The peculiar 
 energetic action of the staphylococcus pyogenes aureus 
 on the tissues of warm-blooded animals would seem 
 to indicate that toxic substances are produced by 
 this organism, which play an important part in its 
 infective properties. Grawitz and De Bary have 
 
PRODUCERS OF ABSCESS, CELLULITIS, ETC. 467 
 
 shown by experiments that cultures of the staphylo- 
 coccus, when sterilized by boiling and injected subcuta- 
 neously into dogs, will produce local abscesses. Leber 
 found also that sterilized cultures introduced into the 
 anterior chamber of the rabbit's eye would bring about 
 a fibro-purulent inflammation, the cornea becoming in- 
 sensible, and perforation alongside of the sclerotic ring 
 finally taking place, followed by the formation of pus 
 in the anterior chamber and recovery. These local 
 changes are the results of the inoculation of small quan- 
 tities only of the dead cultures; but when large amounts 
 are injected into a vein or into the abdominal cavity, 
 toxic effects are produced. Dogs and guinea-pigs thus 
 treated usually die, showing symptoms of poisoning. 
 From the bodies of the bacteria Leber obtained, by 
 treating them with alcohol and ether, a crystalline, 
 chemical substance, which he called phlogosin. This 
 substance, which is an energetic pus-producer, is sup- 
 posed to be the active principle of the staphylococcus 
 aureus. 
 
 Immunization. Immunity against staphylococcus in- 
 fection may be produced in different animal species 
 by the injection of increasing doses of the pure culture, 
 either living or previously sterilized by boiling. Reichel 
 thus succeeded in immunizing dogs against a surely 
 fatal dose of living as well as dead staphylococci. 
 Viquerat claims to have immunized horses in the same 
 way. 
 
 The blood-serum of animals which have been im- 
 munized by means of living or dead cultures possesses 
 slight immunizing and curative effects in other animals, 
 but no practical use of the serum has been attempted 
 in man. 
 
468 BACTERIOLOGY. 
 
 Occurrence in Man. The staphylococcus pyogenes 
 aureus is the commonest pyogenic micro-organism found 
 in man. From the fact that these micrococci are so 
 constantly present in the pus of acute abscesses, as de- 
 monstrated by Ogston, Rosenbach, Passet and others, 
 it was formerly assumed that there could be no pus- 
 formation in the absence of micro-organisms of this 
 class; but it is now well known, from the experiments, 
 that certain chemical substances, such as nitrate of 
 silver, oil of turpentine, strong liquor ammonise, etc., 
 introduced beneath the skin, give rise to pus-formation 
 quite independently of bacteria. Practically all micro- 
 organisms, moreover, have been shown by experiment to 
 produce under certain conditions the formation of pus 
 by their products when inoculated into the animal body ; 
 but, while this has been demonstrated, the extended 
 researches of bacteriologists show that few species are 
 usually concerned in the production of acute abscesses, 
 furuncles, etc., in man. Of these the two most im- 
 portant, by reason of their frequent occurrence and 
 pathogenic power, are staphylococcus pyogenes aureus 
 and streptococcus pyogenes; next to these comes the 
 staphylococcus pyogenes albus. Two or more species 
 are often found in the same abscess; thus, Passet, 
 in 33 cases of acute abscess, found staphylococcus 
 aureus and albus associated in 11, albus alone in 4, 
 albus and citreus in 2, streptococcus pyogenes alone in 
 8, albus and streptococcus in 1, and albus, citreus, and 
 streptococcus in 1. 
 
 As the result of extended researches, however, made 
 by bacteriologists within recent years the golden staphy- 
 lococcus has been demonstrated not only in furuncles 
 and carbuncles, but also in various pustular affections 
 
PRODUCERS OF ABSCESS, CELLULITIS, ETC. 469 
 
 of the skin and mucous membranes impetigo, sycosis, 
 phlyctenular conjunctivitis; in purlent conjunctivitis 
 and inflammation of the lachrymal sac; in acute ab- 
 scesses formed in the lymphatic glands, the parotid 
 gland, the tonsils, the mammae, etc.; in metastatic 
 abscesses and purulent collections in the joints; in em- 
 pyema; in infectious osteomyelitis, in ulcerative endo- 
 carditis, pyelonephritis, etc. It is one of the chief 
 etiological factors in the production of pyaemia in the 
 various pathological forms of that condition of disease. 
 
 Not all persons are equally susceptible to infection 
 by the staphylococcus ; those who are in a cachectic 
 condition or suffering from constitutional diseases, like 
 diabetes, are especially predisposed to infection. In 
 healthy individuals certain parts of the body, as the 
 back of the neck and the seat, are more liable to be 
 attacked than others, with the production of furuncles, 
 carbuncles, etc. In persons in whom sores are readily 
 caused, in consequence of disturbances of nutrition, as 
 in exhausting diseases, the micrococci settle at the points 
 of least resistance. Such conditions are present in the 
 bones of debilitated young children, in fractures, and 
 injuries in general. 
 
 The pyogenic properties of the staphylococcus have 
 been demonstrated upon man by numerous experiments. 
 Garre inoculated a small wound at the edge of one of 
 his finger-nails with a minute quantity of a pure cul- 
 ture, and purulent inflammation extending around the 
 margin of the nail resulted from the inoculation. 
 Staphylococcus aureus was recovered in cultures from 
 the pus thus formed. The same observer applied a 
 considerable quantity of a pure culture obtained from 
 this pus third generation to the unbroken skin of 
 
470 BACTERIOLOGY. 
 
 his forearm, rubbing it well into the skin. At the end 
 of four days a large carbuncle, surrounded by isolated 
 furuncles, developed at the point where the culture had 
 been applied. This ran the usual course, and it was 
 several weeks in healing. No less than seventeen scars 
 remained to testify to the success of the experiment. 
 Bockhart rubbed upon the uninjured skin of the fore- 
 arm a small quantity of an agar culture suspended in 
 salt solution. By gently scratching with a disinfected 
 finger-nail the epithelium was removed in places over 
 the area to which the micrococcus had been applied. 
 Numerous impetigo pustules, and occasionally a gen- 
 uine furuncle, developed as the result of the procedure. 
 Bockhart examined portions of the skin, which he ex- 
 cised for the purpose, under the microscope, and came 
 to the conclusion that the cocci penetrate by way of the 
 hair-follicles, the sebaceous and sudoriparous glands, or, 
 where the epidermis had been removed by scratching, 
 directly to the deeper Jayers of the skin. 
 
 Staphylococcus Pyogenes Albus. 
 
 Isolated by Rosenbach (1884) from the pus of acute 
 abscesses, in which it is sometimes the only micro- 
 organism present, and sometimes associated with the 
 staphylococcus aureus and other pyogenic cocci. 
 
 It is morphologically identical with the staphylococcus 
 pyogenes aureus, and is probably the same organism, 
 which has lost the property of producing pigment. 
 On the average it is somewhat less pathogenic. The 
 surface cultures upon nutrient agar and potato have a 
 milk-white color. Its biological characters are not to 
 be distinguished from the staphylococcus aureus. 
 
PRODUCERS OF ABSCESS, CELLULITIS, ETC. 471 
 
 According to Passet, it is more common than the 
 aureus in man; but the majority of bacteriologists 
 agree with Rosenbach, that the aureus is found at 
 least twice as frequently in human pathological pro- 
 cesses as the albus. 
 
 Staphylococcus Epidermis Albus (Welch). 
 
 Probably identical with the staphylococcus pyogenes 
 albus, but found by Welch on the surface of the body, 
 though often present in parts of the epidermis deeper 
 than can be reached by any known means of cutaneous 
 disinfection save the application of heat. 
 
 With reference to this micrococcus, Welch says : 
 " So far as our observations extend and already they 
 amount to a large number this coccus may be regarded 
 as a nearly, if not quite, constant inhabitant of the 
 epidermis. It is now clear why I have proposed to 
 call it the staphylococcus epidermis albus. It possesses 
 such feeble pyogenic capacity, as is shown by its be- 
 havior in wounds, as well as by experiments on rab- 
 bits, that the designation staphylococcus pyogenes albus 
 does not seem appropriate. Still, I am not inclined to 
 insist too much upon this point, as very probably this 
 coccus, which has hitherto been unquestionably identi- 
 fied by Bossowski and others with the ordinary staphylo- 
 coccus pyogenes albus of Rosenbach, is an attenuated 
 or modified form of the latter organism, although, as 
 already mentioned, it presents some points of difference 
 from the classical description of the white pyogenic 
 coccus ." 
 
 
 
 According to Welch, this coccus differs from the 
 staphylococcus aureus not only in color, but also in 
 the fact that it liquefies gelatin more slowly, does not so 
 
472 BACTERIOLOGY. 
 
 quickly cause coagulation in milk, and is far less viru- 
 lent when injected into the circulation of rabbits. It 
 has been shown by the experiments of Bossowski and 
 of Welch that this micro-organism is very frequently 
 present in aseptic wounds, and that usually it does 
 not materially interfere with the healing of wounds, 
 although sometimes it appears to cause suppuration 
 along the drainage-tube, and it is the common cause 
 of " stitch abscess." 
 
 Staphylococcus Pyogenes Citreus. 
 
 Isolated by Passet (1885) from the pus of acute 
 abscesses, in which it is occasionally found (about 10 
 per cent, of the cases examined) in association with 
 other pyogenic cocci. It is morphologically identical 
 with the Staphylococcus aureus and albus, being distin- 
 guished from the other species only by the formation of 
 a lemon-yellow pigment instead of a golden-yellow, as 
 in the aureus, and a white or colorless deposit, as in the 
 albus. 
 
 THE MICROCOCCUS TETRAGENUS. 
 
 This organism was discovered by Gaffky (1881). It 
 is not infrequently present in the saliva of healthy in- 
 dividuals and in the sputum of consumptive patients. 
 In sputum it is sometimes an evidence of mouth con- 
 tamination rather than lung infection. It has repeatedly 
 been observed in the walls of cavities in pulmonary 
 tuberculosis associated with other pathogenic bacteria, 
 which, though playing no part in the etiology of the 
 original disease, contribute, doubtless, to the progressive 
 destruction of the lung. Its pyogenic character is shown 
 by its occasional occurrence in the pus of acute ab- 
 
PRODUCERS OF ABSCESS, CELLULITIS, ETC. 473 
 
 scesses. Its presence has also been noted in the pus of 
 empyema following pneumonia. 
 
 Morphology. Micrococci having a diameter of about 
 Ijut, which divide in two directions, forming tetrads, 
 and bound together by a transparent, gelatinous sub- 
 stance, enclosing the cell like a capsule. In cultures 
 the cocci are seen in various stages of division as large, 
 round, undivided cells, in pairs of oval elements, and 
 in groups of three and four (Fig. 60). When the divis- 
 
 PlG. 60. 
 
 Micrococcus tetragenus. X 1000 diameters. 
 
 ion is complete they remind one of sarcinae in appear- 
 ance, except that they do not divide in three directions 
 and are not built up like diminutive cotton bales. 
 
 This micrococcus stains readily with the ordinary ani- 
 line dyes; the transparent gelatinous envelope is only 
 feebly stained. It is not decolorized by Gram's method. 
 
 Biological Characters. The growth of this micro- 
 coccus is slow under all conditions. It grows both in 
 the presence and absence of oxygen; it grows best from 
 35 to 38 C., but may be cultivated also at the ordi- 
 nary room-temperature about 20 C. 
 
474 BA GTERIOL OGY. 
 
 Growth on Gelatin. On gelatin plates small, white 
 colonies are developed in from twenty-four to forty- 
 eight hours, which, when examined under a low- 
 power lens, are seen to be spherical or lemon-shaped, 
 grayish-yellow disks, with a finely granular or mul- 
 berry-like surface, and a uniform, but somewhat roughly 
 dentated border. When the colonies push forward to 
 the surface of the gelatin they form white, elevated, 
 drop-like masses, having a diameter of 1 to 2 mm. In 
 gelatin stick cultures the colonies may be either isolated 
 or confluent, in the case forming a thick, white, slimy 
 mass, filling out the fissures and hollow spaces all 
 along the line of puncture; on the surface a broad, 
 thick layer of 4 to 5 mm. in extent is apparent. The 
 gelatin is not liquefied. 
 
 Growth on Agar and Blood-serum. On plate and 
 slant cultures of agar and blood-serum the surface 
 of the growth is moist and glistening. The colonies 
 appear as small, transparent, round points, which have 
 a grayish-yellow color and are slightly elevated above 
 the surface of the medium. 
 
 Pathogenesis. Subcutaneous injections of a culture 
 of this micrococcus in minute quantity is usually fatal 
 to white mice. The animals remain apparently well 
 for a day or two, then become quiet, until death takes 
 place on the third or sixth day. The micrococci are 
 found in comparatively small numbers in the blood of 
 the vessels and heart, but are more numerous in the 
 spleen, lungs, liver, and kidneys. Gray mice are, for 
 the most part, immune to infection by the micrococcus 
 tetragenus. Guinea-pigs at times show only a local 
 reaction after inoculation, and again die from septi- 
 csemic infection. When intraperitoneal injections are 
 
PRODUCERS OF ABSCESS, CELLULITIS, ETC. 475 
 
 given they are followed by purulent peritonitis, beau- 
 tifully formed cocci in groups of four being obtained 
 in immense numbers in the exudate. Rabbits and dogs 
 are not affected by large doses of a culture subcuta- 
 neously or intravenously administered. 
 
 The serum from immunized cases has not been used 
 therapeutically in human infection. 
 
CHAPTER XXVII. 
 
 STREPTOCOCCUS PYOGENES (STREPTOCOCCUS ERYSIPE- 
 LATUS ; STREPTOCOCCUS OF PUS ; STREPTOCOCCUS 
 PATHOGENES LONGUS). 
 
 THIS micrococcus was first observed by Koch in 
 stained sections of tissues attacked by septic processes, 
 and by Ogston in the pus of acute abscesses (1882). 
 It was obtained by Fehleisen (1883) in pure cultures 
 from a case of erysipelas, its cultural and pathological 
 characters studied and demonstrated by him to be 
 capable of producing erysipelas in man. Rosenbach 
 (1884) and Krause and Passet (1885) isolated the 
 streptococcus from the pus of acute abscesses and gave 
 it the name of streptococcus pyogenes. It has since 
 been proved to be one of the chief etiological factors 
 in the production of many suppurative inflammations. 
 Formerly the streptococci of erysipelas, acute abscesses, 
 septicaemia, puerperal fever, etc., were thought to be- 
 long to different species, because they were observed 
 to possess apparent differences in their biological and 
 pathological characteristics, according to the source 
 from which they were obtained. Thus one species of 
 streptococcus was believed to be capable of causing 
 erysipelas only, another only acute abscesses, another 
 sepsis, etc.; but it is now known that the slight differ- 
 ences between the majority of the streptococci growing 
 in long chains are but variations of one and the same 
 
STREPTOCOCCUS PTOGENES. 
 
 477 
 
 species which has been appropriately termed the "strep- 
 tococcus pathogenes longus." Some of the streptococci, 
 at least in so far as their specific products and their 
 reaction in the presence of a curative serum is con- 
 cerned, belong to a species as distinct from the strepto- 
 coccus pyogenes as the pneumococcus. This question 
 has a very practical side, for upon its decision rests our 
 ability to choose a suitable protective serum in cases of 
 streptococcus infection. 
 
 FIG. 61. 
 
 FIG. 62. 
 
 Streptococci in peritoneal fluid, 
 partly enclosed in leucocytes. X 
 1000 diameters. 
 
 Streptococci in throat exudate smeared 
 on cover-glass. X 1000 diameters. 
 
 Morphology. Spherical cocci, when fully developed, 
 having no independent movements, from 0.4// to I/* in 
 diameter, usually larger than the staphylococci, but 
 varying in dimensions in different cultures and even in 
 different parts of a single colony. They multiply by 
 binary division in one direction only, forming chains of 
 eight, ten, twenty, and more elements, being, however, 
 often associated distinctly in pairs. On certain media 
 the cocci occur mostly in diplococci, but usually they 
 grow in longer or shorter chains. Certain cocci fre- 
 
478 BACTERIOLOGY. 
 
 quently exceed their fellows greatly in size, especially 
 in old cultures, when this may be considered to be the 
 result of involution forms. (See Figs. 61, 62, 63, and 
 64.) 
 
 They stain readily by aniline colors and by Gram's 
 method. 
 
 FIG. 63. FIG. 64. 
 
 ?TT> 
 
 T#\v?<& 
 
 ..'jj^fv^,* 
 
 v^^i^'l 
 
 Streptococci from solidified serum cul- Streptococcus growing in long 
 ture appearing mostly as diplococci. chains in bouillon culture. X 1000 
 X 1000 diameters. diameters. 
 
 Biological Characters. Streptococci grow readily in 
 various liquid and solid culture media. The most 
 favorable temperature for their development is from 
 30 to 37 C., but they multiply freely at ordinary 
 room- temperature 18 to 20 C. They are faculta- 
 tive anaerobes, growing both in the presence and ab- 
 sence of oxygen. 
 
 Growth on Gelatin. Tubes of gelatin which have 
 been inoculated with streptococci by puncture with the 
 platinum needle show on the surface no growth beyond 
 the point of entrance. In the depth of the gelatin on 
 the second or third day a distinct, tiny band appears, 
 
STREPTOCOCCUS PYOGENES. 479 
 
 with granular edges or made up of granules. These 
 granules may be very fine or fairly coarse. They are 
 nearly translucent, with a whitish, yellowish, or brown- 
 ish tinge. With characteristic cultures the gelatin is 
 not liquefied, though occasionally, with unusual varie- 
 ties, a certain amount of liquefaction has been observed 
 to take place. 
 
 Growth on Agar. On agar plates the colonies are 
 visible after twelve to thirty hours' growth, and present 
 a beautiful appearance when magnified sufficiently to 
 see the individual cocci in the chain. The colonies 
 from different sources vary in size, thickness, mottling, 
 color, and in the appearance of their borders. The 
 streptococcus growing in short chains in bouillon shows 
 but little tendency to form true loops, but rather pro- 
 jecting rows at the edges of the colonies, while those 
 growing in long chains show beautiful loops, which are 
 characteristic of this organism. The colonies are nearly 
 circular in shape when thinly scattered over the plates, 
 but irregular in form when crowded together. 
 
 Growth in Bouillon. Streptococci grow readily in 
 slightly alkaline bouillon at 37 G., reaching their 
 full development within thirty-six to forty-eight hours. 
 Those which grow in long chains usually give an abun- 
 dant flocculent deposit and leave the liquid clear. The 
 deposit may be in grains, in tiny flocculi, in larger 
 flakes, or in tough, almost membranous masses, the 
 differences depending on the strength of union between 
 the pairs of cocci in the chains. Some of the strepto- 
 cocci growing in long chains, however, cause the broth 
 to become cloudy. This cloudiness may be only tem- 
 porary or it may be lasting. Those growing in short 
 chains, as a rule, cloud the broth; this cloudiness 
 
480 BACTERIOLOGY. 
 
 remaining for days or weeks. A granular deposit 
 appears at the bottom of the tube. 
 
 The development in a mixture of ascitic fluid and 
 bouillon, which is the best medium for the growth of 
 the streptococcus, is more abundant than in bouillon. 
 The liquid is clouded, and a precipitate only occurs 
 after some days, the fluid gradually clearing. 
 
 Growth on Solidified Blood-serum. This is also an 
 excellent medium for the streptococcus. Tiny, grayish 
 colonies appear twelve to eighteen hours after inocu- 
 lation. 
 
 Growth in Milk. All streptococci grow well in milk. 
 As a rule, coagulation of the casein occurs with the 
 production of acid, but this is not always the case. 
 
 The Duration of the Life of Streptococci Outside of 
 the Body. This is not, as a rule, very great. When 
 dried in blood or pus, however, they may live for 
 several months at room-temperature, and longer in an 
 ice-chest; and in gelatin and agar cultures they live 
 for from one week to three months; in bouillon cult- 
 ures they are usually short-lived, the majority dying 
 within two or three days, and very few living over a 
 month; but in serum bouillon they live much longer. 
 In order to keep streptococci alive and virulent, it is 
 best to keep them in serum or ascitic fluid bouillon in 
 small, sealed glass tubes in the ice-chest. The thermal 
 death-point of the streptococcus, according to Stern- 
 berg, is between 52 and 54 C., the time of exposure 
 being ten minutes. 
 
 Von Lingelsheim has reported the following results 
 obtained in an extended series of experiments made to 
 determine the germicidal power of various chemical 
 agents as tested upon this micro-organism (time of ex- 
 
STREPTOCOCCUS PYOGENES. 481 
 
 posure, two hours): Mercuric chloride, 1 : 2500; sul- 
 phate of copper, 1 : 200; trichloride of iodine, 1 : 750; 
 peroxide of hydrogen, i : 50; carbolic acid, 1 : 300; 
 cresol, 1 : 250; lysol, 1 : 300; creolin, 1 : 130. 
 
 Pathogenesis. The majority of test animals are not 
 very susceptible to infection by the streptococcus, and, 
 hence, it is difficult to obtain any definite pathological 
 alterations in their tissues through the inoculation into 
 them of cultures of this organism by any of the methods 
 ordinarily practised. White mice and rabbits, under 
 similar conditions, are the most susceptible, and these 
 animals are, therefore, usually employed for experimen- 
 tation. Streptococci, however, differ greatly in the 
 effects which they produce in inoculated animals, 
 according to their animal virulence, which is very 
 different from human virulence. The most virulent, 
 when injected in the minutest quantity into the circu- 
 lation or into the subcutaneous tissues of a mouse or 
 rabbit, produce death by septicaemia. Those of some- 
 what less virulence produce the same result when in- 
 jected in considerable quantities. Those still less patho- 
 genic produce septicaemia, which may be mild or severe, 
 when injected into the circulation; but when injected 
 subcutaneously, they produce abscess or erysipelas. 
 The remaining streptococci, unless introduced in quan- 
 tities of 20 c.c. or over, produce only a slight redness, 
 or no reaction at all, when injected subcutaneously, 
 and little or no effect when injected directly into the 
 circulation. Many of the streptococci obtained from 
 cases of cellulitis, abscess, empyema, and even septi- 
 caemia belong to this group. 
 
 A number of varieties of streptococci have thus been 
 discovered, differing in virulence and in their growth 
 
 31 
 
482 BACTERIOLOGY. 
 
 on artificial media; but all attempts to separate them 
 into various classes, until recently through the use of 
 specific serum, have failed, because the differences ob- 
 served, though often marked, are not constant, many 
 varieties having been found to lose their distinctive 
 characteristics, and even to apparently change from one 
 class to another. A further objection to any previous 
 classification of streptococci, based on the manner of 
 growth on artificial culture media, is that it has been 
 impossible to make any which would at the same time 
 give even an approximate idea of their virulence. Ex- 
 periments have proved that the streptococci originally 
 virulent may become non-virulent after long cultivation 
 on artificial media, and, again, that they may return to 
 their original properties after being passed through the 
 bodies of susceptible animals. The peculiar type of 
 virulence which they may acquire tends to perpetuate 
 itself, at least for a considerable time. 
 
 One important fact that experience teaches us is, 
 that those streptococci are the most dangerous to any 
 animal which have come immediately from septic con- 
 ditions in the same species of animal, and the more 
 virulent the case the more virulent the streptococci are 
 apt to be in other animals of the same species. There 
 seems also to be a strong tendency for a streptococcus 
 to produce the same inflammation, when inoculated, as 
 the one from which it was obtained; for example, strep- 
 tococci from erysipelas tend to produce erysipelas, from 
 septicaemia to produce septicaemia, etc. Streptococci, 
 however, obtained from different sources (abscesses, 
 puerperal fever, sepsis, erysipelas, etc.) are in many 
 instances capable, under favorable conditions, of pro- 
 ducing erysipelas when inoculated into the ear of a 
 
STREPTOCOCCUS PYOGENES. 483 
 
 rabbit, as has been proved by experiment, provided 
 they possess sufficient virulence (Knorr, Petruschky). 
 If the culture does not have the required virulence to 
 produce this effect the virulence can usually be acquired 
 by passage through animals. Streptococci obtained from 
 the same disease and from the same individual usually 
 show very much the same degree of virulence. 
 
 Occurrence in Man. Streptococci have been found 
 in man as the primary cause of infection in the follow- 
 ing diseases : Erysipelas, acute abscesses, small and 
 large, cellulitis, circumscribed as well as diffused, sep- 
 sis, puerperal infection, lymphatic abscesses, angina, 
 pneumonia, periostitis, otitis media, mastoiditis, men- 
 ingitis, empyema, and endocarditis. Associated with 
 other bacteria in diseases of which they were the specific 
 cause, they have also been found as the secondary or 
 mixed infection in many diseases, such as in pulmonary 
 tuberculosis, bronchopneumonia, septic diphtheria, and 
 diphtheritic scarlatina. In diphtheritic false membranes 
 this micrococcus is very commonly present, and is fre- 
 quently the source of deeper infection, such as abscesses 
 and septicaemia; and in certain cases attended with a 
 diphtheritic exudation, in which the Loffler bacillus 
 has not been found by competent bacteriologists, it 
 seems probable that the streptococcus pyogenes, alone 
 or with other pyogenic cocci, is responsible for the local 
 inflammation and its results. These forms of so-called 
 diphtheria, as first pointed out by Prudden, are most 
 commonly associated with scarlatina and measles, ery- 
 sipelas, and phlegrnonous inflammation, or occur in in- 
 dividuals exposed to these or other infectious diseases. 
 So uniformly are streptococci present in the pseudo- 
 membranous inflammations of patients sick with scarlet 
 
484 BACTERIOLOGY. 
 
 fever that many investigators have suspected them to 
 be the cause of this disease (Kurth, Baginsky, E-oskin). 
 They are found, however, regularly in the secretion of 
 healthy individuals (in 100 examinations by us we found 
 them in 83, and probably could have found them in 
 the others by longer search). Their presence in scarlet 
 fever is most probably due to their increase in the dis- 
 ordered mucous membrane. 
 
 The causal relation of the streptococcus to the above- 
 mentioned diseases has been amply proved by inocula- 
 tion experiments both in man and animals. Fehleisen 
 has inoculated cultures, obtained in the first instance 
 from the skin of patients with erysipelas, into patients 
 in the hospital suffering from inoperable malignant 
 growths lupus, carcinoma, and sarcoma and has 
 obtained positive results, a typical erysipelatous in- 
 flammation having developed around the point of 
 inoculation after a period of incubation of from fifteen 
 to sixty hours. This was attended with chilly sensa- 
 tions and an elevation of temperature. Persons who 
 had recently recovered from an attack of erysipelas 
 proved to be immune. These experiments were under- 
 taken on the ground that malignant tumors had previ- 
 ously been found to improve or entirely disappear in 
 persons who had recovered from accidental erysipelas. 
 During the last few years this fact has been therapeu- 
 tically applied to the treatment of malignant tumors 
 by the artificial production of erysipelas by the inocu- 
 lation of pure cultures of streptococcus or of their toxic 
 products, and in some cases of sarcomata, with con- 
 siderable success. In carcinomata the results have 
 been very slight. In this country the experimental 
 work upon this subject and the actual treatment of 
 
STREPTOCOCCUS PYOGENES. 485 
 
 cases has been largely carried out by or under the 
 direction of Dr. Coley. He has kindly sent me the 
 following notes on his results : 
 
 " The improvement and inhibitory action which the 
 toxins have upon carcinoma have proved to be, in 
 nearly all cases, but temporary, and in no case has 
 the disease remained in abeyance sufficiently long to be 
 regarded as cured. 
 
 " On the other hand, in sarcoma, which is the only 
 form of malignant tumor in which I have advocated 
 the treatment, sufficient time has elapsed to enable us 
 to draw the following conclusions : 
 
 " The toxins injected subcutaneously into the tissues, 
 either into the tumor substance or into parts remote 
 from the tumor, exercise a distinctly inhibitory action 
 upon the growth of all varieties of sarcoma. This 
 action is the least marked in melanotic sarcoma, and 
 tli us far no cases of this form of tumor have disap- 
 peared under the treatment. The influence of the 
 toxins upon round-celled sarcoma is much more pow- 
 erful than it is upon melanotic, although distinctly less 
 than upon the spindle-celled variety. A number of 
 cases of round-celled sarcoma in which the diagnosis 
 was unquestioned disappeared, and the patients have 
 remained well beyond three years. Nearly half of the 
 cases treated showed no appreciable decrease in size; the 
 majority of the others which did show marked improve- 
 ment at first, after decreasing in size for a few weeks, 
 again began to increase and were no longer influenced 
 by the treatment. 
 
 " In half of the cases of spindle-celled sarcoma 
 treated by the toxins the disease has disappeared 
 entirely, and the majority of the successful cases have 
 
486 BACTERIOLOGY. 
 
 remained well sufficiently long to justify their being 
 regarded as cured. It should be distinctly stated that 
 all of the tumors under consideration were inoper- 
 able, as I have never advised treatment except in such 
 cases. 
 
 " It is a curious fact, from the stand-point of path- 
 ology, that the largest percentage of successful cases 
 has occurred in the spindle-celled variety, the very one 
 in which errors of diagnosis are practically impossible. 
 In addition to microscopical examinations by the best of 
 pathologists, the malignancy of the tumors was further 
 confirmed by the characteristic clinical appearances, 
 and in many cases by a history of repeated recurrences. 
 
 ' I have now three cases of spindle-celled sarcoma 
 which have remained well beyond three years; one case 
 of mixed (round and spindle) celled, which, after re- 
 maining well three and one-fourth years, had a return 
 in the abdomen, and died about eight months later. 
 This case certainly would establish the correctness of 
 the early diagnosis. J> 
 
 Dr. Coley would be the first to acknowledge that 
 even the very moderate claims put forward in this 
 communication are disputed by many surgeons, they 
 claiming that the disappearance of the tumors is due 
 to other causes than the treatment. In spite, however, 
 of the treatment being frequently deleterious to the 
 general health, and the occurrence from time to time 
 of the spontaneous disappearance of apparently malig- 
 nant tumors, I think we must allow that the proof is 
 very strong that some sarcomatous tumors have been 
 arrested and caused to disappear by the toxin injec- 
 tions, and that where they are clearly inoperative and 
 progressing the treatment should be tried. 
 
STREPTOCOCCUS PYOGENES. 487 
 
 Production of Toxic Substances. There is no doubt 
 that this micrococcus causes fever, general symptoms 
 of intoxication, and death by means of toxic substances 
 which it forms in its growth; but what these substances 
 are whether they, are due to splitting up of animal 
 proteids, or are secretion-products, or whether they are 
 contained in the cell-bodies of the organism what 
 their composition is and how they are produced in cul- 
 tures we do not know. 
 
 Susceptibility to Streptococcus Infection. As with the 
 other ever-present pus cocci, the staphylococci, which 
 have, as a rule, only slight virulence, the streptococcus 
 is more likely to invade the tissues, forming abscesses 
 or erysipelatous and phlegmonous inflammation in man 
 when the standard of health is reduced from any cause, 
 and especially when by absorption or retention various 
 toxic organic products are present in the body in excess. 
 It is thus that the liability to these local infections, as 
 complications or sequelae of various specific infectious 
 diseases, in the victims of chronic alcoholism, and con- 
 stitutional affections in those exposed to septic emana- 
 tions from sewers, etc.', and probably in many cases 
 from the absorption of toxic products formed in the 
 alimentary canal as a result of the iugestion of im- 
 proper food, or of abnormal fermentative changes in 
 the contents of the intestine, or from constipation, are 
 to be explained. 
 
 Immunity. Knorr succeeded in producing a moder- 
 ate immunity in rabbits against an intensely virulent 
 streptococcus by injections of very slightly virulent 
 cultures. Pasquale was able to immunize these ani- 
 mals partially against septicaemia. Marmorek has 
 immunized sheep, asses, and horses against very large 
 
488 BACTERIOLOGY. 
 
 doses of a streptococcus, which though but slightly 
 virulent for them was intensely so for rabbits. 
 
 In none of the streptococcus inflammations do we 
 notice much apparent tendency to the production of 
 immunizing and curative substances in the blood by a 
 single infection. 
 
 Severe general infections usually progress to a fatal 
 termination after a few days, weeks, or months. It is 
 true, however, that cases of erysipelas, cellulitis, and 
 abscess, after periods varying from a few days to 
 months, tend to recover, and to a certain extent, there- 
 fore, we may assume protective processes have been 
 called forth. In these cases, however, we know from 
 experience that faulty treatment, by lessening the local 
 or general resistance, would, as a rule, cause the sub- 
 siding infection to again progress and that to perhaps a 
 more serious extent than the original attack. Koch 
 and Petruschky tried a most interesting experiment. 
 They inoculated cutaneously a man suffering from a 
 malignant tumor with a streptococcus obtained from 
 erysipelas. He developed a moderately severe attack, 
 whi'ch lasted about ten days. On its subsidence they 
 reinoculated him; a new attack developed, which ran 
 the same course and over the same area. This was 
 repeated ten times with the same results. 
 
 This experiment proved that in this case, at least, 
 little if any lasting curative or immunizing substances 
 were produced by repeated attacks of erysipelas, and 
 that the recovery from each attack was due to local 
 and transitory protective developments. 
 
 The severe forms of infection, such as septicaemia 
 following injuries, operations, and puerperal infections, 
 show little tendency to be arrested after being well 
 
STREPTOCOCCUS PYOGENES. 489 
 
 established. Having, then, in remembrance the above 
 facts, let us consider the results already obtained in the 
 experimental immunization and treatment of animals 
 and men suffering from or in danger of infection with 
 streptococci. One method is now chiefly used for the 
 immunization and attempt to produce curative sub- 
 stances in animals. The living, virulent streptococcus 
 itself is injected in gradually increasing doses. Mar- 
 morek 1 was the first to attempt to produce a curative 
 serum on a large scale. 
 
 Influence of Serum from Animals Immunized Against 
 Streptococcus Infection upon Streptococcus Infections 
 in Other Animals. 
 
 The results reported since Marmorek's commu- 
 nication in 1895 upon the immunizing effects of anti- 
 streptococcic serum in animals have been very vari- 
 able. 
 
 Reliable positive results are, however, more impor- 
 tant than negative ones, since they indicate under 
 proper conditions what can be accomplished. This is 
 certainly true if at the same time we can find good 
 reasons for the failures reported. 
 
 For the data in the following table I am indebted to 
 Anna W.Williams, assistant bacteriologist in the Health 
 Department Laboratories. For the use of the same I 
 wish to express my appreciation, 
 
 In this present table are given the results following 
 the injection of small amounts of a serum which repre- 
 sents in immunizing value what about one-third of the 
 horses are able to produce. In the following experi- 
 
 i Annales de 1'Institut Pasteur, July, 1895. 
 
490 
 
 BACTERIOLOGY. 
 
 ments the serum and culture were injected subcutane- 
 ously in rabbits at the same time, but in opposite sides 
 of the body : 
 
 TABLE SHOWING STRENGTH OF AVERAGE GRADE OF ANTI- 
 
 STREPTOCOCCIC SERUM GIVEN BY SELECTED HORSES AFTER 
 
 SIX MONTHS OF INJECTION OF SUITABLE AMOUNTS OF 
 LIVING STREPTOCOCCI. 
 
 
 Weight 
 of 
 rabbit. 
 
 Amounts 
 inoculated. 
 
 Result. 
 
 Autopsy. 
 
 Serum and culture : 
 
 Grms. 
 
 Serum. Cult. 
 
 
 
 1. Inoculated at same time 
 
 1430 
 
 0.25 c.c. 0.01 c.c. 
 
 Lived 
 
 
 2. 
 
 1350 
 
 0.125" 0.01 " 
 
 " 
 
 
 3. 
 
 1600 
 
 0.25 " 0.01 " 
 
 " 
 
 
 4. Subcutaneously " 
 
 1440 
 
 0.25 " 0.01 " 
 
 " 
 
 
 5. On opposite sides " 
 
 1770 
 
 0.1 " 001 " 
 
 " 
 
 
 6. ' " " " 
 
 1630 
 
 0.1 " 0.01 " 
 
 " 
 
 
 Controls : 
 
 
 
 
 
 1. Rabbits injected with 
 culture only 
 2. 
 
 3 
 
 1750 
 
 1870 
 1820 
 
 0.001 " 
 0.001 " 
 0.01 " 
 
 Died in 
 4 days. 
 Died in 
 24hrs. 
 Died in 
 4 days. 
 
 Strept. in- 
 fection. 
 
 The above results have been repeatedly obtained, and 
 are absolutely conclusive that, as Marmorek and others 
 have claimed, the serum of properly selected animals, 
 which have been repeatedly injected with living strep- 
 tococci in suitable doses, possesses bactericidal properties 
 upon the same streptococcus when it comes in contact 
 with it within the bodies of animals. 
 
 Definite protection from the serum has been obtained 
 by many reliable observers since Marmorek' s first 
 reports. 
 
STREPTOCOCCUS PYOGENES. 491 
 
 Is Protection Afforded by the Same Serum Against All 
 Varieties of Streptococci? 
 
 We have tested the protective value of one serum 
 against five streptococci. First, the streptococcus given 
 us by Marmorek, which was obtained from a case of 
 angina complicating scarlet fever. Its virulence is now 
 such, after having passed through hundreds of rabbits, 
 that 0.000001 c.c. is the average fatal dose. Second, 
 a streptococcus obtained from a case of erysipelas in 
 England. Its virulence is 0.00001 c.c. on the average. 
 Third, a streptococcus obtained from a case of cellu- 
 litis a few weeks ago, its virulence being about 6 c.c. 
 Fourth, a streptococcus sent me by Theobald Smith. 
 Its virulence is such that 0.1 c.c. is the average fatal 
 dose. Fifth, another culture sent me by Smith, which 
 grew in short chains and was obtained from milk; its 
 virulence was similar to No. 4. 
 
 Against the first three streptococci derived from three 
 different varieties of infection existing in three different 
 countries the serum produced in the horse by the strep- 
 tococcus from England had nearly the same value. 
 Against the latter two streptococci, as well as against 
 a pneumococcus, which in ordinary cultures looks like 
 a streptococcus, the serum had no effect. 
 
 The results published by others must also be taken 
 to prove that a serum which protects from infection 
 with one streptococcus may fail against others; but, 
 taking all together, they indicate that the majority of 
 streptococci met with in practice will be influenced by 
 the same serum. Many more streptococci, however, 
 must be obtained from human infections and tested 
 before we can be certain of this. Those obtained from 
 
492 BACTERIOLOGY. 
 
 human sepsis, which are not very virulent in animals, 
 are especially in need of investigation. If those who 
 use the serum will send to the laboratories materials 
 for cultures this can in time be fully determined. 
 
 The Preparation of the Serum. Antistreptococcus 
 serum is obtained from the horse, ass, and sheep after 
 treatment by repeated injections of living streptococcus 
 cultures. The procuring of a serum of the highest 
 potency requires a considerable number of animals, for 
 some produce with the same treatment a more protec- 
 tive serum than others. The serum must be sterile 
 from streptococcus as well as from other contaminations. 
 
 The Stability of the Serum. Unfortunately, after sev- 
 eral weeks or months, the serum, as a rule, at least, loses 
 its protective value. It should be kept in a cold and 
 dark place. Not only ourselves, but others, such as 
 Aronson, have found this to be true. 
 
 To this deterioration can probably be ascribed the 
 failure of Koch, Petruschky and others to find in the 
 serum any power to protect animals from infection. 
 
 The Standardization of the Value of the Serum. The 
 value of the serum is measured by the amount required 
 to protect against a multiple of a fatal dose of a very 
 virulent streptococcus. The dose is usually a thousand 
 times the average fatal amount of a very virulent 
 streptococcus. 
 
 This method gives, as a rule, to those unfamiliar 
 with bacteriology an exaggerated idea of the potency 
 of the serum. 
 
 A thousand times the amount of a very virulent 
 streptococcus culture required to kill an animal by 
 producing septicaemia is still too little to kill by the 
 streptococci injected; it is only their enormous multi- 
 plication in the animal which kills. 
 
STREPTOCOCCUS PYOGENES. 493 
 
 Double the fatal dose of a culture which kills only 
 in a dose of 10 c.c. or over is a more severe test than 
 a thousand times a very virulent one. 
 
 It is entirely different in case of an antitoxin 
 which does not prevent primarily the growth of the 
 germ, but neutralizes a chemical substance its toxin. 
 
 Its Therapeutic Results. To estimate the exact pre- 
 sent and future value of antistreptococcus serum is a 
 matter of the utmost difficulty. Many of the cases 
 reported are of little or no help, because, on account of 
 no cultures having been made, we are in doubt as to the 
 nature of the bacterial infection. Even when bacterio- 
 logical examinations are made during life in cases of 
 septicsemia, they are apt to fail to give us any informa- 
 tion. Under Marmorek's supervision many cases have 
 been injected; thus, even as far back as June, 1895, 
 when his last statistics were published, he had treated 
 96 cases of scarlet fever, 411 cases of erysipelas, 16 
 cases of puerperal fever, and smaller numbers of cases 
 of tonsillitis and of post-operative septicsemia. 
 
 Since then he has treated many forms of phthisis. 
 In all these cases marked improvement is reported to 
 have followed when they were due to streptococci. 
 Thus, in sixteen cases of puerperal fever seven were 
 due to streptococcus alone. All these recovered. 
 Three were due to the streptococcus and colon bacil- 
 lus and one to the colon bacillus alone. These four 
 all died. In five, streptococci were associated with 
 staphylococci. Two of these died, three recovered. 
 
 In phthisis where no cavities have as yet appeared 
 the fever and sweats lessened and all symptoms im- 
 proved. He did not state that any cases were abso- 
 lutely cured. Marmorek's results are by far the best 
 reported, and without casting any doubt upon the in- 
 
494 BACTERIOLOGY. 
 
 tended honesty of his conclusions, it is my conviction 
 that they give undoubtedly too favorable a view of the 
 value of the serum. 
 
 In the comparatively few cases of puerperal fever, 
 wound infection, scarlet fever, and bronchopneumonia 
 that we have seen under the treatment the apparent re- 
 sults have not been uniform. Only occasionally did 
 we see results which appeared to be distinctly due to 
 the serum. 
 
 In a number of cases of septicaemia where chills had 
 occurred daily for days they ceased absolutely or 
 lessened under daily doses of 20 to 50 c.c. The 
 temperature, though ceasing to rise to such high eleva- 
 tions, did not average more than one or two degrees 
 lower than before the injections. The serum treat- 
 ment was kept up for four weeks. Some cases conva- 
 lesced; others after a week or more grew worse and died. 
 
 In some cases the temperature fell immediately upon 
 giving the first injection of serum, and after subse- 
 quent injections remained normal, and the cases seemed 
 greatly benefited. As a rule, in these cases no strep- 
 tococci or any other organisms were obtained from the 
 blood. On bronchopneumonia, laryngeal diphtheria, 
 and in phthisis we have seen absolutely no effect. 
 
 The results obtained here in New York by both 
 physicians and surgeons have not, on the whole, been 
 very encouraging. 
 
 In some of the cases where apparently favorable re- 
 sults were obtained other bacteria than streptococci were 
 found to be the cause of the disease. We believe that 
 the following conclusions will be found fairly accurate : 
 
 A single antistreptococcic serum protects healthy 
 rabbits from infection from most of the streptococci 
 obtained from human sepsis due to the streptococcus, 
 
STREPTOCOCCUS PYOQENES. 495 
 
 but not from all. Failure to do good in human infec- 
 tion cannot, as a rule, be attributed to the variety of 
 streptococcus. The serum will in animals limit an in- 
 fection already started if it has not progressed too far. 
 The apparent therapeutic results in cases of human 
 streptococcus infection are variable. In some cases 
 the disease has undoubtedly advanced in spite of large 
 injections, and here it has not seemed to have had 
 any effect. In other cases good observers rightly or 
 wrongly believe they have noticed great improvement 
 from it. Except rashes, few have noticed deleterious 
 results, although very large doses have been followed 
 in several instances, for a short time, by albuminous 
 urine and even temporary suppression. 
 
 In suitable cases we are, I think, warranted in try- 
 ing it, but we must not expect very striking results. 
 
 For our own satisfaction, and to increase our knowl- 
 edge, we should always have satisfactory cultures made 
 when possible, and the streptococci, if obtained, tested 
 with the serum used in the treatment. In the cases 
 where we want most to use the serum, such as puerperal 
 fever, septicaemia, ulcerative endocarditis, etc., we 
 find that it is very difficult to make a bacteriological 
 diagnosis from the symptoms, and in over one-half of 
 the cases even the bacteriological examination carried 
 out in the most thorough way will fail to detect the 
 special variety of bacteria causing the infection. This 
 is often a great hinderance to the proper use of curative 
 antistreptococcic serum, for it, of course, has no specific 
 effect upon the course of any infection except that due 
 to the streptococcus. 
 
 Care should be taken to get only recently tested 
 serum, for after six weeks the best serum is almost 
 inert; much on the market is worthless, and as it is 
 
496 BACTERIOLOGY. 
 
 weak, and the testing for strength is still very crude, 
 full doses of serum should be given if the case is at all 
 serious, for the dose is limited only by the amount 
 of horse-serum which we feel it safe to give, not be- 
 cause we have sufficient protective substance. 
 
 Bacteriological Diagnosis. Streptococci, using the 
 name in its broadest sense, can often be demonstrated 
 microscopically by simply making a smear preparation 
 of the suspected material and staining with methylene- 
 blue solution or diluted ZiehPs fluid. In order to 
 demonstrate them microscopically in the tissues, the 
 sections are best stained by Kuhne's methylene-blue 
 method. In all cases, even when the microscopical 
 examination fails, the cocci may be found by the culture 
 method on plate agar or slanted agar at 37 C. To 
 obtain them from a case of erysipelas it is best, accord- 
 ing to Fehleisen, to excise a small piece of skin from 
 the margin of the erysipelatous area in which the cocci 
 are most numerous; this is crushed up and part of it 
 transfered to a gelatin tube and to the melted agar in 
 another tube. After shaking thoroughly the contents 
 are poured out into Petri dishes. The gelatin is kept 
 at a temperature of 20 C. At the end of two or 
 three days numerous small colonies develop in the 
 vicinity of the particles of skin. The agar plate is 
 kept at 37 C. for twenty-four hours. It is usually 
 sufficient, however, to make a streak culture on agar in 
 a Petri dish with the crushed excised portion of skin 
 and place this in the incubator at 37 C. 
 
 In septicaemia the culture method is always required 
 to demonstrate the presence of streptococci, as the micro- 
 scopical examination of specimens of blood is not suffi- 
 cient. For this purpose from 3 to 5 c.c. of the blood 
 should be drawn from the vein of the arm aseptically 
 
STREPTOCOCCUS PYOGENES. 497 
 
 by means of a hypodermatic needle, and each c.c. added 
 to a tube of broth, in order to produce an adequate 
 development of the cocci, which are found in small 
 numbers in the bloodvessels. Petruschky is of the 
 opinion that the cocci can best be shown in blood by 
 animal inoculation. Having withdrawn from the 
 patient 10 c.c. of blood by means of a hypodermatic 
 syringe, under aseptic precautions, he injects a portion 
 of this into the abdominal cavity of a mouse, while the 
 other portion is planted in bouillon. Mice thus inocu- 
 lated die from septicaemia when virulent streptococci 
 are present only in very small numbers in the blood. 
 If a successful inoculation takes place we can, through 
 the absence or presence of the development of capsules, 
 often differentiate between the pneumococcus and the 
 streptococcus, which cultures may fail to do. The 
 morphological and cultural characteristics of the strep- 
 tococcus give us, unfortunately, no absolute knowledge 
 as to the influence which the protecting serum will 
 have. The actual test is here our only method. The 
 detection of the streptococcus in the blood is in itself 
 an unfavorable prognostic sign. 
 
 The blood cultures in perhaps the majority of cases 
 of septicaemia give no positive results, for many of 
 these cases develop their symptoms and even die from 
 the absorption of toxins from the local infection, such 
 as an amputation wound or an infected uterus or peri- 
 toneum, and the bacteria never invade the blood. When 
 we get negative results we are, as a rule, utterly unable 
 to test the case with curative serums with any accuracy, 
 for the sepsis may be due to either the streptococcus, 
 colon bacillus, staphylococcus, or a number of other 
 pathogenic varieties of bacteria. 
 
 32 
 
CHAPTER XXVIII. 
 
 MICROCOCCUS LANCEOLATUS (PNEUMOCOCCUS ; MICRO- 
 COCCUS PNEUMONIA CROUPOS2E OF STERNBERG; 
 MICROCOCCUS OF SPUTUM SEPTIC^MIA AND DIPLO- 
 COCCUS OF FRAENKEL; DIPLOCOCCUS PNEUMONIA 
 OF WEICHSELBAUM). 
 
 THIS micrococcus was first observed by Sternberg, in 
 1880, in the blood of rabbits inoculated with his own 
 saliva (and almost simultaneously by Pasteur under 
 similar conditions), whence it was called by Sternberg 
 micrococcus Pasteuri. It was subsequently described 
 by Talamon (1883), and demonstrated by him to be 
 capable of producing fibrinous pneumonia in rabbits 
 when introduced into the parenchyma of the lung of 
 these animals. In 1885 and 1886 this micro-organism 
 was subjected to an extended series of investigations 
 by A. Fraenkel, Sternberg, Weichselbaum, Netter 
 and others, and proved by them to be the chief etio- 
 logical factor in the production of lobar or croupous 
 pneumonia in man. 
 
 Morphology. Very irregular; occurs as spherical or 
 oval cocci, usually united in pairs, but sometimes in 
 longer or shorter chains consisting of from three to six 
 or more elements and resembling the streptococcus. The 
 individual cells, as they commonly occur in pairs, are 
 somewhat oval in shape, being usually pointed at one 
 end hence the name lanceolatus, or lancet-shaped. 
 
MICROCOCCUS LANCEOLATUS. 
 
 499 
 
 When thus united the junction, as a rule, is between 
 the broad ends of the oval, with the pointed ends 
 
 FIG. 65. 
 
 Diplococcus of pneumonia from blood, with surrounding capsule. 
 FIG. 66. 
 
 Pneumococcus from bouillon culture, resembling streptococcus. 
 
 turned outward; but variation in form and arrange- 
 ment of the cells is characteristic of this organism, 
 there being great differences according to the source 
 
500 BACTERIOLOGY. 
 
 from which they are obtained. As observed in the 
 blood of inoculated animals it is usually in pairs of 
 lancet-shaped elements, which are surrounded by a cap- 
 sule. (See Fig. 65.) When grown on culture media 
 longer or shorter chains are frequently formed, which 
 can scarcely be, or even not at all, distinguished from 
 chains of streptococci. The individual cells are almost 
 spherical in shape, and they are rarely surrounded by 
 a capsule. (See Fig. 66.) 
 
 The capsule is best seen in stained preparations from 
 the blood and exudates of fibrinous pneumonia or from 
 the blood of an inoculated animal, especially the mouse, 
 in which it is commonly, though not always, present. 
 It is seldom seen in preparations from cultures. 
 
 It stains readily with ordinary aniline colors; it is 
 not decolorized after staining by Gram's method. The 
 capsule may be demonstrated in blood or sputum either 
 by Gram's or Welch's (glacial acetic acid) method. 
 
 Biological Characters. It grows on almost all the 
 culture media ordinarily employed, but its suscepti- 
 bility is shown not only by its irregularity of form, but 
 also by its slow and comparatively scanty growth and 
 by its rapid loss of virulence and power of reproduction 
 under varying conditions. It grows equally well in 
 the absence as in the presence of oxygen, being thus 
 both aerobic and facultative anaerobic; its parasitic 
 nature is exhibited by the short range of temperature 
 at which it grows viz., from 25 to 42 C. its maxi- 
 mum growth being at about 37 C., or the temperature 
 of the body. Its thermal death-point, as determined 
 by Sternberg, is 52 C., the time of exposure being 
 ten minutes. It loses its vitality in cultures in a 
 comparatively short time, and is very sensitive to the 
 
MICEOCOCCUS LANCEOLATUS. 501 
 
 action of germicidal agents. Its pathogenic power also 
 undergoes attenuation very rapidly when cultivated on 
 artificial media. It is non-motile. 
 
 In the cultivation of this organism one of the most 
 important considerations is the reaction of the media 
 employed. According to Fraenkel, Sternberg and 
 others, it grows only in culture media when they have 
 a slightly alkaline reaction. Kruse and Pansini showed 
 by their investigations that, according to the source 
 from which it was obtained, it grows at times equally 
 well in a slightly alkaline or slightly acid medium. 
 Not infrequently, however, all experimenters have 
 found that no growth at all occurs, irrespective of the 
 composition or reaction of the media employed. The 
 weight of opinion, nevertheless, seems to be in favor of 
 the selection of a slightly alkaline medium. 
 
 The organism grows, as has been said, on all the cul- 
 ture media ordinarily employed for the cultivation of 
 bacteria viz., on agar and gelatin, in bouillon, ascitic 
 fluid, and blood-serum. The best medium for its 
 growth is a mixture of one-third ascitic or pleuritic 
 fluid and two-thirds bouillon. It grows readily in 
 milk, causing coagulation with the production of acid, 
 though this is not constant. 
 
 Growth on Agar. Cultivated on plain nutrient agar, 
 after forty-eight hours in the incubator, there ap- 
 pears a thin, colorless layer composed of dilute non- 
 confluent colonies. If blood-serum or ascitic fluid be 
 added to the agar the individual colonies are larger and 
 closer together, and the growth is more distinct in con- 
 sequence and of a grayish color. The surface colonies 
 resemble those of some of the streptococci growing in 
 short chains; they are almost circular in shape, finely 
 
502 BACTERIOLOGY. 
 
 granular in structure, and have a somewhat darker, 
 more compact centre, surrounded by a paler marginal 
 zone. With high magnification rows of cocci are seen 
 sprouting from the edges. In stick cultures along the 
 line of puncture minute transparent drops appear. 
 
 Growth on Gelatin. The growth on gelatin is slow, 
 if there is any development at all, owing probably to 
 the low temperature viz., 22 to 24 C. at which the 
 gelatin has to be kept. The gelatin is not liquefied. 
 
 Growth on Blood-serum, The growth on Ldffler's 
 blood-serum mixture is very similar to that on agar, 
 but somewhat more vigorous, appearing on the surface 
 as a delicate layer of dew-like drops. 
 
 Growth in Bouillon. In bouillon, at the end of twelve 
 to twenty-four hours in the incubator, a slight cloudi- 
 ness of the liquid will be found to have been pro- 
 duced, due to the development of the micrococci, 
 which on microscopical examination can be seen to be 
 arranged in pairs or longer and shorter chains. After 
 two or three days the medium becomes again trans- 
 parent, owing to the subsidence of the cocci to the 
 bottom of the tube. 
 
 Special Media. Fraenkel was the first to draw at- 
 tention to the fact that this organism soon loses its 
 reproductive power when grown on ordinary culture 
 media, and more particularly solid media. In fluid 
 media the vitality is not quite so quickly lost; but 
 even here it is found advisable in practice to transplant 
 fresh cultures every day. By this method, when bou- 
 illon cultures are used, the vitality may be indefinitely 
 prolonged; but after transplantation through several 
 generations it is found that the cultures begin to lose 
 in virulence, which finally disappears entirely. In order 
 
MICROCOCCUS LANCEOLATUS. 503 
 
 to restore this virulence, or to keep it from becoming 
 attenuated, it is necessary, therefore, to interrupt the 
 transplantation and pass the organism through the 
 bodies of susceptible animals. 
 
 With the view of overcoming some of these obstacles 
 in the way of cultivating this micrococcus, several spe- 
 cial media have been proposed by various experimenters 
 in the place of the ordinary culture media. The best 
 fluid medium for the growth of the pneumococcus is 
 Marmorek's mixture, consisting of bouillon, 2 parts ; 
 ascitic or pleuritic fluid, 1 part. In this fluid pneumo- 
 cocci grow well, and cultures when preserved in a cool 
 place and prevented from drying retain their vitality 
 and also their virulence for a number of months. 
 Lambert has found cultures in this medium alive and 
 fully virulent after eight months. 
 
 Loftier 7 s blood-serum mixture is probably the best 
 solid tube medium for making cultures, and is very 
 convenient and useful at autopsies. One and one-half 
 per cent, fluid nutrient agar mixed with one-third its 
 quantity of warm ascitic fluid makes an excellent plate 
 medium. 
 
 Effects of Germicidal Agents, Light and Drying. The 
 following are the effects of germicidal and antiseptic 
 agents on this organism, according to observations 
 made by Sternberg : Boric add, saturated solution, 
 failed to destroy the vitality after two hours, but a so- 
 lution of 1 : 400 restrained its development; carbolic 
 acid, 1 per cent, solution, destroys the vitality in two 
 hours, and 1 : 500 restrains development; mercuric chlo- 
 ride,! : 20,000, destroys vitality in two hours,! : 40,000 
 restrains development; salicylic acid and sodium bibo- 
 rate, 1 : 400 solution, restrained development. 
 
504 BACTERIOLOGY. 
 
 As to its duration of life outside the body, the 
 researches of Bordoni-Uffreduzzi throw some light. 
 He found that pneumonic sputum attached to clothes, 
 when dried in the air and exposed to diffuse day- 
 light, retained its virulence, as shown by injection 
 in rabbits, for a period of nineteen to fifty-five days. 
 Exposed to direct sunlight the same material retained 
 its virulence after twelve hours' exposure. This reten- 
 tion of virulence for so long a time under these circum- 
 stances is accounted for by the protective influence 
 afforded by the dried albuminous material in which the 
 micrococci were embedded. Thus, Guarnieri observed 
 that the blood of inoculated animals, wheu rapidly dried 
 in a desiccator, retained its virulence for months; and 
 Foa found that fresh rabbit blood, after inoculation and 
 cultivation in the incubator for twenty-four hours, 
 when removed at once to a cool, dark place, retained 
 its virulence for sixty days. There are many condi- 
 tions, therefore, in which the virulence of the micro- 
 coccus is retained for a considerable length of time. 
 
 The Source of Infection. Although, as we have just 
 seen, the pneumococcus may retain its virulence in 
 dried sputa for considerable lengths of time, still such 
 pneumococci are not the only source of contagion, for 
 in the throat secretions of many healthy persons, and 
 in the bronchial and lung discharges of nearly all cases 
 of chronic pulmonary diseases, we have the pneumo- 
 cocci abundantly present. 
 
 Pathogenesis. The micrococcus lanceolatus is quite 
 pathogenic for some animals viz., mice and rabbits 
 less so for others. In mice and rabbits the subcutaneous 
 injection of small quantities of pneumonic sputum in 
 the early stages of the disease, or of a pure, virulent 
 
MICROCOCCUS LANCEOLATUS. 505 
 
 culture of the micrococcus, usually results in the death 
 of these animals in from twenty-four to forty-eight 
 hours. The course of the disease produced and the 
 post-mortem appearances indicate that it is a form of 
 septicaemia what is known as sputum septicaemia. 
 After injection there is loss of appetite and great 
 debility, and the animal usually dies some time during 
 the second day after inoculation. The post-mortem 
 examination shows a local reaction, which may be of 
 a serous, fibrinous, hemorrhagic, necrotic, or purulent 
 character; or there may be combinations of all of these 
 conditions. The most marked pathological lesion is 
 the enlargement of the spleen, which in mice is con- 
 spicuous and common, and in rabbits not so much so. 
 It is sometimes hard, dark colored, and dry, or it may 
 be soft and bright red. The liver also is sometimes 
 dark colored and gorged with blood, but more fre- 
 quently it is paler than normal and rich in fat. The blood 
 of inoculated animals immediately after death often 
 contains the micrococci in very large numbers. For 
 microscopical examination they may be obtained from 
 the blood of the veins, arteries, or cavities of the heart, 
 and usually from the pleural and peritoneal exudations 
 when they are present. 
 
 Mice and rabbits are the most susceptible animals, 
 and are thus usually employed for experimental pur- 
 poses in investigations with this micrococcus; but 
 guinea-pigs, dogs, cats, rats, and sheep are also sus- 
 ceptible. Chickens and pigeons are insusceptible- 
 Young animals, as a rule, are more easily affected 
 than old ones. In dogs subcutaneous injections usually 
 give negative results. True localized pneumonia does 
 not usually result from subcutaneous injections into 
 
506 BACTERIOLOGY 
 
 susceptible animals, but injections made through the 
 thoracic walls into the substance of the lung may 
 induce a typical fibrous pneumonia. This was first 
 demonstrated by Talamon, who injected the fibrinous 
 exudate of croupous pneumonia, obtained after death 
 or drawn during life from the hepatized portions of 
 the lung, into the lungs of rabbits. 
 
 Attenuation of Virulence. The pathological changes 
 above mentioned apply only to the effects produced 
 by fully virulent cultures on susceptible animals. With 
 attenuation of virulence in the cultures or decrease 
 of susceptibility in the animals different effects are 
 produced. When the disease takes a rapid course the 
 local reaction and the changes in the internal organs 
 are comparatively slight; but the longer the process 
 lasts the greater will be the local reaction and patho- 
 logical lesions in the body. Attenuation of virulence 
 may be produced in various ways. The loss of viru- 
 lence which occurs when the micrococcus is trans- 
 planted in cultures through several generations has 
 already been referred to. A similar attenuation of 
 virulence takes place also spontaneously in the course 
 of pneumonia. Patella has shown by daily puncture 
 of the lung in different stages of the pneumonic pro- 
 cess that the virulence of the organism diminished as 
 the disease progressed, and that the nearer the crisis 
 was approached the more attenuated it became a fact 
 which has been confirmed by others. Welch found 
 that the most virulent micrococci were contained in 
 the freshly hepatized portions of the lung. Fraenkel 
 and Weichselbaum showed that the cocci taken from 
 the lung varied in virulence according to the stage of 
 the disease when they were obtained. Attenuation of 
 
MICROCOCCUS LANCEOLATUS. 507 
 
 virulence may also be effected artificially. Banti found 
 that the continued passage through the bodies of guinea- 
 pigs, which are not particularly susceptible, also re- 
 sulted in a loss of virulence. Sanarelli states that the 
 cultivation in human saliva is also attended with an 
 attenuation of virulence. Cultivation in other unfavor- 
 able media, or media to which substances have been 
 added which restrain development, has similar attenu- 
 ating effect on the virulence. 
 
 Restoration and Increase of Virulence. The simplest 
 and perhaps the most reliable method of restoring lost 
 virulence for any animal is by passage through the 
 bodies of highly susceptible animals of the same species. 
 
 Occurrence in Man. The micrococcus lanceolatus is 
 not infrequently present in the saliva of healthy in- 
 dividuals, having been found by Sternberg in the oral 
 cavity of about 20 per cent, of healthy persons exam- 
 ined. It is constantly to be detected in the rusty sputum 
 of patients suffering from acute fibrinous pneumonia. 
 Weichselbaum reports having found it in 94 out of 129 
 cases of pneumonia examined by him; Wolff found it 
 in 65 cases out of 70 examined; Netter in 75 per cent, 
 of his cases, and in the sputum of convalescents from 
 pneumonia in 60 per cent. The more recent the infec- 
 tion the greater is the number of bacteria found in the 
 diseased lung areas. As the disease progresses they 
 decrease in number until finally at the crisis they dis- 
 appear from the tissues, though at this time and long 
 after convalescence they may be present in the sputum. 
 In atypical forms of pneumonia they may remain longer 
 in the tissues, and in walking pneumonia they may be 
 absent in the original centres of infection or present only 
 as attenuated varieties, while the surrounding, newly- 
 
508 BACTERIOLOGY. 
 
 formed foci may contain fully virulent cocci. But 
 lobar pneumonia is not the only form of pneumonia 
 in the production of which this organism is concerned. 
 It has been shown by Netter that more than one-half 
 of the cases of bronchopneumonia, whether primary or 
 secondary to some other disease, as measles and diph- 
 theria, both in children and adults, are due to the 
 micrococcus lanceolatus. The microscopical appear- 
 ances in bronchopneumonia are the same as in lobar 
 pneumonia, the only difference being, according to the 
 observations of Ribbert and Baumgarten, that in the 
 former the infective process is less extended, resulting 
 in the formation of a number of small foci instead of 
 the lung being attacked in toto. 
 
 Beside the affections above mentioned, this micro- 
 coccus is associated with other pathogenic bacteria, 
 producing a secondary or mixed infection. In tuber- 
 culosis, for example, it is often found associated with 
 the tubercle bacillus, taking part with this organism in 
 the destruction of the tubercular tissue of the lungs. 
 Having once reached the lung it may penetrate to dif- 
 ferent organs in the body, producing in them more or 
 less intense inflammatory processes, which are mostly of 
 a purulent character. It may thus cause inflammations 
 of the serous membranes of the endocardium, the peri- 
 cardium, the meuinges, and even of the brain itself. 
 Foremost among the secondary infections which it 
 causes are meningitis, serofibrinous pleurisy, and em- 
 pyema. In 25 cases of purulent meningitis examined 
 by Netter the " pneumococcus " was found in 16; 4 of 
 these cases were complicated with purulent otitis, 6 
 with pneumonia, and 3 with ulcerative endocarditis. 
 In 45 cases collected by Netter from the literature of 
 
MICROCOCCUS LANCEOLATUS. 509 
 
 the subject this micrococcus was present in 27. Monti 
 demonstrated the presence of the same micrococcus in 
 4 cases of cerebro-spinal meningitis. Weichselbaum, 
 in a series of 29 cases of ulcerative endocarditis exam- 
 ined, found <( diplococcus pneumoniae" in 7. It has 
 been found also in acute abscesses in the pus of paro- 
 titis complicating pneumonia it was obtained in pure 
 culture by Testi; in a case of pneumonia, in which 
 there developed a purulent pleuritis and parotitis, and 
 multiple subcutaneous abscesses, it was found in great 
 numbers in all these places in the pus; in a case of 
 tonsillitis resulting in the formation of an abscess it 
 was obtained in pure culture by Gabbi. This micro- 
 coccus has also been found in a number of cases of 
 otitis media in the pus obtained by paracentesis of 
 the tympanic membrane, by Netter in 5 out of 18 cases 
 occurring in children. Monti and Belfanti report cases 
 of arthritis of the wrist-joint, occurring as a complica- 
 tion of pneumonia, in which it was found. Ortmann 
 and Samter, in a case of purulent inflammation of the 
 shoulder-joint following pneumonia and pleurisy, ob- 
 tained the u pneumococcus " in pure culture. It has 
 been found in other cases of inflammation of the knee, 
 ankle, and elbow-joints, and in osteomyelitis and perios- 
 titis. In short, there is scarcely any part of the body 
 in which this organism may not find suitable conditions 
 for existence and in which it does not sometimes occur. 
 How is it conveyed from its original seat in the lungs 
 to distant internal organs? Chiefly by means of the 
 bloodvessels and lymphatics, in both of which it has 
 been found in great numbers. Proof enough of its 
 conveyance through the lymphatics is afforded by the 
 frequent occurrence of inflammations of the serous 
 
510 BACTERIOLOGY. 
 
 membranes complicating pneumonia; but two cases in 
 particular have been reported by Thue of pleurisy and 
 pericarditis following pneumonia in which the lymph 
 capillaries have been found to be chock-full of diplo- 
 cocci, as if injected. Their presence in the blood after 
 death has been amply proved by numerous investiga- 
 tions. In many instances they have been recovered 
 from the blood during life. Lambert, as a rule, found 
 them in all fatal cases twenty-four to forty-eight hours 
 before death. This examination has considerable prog- 
 nostic value, as nearly all cases in which the pneumo- 
 coccus is found end fatally. This micrococcus has 
 been shown experimentally to be capable of producing 
 various forms of septicaemia local phlegm onous in- 
 flammations, peritonitis, pleuritis, and meningitis. A 
 further proof of the transmission of this organism by 
 means of the blood is given by Foa and Bordoni- 
 Uffreduzzi in their investigations into intra-uterine 
 infection in pneumonia and meningitis. These in- 
 vestigators have demonstrated the presence of the 
 micrococcus lanceolatus in foetal and placental blood 
 and in the uterine sinuses in maternal pneumonia. 
 There being no question, therefore, as to the pos- 
 sibility of the conveyance of the infective agent by 
 means of the blood and the lymph to all parts of the 
 body, we need not wonder at the multiplicity of the 
 affections complicating pneumonia which are caused by 
 this micrococcus ; and not only the secondary, but also 
 the primary diseases, as of the brain and the meninges, 
 may be explained in the same way. Knowing that the 
 saliva and nasal secretions under normal conditions so 
 frequently afford a resting-place for the micrococci, we 
 have only to assume the production of a suitable culture 
 
MICROCOCOUS LANCEOLATUS. 511 
 
 medium for these parasites in the body, brought about 
 by an abnormal condition of the mucous membranes 
 from exposure to cold, or a reduction of the vital re- 
 sisting power of the tissue cells in any of the internal 
 organs, caused by disease, traumatism, excesses of vari- 
 ous kinds, etc. , to readily comprehend how an individual 
 may become infected with pneumonia, either primarily 
 affecting the lungs and secondarily other organs in the 
 body, or primarily attacking the middle ear, the pericar- 
 dial sac, the pleura, the serous cavities of the brain, etc. 
 From statistics collected by Netter the following per- 
 centages of diseases were caused by the ' ' diplococcus 
 pneumonias 7 ' : 
 
 Pneumonia 'r- . . . 65.9 per cent, in adults. 
 
 Bronchopneurnonia . . 15.8 ' 
 
 Meningitis. . . .13.0 ' 
 
 Empyema . . . . 8.5 ' " 
 
 Otitis media . . . 2.4 ' " 
 
 Endocarditis . . . 1.2 ' " 
 
 In 46 consecutive pneumococcus infections in chil- 
 dren there were : 
 
 Otitis media . * ' . . . 29 cases. 
 
 Bronchopneumonia . . . . 12 " 
 
 Meningitis . . . . . . 2 ll 
 
 Pneumonia . . . ... 1 " 
 
 Pleurisy . . . . 1 " 
 
 Pericarditis . . . ... 1 " 
 
 Varieties of the Micrococcus Lanceolatus. The ubi- 
 quity of this organism and the irregularity of its 
 behavior under varying conditions have opened a wide 
 field of discussion among bacteriologists. As com- 
 monly found, for instance, in the saliva of different 
 healthy individuals, and even in that of the same in- 
 dividual at different times, it often varies in virulence; 
 
512 BACTERIOLOGY. 
 
 and as obtained from the saliva it differs again 
 from the organism when occurring in pneumonia. 
 When grown on artificial culture media, variations in its 
 morphology have been observed, at one time the indi- 
 vidual cells being oval in shape and united in pairs, and 
 then surrounded by a capsule; at other times spherical 
 and arranged in longer or shorter chains, like strepto- 
 cocci, and then being without a capsule. Variations 
 in virulence have also been noted, both in the animal 
 body in different stages of disease and when grown out- 
 side the body on artificial culture media. These great 
 variations in biological and pathogenic properties have 
 induced some investigators to believe that there were 
 several distinct species of this organism. 
 
 These views, however, have not met with general 
 acceptance. In an exhaustive investigation into the 
 subject by Kruse and Pansini, who obtained the micro- 
 cocci from the most varied sources from the saliva in 
 health and disease, from the nasal secretions, from pneu- 
 monic sputum at different periods and phases of the 
 illness, from the blood of different kinds of animals 
 killed by inoculation, and, finally, from many primary 
 and secondary affections due to this organism after 
 carefully weighing their results from different points 
 of view and comparing the morphological, biological, 
 and pathological characteristics of the various micro- 
 cocci found, these observers have come to the conclu- 
 sion that "it is impossible to distinguish different 
 varieties" of pneumococci. They found numerous 
 quantitative and qualitative variations in virulence, 
 growth, power of resistance, etc., but at the same time 
 such an inconstancy in these variations that they were 
 unable to make any classification into separate varieties. 
 
MICROCOCCUS LANCEOLATUS. 513 
 
 Judging from the streptococcus, the use of a specific 
 bactericidal serum developed from a single pneumo- 
 coccus will probably show that some of the organisms 
 ranked as pneumococci are not influenced by it. 
 
 Immunity. Early in the history of this organism 
 experiments were begun for the production of immunity 
 in animals by means of preventive inoculations. 
 Fraenkel showed that subcutaneous injections of rab- 
 bits with virulent cultures of the diplococcus produced 
 infection in only a small percentage of these animals, 
 which either died from septicaemia or after a time re- 
 covered. In the latter case they were found to be some- 
 what immune to a second infection. Later experiments 
 were conducted on the same principle, the object being 
 to repeatedly slightly infect the animal, and thus to 
 gradually increase its power of resistance to infection. 
 For this purpose either artificially attenuated cultures 
 or material containing naturally attenuated micrococci 
 were used for inoculation. Cultures artificially attenu- 
 ated by heat or several days' growth in the incubator, 
 sputum taken from a pneumonic patient after the crisis, 
 rusty sputum obtained before the crisis and heated to 
 60 C., old pleuritic exudation containing attenuated 
 bacteria, etc., have thus bejen repeatedly employed. 
 
 Another series of experiments were based on the 
 assumption that the immunizing substances are con- 
 tained in the natural or artificial products of the growth 
 of the organism. Thus cultures which were freed from 
 bacteria by filtration, and emulsions of pneumonic 
 sputum, portions of pneumonic lung, pleuritic exuda- 
 tions, etc., were employed by different experimenters. 
 The quantity of material required for inoculation being 
 found inconveniently large, attempts were then made 
 
 33 
 
514 BACTERIOLOGY. 
 
 to obtain the immunizing substances in a more concen- 
 trated form. Foa and Scabia and F. and G. Klein - 
 perer prepared glycerin extracts, after the manner of 
 Koch, calling their extract " pneumoprotein." At 
 present, however, a protective serum is obtained from 
 horses by the repeated injections of fully virulent 
 pneumococci in exactly the same manner as in the pro- 
 duction of antistreptococcus serum. 
 
 Therapeutic Experiments. Curative experiments in 
 man have also recently been made with the blood-serum 
 of immunized animals and of persons who have recov- 
 ered from an attack of pneumonia. The most success- 
 ful of these were conducted by F. and G. Klemperer. 
 These authors hold that in man during the pneumonic 
 process there is a constant absorption into the circula- 
 tion of this toxic albuminous substance produced by 
 the bacteria in the lungs. This continues until event- 
 ually the same antitoxic substance is produced in the 
 circulation that has been seen to occur experimentally. 
 It is then that the crisis occurs. The bacteria are 
 neither destroyed nor is their power to produce pneu- 
 motoxin lessened; but the third factor the antipneumo- 
 toxin now exists and neutralizes the toxic substances 
 as they are produced. They apparently demonstrated 
 that the serum of the blood of patients after the crisis 
 of pneumonia contained antitoxic substance, and was 
 capable, in a fair number of cases, of curing the dis- 
 ease when injected into infected animals. They have 
 made preliminary observations upon patients with a 
 view of inducing the crisis by the injection of the 
 blood-serum of persons convalescent from pneumonia, 
 and which, consequently, contain the antitoxic body. 
 In six pneumonic patients the results were promising. 
 
MICROCOCCUS LANCEOLATUS. 515 
 
 In all there was a decided fall of temperature in from 
 six to twelve hours after subcutaneous injections of from 
 4 to 6 c.c. of the serum. The pulse and respirations 
 were also diminished in frequency. In two cases the 
 temperature fell to 37 C. Twice it fell and remained 
 at normal. In other cases it fell only temporarily. 
 
 The number of cases reported in which the blood- 
 serum of animals artificially immunized against pneu- 
 monic infection has been used for the treatment of the 
 disease, although considerable, is still too few to warrant 
 the expression of any definite opinion as to the final 
 value of this as a therapeutic agent. In the cases we 
 have observed there has been in some a slight imme- 
 diate lowering of the temperature ; in others no ap- 
 parent change. As a rule, the cases did rather better 
 than was expected, but certainly no striking curative 
 effects were apparent. The cases did not develop 
 pnetimococcus blood infection, and it seems probable 
 that the serum may be able to prevent a general infec- 
 tion from taking place from the diseased lung, even 
 though it may fail to influence the local process. It 
 has also been shown that these injections of antipneu- 
 motoxic serum are practically harmless. 
 
CHAPTER XXIX. 
 
 DIPLOCOCCUS INTRACELLULARIS MENINGITIDIS. 
 
 IN the description of the micrococcus lanceolatus 
 reference was made to this organism as the most fre- 
 quent cause of meningitis, especially when it compli- 
 cated pneumonia. In 1887, Weichselbaum discovered 
 another micrococcus in the exudate of cerebro-spinal 
 meningitis in six cases, two of which were not com- 
 plicated by pneumonia. He obtained it in pure cultures, 
 studied its characteristics, and showed that this organism 
 was clearly distinguishable from the micrococcus lanceo- 
 latus, and especially by its usual presence in the interior 
 of pus-cells, on which account he called it diplococcus 
 intracellularis meningitidis. The frequency of the occur- 
 rence of this diplococcus in meningitis and its restric- 
 tion to this disease affords sufficient evidence for the 
 assumption that it was concerned in its production. 
 In 1895, Jaeger and Scheurer found it in the nasal 
 secretions of eighteen living persons suffering from this 
 disease during an epidemic. 
 
 Morphology. This organism occurs as biscuit-shaped 
 micrococci, usually united in pairs, but also in groups 
 of four and in small masses; sometimes solitary and 
 smaller degenerated forms are found. In the exuda- 
 tion, like the gonococcus, to which it bears a close re- 
 semblance in form and arrangement, it is distinguished 
 by its presence within the polynuclear leucocytes. It 
 
INTRA CELL ULARIS MENINGITIDIS. 517 
 
 never appears within the nucleus and rarely within 
 other cells (Fig. 67). 
 
 It stains with all the ordinary aniline colors, but best 
 with Loffler's methylene-blue. According to Weich- 
 selbaum, it is decolorized by Gram's solution; Jaeger 
 states that this is not constantly the case. 
 
 FIG. 67. 
 
 Diplococcus intracellularis meningitidis. X 1100 diameters. 
 
 Biological Characters. It does not grow at room-tem- 
 perature, but only in the incubator, and its develop- 
 ment is usually scanty on the surface of agar, but some- 
 times a few colonies grow quite vigorously. It does 
 not grow at all or scarcely any in bouillon, and very 
 scantily in bouillon plus one-third blood-serum. It 
 grows comparatively well on Loffler's blood-serum 
 medium as used for diphtheria cultures. 
 
 When grown on nutrient agar or glycerin-agar, at 
 the end of forty-eight hours in the incubator, there 
 develops a tolerably good growth, appearing as a flat 
 layer of colonies, about one-eighth of an inch in diam- 
 
518 BACTERIOLOGY. 
 
 eter, grayish- white in color, viscid and non-confluent 
 unless very close together. On Loffler's blood-serum 
 the growth forms round, whitish, shining viscid-look- 
 ing colonies, with smooth and sharply-defined outlines, 
 and may attain diameters of one-eighth to one-sixteenth 
 of an inch in twenty-four hours. The colonies tend to 
 become confluent and do not liquefy the serum. In 
 acute cases, where the organisms are apt to be more 
 abundant, a great many minute colonies may develop 
 instead of a few larger ones. On agar plates the 
 deep-lying colonies are almost invisible to the naked 
 eye; somewhat magnified they appear as finely granular 
 colonies, with a dentated border. On the surface they 
 are larger, appearing as pale disks, almost transparent 
 at the edges, but more compact toward the centres, 
 which are yellowish -gray in color. Cultivated in arti- 
 ficial media it soon loses its vitality within six days 
 and requires, therefore, to be transplanted to fresh 
 material at short intervals at least every two days. 
 
 Pathogenesis. This organism does not show much 
 pathogenic power for animals. It is most pathogenic 
 for mice and guinea-pigs, less so for rabbits and dogs. 
 Subcutaneous injections in animals give negative re- 
 sults; intrapleural or intraperitoneal inoculations in 
 mice and guinea-pigs, when given in large doses, are 
 usually successful. Intravenous injections in rabbits 
 have caused the death of the animal, but no diplococci 
 or pathological changes have been found as a result of 
 the injections. 
 
 When mice are inoculated into the pleural or peri- 
 toneal cavities they usually fall sick and die within 
 thirty-six to forty-eight hours, showing slight fibrino- 
 purulent exudation. In the blood and enlarged spleen 
 
INTRA CELL ULARIS MENINGITIDIS. 519 
 
 . diplococci are found in small numbers and mostly free; 
 in the pleuritic exudation they are present in consider- 
 able quantities, less so in the peritoneal fluid, but then 
 occurring in the interior of pus-cells. 
 
 Certain experiments made by Weichselbaum on dogs, 
 though not entirely successful, are interesting as showing 
 the similarity of the disease produced in them artificially 
 with meningitis as occurring in man. The three dogs, 
 trephined and inoculated subdurally with 0.5 to 2 c.c. of 
 afresh culture, all died: No. 1 within twelve hours, No. 
 2 in three days, and No. 3 in twelve days. In Nos. 1 
 and 2 there were found hypersemia of the meninges, with 
 inflammatory softening of the brain at the point of inocu- 
 lation, which on nearer inspection proved to be a true 
 encephalitic process. In dog No. 2, in which the dis- 
 ease was of longer duration, these changes were the 
 most pronounced. Numerous diplococci were observed 
 in the sections removed, for the most part free, but 
 some few within the pus-cells. In dog No. 3, in 
 which the disease lasted twelve days, between the dura 
 mater and the brain, at the point of inoculation, was 
 found a thick, reddish, purulent liquid; in the brain 
 itself an abscess had formed, about the size of a hazel- 
 nut, filled with tough, yellow pus, while the abscess 
 walls consisted of softened brain -substance infiltrated 
 with numerous hemorrhagic deposits, and simultane- 
 ously the ventricles on that side contained a cloudy, 
 reddish fluid, with flocks of pus; but no diplococci 
 could be demonstrated in the blood or exudations. 
 Weichselbaum suggests that under natural conditions 
 the diplococci gain access to the brain and meninges by 
 way of the nose, ear, and upper air-passages. Cerebro- 
 spinal meningitis, as is well known, is often accorn- 
 
520 BACTERIOLOGY. 
 
 panied by rhinitis and purulent inflammation of the 
 mucous membranes of the nose. In one of his six cases 
 Weichselbaum succeeded in obtaining in pure culture 
 diplococci from the nasal secretion. Scheurer, in his 
 eighteen cases, found the diplococci in the nasal secre- 
 tions of all of them during life. In fifty healthy in- 
 dividuals examined they were found in the nasal secre- 
 tions of only two of them, one being a man suffering 
 at the time from a severe cold. This man, it is inter- 
 esting to note, had been engaged in disinfecting a room 
 which had previously been occupied by a patient with 
 cerebro-spinal meningitis. 
 
 Bacteriological Diagnosis. By means of lumbar punc- 
 ture fluid can be readily obtained without danger from 
 the spinal canal. The microscopical examination will 
 frequently reveal numerous cells crowded with diplo- 
 cocci. When considerable quantities are inoculated 
 upon Loffler's blood-serum mixture or upon glycerin 
 agar, as a rule, a greater or less number of colonies 
 having the characteristics already described will de- 
 velop. The value, clinically, of the examination is 
 that about 40 per cent, of the cases due to this coccus 
 recover, while almost all of those due to the pneumo- 
 coccus and streptococcus die. In fifty-five cases exam- 
 ined by Councilman, Mallory, and Wright, diplococci 
 were found in the fluid removed by lumbar puncture in 
 thirty-eight, either by microscopical examination or 
 cultures. 
 
 The longest time after the onset of the disease in which 
 positive results were obtained by culture was twenty- 
 nine days. In a number of cases examined by us for 
 Northrup a rather smaller percentage of the cases were 
 found to be due to this diplococcus. In many cases 
 
INTEA CELL ULAEIS MENINGITIDIS. 521 
 
 there are very few diplococci present in the spinal fluid, 
 so that a failure to find them in a microscopical examina- 
 tion should not be taken to prove that the disease was 
 not due to this organism. For cultures a considerable 
 amount of fluid must be used, for we have found, as 
 described by Councilman and others, that there may 
 be very few living diplococci even in 1 c.c. of fluid. 
 
 To obtain the fluid the patient should lie on the right 
 side with the knees drawn up and the left shoulder de- 
 pressed. The skin of the patient's back, the hands of 
 the operator, and the large antitoxin syringe should be 
 sterile. The needle should be 4 cm. in length, with a 
 diameter of 1 mm. for children, and longer for adults. 
 
 The puncture is generally made between the third 
 and fourth lumbar vertebrae. The thumb of the left 
 hand is pressed between the spinous processes, and the 
 point of the needle is entered about 1 cm. to the right 
 of the median line and on a level with the thumb-nail, 
 and directed slightly upward and inward toward the 
 median line. At a depth of 3 or 4 cm. in children and 
 7 or 8 cm. in adults the needle enters the subarachnoid 
 space, and the fluid flows out in drops or in a stream. 
 If the needle meets a bony obstruction withdraw and 
 thrust again rather than make lateral movements. 
 Any blood obscures the microscopical examination. 
 The fluid is allowed to drop into absolutely sterile test- 
 tubes or vials with sterile stoppers. From 5 to 15 c.c. 
 should be withdrawn. No ill effects have been ob- 
 served from the operations. 
 
CHAPTER XXX. 
 
 MICROCOCCUS GONORRHOEA (OONOCOCCUS NEISSER). 
 
 THIS micrococcus was first observed by Neisser, in 
 1879, in gonorrhoeal discharges, and described by him 
 under the name of " gonococcus"; but though several 
 attempted to discover a medium upon which it might 
 be cultivated, it was reserved for Bumm, in 1885, to 
 obtain it in pure culture and isolate it and then prove 
 its infective virulence by inoculation into man. Since 
 that time the gonococcus has been cultivated on various 
 media, which, though modifications of Bumm's, are an 
 improvement on his original method, and as the result 
 of various inoculation experiments there now remains 
 no doubt that this organism is the specific cause of 
 gonorrhoea in man. 
 
 Morphological Characters. Micrococci, occurring 
 mostly in the form of diplococci that is, in pairs or 
 in groups of four. The bodies of the diplococci are 
 elongated, and, as shown in stained preparations, have 
 an unstained division or interspace between two flat- 
 tened surfaces facing one another, which gives them 
 their characteristic " coffee-bean " or "biscuit" shape 
 (Fig. 68). The diameter of an associated pair of cells 
 varies from 0.8// to 1.6// in the long diameter 
 average about 1.25// by 0.6/* to 0.8/u. in the cross 
 diameter. In gonorrhoea gonococci are found mostly 
 in small, irregular groups in or upon the pus-cells, and 
 
MIGROGOGGUS GONORRHCEJE. 523 
 
 generally extranuclear. When found in other portions 
 of the field this is mostly due to the mechanical effect 
 of smearing the pus on cover-glass slides, and should 
 not be considered as characteristic. That the gonococci 
 really lie within the protoplasm of the cells is proved 
 by the fact that in carefully made preparations they 
 are usually not found outside of the pus-cells. They 
 appear usually as diplococci, in groups of two or four, 
 
 FIG. 68. 
 
 Smear from pure culture of gonococcus on agar. (HEIMAN.) 
 
 but at times they occur as round, single, and undivided 
 cells. Others, again, are irregular in shape or granular 
 in appearance, involution forms, particularly in older 
 cultures and in chronic urethritis of long standing. 
 The pus-cells containing gonococci are most numerous 
 in the later or purulent stage of the disease, not so fre- 
 quent in the beginning of infection, or as long as the 
 discharge is of a serous character (Fig. 69). 
 
 The gonococcus stains readily with the basic aniline 
 colors, especially with methyl-violet, gentian-violet, and 
 
524 BACTERIOLOGY. 
 
 fuchsin; not so readily with methylene-blue, which is, 
 however, one of the best staining agents for demon- 
 strating its presence in pus. Beautiful double-stained 
 preparations may be made from gonorrhoeal pus by 
 treating cover-glass smears with methyl-violet and 
 eosin. Gonococci are decolorized by Gram's solution; 
 but this cannot be depended upon alone to absolutely 
 distinguish the gonococcus from all other diplococci 
 
 FIG. 69. 
 
 Gonococcus in pus-cells. X 1100 diameters. 
 
 found in the urethra and vulvo-vaginal tract, for espe- 
 cially in the female other diplococci are occasionally 
 found which are also not stained by Gram's method. 
 It serves, however, to distinguish this micrococcus from 
 the common pyogenic cocci, which retain their color 
 when treated in the same way, and in the male urethra 
 it is practically certain, as no organism has been found 
 in that location which in morphology and staining is 
 identical with the gonococcus. It is certainly the most 
 distinctive characteristic of the staining properties of 
 
MICROCOCCUS GONORRHCEjE. 525 
 
 the gonococcus, and it is a test that should never be 
 neglected in differentiating this organism from others 
 which are morphologically similar. 
 
 Biological Characters. The elaborate experiments of 
 Bumm and others have shown that at the ordinary room- 
 temperature no growth of the specific micrococcus occurs 
 on the culture media. Apparently positive results which 
 have been reported are found to be due to other diplo- 
 cocci morphologically almost identical with the gono- 
 coccus. 
 
 Since Bumm's experiments a number of culture 
 methods have been proposed for the gonococcus which 
 are an improvement on Bumm's, partly because the 
 growth produced is more constant and luxuriant and 
 partly because the media employed are more readily 
 prepared. Wertheim (1892) succeeded in developing 
 luxuriant and virulent cultures to many generations on 
 a mixture of placenta blood-serum and 2 per cent, pep- 
 tone-agar. His method is briefly as follows : Several 
 loops of gonorrhoeal pus are diffused through liquid 
 blood-serum warmed to 40 C. contained in a test- 
 tube. Two dilutions are made from this, and an 
 equal quantity of melted 2 per cent, agar cooled to 
 40 C. is added to the three tubes, and the contents, 
 after thorough mixing, poured into Petri dishes. The 
 Petri dishes are placed in an incubating oven at a 
 temperature of 36 to 37 C. At the end of twenty- 
 four hours there will have developed on at least one of 
 the plates distinct colonies; these are translucent, finely 
 granular, with scalloped margin. By transferring such 
 a colony to slant-cultures of serum-agar, pure cultures 
 of the gonococcus are obtained; these are somewhat 
 shining in appearance and of a grayish-white color. 
 
526 BACTERIOLOGY. 
 
 Wertheim demonstrated that the addition of peptone 
 to the culture medium was an important factor in the 
 cultivation of gonococci. Kiefer (1895) proposed a cul- 
 ture medium consisting of one part of hydrothorax or 
 ascitic fluid and one part of a fluid containing 3.5 per 
 cent, agar, 5 per cent, peptone, 2 per cent, glycerin, and 
 0.5 per cent. salt. 
 
 Simultaneously with Kiefer, Heiman recommended 
 a medium made from hydrocele fluid, or from " chest- 
 serum" obtained from a patient suffering from hy- 
 drocele or hydrothorax or acute pleurisy. Having 
 experimented with all the various culture media here- 
 tofore prepared for the cultivation of the gonococctis, 
 Heiman believes this medium to be superior to placenta - 
 serum, sheep blood-serum, or ascitic fluid, because of 
 the large amount of serum albumin which it contains. 
 The medium consists of a 2 per cent, agar, plus 2 per 
 cent, peptone, plus 0.5 per cent, salt and 2 per cent, 
 glucose. Of this mixture two parts are added to one 
 part of chest -serum, which is, if necessary, fractionally 
 sterilized between 65 and 70 C. for one hour for seven 
 consecutive days. The chest-serum-agar should have a 
 neutral reaction. The growth on this medium is thus 
 described : In plate cultures streaked on the surface, 
 growth abundant, colonies circular in shape, edges 
 somewhat irregular, shading off into yellowish- white; 
 texture finely granular in periphery, presenting punc- 
 tated spots of higher refraction in and around the 
 centre of yellowish color (Fig. 70). 
 
 The gonococcus has but little resistant power against 
 outside influences. It is killed by weak disinfecting 
 solutions and by desiccation in thin layers. In com- 
 paratively thick layers, however, as when gonorrhoeal 
 
MICROCOCCUS GONORRHCE^E. 
 
 527 
 
 pus is smeared on linen, it has lived for forty-nine 
 days, and dried on glass for twenty-nine days (Heiman). 
 No development takes place below 25 C. or above 
 39 C.; it is killed at a temperature over 42 C. 
 
 Pathogenesis. Non-transmissible to dogs, monkeys, 
 horses, and rabbits, whether inoculations be made into 
 the urethral, vaginal, or congenital mucous membranes. 
 According to Wertheim, purulent peritonitis, not caus- 
 
 FIG. 70. 
 
 Colonies of gonococci on pleuritic fluid agar. (HEIMAN.) 
 
 ing death, is produced in certain animals by the intro- 
 duction of pieces of serum-agar containing colonies of 
 the gonococcus. This effect was produced constantly in 
 mice, occasionally in guinea-pigs, and rarely, if ever, 
 in dogs, rats, and rabbits. 
 
 Though animal inoculations are thus followed by 
 negative results, the etiological relation of the gono- 
 coccus to human gonorrhoea has been demonstrated 
 beyond question by the infection of healthy men with 
 the disease by inoculation. Thus, Bumm has produced 
 
528 BACTERIOLOGY. 
 
 gonorrhoea in normal urethral mucous membranes by 
 inoculation of a pure culture on blood-serum in the 
 second generation; Wertheim, in the thirtieth; Kiefer, 
 in the sixth, and Heiman in the fifth generation. At 
 the same time the distinctive morphological, staining and 
 biological characters of the organism were carefully noted 
 and confirmed to be those of the gonococcus by these ob- 
 servers; the typical incubation and symptoms of the dis- 
 ease resulted in all cases in the subjects experimented on. 
 According to the observations of the most reliable 
 investigators and those most familiar with the various 
 forms of micrococci which are likely to be mistaken 
 for the gonococcus, affections due to this organism are 
 usually restricted to the mucous membranes of the 
 urethra, conjunctiva, bladder, cervix uteri, and rectum. 
 It rarely, if ever, produces a vaginitis in adults; but 
 occasionally a vulvo- vaginitis in young children. For- 
 merly the presence of gonococci could only be deter- 
 mined microscopically; but since the introduction of the 
 serum-agar the culture method has rendered the diag- 
 nosis of gonorrhoea much more reliable. This method 
 of investigation, moreover, has given valuable infor- 
 mation with regard to the nature of many infections 
 complicating or resulting from gonorrhoea, particularly 
 in affections of the uterus and joints, about which there 
 was heretofore considerable doubt, though the micro- 
 cocci often found in these organs were morphologically 
 identical with the gonococcus. It has now been shown 
 by the culture method that gonococci may occur in the 
 joints in gonorrhoeal arthritis, in the Fallopian tubes in 
 salpingitis, and in ovarian abscesses; and Wertheim 
 asserts that he has found them in the infiltrated con- 
 nective tissue in parametritis. 
 
MICROCOCCUS GONOREHCEjE. 529 
 
 Worthy of special notice in this connection are the 
 cases of endocarditis accompanying gonorrhoea, and some- 
 times terminating fatally, when they are known as en- 
 docarditis gonorrhoeica maligna. The question naturally 
 arises in these cases whether it is the gonococcus or 
 some other coccus or diplococcus which has infected 
 the endocardium. Here, again, it is only by means of 
 the culture method that this question can be settled 
 definitely. Fliigge draws attention to this matter, and 
 states that but few cases have been recorded in which 
 the information given as to the cause of the disease can 
 be unhesitatingly accepted. Weichselbaum mentions 
 a case of endocarditis accompanying gonorrhoea which 
 was shown by the culture method to be due to strepto- 
 coccus infection, proving that so-called gonorrhoeal 
 endocarditis may be a secondary infection. Other cases 
 are recorded by Leyden, Hiss, Councilman, and Wilms 
 which are said to have been most probably of gonor- 
 rhoeal origin; but in these only microscopical examina- 
 tions were made and no culture experiments, or only 
 cultures on gelatin plates, etc., which were inadequate. 
 Welch also reports a case of endocarditis with general 
 septicaemia following gonorrhoea, in which he demon- 
 strated the gonococcus in the blood of a living person 
 in cover-glass and culture medium. No other patho- 
 genic bacteria were found. 
 
 Immunizing Serum. As animals are not infected by 
 the gonococcus they are not very suitable for injections 
 with the cultures for the purpose of producing an anti- 
 toxic or bactericidal serum. Their insusceptibility pre- 
 sents also an almost insurmountable obstacle to the 
 testing of the blood of animals under treatment, so that 
 although it may be possible to bring about an artificial 
 
 34 
 
530 BACTERIOLOGY. 
 
 immunity against gonorrhoeal infection, information on 
 this subject is at present wanting. Immunity in man 
 seems to be similar to that produced after infection with 
 the other pyogenic cocci that is, only slight in amount 
 and for a short period. It is known that a urethra or 
 cervix may contain gonococci which lie dormant and 
 may be innocuous in that person for years, but which 
 may at any time excite an acute gonorrhoea in the one 
 carrying the infection or in another person. 
 
 The Bacteriological Diagnosis of Gonorrhoea. In view 
 of the fact that several non-specific forms of urethritis 
 exist, and also that micrococci morphologically similar 
 to the gonococcus Neisser are often found in the normal 
 urethral and vulvo-vaginal tract, it becomes a matter 
 of great importance to be able to detect gonococci when 
 present and to differentiate these from the non-specific 
 organisms. The gonococci also which occur in old cul- 
 tures and in chronic urethritis of long standing often 
 take on a very diversified appearance sometimes 
 nothing but an irregular, granular mass being seen, 
 which renders their detection difficult. From a medico- 
 legal and social stand-point, therefore, the differential 
 diagnosis of the gonococcus has in certain cases a very 
 practical significance. 
 
 There are two methods of differential diagnosis now 
 available the microscopical and the cultural. Animal 
 inoculations are of little value, as they are not sus- 
 ceptible, and, of course, human inoculations are, except 
 in extremely important cases, generally impossible. In 
 the microscopical diagnosis it should be borne in mind 
 that the specific gonococci in carefully made prepara- 
 tions are found always largely within the pus-cells. 
 Diplococci morphologically similar to gonococci occur- 
 
MICROCOCCUS GONOERHCE^. 531 
 
 ring in other portions of the field and outside of the 
 pus-cells should not be considered specific by this test 
 only. It should also be remembered that thegonococci 
 are decolorized by Gram's method, while other similar 
 micrococci which occur in the urethra are, as a rule, 
 at least, not so decolorized. Organisms having these 
 characteristics can for all practical purposes be con- 
 sidered as certainly gonococci if obtained from the 
 urethra. From the vulvo-vaginal tract the certainty is 
 not so great; here cultures should also be made. Burnm, 
 Heiman and others have shown that other diplococci 
 are occasionally found in gouorrhoeal pus from the vulvo- 
 vaginal tract, and very rarely, indeed, from the urethra, 
 which do not stain by this method. Cover-glass prepa- 
 rations from subacute or chronic cases should be ex- 
 amined, if possible, with a microscope provided with a 
 mechanical stage, and should always be stained by 
 Gram's method and the examination repeated on three 
 consecutive days. Should these specimens prove nega- 
 tive, to exclude any possible doubt in the matter, 
 cultures should then be made on chest-serum-agar, 
 poured in dishes, as proposed by Heiman, also, if with 
 negative results, on three consecutive days. His 
 method of procuring the urine in chronic urethritis 
 is to allow the patient to void his urine either imme- 
 diately into two sterilized centrifugal tubes or first 
 into two sterile bottles. The first tube will contain 
 threads of the anterior urethra; the second tube will 
 be likely to contain secretion from the posterior urethra 
 and from the prostate gland if, while urinating, the 
 patient's prostate be pressed upon with the finger. 
 Tubes containing such urine are placed in the centri- 
 fuge and whirled for three minutes at twelve hundred 
 
532 BACTERIOLOGY. 
 
 or more revolutions per mintue; the threads are thrown 
 down. The " centrifuged" sediment will be found 
 to contain most of the bacteria present, epithelial cells, 
 and at times spermatozoa. Normal urine on being 
 " centrifuged" at this velocity will be found at times 
 slightly turbid at the bottom of the tube. This tur- 
 bidity will be found, on microscopical examination, to 
 consist of epithelial cells, a few leucocytes, and some 
 bacteria. 
 
 Heiman looks upon the decolorization by Gram's 
 method as the only reliable criterion, so far as known, 
 for the gonococcus in discharges from the mucous mem- 
 branes, and it is of material help, also, in determining 
 whether a culture is or is not that of the gonococcus. 
 The careful examination of gonorrhoeal threads with 
 cover-glass by Gram's method is a very tedious affair, 
 as in every instance no less than three cover-glass prep- 
 arations should be looked over before the absence of 
 the gonococcus is proved. It would require many 
 hours upon each and every specimen, especially if the 
 gonococci are present in very small number, before a 
 reliable and conscientious opinion could be rendered. 
 If, after all, a negative opinion is ventured, we still 
 are under the necessity of proving that because the 
 threads which we fished out for the cover-glass exami- 
 nation were free from gonococci the remaining ones 
 were also. For this reason the culture medium is more 
 sensitive for bacteria than is the cover-glass, for we are 
 able to plant each and every thread of the sediment in 
 the centrifugal tube. Fiirbringer, in his work, men- 
 tions the fact that in certain cases the absence of the 
 gonococcus in many examinations of cover-glass prep- 
 arations is not a positive proof that the gonococcus is 
 
MICEOCOCCUS GONORRHCEJE. 533 
 
 not present. Heiman was able to confirm the correct- 
 ness of the above allusion, for on one occasion, in 
 examining threads, when he could not demonstrate the 
 gonococcus in cover-glass preparations, he succeeded in 
 growing it on chest-serum-agar plates, while in all 
 instances in which he found the gonococcus in threads 
 in cover-glass preparations he invariably succeeded in 
 growing it on chest-serum-agar plates. The culture 
 methods, of course, presuppose that one has the facili- 
 ties and knowledge to carry them out successfully, other- 
 wise the microscopical methods are to be used alone. 
 
 In acute cases the specimen for examination may be 
 collected, when the patient is before one, by passing a 
 sterilized platinum wire loop as far up into the urethra 
 as possible and withdrawing some of the secretion. 
 This is a far less satisfactory method than that sug- 
 gested by Heiman, by " centrifuging," except when 
 the pus is abundant. 
 
 The Frequency with which Gonococci are Found in 
 Smears or Cultures in Cases of Chronic Urethritis. 
 Heiman found in 61 cases 14 by cultures and 13 by 
 smears. The following results were obtained by other 
 observers by cover-glass preparations : Goll, accord- 
 ing to his elaborate article, examined 1046 cases of 
 chronic urethritis varying in duration between four 
 weeks to six years or more, finding gonococci in 178 
 cases, the remainder giving negative results. Neisser, 
 out of 143 cases varying in duration between two 
 months and eight years, found gonococci in 80 cases. 
 Weinrich, out of 25 similar cases, obtained 2 positive 
 results. E. Noeggerath, in 1887, deplored the fact 
 that on account of the lack of culture media for 
 the gonococcus we cannot always demonstrate them. 
 
534 BACTERIOLOGY. 
 
 Brose, in 1893, stated that the culture medium is the 
 only reliable agent for the detection of the gonococcus. 
 This latter statement is certainly applicable to chronic 
 urethritis of the male. Neisser, in 1893, stated that 
 in chronic urethritis with slight discharge the examina- 
 tion with a culture medium for gonococci will replace 
 the cover-glass. 
 
CHAPTER XXXI. 
 
 BACILLUS PYOCYANEUS (BACILLUS OF GREEN AND 
 OF BLUE PUS) BACILLUS PROTEUS VULGARIS 
 BACILLUS OF MALIGNANT (EDEMA BACILLUS 
 AEROGENES CAPSULATUS. 
 
 BACILLUS PYOCYANEUS. 
 
 THE blue and green coloration which is occasionally 
 found to accompany the purulent discharges from open 
 wounds is usually due to the action of the bacillus 
 pyocyaneus. According to recent investigations this 
 bacillus appears to be very widely distributed. 
 
 Morphology. Slender rods from 0.3// to 1/j. broad and 
 from 2/^ to 6// long; frequently united in pairs or in 
 chains of four to six elements; occasionally growing 
 out into long filaments and twisted spirals. The bacil- 
 lus is actively motile, a single flagellum being attached 
 to one end. Does not form spores. Stains with the 
 ordinary aniline colors; does not stain with Gram's 
 solution. 
 
 Biological Characters. An aerobic, liquefying, motile 
 bacillus. Capable also of an anaerobic existence, but 
 then produces no pigment. Grows readily on all arti- 
 ficial culture media at the room-temperature, though 
 best at 37 C., and gives to some of them a bright 
 green color in the presence of oxygen. In gelatin 
 plate cultures the colonies are rapidly developed, 
 imparting to the medium a fluorescent green color; 
 liquefaction begins at the end of two or three days, 
 
536 BACTERIOLOGY. 
 
 and by the fifth day the gelatin is usually all lique- 
 fied. The deep colonies, before liquefaction sets in, 
 appear as round, granular masses with scalloped mar- 
 gins, having a yellowish-green color; the surface col- 
 onies have a darker green centre, surrounded by a 
 delicate, radiating zone. In stick cultures in gelatin 
 liquefaction occurs at first near the surface, in the form 
 of a small funnel, and gradually extends downward; 
 later the liquefied gelatin is separated from the solid 
 part of the medium by a horizontal plane, a greenish- 
 yellow color being imparted to that portion which is 
 in contact with the air. On agar a wrinkled, moist, 
 greenish-white layer is developed, while the surround- 
 ing medium is bright green; this subsequently becomes 
 darker in color, changing to blue-green or almost black. 
 In bouillon the green color is produced, and the growth 
 appears as a delicate, flocculent sediment. Milk is coag- 
 ulated with coincident acid reaction. 
 
 There is some difference of opinion with regard to 
 the pigments produced by the bacillus pyocyaneus. 
 Gessard's view is that two pigments are produced by 
 this bacillus one of a fluorescent green and the other 
 (pyocyanin) of a blue color. Pyocyanin is soluble in 
 chloroform, and may be obtained from pure solution 
 in long, blue needles. This pigment, which is thus 
 extracted by chloroform, distinguishes the bacillus 
 pyocyaneus from other fluorescing bacteria. 
 
 Pathogenesis. This bacillus is very widely distributed 
 in nature; it is found on the healthy skin of man, 
 in purulent discharges and in serous wound secre- 
 tions. Its presence in wounds greatly delays the 
 process of repair and may give rise to a general 
 depression of the vital powers from the absorption of 
 
BACILLUS PYOCTANEUS. 537 
 
 its toxic products. Its pathogenic effects on animals 
 have been carefully studied. It is pathogenic for 
 guinea-pigs and rabbits. Subcutaneous or intra-peri- 
 toneal injections of not too small quantities of a 
 recent culture 1 c.c. or more of a bouillon culture 
 usually cause the death of the animal in from twenty- 
 four to thirty-six hours. Subcutaneous inoculations pro- 
 duce an extensive inflammatory oedema and purulent 
 infiltration of the tissues; a serofibrinous or purulent 
 peritonitis is induced by the introduction of the bacillus 
 into the peritoneal cavity. The bacilli multipy in the 
 body, and may be found in the serous or purulent fluid 
 in the subcutaneous tissues or abdominal cavity as well 
 as in the blood and various organs. When smaller 
 quantities are injected subcutaneously the animal usu- 
 ally recovers, only a local inflammatory reaction being 
 set up (abscess), and it is subsequently immune against 
 a second inoculation with doses which would prove 
 fatal to an unprotected animal. Immunity may also 
 be secured by the injection of a considerable amount 
 of a sterilized culture. It is interesting to note that 
 Bouchard, Charrin, and Guignard have shown that in 
 rabbits which have been inoculated with a culture of 
 the bacillus anthracis a fatal result may be prevented 
 by inoculating the same animal soon after with a pure 
 culture of the bacillus pyocyaneus. Similar results 
 have been obtained by Wood head and Wood by the 
 injection of sterilized cultures of this bacillus, made 
 immediately after injection with the anthrax bacillus. 
 Loew and Emmerich have shown that the enzymes 
 produced in the pyocyaneus cultures are capable of de- 
 stroying many forms of bacteria in the test-tube, and 
 have slight protecting value in the body. 
 
538 BACTERIOLOGY. 
 
 Our knowledge of the pathogenic importance of the 
 bacillus pyocyaneus in human diseases has been much 
 increased by recent investigations. Thus cases have 
 been reported in which this bacillus has been obtained 
 in pure culture from pus derived from the tympanic 
 cavity in disease of the middle ear, from cases of oph- 
 thalmia and bronchopneumonia. Kruse and Pasquale 
 have found the same micro-organism in three cases of 
 idiopathic abscess of the liver, in two of them in immense 
 numbers and in pure culture. Ernst and Schiirmayer 
 report the presence of the bacillus pyocyaneus in serous 
 inflammation of the pericardial sac and of the knee- 
 joint. Ehlers gives the history of a disease in two 
 sisters who were attacked simultaneously with fever, 
 albuminuria, and paralysis. It was thought that they 
 would turn out to be typhoid fever or meningitis, but 
 on the twelfth day there was an eruption of blisters, 
 from the contents of which the bacillus pyocyaneus was 
 isolated. Jadkewitsch reports the case of a patient 
 suffering from eczema of the lower extremities, in 
 whom three times during a period of ten years there 
 was eruption of boils containing blue pus, with accom- 
 panying symptoms of poisoning, emaciation, prostra- 
 tion, diarrhoea, and paresis. Krambals refers to seven 
 cases in which a general pyocyaneous infection occurred, 
 and adds an eighth from his own experience. In this 
 the bacillus pyocyaneus was obtained post-mortem from 
 green pus in the pleural cavity, from serum in the peri- 
 cardial sac, and from the spleen in pure culture. 
 Schimmelbush states that a physician injected 0.5 c.c. 
 of sterilized (by heat) culture into his forearm. As a 
 result of this injection, after a few hours he had a 
 slight chill, followed by fever, which at the end of 
 
BACILLUS PROTEUS VULGARIS. 539 
 
 twelve hours reached 38.8 C.; an erysipelatous-like 
 swelling of the forearm occurred, and the glands in 
 the axilla were swollen and painful. Neumann has 
 obtained the bacillus pyocyaneus in pure culture in 
 two cases of hsematemesis and melsena of the new-born 
 from the blood and other organs. Lartigau found it 
 in well-water, and in great abundance in the intestinal 
 discharges of a number of cases made ill by drinking 
 the water. 
 
 We may, therefore, conclude from these facts that 
 the bacillus pyocyaneus, although ordinarily non-patho- 
 genic for man, may under certain conditions become a 
 dangerous source of infection. Children would seem 
 to be particularly susceptible to this infection. 
 
 The differential diagnosis of the pyocyaneus from 
 other fluorescing bacteria is easy enough as long as it 
 retains its pigment-producing property. When an agar 
 culture is agitated with chloroform a blue coloration 
 demonstrates the presence of this bacillus. When the 
 pyocyanin is no longer formed, however, the diagnosis 
 is by no means easy, particularly when the pathogenic 
 properties are also gone. 
 
 BACILLUS PROTEUS VULGAEIS. 
 
 This bacillus, which is one of the most common and 
 widely distributed putrefactive bacteria, was discovered 
 by Hauser (1885) along with other species of proteus 
 in putrefying substances. These bacteria were formerly 
 included under the name a bacterium termo " by previous 
 observers, who applied this name to any minute motile 
 bacilli found in putrefying infusions. 
 
 Morphology. Bacilli varying greatly in size; most 
 commonly occurring 0.6// broad and 1.2/* long, but 
 
540 BACTERIOLOGY. 
 
 shorter and longer forms may also be seen, even grow- 
 ing out into flexible filaments, which are sometimes 
 more or less wavy or twisted like braids of hair. The 
 bacillus does not form spores, and stains readily with 
 f uchsin or gentian-violet. 
 
 Biological Characters. An aerobic, facultative anaer- 
 obic, liquefying, motile bacillus. Grows rapidly in 
 the usual culture media at the room-temperature. 
 
 Growth on Gelatin. The growth upon gelatin plates 
 containing 5 per cent, of gelatin is very characteristic. 
 At the end of ten or twelve hours at room-tem- 
 perature small round depressions in the gelatin are 
 observed, which contain liquefied gelatin and a whitish 
 mass consisting of bacilli in the centre. Under a low- 
 power lens these depressions are seen to be surrounded 
 by a radiating zone composed of two or more layers, 
 outside of which is a zone of a single layer, from which 
 amoeba-like processes extend upon the surface of the 
 gelatin. These processes are constantly undergoing 
 changes in their form and position. The young colo- 
 nies deep down in the gelatin are somewhat more 
 compact, and rounded or humpbacked; later they 
 are covered with soft down; then they form irregular, 
 radiating masses and simulate the superficial colonies. 
 But it is difficult to describe all the forms which the 
 proteus vulgaris takes on in all the stages of its growth 
 on gelatin plates. When the consistency of the medium 
 is more solid, as in 10 per cent, gelatin, the liquefac- 
 tion and migration of surface colonies are more or less 
 retarded. In gelatin stick cultures the growth is less 
 characteristic liquefaction takes place rapidly along 
 the line of puncture, and soon the entire contents of 
 the tube are liquefied. 
 
BACILLUS PROTEUS VULGARIS. 541 
 
 Upon nutrient agar a rapidly spreading, moist, thin, 
 grayish-white layer appears, and migration of the col- 
 onies also occurs. Milk is coagulated, with the pro- 
 duction of acid. 
 
 The cultures in media containing albumin or gelatin 
 have a disagreeable, putrefactive odor, and become alka- 
 line in reaction. Growth is most luxuriant at a tem- 
 perature of 24 C., but is plentiful also at 37 C. It 
 is a facultative anaerobe and grows also in the presence 
 of oxygen, but the proteus then loses its power of lique- 
 fying gelatin. It produces indol and phenol from pep- 
 tone solutions. The proteus develops fairly well in 
 urine, and decomposes urea into carbonate of ammonia. 
 
 Pathogenesis. This bacillus is pathogenic for rabbits 
 and guinea-pigs when injected in large quantities into 
 the circulation, into the abdominal cavity, or subcuta- 
 neously, producing death of the animals with symptoms 
 of poisoning. Hauser has obtained the bacillus proteus 
 vulgaris from a case of purulent peritonitis, from puru- 
 lent puerperal endometritis, and from a phlegmonous 
 inflammation of the hand. Brunner also reports simi- 
 lar infections in which this organism was found asso- 
 ciated with pus cocci, and Charrin describes a case of 
 pleuritis during pregnancy in which the proteus was 
 present and a foul-smelling secretion was produced. 
 Death in this case, which ensued without further com- 
 plication, is said to have been due probably to the 
 poisonous products of the proteus. 
 
 An interesting example of pure toxaemia resulting 
 from the toxin of the proteus is reported by Levy : 
 While conducting some experiments on this organism 
 he had an opportunity of making a bacteriological ex- 
 amination in the case of a man who died after a short 
 
542 BACTERIOLOGY. 
 
 attack of cholera morbus. From the vomited material 
 and the stools he obtained a pure culture of the pro- 
 teus; but the blood, collected at the autopsy, was sterile. 
 In the meantime seventeen other persons who had eaten 
 at the same restaurant were taken sick in the same 
 way. Upon examination at the restaurant it was found 
 that the bottom of the ice-chest in which the meat was 
 kept was covered with a slimy, brown layer, which 
 gave off a disagreeable odor. Cultures from this gave 
 the proteus as the principal organism present. Injec- 
 tions into animals of the pure cultures produced similar 
 symptoms as occurred in the human subjects. Levy 
 concludes that in so-called " flesh-poisoning " bacteria 
 of this group are chiefly concerned, and that the patho- 
 genic effects are due to toxic products evolved during 
 their development. 
 
 Booker, from his extended researches into this sub- 
 ject, concludes that the proteus plays an important part 
 in the production of the morbid symptoms which char- 
 acterize cholera infantuna. Proteus vulgaris was found 
 in the al vine discharge in a large proportion of the cases 
 examined by him, but was not found in the feces of 
 healthy infants. "The prominent symptoms in the 
 cases of cholera infantum in which the proteus bacteria 
 were found were drowsiness, stupor, emaciation, and 
 great reduction in flesh, more or less collapse, frequent 
 vomiting and purging, with watery and generally offen- 
 sive stools." 
 
 Next to the bacillus coli communis the proteus vul- 
 garis appears to be the micro-organism most frequently 
 concerned in the etiology of pyelonephritis. In cases 
 of cystitis and of pyelonephritis this bacillus is often 
 found in pure cultures or associated with other bac- 
 teria. It probably gets into the bladder chiefly through 
 
BACILLUS OF MALIGNANT (EDEMA. 543 
 
 catheterization. From the animal experiments of the 
 authors above mentioned, simple injection of pure cul- 
 tures of proteus into the bladder, without artificial sup- 
 pression of urine, invariably produces severe cystitis. 
 The fact that this organism grows in urine is sufficient 
 to account for the extension of the purulent process 
 finally to the kidneys. 
 
 The proteus vulgaris is, however, a harmles parasite 
 when located in the mucous membrane of the nasal 
 cavities. Here it only decomposes the secretions, with 
 the production of a putrefactive odor. On the whole, 
 considering the very wide distribution of this organism 
 in nature, it is remarkable how few diseases are pro- 
 duced by it. 
 
 BACILLUS OF MALIGNANT (EDEMA. 
 
 This bacillus is widely distributed, being found in 
 the superficial layers of the soil, in putrefying sub- 
 stances, in the blood of animals which have been suffo- 
 cated (by invasion from the intestine), in foul water, 
 etc. It was discovered (1877) by Pasteur in animals 
 after injections of putrefying liquids, and named by 
 him " vibrion septique." He recognized its anaerobic 
 nature, but did not obtain it in pure culture. Koch 
 (1881) carefully studied this micro-organism, described 
 it in detail, and gave it the name " bacillus cedematous 
 maligni." It was isolated first in pure culture by 
 Liborius. 
 
 Morphology. The oedema bacillus is a rod of from 
 0.8/2 to \IJL in width, and of very varying length, from 
 2// to 10// or more, according to .the conditions of its 
 cultivation and growth. It is usually found in pairs, 
 joined end to end, but may occur in chains or long 
 filaments. It forms spores, and these are situated 
 
544 BACTERIOLOGY. 
 
 in or near the middle of the body of the rods. The 
 spores vary in length and are oval in form, being often 
 of greater diameter than the bacilli, to which they give 
 a more or less oval or spindle shape. 
 
 The bacilli stain readily by the usual aniline colors 
 employed, but are decolorized by Gram's method. 
 
 Biological Characters. A strictly anaerobic, liquefy- 
 ing, motile bacillus. Forms spores. It grows, however, 
 in all the usual culture media in the absence of oxygen. 
 Development takes place at the room-temperature, but 
 more rapidly and abundantly at 37 C. 
 
 Growth in Gelatin. This bacillus may be cultivated 
 in ordinary nutrient gelatin, but the growth is more 
 abundant in glucose-gelatin containing 1 or 2 per cent, 
 of glucose. Gas-bubbles are formed and the gelatin 
 liquefies. 
 
 Growth on Agar. On agar plates the colonies appear 
 as dull, whitish points, irregular in outline, and when 
 examined under a low-power lens are seen to be com- 
 posed of a dense network of interlacing threads radi- 
 ating irregularly from the centre toward the periphery. 
 
 Blood-serum is rapidly liquefied, with the production 
 of gas. Cultures of the malignant oedema bacillus give 
 off a peculiar, disagreeable odor. 
 
 Pathogenesis. The bacillus of malignant oedema is 
 especially pathogenic for mice, guinea-pigs, and rabbits, 
 although man, horses, dogs, goats, sheep, calves, pigs, 
 chickens, and pigeons are also susceptible. A small 
 quantity of a pure culture injected beneath the skin of 
 a susceptible animal gives rise to an extensive hemor- 
 rhagic oedema of the subcutaneous connective tissue, 
 which extends over the entire surface of the abdomen 
 and thorax, causing hypersemia and redness of the 
 superficial muscles. There is no odor developed, and 
 
BACILLUS AEROGENES CAPSULATUS. 545 
 
 little, if any, production of gas. In infection with 
 garden earth, owing to the presence of associated ba- 
 cilli, the effused serum is frothy from the development 
 of gas, and possesses a putrefactive odor. The disease, 
 in natural infection caused by the contamination of 
 wounds with earth or feces, runs the course above de- 
 scribed. Simple abrasion of the skin is not sufficient 
 to produce infection; owing to the bacillus being capable 
 only of an anaerobic existence, the poison must pene- 
 trate deep into the tissues. Malignant oedema is con- 
 fined mostly to the domestic animals, but cases have 
 also been reported in man. 
 
 Animals which recover from malignant oedema are 
 subsequently immune. Artificial immunity may be 
 induced in guinea-pigs by injecting filtered cultures of 
 the malignant oedema bacillus in harmless quantities. 
 
 BACILLUS AEROGENES OAPSULATUS. 
 
 Found by Welch in the bloodvessels of a patient 
 suffering with aortic aneurism; on autopsy, made in 
 cool weather eight hours after death, the vessels were 
 observed to be full of gas-bubbles. Since then it has 
 been found in a number of cases in which gas has de- 
 veloped from within sixty hours of death until some 
 hours after death. These cases are, as a rule, marked by 
 delirium, rapid pulse, high temperature, and the devel- 
 opment of emphysema and discoloration of the diseased 
 area, or of marked abdominal distention when the peri- 
 toneal cavity is involved. 
 
 Morphology. Straight or slightly curved rods, with 
 rounded or sometimes square-cut ends; somewhat 
 thicker than the anthrax bacilli and varying in length ; 
 occasionally long threads and chains are seen. The 
 
 35 
 
546 BACTERIOLOGY. 
 
 bacilli in the animal body, and sometimes in cultures, 
 are enclosed in a transparent capsule. 
 
 Biological Characters. An anaerobic, non-motile, non- 
 liquefying bacillus. Does not form spores. Grows at 
 the room-temperature, but more rapidly at 37 C., in 
 the usual culture media in the absence of oxygen, and 
 is accompanied by the production of gas. Nutrient 
 gelatin is not liquefied by the growth of this bacillus, 
 but it is gradually peptonized. In agar colonies are 
 developed which are from 1 to 2 mm. or more in 
 diameter, grayish-white in color, and in the form of 
 flattened spheres, ovals, or irregular masses, beset with 
 hair-like projections. Bouillon is diffusely clouded, and 
 a white sediment is formed. Milk is rapidly coagulated. 
 
 Pathogenesis. Usually non-pathogenic in healthy 
 animals, although Dunham found that the bacillus taken 
 freshly from human infection is sometimes very virulent. 
 When quantities up to 2.5 c.c. of fresh bouillon cultures 
 are injected into the circulation of rabbits and the ani- 
 mals killed shortly after the injection, the bacilli de- 
 velop rapidly, with an abundant formation of gas in 
 the bloodvessels and organs, especially the liver. The 
 following is one of the best methods of obtaining 
 the bacilli : The material suspected to contain the ba- 
 cillus alone or associated with other bacteria is injected 
 into rabbits, which are killed, kept at 37 C., and cultures 
 made twenty-four hours later from their bodies. 
 
 It is suggested by Welch that in some of the cases in 
 which death has been attributed to the entrance of air 
 into the veins the gas found at the autopsy may not 
 have been atmospheric air, but may have been produced 
 by this or some similar micro-organism entering the cir- 
 culation and developing shortly before and after death. 
 The bacillus has been found in the dust of hospitals. 
 
CHAPTER XXXII. 
 
 BACILLUS ANTHRACIS BACILLUS ANTHRACIS SYMP- 
 TOMATICI (ANTHRAX BACILLUS). 
 
 BACILLUS ANTHEACIS. 
 
 ANTHRAX is an acute infectious disease which is 
 very prevalent among animals, particularly sheep and 
 cattle. Geographically and zoologically it is the most 
 wide-spread of all infectious disorders. It is much 
 more common in Europe and in Asia than in America. 
 The ravages among herds of cattle in Russia and Sibe- 
 ria, and among sheep in certain parts of France, Hun- 
 gary, Germany, Persia, and India, are not equalled by 
 any other animal plague. Local epidemics have occa- 
 sionally occurred in England, where it is known as 
 splenic fever. In this country the disease is rare. In 
 infected districts the greatest losses are incurred during 
 the hot months of summer. 
 
 The disease also occurs in man as the result of in- 
 fection, either through the skin, the intestines, or in 
 rare instances through the lungs. It is found in per- 
 sons whose occupations bring them into contact with 
 animals or animal products, as stablemen, shepherds, 
 tanners, butchers, and those who work in wool and 
 hair. Two forms of the disease have been described 
 the external anthrax, or malignant pustule, and the 
 internal anthrax, of which there are intestinal and 
 
548 BACTERIOLOGY. 
 
 pulmonary forms, the latter being known as " wool- 
 sorter's disease." 
 
 Owing to the fact that anthrax was the first infec- 
 tious disease which was shown to be caused by a specific 
 micro-organism, and to the close study which it received 
 in consequence, this disease has probably contributed 
 more to our general knowledge of bacteriology than 
 any other infectious malady. 
 
 Pollender observed in 1849 that the blood of animals 
 suffering from splenic fever always contained minute 
 rod-shaped bacteria. Davaine, in 1863, announced to 
 the French Academy of Sciences the results of his in- 
 oculation experiments, and asserted the etiological rela- 
 tion of the micro-organism to the disease with which 
 his investigation showed it to be constantly associated. 
 For a long time this conclusion was energetically op- 
 posed until, in 1879, Pasteur, Koch and others estab- 
 lished its truth by obtaining the bacillus in pure cultures 
 and showing that the inoculation of these cultures pro- 
 duced anthrax in susceptible animals as certainly as did 
 the blood of an animal recently dead from the disease. 
 
 Morphology, Slender, cylindrical, non-motile rods, 
 having a breadth of \p to 1.25//, and ranging from 2 
 or 3// to 20 or 25// in length. They vary thus very 
 much in their length. Sometimes short, isolated rods 
 are seen, and, again, shorter or longer chains or threads 
 made up of several rods joined end to end. In suitable 
 culture media very long, flexible filaments may be 
 observed, which are frequently united in twisted or 
 plaited, cord-like bundles. (See Fig. 71 and Fig. 13, 
 p. 47, and Fig. 17, p. 207.) These filaments in hang- 
 ing drop cultures, before the development of spores, 
 appear to be homogeneous or nearly so; but in stained 
 
BACILLUS ANTHRACIS. 
 
 549 
 
 preparations they are seen to be composed of a series of 
 rectangular, deeply stained segments. When obtained 
 directly from the blood of an infected animal the free 
 ends of the rods are slightly rounded, but those com- 
 ing in contact with one another are quite square. In 
 cultures the ends are seen to be a trifle thicker than 
 the body of the cell and somewhat concave, giving the 
 appearance of joints of bamboo. At one time much stress 
 
 FIG. 71. 
 
 Anthrax bacillus., X 900 diameters. Agar culture. 
 
 was laid upon these peculiarities as distinguishing marks 
 of the anthrax bacillus; but it has been found that these 
 are the effects of artificial cultivation and not necessarily 
 characteristic of the organism under all conditions. An- 
 other peculiarity of this bacillus is that it is enclosed 
 in a transparent envelope or capsule, which in stained 
 preparations may be distinguished by its taking on a 
 lighter stain than the deeply stained rods which it sur- 
 rounds. 
 
550 
 
 BACTERIOLOGY. 
 
 Under favorable conditions in cultures spores are 
 developed in the bacilli. These spores are elliptical 
 in shape and about one and a half times longer than 
 broad. They first appear as small, refractive gran- 
 ules distributed at regular intervals, one in each rod. 
 As the spore develops the mother-cell becomes less and 
 less distinct, until it disappears altogether, the com- 
 plete oval spore being set free by its dissolution. (Fig. 
 72, Fig. 13, p. 47, and Fig. 17, p. 207). Irregular 
 sporulation sometimes takes place, and occasionally 
 there is no spore-formation, as in varieties of non-spore 
 bearing anthrax. 
 
 FIG. 72. 
 
 Spores heavily stained (in specimen red). Bodies of disintegrating bacilli 
 faintly stained (in specimen blue). X 1000 diameters. 
 
 The anthrax bacillus stains readily with all the aniline 
 colors, and also by Gram's method, when not left too 
 long in the decolorizing iodine solution. In sections 
 good results may be obtained by the employment of 
 Gram's solution in combination with carmine, but when 
 
BACILLUS ANTHRACIS. 551 
 
 only a few bacilli are present this method is not always 
 reliable, as some of the bacilli are generally decolorized. 
 
 Biological Characters. The anthrax bacillus grows 
 easily in a variety of nutrient media at a temperature 
 from 18 to 43 C., 37 C. being the most favorable 
 temperature. Under 12 C. no development takes 
 place, as a rule, though by gradually accustoming the 
 bacillus to a lower temperature it may be induced to 
 grow under these conditions. Under 14 C. and above 
 43 C. spore-formation ceases. The lower limit of 
 growth and sporulation is of practical significance in 
 determining the question whether development can 
 occur in the bodies of animals dead from anthrax 
 when buried at certain depths in the earth. Kitasato 
 has shown that at a depth of 1.5 metres the earth in 
 July has a temperature of 15 C. at most, and that 
 under these conditions a scanty sporulation of anthrax 
 bacilli is possible, but that at a depth of 2 metres sporu- 
 lation no longer occurs. The anthrax bacillus is aerobic 
 that is, its growth is considerably enhanced by the 
 presence of oxygen but it grows also under anaerobic 
 conditions, as is shown by its growth at the bottom of 
 the line of puncture in stick cultures in solid media; 
 but under these conditions it no longer produces the 
 peptonizing ferment which it does with free access of 
 air. Furthermore, the presence of oxygen is absolutely 
 necessary for the formation of spores, while carbonic 
 acid gas retards sporulation. This explains, perhaps, 
 why sporulation does not take place within the animal 
 body either before or after death. 
 
 This bacillus grows best in neutral or slightly alka- 
 line media. It may be cultivated in infusions of meat 
 or of various vegetables, in urine ; etc,, provided the 
 
552 
 
 BACTERIOLOGY. 
 
 reaction be not decidedly acid, which arrests develop- 
 ment. It grows in cow-dung and in more or less 
 contaminated earth. It is also capable of leading a 
 saprophytic existence. The bacillus is non-motile. 
 
 Growth in Gelatin. In gelatin plate cultures, at 
 the end of twenty-four to thirty-six hours at 24 C., 
 small, white, opaque colonies are developed, which 
 
 FIG. 73. 
 
 Colonies of bacillus anthracis upon gelatin plates, a, at the end of twenty- 
 four hours ; 6, at the end of forty-eight hours. X 80. (F. FLtlGGE.) 
 
 under a low-power lens are seen to be dark gray in 
 the centre and surrounded by a greenish, irregular 
 border, made up of wavy filaments. As the colony 
 develops on the surface of the gelatin these wavy 
 filaments spread out, until finally the entire colony 
 consists of a light gray, tangled mass, which has been 
 likened to a Medusa head (Fig. 73). 
 
 At the same time the gelatin begins to liquefy, and 
 the colony is soon surrounded by the liquefied medium, 
 
BA GILL US ANTHRA CIS. 553 
 
 upon the surface of which it floats as an irregular, white 
 pellicle. In gelatin stick cultures at first development 
 occurs along the line of puncture as a delicate white 
 thread, from which irregular, hair-like projections soon 
 extend perpendicularly into the culture medium, the 
 growth being most luxuriant near the surface, but con- 
 tinuing also below. At the end of two or three days 
 liquefaction of the medium commences at the surface 
 and gradually progresses downward. 
 
 Growth on Agar. The growth on agar plate cul- 
 tures in the incubator at 37 C. is similar to that on 
 gelatin, and is still more characteristic and beautiful in 
 appearance. A grayish-white layer is formed on the 
 surface within twenty-four hours, which spreads rapidly 
 and is seen to be made up of interlaced threads. 
 
 In bouillon the growth is characterized by the forma- 
 tion of flaky masses, which sink as a sediment to the 
 bottom of the tube, leaving the supernatant liquid clear. 
 
 Spore formation, as already noted, only takes place 
 in the free presence of oxygen, and at a temperature of 
 15 to 43 C. There is no development of spores at a 
 greater depth than 1.5 metres in the earth, or in the 
 bodies of living or dead animals; but spores may be 
 found in the fluids containing the bacilli when these 
 come in contact with the air, as in bloody discharges 
 from the nostrils or from the bowels of the dead animal. 
 
 There are certain non- spore bearing species of an- 
 thrax. Sporeless varieties have also been produced 
 artificially by cultivating the typical anthrax bacillus 
 under unfavorable conditions. The addition of anti- 
 septics, as carbolic acid, favors these conditions. Vari- 
 eties differing in their pathogenic power may also be 
 produced artificially. Pasteur produced an " attenu- 
 
554 BACTERIOLOGY. 
 
 ated virus " by keeping his cultures for a considerable 
 time before replanting them upon fresh soil. Cham- 
 berland and Roux have shown that cultivation in the 
 presence of certain chemical substances added to the 
 culture medium, as bichromate of potassium, causes an 
 attenuation of virulence. Attenuation of pathogenic 
 power is also effected by cultivation in the body of a 
 non-susceptible animal, like the frog (Lubarsch, Pe- 
 truschky); or in the blood of a rat (Behring); by ex- 
 posure to sunlight (Arloing); to heat, 50 C. for 
 eighteen minutes; and by compressed air (Chauveau). 
 '^Anthrax cultures containing spores retain their vital- 
 ity for years; in the absence of spores the vitality is 
 much more rapidly lost. When grown in liquids rich 
 in albumin the bacilli attain a considerable degree of 
 resistance; thus dried anthrax blood has been found to 
 retain its virulence for sixty days, while dried bouillon 
 cultures only did so for twenty-one days. Dried anthrax 
 spores may be preserved for many years without losing 
 their vitality or virulence. They also resist a com- 
 paratively high temperature. Exposed in dry air they 
 require a temperature of 140 C. maintained for three 
 hours to destroy them; but suspended in a liquid they 
 are destroyed in four minutes by a temperature of 
 100 C. The bacilli, in the absence of spores, are de- 
 stroyed in ten minutes by a temperature of 54 C. 
 Anthrax spores in a desiccated condition are destroyed 
 in four hours when exposed to the action of direct 
 sunlight, only after several weeks in diffuse daylight 
 (Kruse). 
 
 Pathogenesis. The anthrax bacillus is pathogenic for 
 cattle, sheep (except the Algerian race), horses, swine, 
 mice, guinea-pigs, and rabbits. Bats, cats, dogs, chick- 
 
BA GILL US ANTHRA CIS. 555 
 
 ens, owls, pigeons, and frogs are but little susceptible 
 to infection. Small birds the sparrow particularly 
 are somewhat susceptible. Man, though subject to 
 local infection and occasionally to internal forms of the 
 disease, is not as susceptible as some of the lower ani- 
 mals. 
 
 The anthrax bacillus produces in susceptible animals 
 a true septicaemia. Among test animals mice are the 
 most susceptible, succumbing to very minute injections 
 of a slightly virulent virus; next guinea-pigs, and lastly 
 rabbits, both of these animals dying after inoculation 
 with virulent bacilli. Infection is most promptly pro- 
 duced by introduction of the bacilli into the circulation 
 or the tissues, but inoculation by contact with wounds 
 on the skin also cause infection. It is difficult to pro- 
 dace infection by the ingestion even of spores; but it 
 may readily be caused by inhalation, particularly by the 
 inhalation of spores. 
 
 Subcutaneous injections of these susceptible animals 
 results in death in from one to three days. Compara- 
 tively little local reaction occurs immediately at the 
 point of inoculation, but beyond this there is an exten- 
 sive oedema of the tissues. Very few bacilli are found 
 in the blood, but in the internal organs, and especially 
 in the capillaries of the liver, the kidneys, and the 
 lungs, they are present in great numbers. In some 
 places the capillaries will be seen to be stuffed full of 
 bacilli, as in the glomeruli of the kidneys, and hemor- 
 rhages, probably due to rupture of capillaries by the 
 mechanical pressure of the bacilli which are developing 
 within them, may occur. The pathological lesions in 
 animals infected by anthrax are not marked except in 
 the spleen, which, as in other forms of septicaemia, is. 
 
556 BACTERIOLOGY. 
 
 greatly enlarged. The anthrax bacilli in these animals 
 seem to live almost exclusively in the bloodvessels and 
 to leave them only by means of hemorrhages. In this 
 way they reach but only late in the disease -the 
 various secretions of the body, the urine, the intestinal 
 secretions, and occasionally the bile. The passage of 
 the anthrax bacillus from the mother to the foetus in 
 pregnant females is possible, as has been shown by the 
 investigations of Strauss, Chamberlain, and others, but 
 it very rarely occurs. 
 
 Occurrence in Cattle and Sheep. Cattle and sheep 
 are affected chiefly with the intestinal form of an- 
 thrax, infection in these animals commonly resulting 
 from the ingestion of food containing spores. The 
 bacillus itself, in the absence of spores, is quickly de- 
 stroyed by the gastric juice (Koch, Gaffky, Loffler). 
 The disease usually takes a rapid course, and the mor- 
 tality is high 70 to 80 per cent. The pathological 
 lesions consist of numerous ecchymoses, enlargement of 
 the lymphatic glands, serous, fatty, and hemorrhagic 
 infiltration of the mediastinum and mesentery, of the 
 mucous membranes of the pharynx and larynx, and 
 particularly of the duodenum, great enlargement of the 
 spleen, and parenchymatous changes in the lymphatic 
 organs. The blood is very dark and tar-like. Bacilli 
 are present in enormous masses. 
 
 ^Sheep are also subject to external anthrax, infection 
 taking place by way of the skin; cattle are seldom in- 
 fected in this way. At the point of inoculation there 
 develops a hard, circumscribed boil the so-called an- 
 thrax carbuncle; or there may be diffuse oedema, with 
 great 'swelling of the parts. When death occurs the 
 appearances are similar to those in intestinal anthrax, 
 
BA CILL US AN THE A CIS. 557 
 
 except that the duodenum is usually less affected; but in 
 all cases metastasis occurs in various parts of the body, 
 brought about, no doubt, by previous hemorrhages. 
 
 Occurrence in Man. The disease does not occur 
 spontaneously in man, but always results from infec- 
 tion, either through the skin, the intestines, or occasion- 
 ally by inhalation through the lungs. It is usually 
 produced by cutaneous infection through inoculation of 
 exposed surfaces the hands, arms, or face. Infection 
 of the face or neck would seem to be the most danger- 
 ous, the mortality in such cases being 26 per cent.; 
 while infection of the extremities is very rarely fatal 
 in only 5 per cent, of cases (Nassarow and Miiller). 
 
 External anthrax in man is similar to this form of 
 the disease in animals. There are two forms : Malig- 
 nant pustule or carbuncle, and, less commonly, malig- 
 nant anthrax oedema. 
 
 In malignant pustule, at the site of inoculations, a small 
 papule develops, which becomes vesicular. Inflamma- 
 tory induration extends around this, and within thirty- 
 six hours there is a dark brownish eschar in the centre, 
 at a little distance from which there may be a series of 
 small vesicles. The brawny induration may be extreme. 
 There may also be considerable oedema of the parts. In 
 most cases there is no fever; or the temperature at first 
 rises rapidly and the febrile phenomena are marked. 
 Death may take place in from three to five days. In 
 cases which recover the symptoms are slighter. In the 
 mildest form there may be only slight swelling. 
 
 Malignant anthrax cedema occurs in the eyelids, and 
 also in the head and neck, sometimes the hand and arm. 
 It is characterized by the absence of the papule and 
 vesicle forms, and by the most extensive oedema. The 
 
558 BACTERIOLOGY. 
 
 oedema may become so intense that gangrene results; 
 such cases usually prove fatal. 
 
 The bacilli are found on microscopical examination 
 of the fluid from the pustule shortly after infection; 
 later the typical anthrax bacilli are often replaced by 
 involution forms. In this case resort may be had to 
 cultures, animal inoculation, or examination of sections 
 of the extirpated tumor. The bacilli are not present 
 in the blood until just before death. Along with the 
 anthrax bacilli pus cocci are often found in the pustule 
 penetrating into the dead tissue. 
 
 Internal anthrax is much less common in man; it 
 does, however, occur now and then. There are two 
 forms of this : the intestinal form, or mycosis intesti- 
 nalis, and the pulmonic form, or wool-sorter's disease. 
 
 Intestinal anthrax is caused by infection through the 
 stomach and intestines, and results probably from the 
 eating of raw flesh or unboiled milk of diseased animals. 
 That the eating of flesh from infected animals is com- 
 paratively harmless is shown by Gerlier, who states that 
 of 400 persons who were known to have eaten such 
 meat not one was affected with anthrax. On the other 
 hand, an epidemic of anthrax was produced among wild 
 animals, according to Jansen, by feeding them on in- 
 fected horse flesh. It is evident, therefore, that there 
 is a possibility of infection being caused in this way. 
 The recorded cases of intestinal anthrax in man have 
 occurred in persons who were in the habit of handling 
 hides, hair, etc., which were contaminated with spores; 
 in those who were conducting laboratory experiments, 
 and rarely it has been produced by the ingestion of food, 
 such as raw ham and milk. The symptoms produced 
 in this disease are those of intense poisoning chill, fol- 
 
BACILLUS ANTHRACIS. 559 
 
 lowed by vomiting, diarrhoea, moderate fever, and pains 
 in the legs and back. In acute cases there may be dysp- 
 noea, cyanosis, and toward the end convulsions. The 
 pathological lesions are similar to those described in 
 animals. 
 
 Wool-sorter's disease, or pulmonic anthrax, is found 
 in large establishments in which wool and hair are sorted 
 and cleansed, and is caused by the inhalation of dust con- 
 taminated with anthrax spores. The attack comes on 
 with chills, prostration, then fever. The breathing is 
 rapid, and the patient complains of pain in the chest. 
 There may be a cough and signs of bronchitis. The 
 bronchial symptoms in some instances are pronounced. 
 Death may occur in from two to seven days. The path- 
 ological changes produced are swelling of the glands of 
 the neck, the formation of foci of necrosis in the air- 
 passages, oedema of the lungs, pleurisy, bronchitis, en- 
 largement of the spleen, and parenchymatous degener- 
 ations. 
 
 Many theories have been advanced to account for the 
 occurrence of intestinal anthrax among cattle and sheep, 
 which in these animals is the most wide-spread form of 
 the disease. It has been thought that infection was 
 produced mainly by the eating of food contaminated by 
 anthrax spores derived from the bodies of infected ani- 
 mals; but only in rare instances has it been possible to 
 trace the cause of the disease to this source. The 
 grazing of cattle on infected pastures has also been 
 assigned as the cause of the disease; but this does not 
 explain the occurrence of epidemics or the infection of 
 cattle on pastures which have never before been visited 
 by animals affected with anthrax. By some anthrax 
 has been called a miasmatic disease and likened to 
 
560 BACTERIOLOGY. 
 
 malaria. Occurring as it does at the same season of 
 the year viz., from June to October and being con- 
 nected apparently, like malaria, in some way with the 
 condition of the soil, there is a certain analogy between 
 these two diseases. Anthrax is found to occur mostly 
 in low, swampy localities, where the soil is covered with 
 decaying vegetable matter, and subject to overflows and 
 freshets. There is no doubt that this bacillus is able to 
 lead a saprophytic existence for some time, under favor- 
 able conditions, in the superficial layers of the soil, re- 
 maining latent in the form of spores and retaining its 
 vitality; but why an epidemic of anthrax occurs one 
 year at a certain place and at the same place the next 
 year does not, it is not easy to explain. Pasteur believes 
 that the earth-worm plays an important part in bringing 
 to the surface and distributing the spores which have 
 been propagated in the buried carcass of an infected ani- 
 mal; but Koch has shown that this hypothesis is both 
 improbable and superfluous. Apart from the fact that 
 spor ulation does not normally take place inside the bodies 
 of dead animals, the earth-worm is ill adapted for the 
 transportation of anthrax spores, which are unfavorably 
 affected in their intestines. Out of seventy-two earth- 
 worms examined by Ballinger from a notoriously infected 
 locality, only one contained anthrax spores. Further- 
 more, the soil in places where such carcasses lie buried 
 is already saturated with the fluids and other products 
 of decomposition of the body of the dead animal contain- 
 ing bacilli, which under suitable temperature conditions 
 may form spores and thus infect the surface of the land; 
 though it is possible that the earth-worm, in some in- 
 stances, may contribute to the distribution of spores to 
 a certain extent. It would, therefore, seem that the only 
 
BACILLUS ANTHRACIS. 561 
 
 plausible explanation which can be offered, in the light of 
 our present knowledge, for the solution of thib problem, 
 is the supposition that under certain natural conditions 
 unfavorable to the development of the anthrax bacillus 
 an attenuation of its virulence takes place, and then, 
 again, an increase of virulence as the condition becomes 
 more favorable a result which can be produced by arti- 
 ficial means. But whether this actually occurs we do 
 not know. 
 
 Prophylaxis Against Anthrax Infection. Numerous 
 investigations have been undertaken with the object of 
 preventing infection from anthrax. The efforts of Pas- 
 teur to effect immunity in animals by preventive inocu- 
 lations of " attenuated virus" of the anthrax bacillus, 
 opened a new field of productive original research. Fol- 
 lowing in his wake many others have prepared methods 
 of immunization against anthrax infection; but the one 
 adopted by Pasteur, Chamberland and Roux has alone 
 been practically employed on a large scale. According 
 to these authors, two anthrax cultures of different de- 
 grees of virulence, attenuated by cultivation at 42 to 
 43 C., are used for inoculation. Vaccine No. 1 kills 
 mice, but not guinea-pigs; Vaccine No. 2 kills guinea- 
 pigs, but not rabbits, according to Koch, Gaffky, and 
 Loffler. The animals to be inoculated viz., sheep 
 and cattle are first given a subcutaneous injection 
 of one to several tenths of a cubic centimetre of 
 a four-day-old bouillon culture of Vaccine No. 1; 
 after ten to twelve days they receive a similar dose 
 of Vaccine No. 2. Prophylactic inoculations given 
 in this way have been widely employed in France, 
 Hungary, and Russia. Statistics collected by Cham- 
 berland of the results of twelve years 7 use of this 
 
 36 
 
562 BACTERIOLOGY. 
 
 method in France show that 3,300,000 sheep have 
 been thus inoculated. Of these 1 per cent, only have 
 died from anthrax, either during or after treatment; 
 whereas the mortality previous to the introduction of 
 this method was 10 per cent, on the average. Of 
 438,000 cattle inoculations only 0.33 per cent, have 
 died; the previous mortality from anthrax was 5 per 
 cent. These figures would seem to indicate the prac- 
 tical value of Pasteur's method of inoculation, notwith- 
 standing the arguments which have been put forward in 
 opposition to it. It is, however, not unattended with 
 danger, as some of the animals succumb to the after- 
 effects of the attenuated culture. 
 
 Differential Diagnosis. The differential diagnosis of 
 the anthrax bacillus is ordinarily not difficult, as this 
 organism presents morphological, biological, and patho- 
 gen ical characteristics which distinguish it from all other 
 bacteria. In the later stages of the disease, however, 
 the bacilli may be absent or difficult to find, and culti- 
 vation on artificial media and experimental inoculation 
 in animals are not always followed by positive results. 
 Even in sections taken from the extirpated pustule it is 
 sometimes difficult to detect the bacilli. In such cases 
 only a probable diagnosis of anthrax can be made. It 
 should be remembered that the bacilli are not found in 
 the blood until shortly before death, and then only in 
 varying quantity; thus blood examinations often give 
 negative results, though the bacilli may be present in 
 large numbers in the spleen, kidneys, and other organs 
 of the body. The suspected material should be inocu- 
 lated in nutrient gelatin and agar in Petri plates and 
 in mice. 
 
 Among other bacteria which may possibly be mis- 
 
BACILL US ANTHRA CIS SYMPTOMA T1CL 563 
 
 taken for anthrax bacilli are the bacillus subtilis and 
 the bacillus of malignant oedema. The former is dis- 
 tinguished by its motility, by various cultural peculi- 
 arities, and by being non-pathogenic. The latter differs 
 from the anthrax bacillus in form and motility, in being 
 decolorized by Gram's solution, in being a strict anaer- 
 obe, and in various pathogenic properties. 
 
 The diagnosis of internal anthrax in man is by no 
 means easy, unless the history points definitely to infec- 
 tion in the occupation of the individual. In cases of 
 doubt cultures should be made and inoculations per- 
 formed in animals. According to Cornil and Babes, 
 some of these cases may possibly be caused by organ- 
 isms other than the bacillus of anthrax. 
 
 BACILLUS ANTHRACIS SYMPTOMATICI. 
 
 (Bacillus of Symptomatic Anthrax.) 
 
 Like the bacilli of anthrax and of malignant oedema, 
 both of which it resembles in other respects also, the 
 bacillus of symptomatic anthrax is an inhabitant of the 
 soil. It is found as the chief cause of the disease in 
 animals principally cattle and sheep affected with 
 what is known as " black leg," " quarter evil," or 
 symptomatic anthrax (German, rauschbrand; French, 
 charbon symptomatique), a disease which prevails in 
 certain localities in summer, and is characterized by a 
 peculiar emphysematous swelling of the subcutaneous 
 tissues and muscles, especially over the quarters. 
 
 Morphology. Bacilli having rounded ends, from 
 0.5/j. to O.Qfj. broad and from 3// to 6/J. long; mostly 
 isolated, also occurring in pairs, joined end-to-end, 
 but never growing out into long filaments, as the an- 
 
564 SA CTERIOL OGY. 
 
 thrax bacilli in culture and the bacilli of malignant 
 oedema in the bodies of animals are frequently seen to 
 do. Under the hanging drop the bacilli are observed 
 to be actively motile, and in stained preparations flagella 
 may be demonstrated surrounding the periphery. The 
 spores are elliptical in shape, usually thicker than the 
 bacilli, lying near the middle of the rods, but rather 
 toward one extremity. This gives to the bacilli con- 
 taining spores a somewhat spindle shape. 
 
 Stains with the ordinary aniline dyes, but not with 
 Gram's method, or only with difficulty and after long 
 treatment or intense colors. 
 
 Biological Characters. Like the bacillus of malignant 
 oedema this is also a strict anaerobe, and cannot be cul- 
 tivated in an atmosphere in which oxygen is present. 
 It grows best under hydrogen, and does not grow under 
 carbonic acid. This bacillus develops at the room-tem- 
 perature in the usual culture media, in the absence of 
 oxygen, but it grows best in those to which 1.5 to 2 per 
 cent, of glucose or 5 per cent, of glycerin has been added. 
 
 Growth on Agar. The colonies on agar are some- 
 what more compact than those of malignant oedema, but 
 they also send out projections very often. In agar stick 
 cultures, in the incubator, growth occurs after a day or 
 two also some distance below the surface, and is accom- 
 panied by the production of gas and a peculiar disagree- 
 able acid odor. 
 
 Pathogenesis. The bacillus of symptomatic anthrax 
 is pathogenic for cattle (which are immune against ma- 
 lignant oedema), sheep, goats, guinea-pigs, and mice; 
 horses, asses, and white rats when inoculated with a cul- 
 ture of this bacillus present only a limited reaction; and 
 rabbits, swine, dogs, cats, chickens, ducks, and pigeons 
 
BACILLUS ANTHEACIS SYMPTOMATIC!. 565 
 
 are, as a rule, naturally immune to the disease. The 
 guinea-pig is the most susceptible of test animals. 
 When susceptible animals are inoculated subcutane- 
 ously with pure cultures of this organism, with spores 
 attached to a silk thread, or with bits of tissue from 
 the affected parts of another animal dead of the disease, 
 death ensues in from twenty-four to thirty-six hours. 
 At the autopsy a bloody serum is found in the subcuta- 
 neous tissues extending from the point of inoculation 
 over the entire surface of the abdomen, and the muscles 
 present a dark-red or black appearance, even more in- 
 tense in color than in malignant oedema, and there is a 
 considerable development of gas. The lymphatic glands 
 are markedly hypenemic. 
 
 The disease occurs chiefly in cattle, more rarely in 
 sheep and goats; horses are not attacked spontaneously 
 i. e. y by accidental infection. In man, infection has 
 never been produced, though ample opportunity, by 
 infection through wounds in slaughter-houses and by 
 the ingestion of infected meat, has been given. The 
 usual mode of natural infection by symptomatic an- 
 thrax is through wounds which penetrate not only the 
 skin but the deep intercellular tissues; some cases of 
 infection by ingestion have been observed. The patho- 
 logical findings present the conditions above described 
 as occurring in experimental infection. 
 
 Symptomatic anthrax, like anthrax and malignant 
 oedema, is a disease of the soil, but it shows a more 
 limited endemic distribution than the first, and is differ- 
 ently distributed over the earth's surface than the sec- 
 ond of these diseases, being confined especially to places 
 over which infected herds of cattle have been pastured. 
 It is doubtful whether the bacilli are capable of devel- 
 
566 BACTERIOLOGY. 
 
 opment outside of the body like anthrax. In the form 
 of spores, however, reproduction may take place; and 
 by contamination with these, through deep wounds ac- 
 quired by animals in infected pastures, the disease is 
 spread. Possibly also it may originate through infec- 
 tion of the mouth and by feeding which would account 
 for the cases of symptomatic anthrax occurring in stall- 
 fed cattle (Hafner). 
 
 It is well known to veterinarians that recovery from 
 one attack of symptomatic anthrax protects an animal 
 against a second infection. Artificial immunity to in- 
 fection can also be produced in various ways : by intra- 
 venous inoculations; or, in guinea-pigs, by inoculations 
 with bouillon cultures which have been kept for a few 
 days and as a result have lost their original virulence, 
 or with cultures kept in an incubating oven at a tem- 
 perature of 42 to 43 C.; or by inoculations made into 
 the extremity of the tail; or by inoculations with filtered 
 cultures, or with cultures sterilized by heat. For the 
 production of immunity in cattle, Arloing, Cornevin, 
 and Thomas recommend the use of a dried powder of 
 the muscles of animals which have succumbed to the 
 disease, and which have been subjected to a suitable 
 temperature to ensure attenuation of the virulence of 
 the spores contained in it. Two vaccines are prepared, 
 as in anthrax a stronger vaccine by exposure of a por- 
 tion of the powder to a temperature of 85 to 90 C. 
 for six hours, and a weaker vaccine by exposure for the 
 same time to a temperature of 100 to 104 C. Inocu- 
 lations made with this attenuated virus (into the end of 
 the tail) the weakest first and later the stronger give 
 rise to a local reaction of moderate intensity, and the 
 animal is subsequently immune from the effects of the 
 
BA CILL US ANTHRA CIS S YMPTOMA TICI. 567 
 
 most virulent material and from the disease. Fourteen 
 
 
 
 days are allowed to elapse between the two inoculations. 
 The results obtained from this method of preventive in- 
 oculation seem to have been very satisfactory. Accord- 
 ing to the statistics of Hafner, Luchanka, Hess, Strebel, 
 etc. (1885-93), including many thousand cattle treated, 
 the mortality, which among 22,300 non-inoculated cattle 
 was 2.20 per cent., has been reduced to 0.16 per cent, 
 in 14,700 animals inoculated. 
 
 To recapitulate briefly, the principal points of differ- 
 entiation between this bacillus and the bacillus of malig- 
 nant oedema, which it closely simulates, are smaller; it 
 does not develop into long threads in the tissues; it is 
 more actively motile, and forms spores more readily in 
 the animal body than does the bacillus of malignant 
 oedema. It is pathogenic for cattle, while malignant 
 oedema is not; and swine, dogs, rabbits, chickens, and 
 pigeons, which are readily infected with malignant 
 oedema, are not, as a rule, susceptible to symptomatic 
 anthrax. 
 
CHAPTER XXXIII. 
 
 SPIRILLUM CHOLERA ASIATICS (KOCH ? S COMMA 
 BACILLUS OF ASIATIC CHOLERA). 
 
 IN 1883, Koch separated a characteristically curved 
 organism from the dejecta and intestines of cholera 
 patients the so-called " comma bacillus." This he 
 declared to be absent from the stools and intestinal con- 
 tents of healthy persons and of persons suffering from 
 other affections. The organism was said to possess cer- 
 tain morphological and biological features which readily 
 distinguished it from all previously described organisms. 
 It was absent from the blood and viscera, and was found 
 only in the intestines; and in greater number, it was 
 said, the more acute the attack. Koch also demonstrated 
 an invasion of the mucosa and its glands by the comma 
 bacilli. The organisms were found in the stools on 
 staining the mucous flakes or the fluid with methylene- 
 blue or fuchsin, and sometimes alone; by means of cul- 
 tivation on gelatin they were readily separated from the 
 stools. During his stay in India, in Egypt, and at Tou- 
 lon, Koch had examined over one hundred cases, and 
 other investigators confirmed his statements. Numer- 
 ous control observations made upon other diarrhoeic de- 
 jecta and upon normal stools were negative; the comma 
 bacillus was found in choleraic material only, or in mate- 
 rial contaminated by cholera. Soon other observers, 
 however, described comma-shaped organisms of non- 
 
SPIRILLUM CHOLERA ASIATICS. 569 
 
 choleraic origin; Finkler and Prior, for instance, found 
 them in the diarrhceic stools of cholera nostras, Deneke 
 in cheese, Lewis and Miller in saliva. All of these 
 organisms, however, differed in many respects from 
 Koch's comma bacillus, and since then it has been 
 proved that none of them was affected by the specific 
 serum of animals immunized to cholera; and gradually 
 the exclusive association of Koch's vibrio with cholera 
 
 FIG. 74. FIG. 75. 
 
 Contact smear of colony of Contact spirilla preparation from plate 
 
 cholera spirilla from agar. culture of cholera. X 800 diameters. 
 
 X 700 diameters. (DUNHAM.) (DUNHAM.) 
 
 became almost generally acknowledged. It is now re- 
 garded by bacteriologists everywhere to be the specific 
 cause of Asiatic cholera. 
 
 Morphology. Curved rods with rounded ends which 
 do not lie in the same plane, from 0.8// to 2/* in length 
 and about 0. 4// in breadth. The curvature of the rods 
 may be very slight, like that of a comma, or distinctly 
 marked, particularly in fresh unstained preparations 
 of full-grown individuals, presenting the appearance 
 of a half -circle. By the junction of two vibrios 
 S-shaped forms are produced, and under unfavorable 
 
570 BACTERIOLOGY. 
 
 conditions of growth they may develop into long, spiral 
 filaments, which may consist of numerous spiral turns 
 in which it is impossible to recognize any connection 
 with the individual elements of which they are made 
 up. In stained preparations the spiral character of the 
 long filaments is often obliterated, or nearly so. Under 
 favorable conditions of growth that is, when the 
 growth is rapid the short-curved or almost straight 
 forms are commonly observed (Figs. 74 and 75). In 
 old cultures involution forms are frequent. 
 
 Stains with the aniline colors usually employed, but 
 not as readily as many other bacteria; an aqueous solu- 
 tion of carbol-fuchsin is recommended as the most reli- 
 able staining agent with the application of a few minutes' 
 heat. It is decolorized by Gram's method. The motile 
 organs exhibit one or two long, fine, spiral flagellse 
 attached to one end of the rods. 
 
 Biological Characters. An aerobic (facultative ana- 
 erobic), liquefying, motile spirillum. Grows readily in 
 the ordinary culture media, best at 37 C., but also at 
 the room-temperature (22 C.); does not grow at a tem- 
 perature above 42 or below 8 C. Does not form 
 spores. 
 
 In gelatin plate cultures, at 22 C., at the end of 
 twenty-four hours, small, round, yellowish-white to yel- 
 low colonies may be seen in the depths of the gelatin, 
 which later grow toward the surface and cause liquefac- 
 tion of the medium, the colonies lying at the bottom of 
 the holes or pocket thus formed. The zone of lique- 
 faction, which increases rapidly, remains at first clear, 
 then becomes cloudy, mostly gray, as the result of the 
 growth of the colonies. In many cases after a time 
 concentric rings, which increase from day to day, appear 
 
SPIRILLUM CHOLERA ASIATICS. 571 
 
 in the zone of liquefaction. (See Figs 76 and 77.) 
 Examined under a low-power lens, at the end of sixteen 
 to twenty-four hours, the colonies appear as small, light 
 yellow, round, coarsely granular disks, with a more or 
 less irregular outline. In many cases at this stage an 
 
 FIG. 76. FIG. 77. 
 
 Cholera colonies in gelatin ; 
 Cholera colonies in gelatin; twenty-four thirty-six to forty-eight hours' 
 hours' growth. (DUNHAM.) growth. X about 30 diameters. 
 
 ill-defined halo is seen to surround the granular colony, 
 which by transmitted light has a peculiar reddish tint. 
 The older the colonies become the more the granular 
 structure increases, until a stage is reached when the 
 surface looks as if it were covered with little fragments 
 of broken glass (Koch). Liquefaction continues around 
 the colonies, their structure appears fissured and coarsely 
 granular in texture, and occasionally a hair-like border 
 is formed at the periphery (Fig. 78), or a gray trans- 
 parent zone, until the entire colony breaks up into frag- 
 ments. Sometimes the colonies may be retained as com- 
 pact masses in the zone of liquefaction, and then they are 
 
572 BACTERIOLOGY. 
 
 dark-yellow or brown in color, and forms occur which 
 are absolutely unlike the typical cholera colonies. In 
 gelatin stick cultures the growth is at first thread-like 
 and uncharacteristic. At the end of twenty-four to 
 thirty-six hours a small, funnel-shaped depression ap- 
 pears on the surface of the gelatin, which soon spreads 
 out in the form of an air-bubble above, while below 
 this is a whitish, viscid mass. Later, the funnel in- 
 creases in depth and diameter, and at the end of from 
 four to six days may reach the edge of the test-tube; 
 
 PIG. 78. 
 
 Cholera colony in gelatin. X 30 diameters. (DUNHAM.) 
 
 in from eight to fourteen days the upper two-thirds of 
 the gelatin is completely liquefied. (See Fig. 79 and 
 Fig. 31, page 230.) Freshly isolated cholera vibrios 
 liquefy gelatin more rapidly than old laboratory cul- 
 tures; a certain variation in the characteristic liquefac- 
 tion of the gelatin even in fresh cultures under some 
 circumstances should be borne in mind in making a 
 diagnosis. Such variations in cultural peculiarities 
 occur also with other bacteria, and only the sum of all 
 the characteristics taken together enables a positive 
 diagnosis to be established. 
 
 Upon the surface ofagar the comma bacillus develops 
 a moist, shining, grayish-yellow layer. Blood-serum 
 is rapidly liquefied at the temperature of the incu- 
 
SPIRILLUM CHOLERA ASIATICS. 
 
 573 
 
 bator. In bouillon the growth is rapid and abun- 
 dant; in the incubator at the end of ten to sixteen hours 
 the liquid is diffusely clouded, and on the surface a 
 wrinkled membranous layer is often formed. In gen- 
 
 FIG. 79. 
 
 A characteristic series of cholera cultures in gelatin ; one, two, three, four, 
 and six days' growth. (DUNHAM.) 
 
 eral the spirillum grows in any liquid containing a 
 small quantity of organic matter and having a slightly 
 alkaline reaction. An acid reaction of the culture me- 
 dium prevents its development, as a rule; but it has the 
 power of gradually accommodating itself to the pres- 
 ence of vegetable acids, and grows upon potatoes, in 
 the incubator only, which have a slight acid reaction. 
 Abundant development occurs in bouillon which has 
 been diluted with eight to ten parts of water and in 
 
574 BACTERIOLOGY. 
 
 simple peptone solution, and it has been shown by ex- 
 periment that it also multiplies to some extent in steril- 
 ized river or well-water, and preserves its vitality in 
 such water for several weeks or even months. Koch 
 found in his early investigations that rapid multiplica- 
 tion may occur upon the surface of moist linen, and also 
 demonstrated the presence of this spirillum in the foul 
 water of a tank in India which was used by the natives 
 for drinking purposes. 
 
 The comma bacillus belongs to the class of aerobic 
 organisms, inasmuch as it grows readily only in the pres- 
 ence of oxygen, and that it develops active motility 
 only when a certain amount of oxygen is present. It 
 does not grow in the total absence of oxygen, but a 
 small quantity of oxygen is all that is required for its 
 development, as in the intestines. 
 
 Temperature is also of considerable importance in the 
 growth of cultures. Active growth does not begin 
 until a temperature of 22 to 25 C. is reached, though 
 the optimum growth is between 30 and 40 C. 
 
 The vitality of cultures of the comma bacillus is 
 quickly destroyed by desiccation. If a culture be 
 spread on a cover-glass and exposed to the action of 
 the air at room-temperature the bacilli are dead at the 
 end of two or three hours, unless the layer of culture 
 is very thick, when it may take twenty-four hours or 
 more to kill all the bacilli. This fact indicates that 
 infection is not produced by means of dust or other dried 
 objects contaminated with cholera bacilli. The trans- 
 misson of these organisms through the air, therefore, 
 can only take place for short distances, as by a spray of 
 infectious liquids by mechanical means as, for instance, 
 
SPIRILLUM CHOLERA ASIATICS. 575 
 
 the breaking of waves in a harbor, on water-wheels, 
 etc., or in moist wash of cholera patients. 
 
 The cholera bacillus is also injuriously affected by the 
 abundant growth of saprophytic bacteria. It is true 
 that when associated with other bacteria, if present in 
 large numbers, and if the conditions for their develop- 
 ment are particularly favorable, the cholera bacillus 
 may at first gain the upper hand, as in the moist linen 
 of cholera patients, or in soil impregnated with chol- 
 era dejecta; but later, after two or three days, even in 
 such cases, the bacilli die off and other bacteria gradu- 
 ally take their place. Thus Koch found that the fluid 
 contents of privies twenty-four hours after the introduc- 
 tion of comma bacilli no longer contained the living 
 organisms; in Berlin canal-water they were not demon- 
 strable for more than six to seven days, as a rule. In 
 the dejecta of cholera patients they were found usually 
 only for a few days (one to three days), though rarely 
 they have been observed for twenty to thirty days, and 
 on one occasion for one hundred and twenty days. In 
 unsterilized water they may also retain their vitality for 
 a relatively long time ; thus in stagnant well-water they 
 have been found for eighteen days, and in an aquarium 
 containing plants and fishes, the water of which was 
 inoculated with cholera germs, they were isolated sev- 
 eral months later from the mud at the bottom. In 
 running river-water, however, they have not been ob- 
 served for over six to eight days. Even in cultures the 
 comma bacillus is one of the shorter-lived bacteria. 
 They have been observed, however, in pure bouillon 
 cultures for three to four months, and in agar cul- 
 tures for six months, and occasionally in one-year- 
 old cultures when they were protected from desicca- 
 
576 BACTERIOLOGY. 
 
 tion. In these they occurred only in involution 
 forms. 
 
 The comma bacillus is killed by exposure to moist 
 heat at 60 C. in ten minutes. The bacilli have been 
 found alive in ice kept for a few days, but ice which 
 has been preserved for several weeks does not contain 
 living bacilli. 
 
 Chemical disinfectants readily destroy the vital- 
 ity of cholera vibrios. For disinfection on a small 
 scale, as for washing the hands when contaminated with 
 cholera infection, a 0. 1 per cent, solution of bichloride 
 of mercury or a 2 to 3 per cent, solution of carbolic acid 
 may be used. For disinfection on a large scale, as for 
 the disinfection of cholera stools, strongly alkaline milk 
 of lime, according to PfuhFs experiments, is an excel- 
 lent agent. The wash of cholera patients, contaminated 
 furniture, floors, etc., may be disinfected by a solution 
 of 5 per cent, carbolic acid and soap-water. The dis- 
 infecting action of mineral acids, particularly of sul- 
 phuric acid, has been advantageously employed for the 
 disinfection of entire systems of water-works into which 
 cholera bacilli had gained access. 
 
 Pohl, Bujivid, and Dunham have shown that when 
 a small quantity of chemically pure sulphuric acid is 
 added to a twenty-four-hour bouillon culture of the 
 cholera bacillus containing peptone a reddish-violet 
 color is produced. Brieger separated the pigment 
 formed in this reaction the so-called cholera-red and 
 showed that it was indol, and that the reaction was 
 nothing more than the well-known indol reaction. Sal- 
 kowski and Petri then demonstrated that the cholera 
 bacilli produced in thin bouillon cultures, along with 
 indol, nitrites by reduction from the nitrates con- 
 
SPIRILLUM CHOLERA ASIATICS. 577 
 
 tained in small quantities in the culture media; and 
 showed that it is the setting free of nitric acid, upon 
 the addition of sulphuric acid to the culture, which 
 gives with indol the red body upon which the cholera 
 reaction depends. For a long time it was believed that 
 this nitroso-indol reaction was peculiar to the cholera 
 bacillus, and great weight was placed on it as a diag- 
 nostic test. It has since been shown, however, that 
 there are a number of other vibrios which, under sim- 
 ilar conditioDS as the cholera vibrio, give the same red 
 reaction. The reaction is, nevertheless, a constant and 
 characteristic peculiarity of this spirillum, and is of 
 unquestionable value. It is even more valuable as a 
 negative than as a positive test, as the absence of the 
 reaction enables one to say of a suspected organism that 
 it is not the cholera spirillum. There are, however, 
 certain precautions to be observed in its use. It has 
 been shown that the reaction may be absent, for in- 
 stance, when the culture contains either too much or 
 too little nitrate. It is, therefore, advisable not to em- 
 ploy a bouillon culture the composition of which is 
 uncertain, but a distinctly alkaline solution of peptone, 
 containing 1 per cent, pure peptone and 0.5 per cent, 
 of pure chloride of sodium (Dunham's solution). With 
 such a solution constant results can be obtained. 
 
 Pathogenesis. Since none of the lower animals is nat- 
 urally subject to cholera, nor has ever contracted the 
 disease during the prevalence of an epidemic or as the 
 result of the iugestion of food contaminated with chol- 
 eraic excreta, there is no reason to expect that inocula- 
 tions of pure cultures of the spirillum, either subcuta- 
 neously or by the mouth, will give rise in animals to a 
 typical attack of cholera. It has been shown, more- 
 
 37 
 
578 BACTERIOLOGY. 
 
 over, that the comma bacillus is extremely sensitive to 
 the action of acids, and is quickly destroyed by the acid 
 secretions of the stomach of man or the lower animals 
 when these secretions are normally produced. Despite 
 the small prospects of success, however, from animal 
 experiments, these have been undertaken again and 
 again, until finally a method was found by which at 
 least similar processes have been produced in test ani- 
 mals by inoculation of pure cultures of the cholera 
 vibrio. Koch sought to produce infection in guinea- 
 pigs per vias naturales by first neutralizing the con- 
 tents of the stomach with a solution of carbonate of 
 soda 5 c.c. of a 5 per cent, solution injected into the 
 stomach through a pharyngeal catheter and then after 
 a while administered through a similar catheter 10 c.c. 
 of a liquid into which had been put one or two drops 
 of a bouillon culture of the comma bacillus. The ani- 
 mal then receives a dose of 1 c.c. of tincture of opium 
 per 200 grammes of body-weight introduced into the 
 abdominal cavity, for the purpose of controlling the 
 peristaltic movements. As a result of this treatment 
 the animals are completely narcotized for about half an 
 hour, but recover from it without showing any ill effects. 
 On the evening of the same or following day the ani- 
 mal shows an indisposition to eat and other signs of 
 weakness, its posterior extremities become weak and 
 apparently paralyzed, and, as a rule, death occurs 
 within forty-eight hours with the symptoms of collapse 
 and fall of temperature. At the autopsy the small 
 intestine is found to be congested and filled with a 
 watery fluid containing the spirillum in great numbers. 
 Koch experimented in this way on about one hundred 
 guinea-pigs. These results, however, are somewhat 
 
SPIRILLUM CHOLERA ASIATICS. 579 
 
 weakened by the fact that experiments made with 
 some other bacteria viz., those isolated by Finkler and 
 Prior, Deneke, and Miller, and morphologically similar 
 to the comma bacillus of Koch occasionally pro- 
 duced death when introduced in the same way into 
 the small intestines of guinea-pigs; but while only 
 twelve out of fifty-one animals died when injected 
 with cultures of these last-mentioned bacteria, in the 
 cholera experiments there was 90 per cent, of deaths, 
 and when larger doses were administered all of the 
 animals died. Control experiments made with many 
 other bacteria gave negative results. Intraperitoneal 
 injections of larger quantities of pure cholera cultures 
 also often produce death in rabbits and mice. 
 
 There are several cases on record which furnish the 
 most satisfactory evidence that the cholera bacillus is 
 able to produce the disease in man. In 1884, a student 
 in Koch's laboratory in Berlin, who was taking a course 
 on cholera, became ill with a severe attack of cholera. 
 At that time there was no cholera in Germany, and the 
 infection could not have been produced in any other 
 way than through the cholera cultures which were being 
 used for the instruction of students. In 1892, Petten- 
 kofer and Emmerich experimented on themselves by 
 swallowing small quantities of fresh cholera cultures 
 obtained from Hamburg. Pettenkofer was affected 
 with a mild attack of cholerine or severe diarrhoea, from 
 which he recovered in a few days without any serious 
 effects; but Emmerich became very ill. On the night 
 following the infection he was attacked by frequent evac- 
 uations of the characteristic rice-water type, cramps, 
 tympanitis, and great prostration. His voice became 
 hoarse, and the secretion of urine was somewhat dimiu- 
 
580 BACTERIOLOGY. 
 
 ished, this condition lasting for several days. In both 
 cases the cholera spirillum was obtained in pure cul- 
 ture from the dejecta. Another instance is reported 
 by Metschnikoff, in Paris, of a man who became 
 infected experimentally. In this case the algid stage 
 of cholera was produced, with complete suppression of 
 urine, cramps in the legs, contraction of the extrem- 
 ities, and collapse, the man's life being saved only with 
 difficulty. Finally, there is the case of Dr. Oergel, 
 of Hamburg, who accidentally, while experimenting on 
 a guinea-pig, had some of the infected peritoneal fluid 
 to squirt into his mouth. He was taken ill and died 
 a few days afterward of typical cholera, though at the 
 time of his death there was no cholera in the city. 
 These accidents and experiments would certainly seem 
 to prove conclusively the capability of pure cholera cul- 
 tures of producing the disease; and yet Strickler and 
 Hasterlick (Vienna, 1893) report negative results from 
 experiments on the human subject. This only goes to 
 show, however, that in cholera, like other infectious 
 diseases, there is an individual susceptibility. It is 
 also possible that the cultures used for experimentation 
 may have lost in virulence, as cholera cultures are so 
 liable to do when kept for any length of time. 
 
 Cholera Toxin. Koch was the first to assume, as the 
 result of his investigations, that the severe symptoms 
 of the algid stage of cholera were due to the effects of 
 a toxin produced by the growth of the comma bacillus 
 in the intestines. 
 
 In 1892, Pfeiffer published an account of his elabo- 
 rate researches relating to the cholera poison. He finds 
 that recent aerobic cultures of the cholera spirillum con- 
 tain a specific toxic substance which is fatal to guinea- 
 
SPIRILLUM CHOLER^E ASIATICS. 581 
 
 pigs in extremely small doses. This substance stands in 
 close relation with the bacterial cells, and is perhaps an 
 integral part of them. The spirilla may be killed by 
 chloroform, thymol, or by desiccation without apparent 
 injury to the toxic power of this substance. It is de- 
 stroyed, however, by absolute alcohol, by concentrated 
 solutions of neutral salts, and by the boiling tempera- 
 ture. Secondary toxic products are formed which have 
 a similar physiological action, but are from ten to twenty 
 times less potent. Similar toxic substances were ob- 
 tained by Pfeiffer from cultures of Finkler and Prior's 
 spirillum and from the spirillum Metschnikovi. 
 
 Cholera Immunity. Koch found in his animal experi- 
 ments that recovery from an intraperitoneal infection 
 with small doses of living cholera vibrios produced a 
 certain immunity against larger doses, though the ani- 
 mals inoculated were not very much more resistant to 
 the cholera poison than they were originally. In 1892 
 Lazarus observed that the blood-serum of persons who 
 had recently recovered from an attack of cholera pos- 
 sessed the power of preventing the development in 
 guinea-pigs of cholera bacilli, which in these animals 
 are rapidly fatal when injected intraperitoneally ; while 
 the serum of healthy individuals had no such effect. 
 He attributed this to the presence in the serum of con- 
 valescents from cholera of antitoxic substances which 
 neutralized the poison produced by the cholera vibrios, 
 in the same manner as the antitoxins of diphtheria and 
 tetanus. Pfeiffer, on the other hand, maintained that 
 the serum contained bactericidal substances which killed 
 the bacilli so rapidly when injected into the animal that 
 they did not have time to produce their specific poison, 
 and that thus the death of the animal was prevented. 
 
582 BACTERIOLOGY. 
 
 The serum is now known to be feebly antitoxic and 
 strongly bactericidal. This specific change in the blood 
 is observed to take place from eight to ten days after 
 the termination of an attack of cholera and reaches its 
 maximum during the fourth week of convalescence, 
 after which it declines rapidly and disappears entirely 
 in about two or three months. Similar antitoxic or 
 bactericidal substances exist also in the serum of guinea- 
 pigs, rabbits, and goats, when these animals are immu- 
 nized artificially against cholera by subcutaneous or 
 intraperitoneal injections of living or dead cultures. 
 These specific substances present in the blood of chol- 
 era-immune men and animals act only upon organisms 
 similar to those with which they were infected ; but, 
 as Pfeiffer showed, this specific relation, which is found 
 to exist between the antibacterial and protective sub- 
 stances produced during immunization and the bacteria 
 employed to immunize the animals, is not confined 
 alone to cholera. The discovery, moreover, of this 
 specific reaction of the blood-serum of immunized men 
 and animals when brought in contact with the spirilla, 
 has given us an apparently reliable means of distin- 
 guishing the cholera from all other vibrios, and the 
 disease cholera from other similar affections, both of 
 which have proved to be of great value, particularly in 
 obscure or doubtful cases, in which heretofore the only 
 method of differential diagnosis available viz., by 
 cultural tests was often unsatisfactory. 
 
 Cholera in man is an infective process of the epithe- 
 lium of the intestine, in which the spirilla clinging to 
 and between the epithelial cells produce a partial or 
 entire necrosis and final destruction of the epithelial 
 covering, which thus renders possible the absorption of 
 
SPIRILLUM CHOLERA ASIATICS. 583 
 
 the cholera toxin formed by the growth of the spirilla. 
 The larger the surface of the mucous membrane infected, 
 the more luxuriant the development of bacilli and the 
 production of toxin, the more pronounced will be the 
 poisoning, ending fatally in a toxic paralysis of the 
 circulatory and thermic centres. On the other hand, 
 however, there may be cases where, in spite of the large 
 number of cholera bacilli present in the dejecta, severe 
 symptoms of intoxication may be absent. In such cases 
 the destruction of epithelium is then either not pro- 
 duced or so slight that the toxic substance absorbed is 
 not in sufficient concentration to give rise to the algid 
 stage of the disease, or for some reason the toxin is 
 not produced to any extent by the spirilla. In no 
 stage of the disease are living cholera spirilla found in 
 the organs of the body or in the secretions. 
 
 From this fact and other known properties of the 
 cholera bacillus, which have already been referred to, 
 several important deductions may be made with regard 
 to the mode of transmission of cholera infection. In 
 the first place the bacilli evidently leave the bodies of 
 cholera patients, chiefly in the dejections during the early 
 part of the disease (they have usually disappeared after 
 the fourth to the fourteenth day), and only these dejec- 
 tions, therefore, and objects contaminated by them, such 
 as bed and body wash, floors, vaults, soil, well-water 
 and river-water, etc., can be regarded as possible sources 
 of infection. There is a special limitation even in 
 these sources of infection, owing to the fact that this 
 spirillum is so easily destroyed by desiccation and 
 crowded out by saprophytic organisms. Thus, as a 
 rule, only fresh dejections and freshly contaminated 
 objects are liable to convey infection; after they have 
 
584 BACTERIOLOGY. 
 
 become completely dry there is little danger. Farther, 
 we must conclude from the distribution of the cholera 
 bacillus in the body and from experiments upon ani- 
 mals that the commonest mode of infection is by way 
 of the mouth, and chiefly by means of water used for 
 drinking purposes, for the preparation of food, etc. In 
 recent times cholera spirilla have been found not infre- 
 quently in water (wells, water-mains, rivers, harbors, 
 and canals) which have become contaminated by the 
 dejections of cholera patients. 
 
 But, like other infectious diseases, not everyone who 
 is exposed to infection is attacked by cholera. The 
 bacilli have been found during cholera epidemics in the 
 dejections of healthy individuals without any patholog- 
 ical symptoms. Abel and Claussen found, for example, 
 in 14 out of 17 persons belonging to the families of 7 
 cholera patients, cholera vibrios, in some of them for a 
 period of fourteen days. In Hamburg there were 28 
 such cases of healthy choleraic individuals with abso- 
 lutely normal stools. It is evident, therefore, that an 
 individual susceptibility is requisite to produce the dis- 
 ease. In the normal healthy stomach the hydrochloric 
 acid of the gastric secretions may destroy the spirilla; 
 and, finally, the normal vital resistance of the tissue 
 cells to the action of the cholera poison may be taken 
 into consideration. According to the greater or less 
 power of this vital resistance of the body the same 
 infectious matter may give rise to no disturbance what- 
 ever, a slight diarrhoea, or it may lead to serious results. 
 Furthermore, it may be accepted as an established fact, 
 that recovery from one attack of cholera produces per- 
 sonal immunity to a second attack for a considerable 
 length of time. This does not appear to depend upon 
 
SPIRILLUM CHOLERA ASIATICS. 585 
 
 the severity of the attack; for cases are recorded of 
 persons who were apparently not sick at all, and yet 
 in whom an acquired immunity was produced. How 
 long this immunity lasts is not positively known, but 
 probably for a month or more, so that the same person 
 is not likely to be taken ill again with cholera during 
 an epidemic. 
 
 Within the last few years Haffkine, in India, has 
 succeeded in producing an artificial immunity against 
 cholera infection by means of subcutaneous injections of 
 cholera cultures. In over 200,000 persons whom he 
 has inoculated the results obtained would undoubtedly 
 seem to show a distinct protective influence in the pre- 
 ventive inoculations. And Kolle has found that the 
 blood-serum of persons inoculated by Haffkine' s method 
 gave a similar reaction to that of persons who had re- 
 covered from cholera. 
 
 On the other hand, we may take it for granted that 
 susceptibility to cholera may be acquired or increased. 
 For instance, there is no doubt that gastric and intes- 
 tinal disorders produced by overeating, etc., may act 
 as contributing causes to the disease. Other predispos- 
 ing causes are general debility from poverty, hunger, 
 disease, etc. 
 
 Varieties and Variations of the Cholera Bacillus. Cun- 
 ningham, as a result of researches made in Calcutta 
 (1891), arrives at the conclusion that Koch's comma 
 bacillus cannot be accepted as the specific etiological 
 agent in this disease: First, as in many undoubted cases 
 of cholera he has failed to find comma bacilli; second, 
 because in one case he found three different species; 
 and, third, because in one case the indol reaction could 
 not be obtained. Since Cunningham's investigations 
 
586 BACTERIOLOGY. 
 
 many other observers have reported finding varieties of 
 the comma bacillus. Only a few of these can be here 
 mentioned, of which there is any certainty that they 
 were derived from true cholera cases. Thus Friedreich 
 has accurately described and photographed a series of 
 forms which, however, vary but little from the original 
 type. But more interesting than the reports of varie- 
 ties are the observations of the variability of the cholera 
 bacillus. Claussen, in Esmarch' s institute, isolated from 
 fresh cholera stools vibrios which presented in plate 
 cultures a different appearance of the colonies, which 
 showed a tendency to disintegrate and having an irreg- 
 ular border. The nitrosoindol reaction was absent; 
 bouillon cultures were non-pathogenic to guinea-pigs, 
 and stick cultures grew slowly and uncharacteristically. 
 On repeated inoculation, however, a guinea-pig died 
 after the injection of 1 c.c. of a bouillon culture; in 
 the peritoneal exudate and even in the blood character- 
 istic bacilli were found and the cultures gave the indol 
 reaction. Celli and Santori, in Rome (1893), isolated 
 from the stools of many typical cholera cases a vibrio 
 which they called vibrio romanus, which was non-patho- 
 genic for animals, gave no indol reaction, did not coag- 
 ulate milk, and at 37 C. grew neither in bouillon nor 
 on agar. After cultivation for nine months it gave the 
 indol reaction and grew at 37 C., but was still almost 
 non-pathogenic. Bordoni-Uffreduzzi and Abb culti- 
 vated from a typical cholera case a short vibrio which 
 liquefied gelatin very rapidly and presented an abnor- 
 mal growth, and gave a yellow growth on potato, but 
 which on continued cultivation became more and more 
 like the cholera spirillum. Variations even greater than 
 occur in these varieties of cholera spirilla are met with 
 among diphtheria bacilli. 
 
SPIRILLUM CHOLERJE ASIATICS. 587 
 
 Plan of Procedure for the Biological Diagnosis of the 
 Cholera Vibrio. A. Dejecta (fluid) or intestinal con- 
 tents of a cholera patient or cholera suspect. 
 
 1. Use one drop (one platinum loop) for gelatin plate- 
 cultures, making two dilutions. Do this in duplicate 
 or triplicate. Cultivate at 22 C. 
 
 2. Inoculate a couple of bouillon tubes and a couple 
 of Dunham's 1 per cent, peptone solution with one drop 
 each, and place them in the incubator (37 to 38 C.) 
 for six to eight hours. 
 
 3. Examine a drop of the dejecta in the hanging drop. 
 
 4. Examine a drop of the dejecta in stained cover- 
 glass preparation. 1 
 
 5. Make gelatin plates from one drop taken from 
 the surface of each of the bouillon and peptone solution 
 tubes and cultivate at 22 C. 
 
 6. As soon as the plates (see 1 and 5) are sufficiently 
 developed (thirty-six to forty-eight hours) fish the 
 suspected cholera colonies and use the material for the 
 following procedures : 
 
 7. Inoculate six or eight peptone tubes (1 per cent, 
 peptone, 0.5 per cent. NaCl in distilled water) and 
 place them at once in the incubator. Note the time. 
 
 8. Examine hanging drop for form, size, and motility 
 (and arrangement). 
 
 9. Make stained cover-glass preparations and exam- 
 ine. 
 
 1 These direct microscopical examinations of the intestinal contents are, as 
 a rule, very unsatisfactory, at least in those in which the symptoms are not 
 marked. In a few the spirals will make up from 50 to 100 per cent, of the 
 bacteria present. In most of the cases during the last epidemic in New York 
 Dunham found abundance of columnar epithelium from the intestinal mucous 
 membrane, numerous straight, thick bacilli, and only a few curved bacilli or 
 segments of spirals too few to identify. Plate cultures from these showed 
 from 20 to 80 per cent, of all the colonies developing to be cholera spirilla. 
 
588 BACTERIOLOGY.. 
 
 10. Then try indol reacton with the same tubes. 
 
 11. While these tubes are incubating use material 
 from the suspected colonies on the plates (1 and 5) for 
 hanging drop cultures. 
 
 12. Meanwhile make stained cover-glass prepara- 
 tions from other colonies of suspected cholera on the 
 plates (1 and 5). 
 
 13. Make gelatin tube cultures from colonies on 
 plates (1 and 5). 
 
 14. Make gelatin tube cultures daily for five or six 
 days, to study shape of growth along the line of punc- 
 ture to preserve the culture. 
 
 B. Suspected water. 
 
 Add to 500 c.c. or 1 litre of the water to be exam- 
 ined in a flask half -full enough peptone-salt solution 
 (20 per cent, peptone and 10 per cent. NaCl) to make 
 a 1 per cent, solution of peptone. Then proceed as in A. 
 
 PFEIFFER'S SERUM REACTION. All authors now 
 agree that the differentiation of the cholera vibrio from 
 other similar vibrios cannot always be made by the cul- 
 tural method, nor is the usual inoculation of animals 
 sufficient. For this purpose serum is employed either 
 by making intraperitoneal injections of a surely fatal 
 dose of the suspected spirillum along with the serum 
 of animals immunized to undoubted cholera cultures, 
 or by watching the action of the spirillum in the hang- 
 ing drop when added to a dilution of the above men- 
 tioned serum, so as to note whether immobilization 
 and clumping occurred. 
 
CHAPTER XXXI Y. 
 
 SPIRILLA RESEMBLING THAT OF CHOLERA THE 
 SPIRILLUM OF RELAPSING FEVER. 
 
 SPIRILLUM OF FINKLER AND PRIOR. 
 
 FINKLER and Prior, in 1884, obtained from the feces 
 of patients with cholera nostras, after allowing the 
 dejecta to stand for some days, a sprillum which is 
 of interest mainly because it simulates the comma 
 bacillus of Koch, but differs from it in several cultural 
 peculiarities. 
 
 Morphology. More or less curved rods with an aver- 
 age length of 2.4/z and a breadth of 0.4 to 0.6//, some- 
 what longer and thicker than the spirillum of Asiatic 
 cholera and not so uniform in diameter, the central por- 
 tion being usually wider than the pointed ends; forms 
 sometimes S-shaped and spiral filaments, which are not 
 as numerous, and are usually shorter than those formed 
 by the cholera spirillum. Examined in the hanging 
 drop they are seen to be actively motile. A single 
 flagellum is attached to one end of the curved segments. 
 In unfavorable media involution forms are common. 
 
 Stains with the usual aniline colors. 
 
 Biological Characters. An aerobic and facultative 
 anaerobic, liquefying spirillum. Does not form spores. 
 Upon gelatin plates small, white, punctiforrn colonies 
 are developed at the end of twenty-four hours, which 
 
590 
 
 BACTERIOLOGY. 
 
 under the microscope are seen to be finely granular and 
 yellowish or yellowish-brown in color; the colonies are 
 round with more sharply denned border, less coarsely 
 granular and darker in color than those of the comma 
 bacillus. Liquefaction of the gelatin around these col- 
 onies progresses rapidly, and at the end of forty-eight 
 hours is usually complete in plates where they are numer- 
 The surface colonies sink quickly into the gelatin 
 
 ous. 
 
 FIG. 80. 
 
 Spirillum of Finkler and Prior. X 1100 diameters. 
 
 and present a darker peripheral zone. The differenti- 
 ation between the Finkler and Prior and cholera spirilla 
 can readily be made in the earlier stages of their growth. 
 Later on, and especially when the cholera colonies are 
 the older, the diagnosis is not so easy. In gelatin stick 
 cultures liquefaction progresses much more rapidly than 
 in similar cultures of the cholera spirillum, and a stock- 
 ing-shaped pouch of liquefied gelatin is already seen 
 after forty-eight hours, which is filled with a cloudy 
 liquid. There is no bubble formation. The liquefac- 
 
SPIRILLUM OF FINKLER AND PRIOR. 591 
 
 tion increases, and in twenty-four hours more reaches 
 the sides of the tube in the upper part of the medium ; 
 by the end of the week the gelatin is usually completely 
 liquefied. Upon the surface of the liquefied medium a 
 whitish film is seen. Upon agar there is a somewhat 
 more luxuriant growth than with the cholera vibrio; a 
 slimy, whitish-yellow layer covering the entire surface 
 is quickly developed. Upon potato this spirillum grows 
 at the room-temperature and produces a slimy, grayish- 
 yellow, glistening layer which soon extends over the 
 entire surface. The cholera spirillum does not grow at 
 room-temperature, and in the incubator produces a thin, 
 brownish layer. Cultures of the Finkler and Prior 
 spirillum give off a strong putrefactive odor; in media 
 containing sugar, according to Buchner, an acid reac- 
 tion is produced as a result of their growth; they do 
 not form indol in peptone solutions; and they have a 
 greater resistance to desiccation than the cholera spiril- 
 lum. The absence of agglutination with a dilution of 
 the serum of an animal immunized to the cholera 
 spirillum is a valuable differential sign. 
 
 Pathogenesis. When injected into the stomach of 
 guinea-pigs, after previous injection of a soda solution 
 and opium, the Finkler and Prior spirillum is some- 
 what pathogenic for these animals; but a smaller pro- 
 portion die from such injections than from those of 
 the fresh cultures of cholera. At the autopsy the in- 
 testine is pale, and its watery contents, which contain 
 the spirilla in great numbers, have a putrefactive odor. 
 
 This organism has been found in the dejections of 
 some healthy persons and of persons affected with diar- 
 rhoea or cholera nostras. It does not seem to have any 
 etiological relation, however, with this disease in man, 
 
592 BACTERIOLOGY. 
 
 as since its disco verey, though repeatedly sought for, it 
 has seldom been found by subsequent investigators. 
 
 In 1884, Miller observed a curved bacillus in a hollow 
 tooth, which from its behavior in microscopical prepa- 
 rations, in cultures and animal experiments, is probably 
 identical with the Finkler and Prior spirillum; and 
 other very similar spirilla have been found by others. 
 
 DENEKE'S CHEESE SPIRILLUM. 
 
 Obtained by Deneke (1885) from old cheese, but since 
 then rarely met with. Morphologically and culturally 
 it shows greater similarity to Koch's comma bacillus 
 than the Finkler and Prior spirillum, but can be read- 
 ily differentiated from it also. 
 
 Morphology. Curved rods and long spiral filaments, 
 somewhat more slender than the cholera spirillum, the 
 turns in the spiral threads being lower and closer 
 together. Has a single flagelluin attached to one end. 
 
 Stains with the usual aniline colors. 
 
 Biological Characters. An aerobic and facultative 
 anaerobic, liquefying, motile spirillum. Does not form 
 spores. Upon gelatin plates small, punctiform colonies 
 are formed at the end of twenty-four hours, which when 
 slightly magnified are seen to be circular in shape, with 
 sharply defined border and of a greenish-brown color 
 in the centre and paler toward the margins. Later, 
 when liquefaction has commenced, the sharp contour is 
 often lost. The liquefaction progresses more rapidly 
 than with the cholera bacillus, but not so energetically 
 as with the spirillum of Finkler and Prior. In gelatin 
 stick cultures after forty-eight hours a stocking-like 
 pouch is developed, the spirilla sinking to the bottom 
 
SPIRILL UM METSCHNIKO VI. 593 
 
 of the liquefied gelatin in the form of a coiled mass, 
 while a thin, yellowish layer forms upon the surface ; 
 complete liquefaction usually occurs in about two weeks. 
 Upon the surface of agar a thin, yellowish layer is de- 
 veloped. Blood-serum is rapidly liquefied. The indol 
 reaction in peptone solutions is absent. 
 
 Pathogenesis. Somewhat pathogenic for guinea-pigs 
 when inoculated by Koch's method with previous ad- 
 ministration of soda solution and laudanum. 
 
 It is probable that this organism, from the locality in 
 which it is found and its behavior, is a saprophyte. 
 
 SPIRILLUM METSCHNIKOVI. 
 
 Discovered in 1888, in Odessa, by Gamalei'a in the 
 intestinal contents of fowls dying of an infectious dis- 
 ease which prevails in certain parts of Russia during 
 the summer months, and which presents symptoms 
 resembling fowl-cholera. GamaleiVs experiments show 
 that this organism is the cause of the disease mentioned. 
 It has since been found by Pf uhl and Pfeiffer in the water 
 of the Spree at Berlin, and in the Lahn by Kutchler. 
 
 Morphology. Morphologically this spirillum is almost 
 identical with the cholera spirillum; it forms curved 
 rods with rounded ends and spiral filaments, the curved 
 segments being somewhat thicker, shorter, and often 
 more decidedly curved than the comma bacillus. In 
 the blood of inoculated pigeons the diameter is some 
 times twice as great as that of the cholera spirillum, 
 and almost coccus-like forms are often found. A single, 
 long, undulating flagellum is attached to one end of the 
 spiral filaments or curved rods. In old cultures beau- 
 tiful long spiral filaments may be seen. 
 
 38 
 
594 BACTERIOLOGY. 
 
 Stains with the usual aniline colors, but not by 
 Gram's method. 
 
 Biological Characters. An aerobic, liquefying, motile 
 spirillum. Upon gelatin plates the vibrio Metschnikovi 
 grows considerably faster than the cholera vibrio; small, 
 white punctiform colonies are developed at the end of 
 twelve hours; these rapidly increase in size and cause 
 liquefaction of the gelatin within twenty-four to thirty 
 hours. At the end of three days large, saucer-like 
 areas of liquefaction may be seen, the contents of which 
 are turbid, as a rule. Under the microscope the 
 colonies appear as yellowish-brown granular masses, 
 which are in active movement, and the margins are 
 surrounded by a border of radiating filaments. In 
 gelatin stick cultures the growth is almost twice as rapid 
 as the cholera bacillus. In bouillon at 37 C. devel- 
 opment is very rapid, and the liquid becomes clouded 
 and opaque, and a thin, wrinkled film forms upon the 
 surface. On the addition of pure sulphuric acid to 
 twenty-four-hour peptone cultures a distinct nitroso- 
 indol reaction is produced. Milk is coagulated and 
 acquires a strongly acid reaction. The spirillum does 
 not clump and lose its motility with the diluted serum 
 from an animal immunized to cholera. 
 
 Pathogenesis. The vibrio Metschnikovi is pathogenic 
 for fowls, pigeons, and guiaea-pigs. A small quantity 
 of a culture injected into the breast muscles of chickens 
 and pigeons causes their death with the local and gen- 
 eral symptoms of fowl cholera. At the autopsy the 
 most constant appearance is hypersemia of the entire 
 alimentary canal. A grayish-yellow liquid, more or 
 less mixed with blood, is found in considerable quan 
 tity in the small intestine; the spleen is not enlarged, 
 
SPIRILL UM METSCHNIKO VI. 595 
 
 rather diminished in size, and the organs generally are 
 normal in appearance. In the watery fluid large num- 
 bers of spirilla are found; they are found in the blood 
 of pigeons always, but only in the blood of young fowls. 
 A few drops of a pure culture inoculated subcutane- 
 ously in pigeons cause their death in eight to twelve 
 hours. According to Gamale'ia, fowls may be infected 
 by giving them food contaminated with the cultures of 
 the spirillum, but pigeons resist infection in this way. 
 Infection may also be produced by way of the mouth 
 by Koch's method, a solution of carbonate of soda and 
 laudanum having been previously administered. The 
 animals then die with symptoms of acute gastro- 
 enteritis; the intestines are found to be highly inflamed 
 and their liquid contents contain numerous spirilla. 
 In contradistinction to the pathogenic virulence of 
 these spirilla for pigeons and guinea-pigs, the cholera 
 spirillum is much less pathogenic. Pigeons are not 
 killed by the intramuscular inoculation of pure fresh 
 cultures of the vibrio cholerse. Gamale'ia has claimed 
 that by passing the cholera spirillum of Koch through 
 a series of pigeons, by successive inoculation, its path- 
 ogenic power is greatly increased, and that when steril- 
 ized cultures of this virulent variety of the comma 
 bacillus are injected into pigeons they become immune 
 against the pathogenic action of the vibrio Metschni- 
 kovi, and the reverse. But Pfeiffer has shown that 
 this statement is not founded upon fact. The patho- 
 genic action of the vibrio Metschnikovi upon pigeons 
 and guinea-pigs, producing in these animals general 
 septicsemia and death, is, therefore, a characteristic point 
 of difference between this and the spirillum of Asiatic 
 cholera. 
 
596 BACTERIOLOGY. 
 
 Within recent years numerous other vibrios, the so- 
 called " water vibrios," have been found while looking 
 for the cholera bacillus, the identity or variation of 
 which from the spirillum of cholera it has been ex- 
 tremely difficult to determine, as morphological, biolog- 
 ical, and pathogenical examinations have led to no posi- 
 tive results. 
 
 SPIRILLUM OBERMEIERI (Spirillum of Relapsing Fever). 
 
 First observed by Obermeier (1873) in the blood of 
 persons suffering from relapsing fever. 
 
 Morphology. Long, slender, flexible, spiral, or wavy 
 filaments, with pointed ends, from 16//to40// in length 
 and from one-quarter to one-third the thickness of the 
 cholera spirillum. 
 
 Stains readily with the ordinary aniline colors, es- 
 pecially with fuchsin, Loffler's solution of methylene- 
 blue and Bismarck-brown. Does not stain by Gram's 
 method. 
 
 Biological Characters. A motile spirillum which 
 has not been cultivated in artificial media. Spore 
 formation has not been demonstrated. In fresh prep- 
 arations from the blood the spirillum exhibits active 
 progressive movements accompanied by very rapid 
 rotation in the long axis of the spiral filaments or by 
 undulating movements. The spirilla are found exclu- 
 sively in the blood and spleen of persons suffering from 
 relapsing fever, never in the secretions, and only during 
 the fever, not in the intermissions, or at most singly at 
 the beginning of an attack. When preserved in blood- 
 serum or a 0. 5 per cent, solution of salt they continue 
 to exhibit active movements for a considerable time. 
 
SPIRILLUM OBERMEIERI. 597 
 
 Efforts to cultivate this spirillum in artificial culture 
 media have thus far been unsuccessful, although Koch 
 has observed an increase in the length of the spirilla 
 and the formation of a tangled mass of filaments. 
 
 Pathogenesis. Inoculation experiments have been suc- 
 cessfully made on man and monkeys. Monkeys when 
 inoculated with human blood containing the spirilla 
 take sick after about three and a half days, but show 
 only the initial febrile attack; no relapses, such as are 
 characteristic of the disease in man. The organisms 
 are found in the blood, and at the height of the fever 
 in the other organs on autopsy. Extirpation of the 
 spleen renders the disease more dangerous for monkeys. 
 
 Blood from one animal, taken during the attack, in- 
 duces a similar febrile paroxysm when inoculated in 
 another monkey. One attack does not preserve the 
 animal experimented on from a second attack (Koch 
 and Carter). 
 
 Very little is known bacteriologically of this disease, 
 but from the fact that these peculiarly shaped organ- 
 isms are constantly and exclusively found in relapsing 
 fever, and that the disease can be transmitted to mon- 
 keys by inoculating them with the blood containing the 
 spirilla, it may be assumed that they are the true cause 
 of the disease. 
 
CHAPTER XXXV. 
 
 GLANDERS BACILLUS. 
 
 BACILLUS MALLEI (Bacillus of Glanders). 
 
 THIS bacillus was discovered and proved to be the 
 cause of glanders by isolation in pure culture and com- 
 munication to animals by inoculation, by several bacte- 
 riologists almost at the same time (1882), viz., by the 
 investigations of Loftier, Schiitz, Israel, Bouchard, 
 Charrin, Weichselbaum, Kauzfeld, and Kitt. It is 
 found in the recent nodules in animals affected with 
 glanders, and in the discharge from the nostrils, pus 
 from the specific ulcers, etc., and occasionally in the 
 blood. 
 
 Morphology. Small bacilli with rounded or pointed 
 ends, from 0.25/* to 0.4^ broad and from 1.5// to 3// 
 long; usually single, but sometimes united in pairs, or 
 growing out to long filaments, especially in potato cul- 
 tures. Frequently breaks up into short, almost coccus- 
 like elements (Fig. 81). 
 
 The bacillus mallei stains with difficulty with the 
 aniline colors, best when the aqueous solutions of these 
 dyes are made feebly alkaline; it is decolorized by 
 Gram's method. This bacillus presents the peculiarity 
 of losing very quickly in decolorizing solutions the color 
 imparted to it by the aniline staining solutions. For 
 this reason it is difficult to stain in sections. Loffier 
 recommends his alkaline methylene-blue solution for 
 
GLANDERS BACILLUS. 599 
 
 staining sections, and for decolorizing a mixture con- 
 taining 10 c.c. of distilled water, 2 drops of strong 
 sulphuric acid,, and 1 drop of a 5 per cent, solution of 
 oxalic acid; thin sections to be left in this acid solution 
 for five seconds. 
 
 FIG. 81. 
 
 Glanders bacilli. Agar culture. X 1000 diameters. 
 
 Biological Characters. An aerobic, non-motile bacil- 
 lus, whose molecular movements are so active that they 
 have often been taken for motility. It grows on vari- 
 ous culture media at 37 C. Development takes place 
 slowly at 22 C. and ceases at 43 C. It does not form 
 spores. Exposure for ten minutes to a temperature of 
 55 C., or for five minutes to a 3 to 5 per cent, solution 
 of carbolic acid, or for two minutes to a 1 : 5000 solu- 
 tion of mercuric chloride, was effectual in destroying 
 its vitality. As a rule, the bacilli do not grow after 
 having been preserved in a desiccated condition for a 
 week or two; in distilled water they are also quickly 
 destroyed. The bacillus does not grow in infusions of 
 
BA CTERIOL OOY. 
 
 hay, straw, or horse-manure, and it is doubtful whether 
 it finds conditions in nature favorable to a saprophytic 
 existence. It grows well in the incubating oven on 
 glycerin-agar. Upon this medium at the end of twenty- 
 four to forty-eight hours, whitish, transparent colonies 
 are developed, which in six or seven days may attain a 
 diameter of 7 or 8 mm. On blood-serum a moist, 
 opaque, slimy layer develops, which is of a yellowish- 
 brown tinge. The growth on cooked potato is especially 
 characteristic. At the end of twenty-four to thirty-six 
 hours at 37 C. a moist, yellow, transparent layer de- 
 velops; this later becomes deeper in color, and finally 
 takes on a reddish-brown color, and the potato about it 
 acquires a greenish-yellow tint. In bouillon it causes 
 diffuse clouding, with ultimately the formation of a 
 more or less ropy tenacious sediment. Milk is coagu- 
 lated with the production of acid. It grows on media 
 possessing an acid reaction, and both with and without 
 oxygen. 
 
 Pathogenesis. The bacillus of glanders is pathogenic 
 for a number of animals. Among those which are most 
 susceptible are horses, asses, guinea-pigs, cats, dogs, 
 ferrets, moles, and field mice; sheep, goats, swine, rab- 
 bits, white mice, and house mice are much less suscep- 
 tible; cattle are immune. Man is susceptible, and in- 
 fection not infrequently terminates fatally. 
 
 When pure cultures of the bacillus mallei are injected 
 into horses and other susceptible animals true glanders 
 is produced. The disease is characterized in the horse 
 by the formation of ulcers upon the nasal mucous mem- 
 brane, which have irregular, thickened margins, and 
 secrete a thin, virulent mucus; the submaxillary lym- 
 phatic glands become enlarged and form a tumor, which 
 
GLANDERS BACILLUS. 601 
 
 is often lobulated; other lymphatic glands become in- 
 flamed, and some of them suppurate and open exter- 
 nally, leaving deep, open ulcers; the lungs are also 
 involved, and the breathing becomes rapid and irreg- 
 ular. In farcy, which is a more chronic form of the 
 disease, circumscribed swellings, varying in size from 
 a pea to a hazel-nut, appear on different parts of the 
 body, especially where the skin is thinnest; these sup- 
 purate and leave angry- looking ulcers with ragged 
 edges, from which there is an abundant purulent dis- 
 charge. The bacillus of glanders can easily be obtained 
 in pure cultures from the interior of suppurating nod- 
 ules and glands which have not yet opened to the sur- 
 face, and the same material will give successful results 
 when inoculated into susceptible animals; but the dis- 
 charge from the nostrils or from an open ulcer contains 
 comparatively few bacilli, and these being associated 
 with other bacteria which grow more readily on the 
 culture media than the bacillus mallei, it is not easy to 
 obtain pure cultures by the plate method from such 
 material, and here animals are useful. 
 
 Of test animals guinea-pigs and field-mice are the 
 most susceptible. In guinea-pigs subcutaneous injec- 
 tions are followed in four or five days by swelling at 
 the point of inoculation, and a tumor with caseous con- 
 tents soon develops; then ulceration of the skin takes 
 place, and a chronic purulent ulcer is formed. The 
 lymphatic glands become inflamed and general symp- 
 toms of infection are developed in from two to four 
 weeks; the glands suppurate and in males the testicles 
 are involved; finally purulent inflammation of the joints 
 occur, and death ensues from exhaustion. The forma- 
 tion of the specific ulcers upon the nasal mucous mem- 
 
602 BA CTER10L OGY. 
 
 brane, which characterize the disease in the horse, rarely 
 result from inoculation of the guinea-pig. The process 
 is often prolonged, or it remains localized on the skin. 
 Guinea-pigs succumb more rapidly to intraperitoneal 
 injection, usually in from eight to ten days, and in 
 males the testicles are invariably affected. 
 
 Glanders occurs as a natural infection only in horses 
 and asses; the disease is occasionally communicated to 
 man by contact with affected animals, and usually by 
 inoculation on an abraded surface of the skin. The 
 contagion may also be received on the mucous mem- 
 brane. Infection has sometimes been produced in 
 bacteriological laboratories. In the horse, the disease 
 may be localized in the nose (glanders) or beneath the 
 skin (farcy). The essential lesion is the granulomatous 
 tumor, characterized by the presence of numerous lym- 
 phoid and epithelioid cells, among and in which are seen 
 the glanders bacilli. These nodular masses tend to break 
 down rapidly, and on the mucous membrane form ulcers, 
 while beneath the skin they form abscesses. The glan- 
 ders nodules may also occur in the internal organs. An 
 acute and chronic form of glanders may be recognized 
 in man, and an acute and a chronic form of farcy. The 
 disease is fatal in a large proportion of cases. It is 
 transmissible also from man to man. Washer-women 
 have been infected from the clothes of a patient. The 
 infective material exists in the secretions of the nose, 
 in the pus of glanders nodules, and sometimes in blood; 
 it may occasionaly be found in the secretions of healthy 
 glands, as in the urine, milk, and saliva, and also in 
 the foetus of diseased animals (Bonome). From recent 
 observations it appears that glanders is by no means 
 an uncommon disease among horses, particularly in 
 
GLANDERS BA GILL US. 603 
 
 southern countries, sometimes taking a mild course and 
 remaining latent for a considerable time (Semmer and 
 Babes). Apparently healthy horses, therefore, may pos- 
 sibly spread the disease. 
 
 Attenuation of virulence occurs in cultures which 
 have been kept for some time, and inoculations with 
 such cultures may give a negative result; or, when con- 
 siderable quantities are injected, may produce a fatal 
 result at a later date than is usual when small amounts 
 of a recent culture are injected into susceptible animals. 
 
 Several attempts have been made by investigators to 
 produce artificial immunity against glanders, but so far 
 with unsatisfactory results. According to Strauss, dogs 
 may be protected by intravenous inoculations of small 
 quantities of living bacilli against an injection with 
 large quantities which usually kill them. Fenger has 
 found that animals inoculated with glanders bacilli 
 react less powerfully to fresh injections; and that rab- 
 bits which have recovered from an injection of glanders 
 are subsequently immune, the immunity lasting for from 
 three to six weeks. Ladowski has obtained positive re- 
 sults also in rabbits and cats by intravenous injections 
 of sterilized cultures. Other observers have reported 
 not only the production of immunity, but also cures, by 
 the use of mallein. Mallein is produced by evaporat- 
 ing a six-weeks 7 old culture of the glanders bacillus in 
 5 per cent, glycerin nutrient veal bouillon to 10 per 
 cent, of its original bulk. It is made in the same way 
 as Koch's crude tuberculin from the tubercle bacillus 
 cultures. j % . 
 
 Differential Diagnosis. It is often difficult to demon- 
 strate microscopically the presence of the bacillus of 
 glanders in the nodules which have undergone purulent 
 
CHAPTER XXXVI. 
 
 BUBONIC PLAGUE BACILLUS YELLOW FEVER 
 BACILLUS WHOOPING-COUGH BACILLUS. 
 
 BACILLUS OF BUBONIC PLAGUE (Bacillus Pestis 
 Bubonicse Kitasato ; Bacterium Pestis). 
 
 DISCOVERED simultaneously by Kitasato and Yersin 
 (1894) during an epidemic of the bubonic plague in 
 China. It is found in large numbers in the pus from 
 
 FIG. 82. 
 
 Bacillus of bubonic plague. X 1000 diameters. 
 
 the buboes characteristic of this disease and in the 
 lymphatic glands; more rarely in the internal organs 
 and in the blood, in which it occurs in acute hemor- 
 
BACILLUS OF BUBONIC PLAGUE. 607 
 
 rhagic cases and shortly before death. It also occurs 
 in the feces of men and animals. 
 
 Morphology. Short thick rods, with rounded ends, 
 frequently occurring in short chains and often sur- 
 rounded by a capsule. When obtained from cultures 
 the bacilli present considerable spherical enlargement 
 (Fig. 82). 
 
 Stains readily with the ordinary aniline dyes, the 
 ends being usually more deeply colored than the central 
 portion; does not stain by Gram's method. 
 
 Biological Characters. An aerobic, non-motile bacil- 
 lus. Does not form spores. Grows on the usual culture 
 media. Does not liquefy gelatin. Grows best on blood- 
 serum in the incubator, the growth appearing on the 
 surface after twenty-four to forty-eight hours, in the 
 form of white, moist, transparent and iridescent col- 
 onies. It grows rapidly on glycerin-agar, forming a 
 grayish-white surface growth. In bouillon a very char- 
 acteristic appearance is produced, the culture medium 
 remaining clear while a granular or grumous deposit 
 forms on the walls and on the bottom of the tube. 
 
 Pathogenesis. This bacillus is pathogenic for rats, 
 mice, guinea-pigs, monkeys, rabbits, flies, and other 
 insects, which usually die within two or three days 
 after inoculation. Then at the point of inoculation is 
 found a somewhat hemorrhagic infiltration and rcdema, 
 with enlargements of the neighboring lymph-glands, 
 hemorrhages into the peritoneal cavity and parenchy- 
 matous congestion of the organs. The spleen sometimes 
 shows minute nodules resembling miliary tubercles. 
 Microscopically the bacilli are found in all the organs 
 and in the blood. The disease is rapidly communicated 
 from one animal to another, and thus its extension is 
 
CHAPTER XXXVI. 
 
 BUBONIC PLAGUE BACILLUS YELLOW FEVER 
 BACILLUS W HOOPING-COUG H B A CILLUS. 
 
 BACILLUS OF BUBONIC PLAGUE (Bacillus Pestis 
 Bubonicae Kitasato ; Bacterium Pestis). 
 
 DISCOVERED simultaneously by Kitasato and Yersin 
 (1894) during an epidemic of the bubonic plague in 
 China. It is found in large numbers in the pus from 
 
 FIG. 82. 
 
 Bacillus of bubonic plague. X 1000 diameters. 
 
 the buboes characteristic of this disease and in the 
 lymphatic glands; more rarely in the internal organs 
 and in the blood, in which it occurs in acute hemor- 
 
BACILLUS OF BUBONIC PLAQUE. 607 
 
 rhagic cases and shortly before death. It also occurs 
 in the feces of men and animals. 
 
 Morphology. Short thick rods, with rounded ends, 
 frequently occurring in short chains and often sur- 
 rounded by a capsule. When obtained from cultures 
 the bacilli present considerable spherical enlargement 
 (Fig. 82). 
 
 Stains readily with the ordinary aniline dyes, the 
 ends being usually more deeply colored than the central 
 portion; does not stain by Gram's method. 
 
 Biological Characters. An aerobic, non-motile bacil- 
 lus. Does not form spores. Grows on the usual culture 
 media. Does not liquefy gelatin. Grows best on blood- 
 serum in the incubator, the growth appearing on the 
 surface after twenty-four to forty-eight hours, in the 
 form of white, moist, transparent and iridescent col- 
 onies. It grows rapidly on glycerin-agar, forming a 
 grayish- white surface growth. In bouillon a very char- 
 acteristic appearance is produced, the culture medium 
 remaining clear while a granular or grumous deposit 
 forms on the walls and on the bottom of the tube. 
 
 Pathogenesis. This bacillus is pathogenic for rats, 
 mice, guinea-pigs, monkeys, rabbits, flies, and other 
 insects, which usually die within two or three days 
 after inoculation. Then at the point of inoculation is 
 found a somewhat hemorrhagic infiltration and oedema, 
 with enlargements of the neighboring lymph-glands, 
 hemorrhages into the peritoneal cavity and parenchy- 
 matous congestion of the organs. The spleen sometimes 
 shows minute nodules resembling miliary tubercles. 
 Microscopically the bacilli are found in all the organs 
 and in the blood. The disease is rapidly communicated 
 from one animal to another, and thus its extension is 
 
608 BACTERIOLOGY. 
 
 facilitated. During epidemics, rats, mice, and flies, in 
 large numbers, become infected and die, and the disease 
 is apparently transmitted through them to man. The 
 organism is found in the feces of sick animals, in the 
 dust of infected houses, and in the soil. 
 
 The virulence of the bacilli in cultures and in nature 
 seems to vary considerably, and is rather rapidly lost 
 when grown on artificial media. The growth in cul- 
 tures becomes more abundant after frequent transplan- 
 tation. The virulence of the organism is increased by 
 successive inoculation in certain animal species, and 
 then its pathogenic properties for other species are less 
 marked. 
 
 Yersin, Calmette, and Borrel have succeeded in im- 
 munizing animals against the bacillus of bubonic plague 
 by inoculation, by the intravenous or intraperitoneal 
 injection of dead cultures, or by repeated subcutaneous 
 inoculation. They also succeeded in immunizing rab- 
 bits and horses, so that the serum afforded protection 
 to small animals, after subcutaneous injection of viru- 
 lent cultures, and even cured those which had been 
 inoculated, if administered within twelve hours after 
 injection. The serum has considerable antitoxic as 
 well as bactericidal properties. More recently this 
 serum has been applied by Yersin to the treatment of 
 bubonic plague in man, with very promising results. 
 Experience has shown that the treatment is more 
 efficacious the earlier the stage of the disease. When 
 treatment is begun in the first day of the attack, fever 
 and all alarming symptoms usually disappear with 
 astouishing rapidity. In cases treated at a later stage 
 larger doses of the serum are required, and even in the 
 favorable cases suppuration of the buboes is not always 
 
BACILLUS ICTEROIDES. 609 
 
 prevented. In some of the early cases and in many of 
 the rather late ones the serum fails. When the disease 
 is far advanced the serum is powerless. For immu- 
 nizing purposes the serum should be valuable, and a 
 single injection would probably give protection for 
 several weeks. 
 
 Haffkine, in India, has recently applied his method 
 of preventive inoculation to the bubonic plague, as he 
 previously did with cholera and apparently with equally 
 good results. This method consists in an inoculation 
 of dead cultures, and is essentially a protective rather 
 than a curative treatment. It gives after six to ten 
 days a considerable immunity, lasting a month or more. 
 By means of these two methods of inoculation, along 
 with strict quarantine regulations, it is to be hoped 
 that this disease which under the name of Black 
 Death once decimated the populations of the earth, and 
 which in the East still causes great mortality at times, 
 may finally be greatly restricted or even stamped out 
 altogether. 
 
 BACILLUS ICTEROIDES (Bacillus of Yellow Fever). 
 
 In 1897 Sanarelli announced the discovery of a micro- 
 organism which he claimed to be the specific cause of 
 yellow fever. This he called the " bacillus icteroides." 
 It is found in the circulating blood and in the tissues of 
 yellow fever patients. 
 
 Morphology. Short rods with rounded extremities, 
 single or united in pairs in cultures and in groups in 
 the tissues, from I/* to 2// in length, and generally two 
 to three times longer than broad. Somewhat polymor- 
 phous. It resembles the colon bacilli. 
 
 39 
 
610 BACTERIOLOGY. 
 
 Stains readily with the ordinary aniline dyes, but 
 not by Gram's method. 
 
 Biological Characters. A motile, facultative anaero- 
 bic, non-liquefying bacillus. Does not form spores as 
 far as known. Grows readily in all the ordinary cul- 
 ture media, at the room-temperature, but best at 37 C. 
 iu the incubator. On yelatin plates it forms rounded 
 colonies, transparent and granular It never liquefies 
 gelatin. In bouillon the bacillus grows quickly, without 
 forming either a pellicle or deposit. On blood-serum 
 its growth is almost imperceptible. Cultures on agar 
 are characteristic, according to Sanarelli. When the 
 colonies grow in the incubator they present an appear- 
 ance that does not differ from many other species; they 
 are rounded, of a slightly iridescent gray color, trans- 
 parent, even in surface, and regular in outline. Grown 
 at the room-temperature from 20 to 22 C., they ap- 
 pear like drops of milk, opaque, projecting, and with 
 pearly reflections, completely distinct from those grown 
 in the incubator. These different modes of evolution 
 Sanarelli considers to be an important diagnostic point; 
 first exposing the cultures for from twelve to sixteen 
 hours to the temperature of the incubator, and after- 
 ward for twelve to sixteen hours more to the tempera- 
 ture of the air. 
 
 The bacillus icteroides ferments glucose and sacch- 
 arose, but does not coagulate milk; produces little 
 indol, and is quite resistant to desiccation; it dies in 
 water at 60 C., or after being exposed for seven hours 
 to the sunlight, and lives for a long time in sea-water. 
 
 Pathogenesis. It is pathogenic for the greater num- 
 ber of the domestic animals; but birds are completely 
 refractory. According to the discoverer the dog lends 
 
BA GILL US ICTEROIDES. 61 1 
 
 itself particularly to experimentation with this organ- 
 ism. The virus should be injected into a vein. The 
 lesions found after death are said to be almost identical 
 with those in human yellow fever cadavers. There is 
 fatty degeneration of the liver and kidneys, accompa- 
 nied by acute parenchymatous nephritis. The digestive 
 apparatus shows lesions of hemorrhagic gastro-enteritis. 
 The bacilli are found in the blood and the organs in 
 variable quantity and in a state of absolute purity; at 
 times, they may be associated with the B. coli and the 
 streptococcus. 
 
 The disease may be transmitted experimentally even 
 by the respiratory tract to rabbits and guinea-pigs; the 
 bacteriological examination of these cases shows, at 
 least, the existence of toxic processes similar with that 
 which takes place in man. The toxin is obtained by 
 filtering twenty to twenty-five days' old cultures in 
 broth. It withstands a temperature of 70 C., but is 
 sensibly weakened by boiling. 
 
 According to Sanarelli, infection in the human sub- 
 ject does not take place by the digestive but by the 
 respiratory tract; and he suggests that the common 
 moulds of the atmosphere may constitute protectors of 
 the bacillus icteroides. 
 
 Sanarelli has also prepared a protective or curative 
 serum for the treatment of the disease, which he calls 
 " anti-amarylic serum." This serum has not been 
 sufficiently tested as yet to form any definite conclu- 
 sions as to its value ; but because of its not being an 
 antitoxin, it does not tend to overcome the toxins of 
 yellow fever produced in the system, and depends for 
 its curative and prophylactic properties upon its germi- 
 cidal influence. Hence, it is argued by Sanarelli that 
 
612 BACTERIOLOGY. 
 
 its use will be absolutely negative in cases in which an 
 amount of toxin has been produced sufficient to destroy 
 life. He, therefore, insists upon the early use of the 
 serum, and thus the destruction of the organism before 
 it has elaborated the fatal proportion of its toxin. It 
 is claimed that in thirty-one cases treated with the serum 
 the mortality was only 32 per cent., whereas in South 
 America, where the treatment was applied, the mortality 
 of yellow fever often rises to 50 per cent. (Sanarelli). 
 
 Since Sanarelli' s supposed discovery a number of 
 investigations have been made into the causal relation of 
 this organism to the disease, some of which seem to 
 cast considerable doubt upon its being the specific 
 cause of yellow fever, while others are in its favor. 
 Novy, in a recent paper (September, 1898), comes to 
 the conclusion, after an exhaustive examination into 
 this subject, that the Sanarelli bacillus belongs to the 
 typhoid group, and that it is not the cause of yellow 
 fever, which is yet to be worked out. 
 
 Novy's chief objection to this bacillus rests upon 
 the fact that yellow fever is stopped by a frost and that 
 this bacillus is not injured by much greater cold. It is 
 perfectly possible, however, that the infection is carried 
 in some indirect way, as by insects, and that the 
 carriers of infection are affected by the cold, and so the 
 dissemination of the poison is prevented. The long 
 immunity conferred by an attack and the peculiar effect 
 of cold on the spread of the disease are nevertheless 
 difficult to explain by means of the known character- 
 istics of this bacillus. In September a report by 
 Geddings and Wasdin appeared which favored the 
 claims of Sanarelli, they finding the bacilli in almost 
 every case of yellow fever, and not in any which were 
 
THE BACILLUS OF WHOOPING-COUGH. 613 
 
 not yellow fever. According to them the lungs were 
 the earliest and the chief seat of the lesions. Their 
 report adds to the mystery of the effect of cold on 
 stopping the spread of the disease, for nearly all respi- 
 ratory diseases due to bacteria tend to increase in cold 
 weather, and certainly their spread is not stopped im- 
 mediately. This bacillus can as yet be considered as 
 only the possible cause of yellow fever. 
 
 THE BACILLUS OF WHOOPING-COUGH. 
 
 From lime to time observers have found in the 
 sputum of persons suffering from whooping-cough 
 small bacilli, often in great numbers. These have been 
 studied lately especially by Koplik 1 and Czaplewski 2 
 and Hensel, who believe that these bacilli are the cause 
 of the disease. They are small bacilli of about the size 
 of the influenza bacillus, and grow on blood-serum and 
 nutrient agar in tiny colonies. Mice and rabbits die 
 after intravenous inoculations. No symptoms similar 
 to those in man are noted. The observers differ as to 
 the description of the bacilli, and those interested are 
 referred to the original articles. The examination for 
 these bacilli, if they prove to be the true cause of the 
 disease, may prove of diagnostic importance, and also 
 be of use in detecting sources of contagion. 
 
 i Centralblatt fur Bact. Abth., 1 Bd. 22, p. 222 * Ibid., p. 641. 
 
APPENDIX. 
 
 BRIEF DESCRIPTIONS OF A FEW REPRESENTATIVE 
 
 PATHOGENIC MICRO-ORGANISMS WHICH 
 
 ARE NOT BACTERIA. 
 
 CHAPTER XXX VI T. 
 
 THE STREPTOTHKIX GROUP FAVU8 AND RINGWORM 
 FUNGI. 
 
 THE varieties of the streptothrix group have as yet 
 not beeu clearly described. Some at least are patho- 
 genic. This group of micro-organisms while having 
 many affinities with the bacteria, yet differs from them 
 in many important respects which link them with the 
 fungi. They develop from spore-like bodies into 
 cylindrical dichotomously branching threads which 
 grow into colonies, the appearance of which suggests a 
 mass of radiating filaments. Under favorable condi- 
 tions certain of the threads become fruit-hypse, and 
 these break up into chains of round spore-like bodies, 
 which do not, however, have the same staining reac- 
 tions nor resisting powers as true spores. The tubercle 
 grass and diphtheria bacilli are by some believed to 
 properly belong in the streptothrix group on account 
 of the true branching forms developed by them under 
 certain conditions. The best known of the strepto- 
 thrix group is the actinomyces fungus. 
 
APPENDIX. 
 
 STREPTOTHRJX ACTINOMYCES (Actinomyces Fungus ; 
 Ray Fungus). 
 
 This micro-organism was first described by Bollinger 
 (1877) in the ox, in which it forms the affection known 
 as " big-jaw. " In man the disease was first described 
 by J. Israel (1885), and subsequently Ponfick insisted 
 upon the identity of the disease in man and cattle. To 
 Bostrom we owe the most elaborate and accurate account 
 of the structure and development of this organism. 
 
 Morphology. In both man and animals it can be seen 
 in the pus from the affected regions as small yellowish 
 granules from 0.5 to 2 mm. in diameter. Microscopi- 
 cally these bodies are seen to be made up of threads 
 which radiate from a centre and present bulbous, club- 
 like terminations. These club -like terminations are 
 characteristic of the actinomyces. They are generally 
 arranged in pairs, closely crowded together, and are very 
 glistening in appearance. The threads which compose 
 the central mass of the granules are from 0.3// to 0.5// 
 in diameter; the clubs are from 6// to 8// in diameter. 
 
 The organism is stained with the ordinary aniline 
 colors, also by Gram's solution; when stained with 
 gentian- violet and by Gram's method the threads ap- 
 pear more distinct than when stained with methylene- 
 blue. 
 
 Biological Characters. It grows in all the ordinary 
 artificial culture media, but often several cultures have 
 to be made before getting a satisfactory one. It de- 
 velops at the room-temperature, and grows both with 
 and without oxygen, but best with access of air and at 
 the temperature of the body. 
 
 Growth on Blood-serum and Agar. Isolated colonies 
 at first develop on the surface of these media, but on 
 
STREPTOTHRIX A CTINOMYCES. 617 
 
 keeping the cultures for a week or more the colonies 
 run together and form a thick, wrinkled mass which 
 sinks into the media. The individual colonies are 
 yellowish to red in color, and are covered by a whit- 
 ish, fluffy down, consisting of cobweb-like threads. 
 On touching the colonies they will be found to cling 
 close to the medium, and on forcible removal they go 
 to pieces. On making a smear preparation the thread- 
 like structure will be seen. In stick cultures the growth 
 usually presents a tree-like appearance, but it varies 
 very considerably; there may be no reddish pigmenta- 
 tion, and the cobweb-like threads are not always devel- 
 oped on the surface. Occasionally the culture on agar 
 is colored brown. 
 
 The Growth in Bouillon. When the medium is allowed 
 to stand perfectly still a distinct granular growth occurs, 
 but on agitation these grains are broken up, though the 
 liquid is never clouded. At times large flakes or a mem- 
 branous film form on the surface of the medium, upon 
 which develops the fluffy down previously described. 
 
 The Growth on Potato, On this medium the growth 
 is somewhat slower, resulting in a thick, viscid, mem- 
 branous deposit on the surface of the potato on which 
 the same cobweb-like threads are developed. The color 
 is yellowish-red. 
 
 The cultures are quite resistant to outside influences; 
 dried they may be kept for a year or more ; they are 
 killed by a temperature of 75 C., the time of exposure 
 being five minutes. 
 
 Occurrence in Animals. Actinomycosis is quite prev- 
 alent among cattle, in which it occurs endemically; 
 it is more rare among swine and horses, and is some- 
 times found in man. The disease is probably not con- 
 
618 . APPENDIX. 
 
 tagious, but infection may result from the ingestion of 
 vegetable products which contain the fungus. The 
 cereal grains, which from their nature are capable of 
 penetrating the tissues, have been repeatedly found in 
 centres of actinomycotic infection. This usually occurs 
 in the vicinity of the mouth, where injuries have been 
 accidentally caused. The micro-organism may also be 
 introduced by means of carious teeth. Cutaneous in- 
 fection has been produced by wood-splinters, and infec- 
 tion of the lungs by aspiration of fragments of teeth 
 containing the fungus. The further distribution of the 
 fungus after it is introduced into the tissues is effected 
 partly by its growth and partly by conveyance by 
 means of the lymphatics and leucocytes. Not infre- 
 quently a mixed infection with the pyogenic cocci 
 occurs in actinomycosis. 
 
 In the earliest stages of its growth the parasite gives 
 rise to a small granulation tumor, not unlike that pro- 
 duced by the tubercle bacillus, which contains, in addi- 
 tion to small round cells, epithelial elements and giant 
 cells. After it reaches a certain size there is great pro- 
 liferation of the surrounding connective tissue, and the 
 growth may, particularly in the jaw, look like, and was 
 long mistaken for, osteosarcoma. Finally, suppuration 
 occurs, which, according to Israel, may be produced 
 directly by the fungus itself. 
 
 The experimental production of actinomycosis in ani- 
 mals has been followed by negative or very unsatisfac- 
 tory results. When artificially introduced into the 
 tissues the organism is either absorbed or encapsulated. 
 If introduced in large quantities multiple nodules are 
 apparently formed in some cases, which may suggest 
 the production of a general infective process; but on 
 closer inspection of these nodules the thread-like por- 
 
THE FUNGI. 619 
 
 tion of the fungus are found to have disappeared, leav- 
 ing only the remains of the club-like ends, thus showing 
 that no growth has taken place. Ponfick, Johne, Rot- 
 ter, Liming, and Hanan claim to have obtained positive 
 results in animals, but according to Bostrom these re- 
 sults are not conclusive. The animals used for experi- 
 mentation have been calves, swine, dogs, rabbits, and 
 guinea-pigs, the places of inoculation being the anterior 
 chamber of the eye, the subcutaneous intercellular tissue, 
 the peritoneum, and the blood; and the material em- 
 ployed for inoculation, pus from the infected regions in 
 animals and man; very rarely cultures. 
 
 A number of other streptothrices have been described 
 in connection with pathogenic processes, but most of 
 them are not well defined. They have been found in 
 brain abscess, cerebro-spinal meningitis, pneumonic 
 areas, and in other pathological conditions. Eppinger 
 injected cultures into guinea-pigs and rabbits, and ob- 
 served that it caused a typical pseudotuberculosis. Con- 
 solidation of portions of both lungs, thickening of the 
 peritoneum, and scattered nodules resembling tubercles, 
 were noted in a case of human infection as due to a 
 streptothrix by Flexner, in which the pathological 
 picture of the disease resembled so nearly tuberculosis 
 in human beings that the two diseases could be sepa- 
 rated only by the causative micro organism in each case. 
 
 THE FUNGI. 
 
 Most of the fungi are not pathogenic and interest us 
 merely as organisms which are apt to infect our bac- 
 teriological media. Some are, however, true parasites, 
 and already we know that ringworm, favus, thrush, and 
 pityriasis versicolor are caused by fungi. Only those 
 causing ringworm and favus can be touched on here. 
 
620 APPENDIX. 
 
 TRICHOPHYTON (Ringworm Fungus). 
 
 Kingworm of the body or hairless parts of the skin, 
 tinea circinata, and ringworm of the hairy parts, tinea 
 tonxurans and tinea barbce or tinea sycosis are due to 
 the fungus trichophyton, discovered by Gruby in the 
 human hair, and between the epidermal cells by Hebra, 
 and obtained in free cultures by gravity. 
 
 FIG. 83. 
 
 Hair riddled with ringworm fungus. Megalosporon variety. 
 
 According to Sabouraud, whose conclusions are based 
 on an extensive series of microscopical examinations of 
 cases of tinea in man and animals, of cultivation in 
 artificial media, and of inoculation on man and animals, 
 there are two distinct types of the fungus trichophyton 
 causing ringworm in man one with small spores (2 to 
 3 mm.), which he calls "T. microsporon," and one 
 with large spores (7 to 8 mm.), which he calls "T. 
 megalosporon." They differ in their mode of growth 
 on artificial media and in their pathological effects on 
 
TRICHOPHYTON. 621 
 
 the human skin and its appendages. T. microsporon 
 is the common fungus of tinea tonsurans of children, 
 especially of those cases which are rebellious to treat- 
 ment, and its special seat of growth is in the substance 
 of the hair. T. megalosporon (Fig. 83) is essentially 
 the fungus of ringworm of the beard and of the smooth 
 parts of the skin; the prognosis as regards treatment is 
 good. One-third of the cases of T. tonsurans of chil- 
 dren are due to trichophyton megalosporon. The spores 
 of T. microsporon are contained in a mycelium; but 
 this is not visible, the spores appearing irregularly 
 piled up like zoogloea masses; and, growing outside, 
 they form a dense sheath around the hair. The spores 
 of T. megalosporon are always contained in distinct 
 mycelium filaments, which may either be resistant when 
 the hair is broken up, or fragile and easily separating 
 up into spores. The two types when grown in artificial 
 cultures show distinct and constant characters. The 
 cultures of T. microsporon show a downy surface and 
 white color; those of T. megalosporon a powdery sur- 
 face, with arborescent peripheral rays, and often a yel- 
 lowish color. Although the morphological appearances, 
 mode of growth, and clinical effects of each type of tri- 
 chophyton show certain characters in general, yet there 
 are certain constant minor differences which point to 
 the fact that there are several different kinds or species 
 of fungus included under each type. The species in- 
 cluded under T. microsporon are few in number, and, 
 with the exception of one which causes the common 
 contagious " herpes" of the horse, almost entirely 
 human. The species of T. megalosporon are numer- 
 ous and fall under several natural groups, the members 
 of which resemble one another both from clinical 
 
622 
 
 APPENDIX. 
 
 and mycological aspects (Fig. 84). Many animals are 
 subject to the growth upon their skins of particular 
 species of T. megalosporon. 
 
 FIG. 84. 
 
 These two half-plates show three- mouths' growth on peptone maltose agar of 
 two megalosporon varieties of the ringworm fungus. Natural size. 
 
 ACHORION SCHGENLEINII (Favus). 
 
 Favus is due to a fungus discovered by Schoenlein in 
 
 1839, and called by Remak Achorion schcenleinii. The 
 
 disease is communicated by contagion, the fungus being 
 
 often derived from animals, especially cats, mice, rab- 
 
A CHORWN SCHCENLEINIL 623 
 
 hits, fowls; and dogs are also subject to it. It grows 
 much more slowly than the ringworm fungus, and is, 
 therefore, not so easily transmitted. Want of cleanli- 
 ness is a predisposing factor. The fungus seems to 
 find a more favorable soil for its development on the 
 skin of persons in weak health, especially from phthisis, 
 than in others. 
 
 Pathologically, the disease represents the reaction of 
 the tissues to the irritation caused by the growth of the 
 fungus. The spores generally find their way into the 
 
 FIG. 85. 
 
 A portion ot a favus infected hair. Magnified. 
 
 hair-follicles, where they grow round the hair-seat 
 (Fig. 85). The favus fungus grows in the epidermis, 
 the density of the growth causing pressure on the parts 
 below, thus crushing out the vitality of the hair and 
 giving rise to atrophic scarring. The disease shows a 
 marked preference for the scalp, but no part of the 
 skin is exempt, and even the muco-us membranes are 
 liable to be attacked. Kaposi has reported a case in 
 which a patient suffering from universal favus died, 
 with symptoms of severe gastro intestinal irritation, 
 which was found after death to be due to the presence 
 
6 24 APPENDIX. 
 
 of the favus fungi in the stomach and intestine. On 
 the scalp it first appears as a tiny sulphur-yellow 
 disk or scutulum, depressed in the centre like a cup and 
 pierced by a hair. This is the characteristic lesion. The 
 cup-shape is attributed by Unna to growth at the sides 
 proceeding more vigorously than at the centre. 
 
 There is some difference of opinion as to whether 
 there is only one or several varieties of favus fungus. 
 It was suggested by Quincke that there are three dif- 
 ferent species of favus fungus. Later investigations 
 
 FIG. 86. 
 
 Five-months' old colony of favus on peptone maltose agar. Actual size. 
 
 have apparently shown, however, that the achorion 
 Schoenleinii is the only fungus of favus. 
 
 The favus fungus is readily cultivated at the body- 
 temperature, and also at room-temperature, in the or- 
 dinary culture media, as agar, blood-serum, gelatin, 
 bouillon, milk, infusion of malt, eggs, potato, etc. 
 (Fig. 86). The growth develops slowly and shows a 
 preference to grow beneath the surface of the medium 
 except on potato, upon which it develops on the surface 
 in layers. The characteristic form of growth is that of 
 moss-like projections from a central body. The color 
 
YEASTS. 625 
 
 is at first grayish-white, then yellowish. As seen 
 under the microscope, ray-like mycelium filaments are 
 developed, which divide into branches. The ends are 
 often swollen or club-shaped, and there are various 
 enlargements along the body of the filament. 
 
 YEASTS (Saccharomyces). 
 
 These micro-organisms are of the greatest importance 
 in brewing and baking, but as yet no important patho- 
 logical lesions in man have been attributed to them, 
 although certain recent experiments have shown that 
 some varieties when injected are capable of producing 
 tumors and many are pathogenic for mice. They are 
 are not uncommonly present in the air and in cultures 
 made from the throat. They consist of round or oval 
 cells, usually many times larger than the bacteria. 
 They usually reproduce themselves by budding, a por- 
 tion of the protoplasm budding, and finally being cut 
 off to form a new individual. 
 
 40 
 
CHAPTER XXXVIII. 
 
 PLASMODIUM MALARI^E (MALARIAL PARASITES ; 
 LAVERANIA) AMCEBA COLI (AMCEBA DYSEN- 
 TERIC OF COUNCILMAN AND LAFLEUR ; DYSEN- 
 TERIC AMCEBA). 
 
 MANY attempts have been made from time to time 
 to discover a specific organism in malaria. As early as 
 1846, according to Marchiafava and Bignami, an Ital- 
 ian observer (Risori) suggested the possible parasitic 
 nature of the disease. In 1880, Laveran announced the 
 discovery of certain parasitic bodies in the blood of 
 patients with malarial fever. He recognized that they 
 were parasites, and raised the question whether they 
 were amoebae. Subsequently, influenced no doubt by 
 the presence of the motile filaments, he suggested the 
 term osdllaria malarice. Marchiafava and Celli de- 
 scribed with great accuracy the intracorpuscular amoe- 
 boid form, to which they gave the name plasmodium. 
 The most important additional fact was added by Golgi, 
 who pointed out the association of the paroxysm with 
 the segmentation of a group of the malarial organisms. 
 Laveran' s work and the differentiation by the Italian 
 observers of varieties of the parasite in different clinical 
 forms of the disease have since received full confirma- 
 tion, and the testimony is now unanimous in France, 
 England, India, America, Italy, and Germany that 
 these bodies are always present in the malarial fevers. 
 
PLASMODIUM MALARIA. 627 
 
 There is still much uncertainty with regard to the 
 classification of the parasites. Many authors place them 
 among the sporozoa in the order of the hoemosporidia of 
 Danilewsky; others place them in the aarcodinia, and 
 speak of them as hcemamcebce. Until the matter is settled, 
 however, they may be considered to belong to the gen- 
 eral order of protozoa and to that group of organisms 
 known as hcematozoa. Parasites of the red blood-cor- 
 puscles have been met with abundantly in the blood of 
 fish, turtles, and many species of birds. 
 
 The relation of the different forms of the malarial 
 parasite to each other and to the varieties of the disease 
 are still under discussion. Galgi, Marchiafava and 
 other Italian observers hold that they are distinct 
 varieties, not interchangeable, though closely allied 
 biologically. Laveran, on the other hand, contends for 
 the unity of the forms, which he regards as modifica- 
 tions of one polymorphic parasite. But with the 
 present imperfect knowledge of the full life-history of 
 the parasite the question cannot be considered as settled. 
 
 The following varieties are associated with the differ- 
 ent forms of malarial fever : 
 
 I. Parasite of the Simple Intermittent Fever, (a) TER- 
 TIAN PARASITE (see Plate II.). If the blood of a 
 patient be examined during or shortly after the chill 
 in tertian fever, inside the red blood-corpuscles, or less 
 often free in the plasma, will be seen small, pale, hya- 
 line amoebae which undergo rapid changes in shape, 
 often assuming the form of a star or of a cross. There 
 may be no pigment visible, and to these hyaline bodies 
 Marchiafava and Celli gave the name plasmodia. In a 
 few examples scattered pigment granules may be seen 
 in the amcebse, usually placed near the periphery. In 
 
628 APPENDIX. 
 
 dry specimens these bodies stain deeply with methylene- 
 blue, and they are solid or vesicular in form. If the 
 examination be made within twelve to eighteen hours 
 after the chill the hyaline bodies are seen to have 
 grown to occupy one-fourth to one-third of the bodies 
 of the red cells. They are more pigmented, and the 
 corpuscles containing them have become gradually paler 
 and somewhat expanded. The pigment granules, which 
 at first are small, increase in size, and the organisms 
 show very active amoeboid movements. At the end of 
 forty-eight hours they occupy entire corpuscles, are very 
 sluggish in their movements, and look like thin, trans- 
 lucent shells, and are usually devoid of color. Many 
 of the organisms then undergo the remarkable change 
 known as segmentation, which precedes and is asso- 
 ciated with chills and fever. The amoeboid movement 
 ceases as well as that of the pigment granules. The 
 latter gradually collect toward the centres of the amoebae 
 until they are in the form of closely packed, more or less 
 central clumps. The protoplasm becomes more finely 
 granular, and indistinct lines of striation are seen, which 
 begin at the periphery. At this stage the organisms 
 may present the appearance of rosettes. The segmen- 
 tation progresses until the entire protoplasm is divided 
 into twelve to eighteen or twenty spheres. The shell 
 of the corpuscles containing a parasite usually bursts, 
 and the small, rounded, hyaline bodies are set free. 
 Each one of these little bodies consists of a translucent 
 protoplasm, with a central, more highly refractile spot. 
 In stained preparations, during the segmenting process, 
 the reticulum becomes denser and sharper, and then 
 breaks up into fifteen to twenty small spheroidal 
 spores. 
 
PLASMODIUM MALARIJE. * 629 
 
 The segmentation is regarded as a reproductive pro- 
 cess, and these small spherical bodies are believed to 
 be the spores which penetrate a new set of corpuscles, 
 and so begin a new cycle of development. The pig- 
 ment is discharged into the plasma, and partly taken 
 up by the leucocytes. It is finally lodged chiefly in 
 the spleen, liver, and lymphatic organs. The pres- 
 ence of the segmenting forms is invariably associated 
 with the paroxysm. On finding them in the blood it 
 can be predicted with certainty that a paroxysm is 
 imminent. In quotidian fever we have to deal with 
 two groups of tertian (or three groups of quartan) para- 
 sites, maturing on successive days; and the full-grown 
 segmenting forms of to-day's paroxysms and the half- 
 grown organisms of to-morrow's attack are to be found 
 in the blood. 
 
 (b) QUARTAN PARASITE (see Plate II.). The early 
 forms within the red blood-corpuscles are amoeboid 
 bodies, similar to those of tertian fever. Soon, how- 
 ever, it is noticed that the pigment is different ; the 
 granules are larger and blacker, and the amoeboid 
 movements are not so active. In their growth the 
 parasites do not decolorize the corpuscles, which some- 
 times have a greenish, brassy look. From the sixty- 
 fourth to the seventy-second hour the amoebae have 
 reached their full development, occupying the greater 
 portion of the affected corpuscles ; but a thin rim of 
 colored stroma can usually be seen. Some of the cor- 
 puscles are completely filled by the parasites. The 
 cells, as a rule, appear shrunken rather than swollen. 
 Even at this stage a skilled observer can usually 
 recognize the quartan from the tertian organism. The 
 pigment granules then collect toward the centre, and 
 
630 APPENDIX. 
 
 in so doing usually form distinct rays. Then, as in 
 the tertian form, the organism begins to segment ; a 
 marginal indentation is first seen, with lines of radia- 
 tion, and a beautiful rosette is formed, which segments 
 into from six to ten, occasionally twelve, small, spher- 
 ical or ovoid bodies. The character of the pigment, 
 the smaller size of the organism, and the development 
 are differences which separate the quartan from the 
 tertian variety. 
 
 In the quartan malarial fever the blood may show 
 two or more groups of parasites. There may be two 
 groups which reach maturity on successive days, with 
 one day interval double quartan fever; or there may 
 be three groups of organisms maturing on successive 
 days, causing daily paroxysms triple quartan fever. 
 
 II. The -ffistivo-autumnal Parasite (see Plate II.). 
 In the more irregular and as a rule pernicious types 
 of malarial infection which are met with in the autumn 
 months a third variety of organism may be recognized, 
 which has been specially studied by the Italian obser- 
 vers. The youngest forms of this parasite are small 
 hyaline bodies about one-sixth the diameter of the red 
 cell. At first they are quiescent, but later develop 
 active amoeboid movement. They are at this stage 
 not unlike those of the tertian varieties; but the hya- 
 line body is more signet ring-like, more highly refrac- 
 tile, and the central part often looks shaded, as if a 
 more solid body were enclosed within a vacuole. As 
 this form increases the amoeboid movements are well 
 seen. The pigment is in small amount, at first in the 
 form of one or two very dark granules at the margin 
 of the amoebae, and the pigment never becomes so 
 abundant as in the tertian or quartan forms. The 
 
PLASMODIUM MALARIJE. 631 
 
 organism rarely occupies more than about one-third of 
 the corpuscle, the stroma of which is never entirely de- 
 colorized. On the contrary, it often presents a curious 
 brassy-green appearance, and looks shrunken or crum- 
 pled. The cycle of development of this form is rarely 
 carried out entirely in the circulating blood, but the 
 bodies with centrally placed pigment are not uncom- 
 mon. The observations of the Italian observers seem 
 to show conclusively that the segmentation takes place 
 in the spleen and in the bone-marrow and internal 
 organs. The length of its cycle of development has not 
 been determined. Probably different groups mature at 
 varying intervals of time, from twenty-four hours or 
 less to forty-eight or more (Welch). The fever associ- 
 ated with this organism is characterized by irregularity, 
 the paroxysms are not at definite periods, and the 
 pyrexia may be more or less continuous, with remissions. 
 This form is associated with the severer types of the 
 malaria seen in late summer and autumn the sestivo- 
 autumnal fevers of Cuba, Italy, etc. 
 
 There are several other points of interest about the 
 parasites. A corpuscle containing a half -grown organism 
 may suddenly rupture; the haemoglobin diffuses, and 
 the pigmented parasite is set free. The parasite may 
 break up into two or three portions, perhaps from 
 pressure on the slide, and slight amoeboid changes may 
 be seen. In other instances, apparently from certain 
 free extra-corpuscular organisms, the remarkable flagel- 
 late form develops itself. The pigment becomes more 
 central, and the granules dance with great activity. 
 Suddenly, long, thread-like processes extend from the 
 body of the parasite and display remarkable move- 
 ments, thrashing about over the corpuscle with extra- 
 
632 APPENDIX. 
 
 ordinary rapidity. A flagellum may break off from 
 the main body and move about independently among 
 the corpuscles. While these flagellate bodies appear 
 in both the tertian and quartan fevers they are very 
 much more numerous in the irregular malaria. The 
 significance of the flagellate form is still under discus- 
 sion. By some it has been regarded as a degenerate 
 form. 
 
 In the sestivo-autumnal, quotidian, or pernicious mala- 
 rial fevers there is developed also a very striking body, 
 to which much attention has been paid, viz., the <e cres- 
 cent " of Laveran. In any case of irregular malarial 
 fever which has lasted a week or more these bodies are 
 to be found. They are developed within the red blood- 
 corpuscle, the margin of which may usually be seen on 
 the concave surface of the crescent. The border is very 
 sharply defined, the protoplasm uniform, homogenous, 
 with coarse pigment granules, often in the form of rods, 
 which are collected about the centre. Bodies similar 
 in structure, but differing in form, being ovoid and 
 rounded, are also met with ; and the change of a cres- 
 cent into an ovoid or rounded body can be traced, which, 
 in turn, may in some instances be seen to project flagella 
 or form a flagellated body similar to that derived from 
 the extracorpuscular organisms above referred to. 
 Most authors say that both kinds of flagellate bodies 
 do not develop unless the blood be exposed to the air, 
 but an exposure of one or two minutes gives the best 
 results. It would seem that they do not exist as flagel- 
 late forms in the circulation. (Osier, in Allbutt's Sys- 
 tem of Medicine.) 
 
 Pigmented Leucocytes. Typical pigmented leucocytes 
 are very characteristic signs in malarial blood, and on 
 
PLASMODIUM M ALARMS. 633 
 
 their presence alone the diagnosis must often rest: (1) 
 In severe acute cases after the administration of much 
 quinine; (2) in remittent malarial fevers; and (3) in 
 chronic malarial fever and cachexia. They persist in 
 the blood long after all traces of parasites have dis- 
 appeared. The identification of free malarial pigment 
 is usually hazardous, and the diagnosis of malaria 
 should never be based on its presence alone (Ewing). 
 
 Inoculation Experiments. Malarial infection can be 
 transmitted directly from man to man by subcutaneous 
 or intravenous inoculation of malarial blood. This was 
 shown first by Gerhardt in 1884. Later experiments, 
 chiefly by Italians observers, have confirmed Gerhardt 7 s 
 investigations, and almost in every instance the variety 
 of organism introduced has been reproduced. It has 
 also been experimentally shown that the ague paroxysm 
 is associated with the segmentation of enormous groups 
 of intracorpuscular amoeba?, the symptoms being prob- 
 ably due, as Bacelli suggests, to toxins liberated during 
 sporulation or to substances set free in the blood by 
 the rapid destruction of a large number of its corpuscles. 
 The period of incubation is from eleven to twelve days 
 in the regular intermittents and from two to five days 
 in the irregular autumnal fever. 
 
 Active phagocytosis goes on in all forms of malarial 
 infection, but its true significance is still undetermined. 
 That many parasites are devoured by the leucocytes, 
 especially in the spleen, is certain. This apparently 
 takes place during or after sporulation. But sponta- 
 neous recovery may also be due to the death of the 
 plasmodia. It is not improbable, however, that the 
 phagocytes contribute to the process of recovery, even 
 if they are not the chief factors in it. 
 
634 APPENDIX. 
 
 With regard to immunity, we know that one attack 
 of malaria may linger a long time, and seems rather to 
 favor than to prevent a new infection. There is, 
 however, a natural susceptibility to the disease which 
 is very variable. Different races of men especially 
 seem to possess in variable degree the power of resist- 
 ance to malarial infection. This is shown not only in 
 a diminished tendency to contract the disease, but also 
 in the form by which they are affected. For instance, 
 the negroes in the Southern parts of the United States 
 are much less liable to contract malaria than the whites; 
 and Martin reports that the Europeans living in Suma- 
 tra are far more frequently and severely affected by 
 malaria than the natives, who, if they are attacked at 
 all, it is only with the simple intermittent tertian and 
 quartan fevers. 
 
 The Action of Quinine on the Parasites. Laveran 
 showed that a solution of 1 to 10,000 of quinine, run 
 under the cover-glass, would check at once the move- 
 ments of malarial organisms. As demonstrated by 
 Marchiafava and Celli, however, a like effect is pro- 
 duced either by the water or by the salt solution in 
 which the quinine is dissolved, and we meet with an 
 almost insuperable difficulty in the study of the direct 
 action of the drug upon the parasites themselves. 
 
 Many careful experiments have been made to deter- 
 mine the effect of quinine on the parasites circulating 
 in the blood, and Romanowsky, Golgi and others have 
 reported a diminution in the activity of the amoeboid 
 movements. Osier stated that, as a result of careful 
 hourly examinations made in a series of cases with a 
 view of ascertaining the direct influence of full doses of 
 quinine, he was unable to make up his mind that any 
 
PLASMODIUM MALARIA. 635 
 
 particular change took place in the intracorpuscular 
 tertian parasite while undergoing destruction by the 
 specific. 
 
 The following points, nevertheless, about the action 
 of quinine on the parasites seem to be well established : 
 First, that under its use the intracorpuscular varieties, 
 whether tertian, quartan, or sestivo-autumnal, rapidly 
 disappear from the circulating blood; second, that 
 quinine administered some hours before a paroxysm 
 will not interrupt the cycle of their development, but 
 will usually destroy the products of segmentation, and 
 so check the succeeding paroxysm; third, that the cres- 
 centic and ovoid bodies which develop in sestivo- 
 autumnal fevers are very slightly affected by the action 
 of quinine. 
 
 Mixed Infection in Malarial Fever. It is now a well- 
 known fact that along with a malarial infection there 
 may exist another due to the typhoid bacillus, to one 
 of the pyogenic cocci, or to other micro-organisms. 
 Such mixed infection may make a complete diagnosis 
 a very difficult matter. 
 
 Diagnosis. The diagnosis of malaria in all its forms 
 has been greatly simplified by Laveran's discovery. 
 This is not a matter of so much importance in the 
 simple typical intermittents, but in the atypical forms 
 of the disease, and especially in pernicious malaria, the 
 symptoms of which are readily overlooked, serious 
 errors in diagnosis may be made. Moreover, par- 
 oxysms of intermitting fever, which are common in 
 other diseases, may be mistaken for those of malaria 
 such as occur in the early stages of tuberculosis, in 
 ulcerative endocarditis, in suppuration associated with 
 septicaemia or pyaemia, in pyelitis; etc. In all such 
 
636 APPENDIX. 
 
 cases, and in cases of mixed malarial infection occur- 
 ring in malarial regions, a careful blood examination 
 enables a positive diagnosis to be made in a large 
 majority. 
 
 Technique of Blood Examinations for Malaria. The 
 finding of the parasite should not prevent us from 
 seeking farther in doubtful cases by means of the 
 Widal reaction and blood cultures for other infections 
 which may exist along with the malaria. 
 
 The parasites require a proper technique and a 
 certain experience for their recognition. The fresh 
 blood, when it can be obtained, should be examined, 
 but if no bodies be found, stained preparations should 
 always be later searched through; the drops may be 
 taken either from the tip of the finger or from the 
 lobe of the ear. It is important to have a perfectly 
 clean cover-glass and slide, and to cleanse the skin 
 thoroughly and to wipe it dry, so as to avoid dirt and 
 perspiration. A very small drop should be taken, and 
 care must be exercised that the cover-slips, when pressed 
 against the blood-drop, do not touch the skin. The 
 drop should be so small that the corpuscles are spread 
 out in a uniform layer and are not in rolls when the 
 cover-glass is laid upon the slide, for the intracorpus- 
 cular form cannot be well seen unless the blood-disk 
 presents the flattened surface. For making permanent 
 preparations the blood is collected upon cover-glasses 
 in very thin films, which should be instantly dried. 
 The blood-cells are fixed by immersion in equal parts 
 of alcohol (95 per cent.) and ether for fifteen minutes, 
 or by exposing for five minutes over a wide-mouthed 
 bottle containing 25 per cent, solution of formalin, or 
 by heating to 120 C. for ten minutes. Ewing advises 
 
PLASMODIUM MALARIA. 637 
 
 the alcohol and ether method. The preparations are 
 stained with methylene-blue, or, if desired, with a 
 double stain of methylene-blue and eosin. The prep- 
 aration is covered with a mixture made of equal parts 
 of a saturated alcoholic solution of eosin and water for 
 one minute ; wash in water and dry in air. The prep- 
 aration is then covered by a saturated watery sol ution of 
 methylene-blue for a minute or two, washed in water, 
 dried, mounted, and examined with the immersion lens. 
 Thorough drying after the eosin staining makes the 
 blue stain of the parasites sharper (Ewing). 
 
 In some cases of sestivo-autumnal fever the para- 
 sites are chiefly in the spleen, liver, and bone-marrow. 
 The blood withdrawn directly from the spleen may 
 show large numbers, although in the circulating blood 
 they may be scanty. In these cases puncture of the 
 spleen and examination of the blood withdrawn may 
 render the diagnosis more certain, but in acute splenic 
 tumor the procedure is not without risk. The finding 
 of malarial parasites in the blood not only separates the 
 intermittent, continued, and remittent malarial fevers 
 from all other diseases in which similar fevers may 
 occur, but the variety of parasites found influences the 
 prognosis of the malarial infection. The number of 
 parasites observed on examination also influences the 
 prognosis to a certain degree, though too great weight 
 should not be laid on this point, particularly as the 
 result of a single examination. Whether there are any 
 forms of malarial infection in which there are no plas- 
 modia present in the circulating blood is a question 
 for future determination. We know that in all severe 
 seizures, if the blood is examined within twenty-four 
 hours of the beginning of the paroxysms and before 
 
638 APPENDIX. 
 
 much quinine is given, the plasm odia can readily be 
 found, usually in considerable numbers. In some very 
 mild initial paroxysms the plasm odia may be difficult 
 to find. In aestivo-auturnnal malaria, while quinine is 
 being administered, there may be no organisms during 
 the period between the second and fourth day, but on 
 the fourth or fifth day the crescents almost always make 
 their appearance, notwithstanding the use of quinine 
 (Ewing). 
 
 Mode of Infection. It is generally acknowledged 
 that the most common mode of infection in malaria 
 is through the air. Whether the disease may be 
 directly conveyed by water has been much disputed. 
 Many favor the view, but experimental evidence is 
 distinctly against it. Persons have been allowed to 
 drink water from the Pontine marshes without ill 
 effects, and in Bacelli's clinic at Rome experiments 
 were made in thirty cases with water from malarial 
 districts without a single positive result. Grassi could 
 not produce the disease with dew from malarial regions 
 or by allowing healthy men to drink blood from mala- 
 rial patients. We may therefore assume that malarial 
 infection is not produced, as a rule, by way of the in- 
 testines. Numerous experiments have shown, oir the 
 contrary, that the infection may be induced by subcu- 
 taneous inoculation. It is quite conceivable, therefore, 
 that under natural conditions malarial infection may be 
 produced by way of the skin, and possibly by the bites 
 of insects. This is all the more probable, as certain 
 varieties of mosquitoes, in malarial regions, have been 
 found to be laden with the plasmodia. In another wide- 
 spread disease produced by blood parasites Texas fever 
 in cattle it has been shown that the amoebae are con- 
 
PLASMODIUM MALARIJE. 639 
 
 veyed by means of the cattle tick from animal to animal. 
 The further the investigations have been pushed the 
 closer becomes the connection between mosquitoes and 
 malarial infection in man. So far as we know, a few 
 varieties of mosquitoes and man are the only places 
 where the malarial parasites develop, and Koch, fol- 
 lowing lines suggested by the work of others, has 
 now shown that the fresh cases of infection with 
 malaria occur only in warm weather when the parasites 
 can develop in the mosquitoes. Koch's idea is that 
 human beings having chronic malaria preserve in their 
 blood the malarial parasites during the cool months. 
 In the warm weather mosquitoes become infected, the 
 parasites develop in them and are present in their 
 poison sacs. These mosquitoes bite and infect fresh 
 human cases through subcutaneous inoculation. He 
 believes if we would treat all chronic malarial patients 
 with quinine so as to prevent the development of the 
 parasites and thus the infection of the new crop of 
 mosquitoes, we would prevent most, at least, of human 
 infection. 
 
 Blood parasites are extremely common in cold- 
 blooded animals, fish, reptiles, and in birds. .Birds 
 appear to suffer from malarial infection similar to that 
 in man, and the parasites found in the blood-corpuscles 
 are closely allied to those of human malaria. But in 
 birds infection cannot be produced by subcutaneous or 
 intravenous inoculation with parasites from human 
 blood, nor can infection be transmitted from birds to 
 man. The blood parasites found in fish and reptiles, 
 though similar to, are not identical with, those found 
 in man, and they are not transmissible to man. This 
 source of infection may, therefore, be excluded. Ex- 
 
640 APPENDIX. 
 
 perience shows that the disease is Dot contagious, in 
 the ordinary sense of the word, and that it is not 
 directly transmitted from man to man. 
 
 AMCEBA COLI (Amoeba Dysenteriae of Councilman and 
 Lafleur; Dysenteric Amoeba). 
 
 In 1875, Losch, of St. Petersburg, gave the first 
 accurate description of an amoeboid organism which 
 he found in the stools of a dysenteric patient, and to 
 it he gave the name amoeba coli. He claimed that this 
 organism is the cause of dysentery, and he succeeded in 
 producing a superficial ulceration of the large intestine 
 in one of four dogs which had received rectal injec- 
 tions of the dysenteric stools. Losch' s observation 
 has been confirmed by various researches in different 
 countries. 
 
 Morphology. The amoeba is a unicellular organism 
 belonging to the class of rhizopada of the protozoa, 
 and consists of slightly differential masses of proto- 
 plasm, which, under favorable circumstances, exhibits 
 spontaneous movements. In a state of rest the amoeba 
 assumes a spherical shape which appears discoid in the 
 field of the microscope. It may generally be distin- 
 guished from the other cellular elements found in the 
 feces by its pale greenish tint and by its stronger 
 refraction of light. Its diameter varies within wide 
 limits, 6// to 35// 7 more commonly between 12// and 
 26//. It is noteworthy that such differences in size 
 are found, as a rule, in different cases of the disease, 
 while the amoebae in any individual case are nearly 
 uniform in diameter. The body of a resting amoeba 
 has a well-defined, regular body, which, under ordi- 
 nary conditions, appears as a thin, single, dark line. 
 
PLATE II. 
 
 
 v 
 
 o 
 
 
 
 
 Figs, i, 2, and 3 show three phases of the parasite of tertian fever. 
 Fig. i, ring form, showing beginning pigment formation. Fig. 2, full- 
 grown parasite. Fig. 3, segmenting bodies. (WELCH and THAYER.) 
 
 Figs. 4, 5, and 6 show the parasite of quartan fever at different stages 
 of growth. Fig. 4, moderately developed intracorpuscular parasite. Fig. 5, 
 large swollen extracorpuscular form. Fig. 6, flagellate body. 
 
 (WELCH and THAYER.) 
 
 Figs. 7, 8, and 9 illustrate the sestivo-autumnal parasite. Fig. 7, ring-like 
 body, with a few pigmented granules. Fig. 8, crescent still in blood-cor- 
 puscle. Fig. 9, vacuolation of crescent. (WELCH and THAYER.) 
 
 Fig. 10. Amoeba from section of intestine hardened in alcohol and 
 stained with methylene blue. (COUNCILMAN and L,AFLEUR.) 
 
AMCEBA COLL 641 
 
 The body consists of two portions: the inner one, 
 which is more or less granular and of a darker color, 
 is known as the entoplasma; the outer one, which is 
 homogeneous and of a lighter color, as the ectoplasma 
 (see Plate II. , Fig. 10). This division into two zones 
 cannot always be made out, and is more evident in the 
 motile than in the resting amoeba. 
 
 The entoplasma constitutes the greater portion of the 
 body of the amoeba, being usually centrally situated, 
 but occasionally slightly eccentric. In the smaller 
 forms of amoebae it is finely granular, and may show 
 no other structure. In the larger forms it is more 
 coarsely granular, and often contains clear, circular, 
 and slightly oval spaces known as vacuoles. These 
 are extremely variable in number and size. 
 
 The edoplasma is quite homogeneous, forming a zone 
 of variable thickness around the entoplasrn. It has 
 the appearance of finely ground glass of a distinctly 
 pale green tint. 
 
 In most amoebae a nucleus can be seen. Its detection 
 is not always possible in fresh or motile amoebae, but 
 under certain conditions in the motionless or dead 
 amoebae the nucleus becomes evident, and it may be 
 easily shown by appropriate staining reagents. It is 
 situated eccentrically, at the edge of the entoplasm, 
 and appears as a discoid body, about 6// in diameter, 
 with a sharp contour, which, though occasionally 
 broken and irregular, is generally even; it may be 
 distinguished from vacuoles of the same size by its 
 higher refracting power. A nucleolus can seldom be 
 observed, and in stained specimens only. 
 
 Foreign bodies are frequently seen in the amoebae, 
 especially red blood-cells. These are sometimes so 
 
 41 
 
642 APPENDIX. 
 
 numerous that the whole body of the amoebae is filled 
 with them; they may be in a perfect state of preserva- 
 tion, or quite decolorized, or only recognizable by 
 their outline. The amoeba rarely contains leucocytes 
 or fat-globules. Various forms of bacteria are more 
 or less frequent inclusions, and black pigment gran- 
 ules and irregular brownish masses of pigment have 
 been noted by some observers. 
 
 Biological Characters. The most striking and char- 
 acteristic feature of the amoeba is its motility. This 
 may consist either in an alteration of its shape or in 
 an actual change of place. Both of these phenomena 
 are produced through the mechanism of pseudopodia. 
 These latter are rounded, blunt, and homogeneous 
 processes formed by the more or less gradual protru- 
 sion of a portion of the ectoplasm at some part of the 
 periphery of the amoeba. The motion is sometimes 
 quite gradual and continuous, at others sudden and 
 jerky. The progressive movement that is, actual loco- 
 motion is brought about by the protrusion of pseudo- 
 podia, and into these, when they have reached a certain 
 size, the granular or vacuolated entoplasm, with its 
 other contents, flows with a more rapid movement 
 than that by which the pseudopodia themselves were 
 formed. Locomotion is generally observed to take 
 place in the direction of least resistance, a group of 
 cellular elements or some detritus being sufficient to 
 divert the course of the amoeba. The amoeboid move- 
 ments are also influenced by various factors, particu- 
 larly by variations of temperature. They are most 
 active at the mean temperature of the human body, 
 becoming less active as the temperature falls or rises 
 above this mean. They become motionless in a tern- 
 
AMGEBA COLL 643 
 
 perature lower than 75 F. The amoeba does not 
 take the stain of various coloring solutions until the 
 movements cease, presumably on the death of the 
 organism. 
 
 Practically nothing is known of the conditions of 
 nutrition, respiration, and reproduction of the amoeba, 
 as no observations on these points are recorded. 
 
 Occurrence of Amoebae in Man. Amcebse were found 
 in the stools by Kruse and Pasquale in forty out of 
 fifty cases of the amoebic type of dysentery; by 
 Kartulis in every case in nearly 500 observations; and 
 by Councilman and Lafleur in thirteen out of fifteen 
 cases; while in their remaining two cases the amoeba 
 was found post-mortem, either in the material scraped 
 from the base of the intestinal ulcers or in sections of 
 the latter. The number found is very variable. In 
 some cases actively moving amoebae are found in great 
 numbers in every stool examined throughout the course 
 of the illness, while in other cases they can be detected 
 only in a long and careful search. As a general rule 
 they are more numerous and more frequently present 
 in the acute cases or in the earlier stages of the disease, 
 or in the periods of exacerbations of chronic dysentery ; 
 and they disappear more or less gradually from the 
 stools during convalescence. Occasionally the intestinal 
 ulceration is latent, the motions being quite formed, 
 with but small flakes of mucus adherent to them, in 
 which no amoebae may be found. In these cases the 
 existence of dysentery is not suspected until an abscess 
 of the liver occurs in which actively motile amoebae are 
 found, either by exploratory puncture or in the sputa 
 if the abscess evacuates itself spontaneously through 
 the bronchi. 
 
644 APPENDIX. 
 
 Numerous investigations have demonstrated conclu- 
 sively that amoebae may be present in the feces of 
 healthy persons. They have also been found in cases 
 of chronic diarrhoea, cholera, intestinal tuberculosis, 
 typhoid fever, hemorrhoids, and other diseases; chiefly 
 in such as are accompanied by looseness of the bowels. 
 Some of the cases cited as chronic enteritis or chronic 
 diarrhoea were in all probability examples of the more 
 chronic form of amoebic dysentery, but not all of them, 
 of course. Temporary looseness of the bowels in other- 
 wise healthy persons, either as the result of slight indis- 
 position or of medication, seems to be a condition of the 
 presence of amoebae in the stools. Thus, Schulberg 
 found these organisms in ten out of twenty loose stools 
 produced by the administration of Carlsbad salts, and 
 concluded that the amoeba is a normal and harmless 
 parasite of the intestines, the reason for its non-appear- 
 ance in ordinary fecal evacuations being the solidity and 
 acid reaction of the contents of the lower bowel, which 
 soon destroy it. The question naturally arises whether 
 more than one species of amoeba is found in the human 
 intestinal tract. So far no definite morphological dif- 
 ferences ha ye been found between the amoeba occurring 
 in the stools of healthy persons and that Jn patients 
 suffering from dysentery; nor can any deductions be 
 drawn from the attempts to cultivate the amoeba, for no 
 one yet has succeeded in producing pure cultures of it. 
 
 Pathogenesis. It is evident that, in the absence of 
 artificially produced pure cultures of amoebae, inocula- 
 tion experiments must be made wtih material such as 
 dysenteric stools or the contents of hepatic abscesses. 
 In a few cases such material from hepatic abscesses 
 which was found to contain no organisms other than 
 
AMCEBA COLL 645 
 
 amoebae, the inoculations have been made in three 
 ways: by feeding animals with material containing the 
 amoeba, by inoculation of the small intestine after a 
 preliminary laparotomy, and, finally, by rectal injec- 
 tions with or without suture of the anal orifice. The 
 first method has always proved unsuccessful except 
 when encysted forms were present. To the second 
 method the objection has been raised that the manipu- 
 lation of the intestines and the use of antiseptic solu- 
 tions during the course of the operation are in them- 
 selves a source of irritation to the bowel, and in some 
 cases have produced an enteritis. The third method 
 is the simplest, and has given positive results in the 
 hands of Losch, Kruse, Pasquale and others. 
 
 The results of the last two observers were as fol- 
 lows : Dysenteric stools, or material from hepatic 
 abscesses containing amoebae, were injected into the 
 rectum of various animals, with or without subsequent 
 closure of the anus, for twenty-four or forty-eight 
 hours. In some cases, chiefly those in which motion- 
 less amoebae were injected, no abnormal result followed; 
 in others, blood-tinged mucus, containing actively 
 moving amoebae, appeared in the evacuations from the 
 second day or thereabouts, but the animals did not 
 appear to be ill; in a third series, with evacuations of 
 a like character, the animals wasted and died after 
 a variable number of days. In both the second and 
 third series of cases post-mortem examination showed 
 pathological changes in the large intestine, proportion- 
 ate, as a rule, to the severity of the symptoms. Of 
 especial interest are the experiments made with material 
 from liver abscesses which were proved to contain no 
 other organism than the amoeba. Three such cases are 
 
APPENDIX. 
 
 recorded in cats, in all of which an experimental dysen- 
 tery was produced. The lesions found are reddening 
 and swelling of the intestinal mucosa, chiefly of the 
 lower half of the large bowel, with here and there 
 ecchymoses, small, superficial areas of necrosis, and 
 shallow ulcerations. The mesenteric glands and the 
 solitary lymphoid follicles are often swollen. In the 
 blood-tinged mucus covering the mucous membranes 
 amoebae are found in greater or less numbers. Micro- 
 scopical examination of sections of the intestine shows 
 that the necrosis is limited, as a rule, to the mucosa, 
 and that beneath it the submucosa is thickened and 
 oedematous and its vessels engorged; there is also small- 
 celled infiltration. Amoebae are found in the borders 
 of the ulcers, chiefly in the follicles of Lieberkiihn; 
 in the base of the ulcers they rarely penetrate more 
 deeply than the upper layers of the submucosa. With 
 the amoebae are found many bacteria, chiefly streptococci. 
 From a comparison with the lesions of amoebic dysen- 
 tery in man it will be seen that while the processes in man 
 and in the cat are not identical, more especially as re- 
 gards the depth and extent of the ulceration, yet in many 
 points the resemblance is striking. A series of control 
 experiments was undertaken, by the authors quoted, 
 with amoebae from the stools of healthy individuals and 
 the straw-infusion amoeba of Kartulis obtained in his 
 culture experiments. In neither of these cases could 
 an experimental dysentery be produced in any of the 
 animals inoculated. They conclude that it is proper 
 to designate the pathogenic amoeba as the amoeba dys- 
 enterice (Councilman and Lafleur), and to retain the name 
 amoeba coli (Losch) for the non-pathogenic amoeba of 
 the normal healthy intestine. 
 
AMCEBA COLL 647 
 
 Concerning the source of the amoeba and the mode of 
 infection little can be' positively stated. It is reason- 
 able to suppose, however, that the mouth must be the 
 usual path of infection, and that the amoeba, in all 
 probability, is taken with drinking water. 
 
 With regard to other organisms found in amoebic 
 dysentery, a great number of bacteria of various kinds 
 and some flagellated infusorial organisms are associated 
 with the amoeba in dysenteric stools. 
 
CHAPTER XXXIX. 
 
 THE MICRO-ORGANISM OF SMALLPOX AND COWPOX. 
 
 No bacteria have been found in smallpox which 
 seem to have any relation to the disease except as 
 secondary infections. The same is true of vaccinia. 
 In both the smallpox and vaccinia papules, vesicles, 
 and pustules, L. Pfeiffer and others have constantly 
 found small, homogeneous bodies in the epithelial cells 
 surrounding the lesions. These little bodies are in the 
 cell substance, not in the nucleus, and usually but one 
 or two exist in any one cell. They are regularly 
 missed in the skin when vaccination has failed, and 
 also in similarly appearing papules and pustules in 
 pyaemia, acne, etc. They apparently belong to the 
 class of protozoa, and from their constant presence are 
 believed to be the probable specific micro-organisms of 
 both diseases. They are at first about double the size 
 of the staphylococcus and increase to double that size 
 (see Fig. 87, p. 651). Similar bodies have been noted 
 in the blood. In a great many specimens of skin from 
 cases of variola and vaccinia examined by Williams in 
 the health department laboratories these bodies have 
 never been entirely missed in the epithelial cells sur- 
 rounding the lesions 
 
 The Connection Between Smallpox and Cowpox. The 
 inoculation of the virus of smallpox into calves 
 produces, when successful, in the first series moderate 
 
SMALLPOX AND COWPOX. 649 
 
 redness and swelling at the point of inoculation, with 
 some general disturbance. After the passage through 
 several animals an affection exactly similar to cowpox 
 occurs. The successful inoculation of the first series 
 of cattle from smallpox is a matter of great difficulty, 
 but so many experimenters have asserted that they 
 have produced lesions similar to cowpox from small- 
 pox that there seems no possibility of doubt that it 
 has been done. In the laboratory we have failed in 
 several attempts. 
 
 Experiments have demonstrated that children vacci- 
 nated with cowpox vaccine are not susceptible to inocu- 
 lation with smallpox lymph, and also that those who 
 have passed through smallpox cannot be inoculated 
 successfully with cowpox vaccine. The mutual immu- 
 nity conferred by inoculation with either, the similar 
 appearance of the bodies in the cells about the vesicles 
 of both, and the statements from reliable sources that 
 smallpox virus has produced in cattle a disease indis- 
 tinguishable from cowpox, leaves hardly any doubt that 
 the two are due to the same micro-organism, which has 
 become modified by transmission through cattle. Why 
 such passage should produce a permanent change in the 
 virulence of the organism is undoubtedly a difficult 
 matter to explain, but we must remember that we 
 know practically nothing about the life-processes of 
 this form of micro-organisms, and changes once pro- 
 duced in them may tend to become fixed. 
 
 The Duration of the Immunity Conferred by Vaccina- 
 tion. The immunity caused by successful vaccination 
 is not permanent, and varies in its duration in different 
 individuals. Although it may give some protection 
 from smallpox for ten or fifteen years, it is not well 
 
650 APPENDIX. 
 
 to count on immunity for more than two years, and 
 whenever we are liable to exposure it is well to be 
 vaccinated. If it was unnecessary it will not be suc- 
 cessful, while if it is successful we have reason to 
 believe we were liable to at least a mild smallpox 
 infection. 
 
 Protective Substances Present in the Serum of Animals 
 After Successful Vaccination. It has been repeatedly 
 shown that the blood-serum of a calf several weeks 
 after successful vaccination possesses feeble protective 
 properties, so that the injection of one to two litres of 
 it into a susceptible calf would prevent a successful 
 vaccination. A further and more convincing fact has 
 been demonstrated in the laboratory by Huddleston 
 namely, that when equal parts of a serum taken from 
 a calf, two weeks after successful vaccination, and of 
 an active vaccine are mixed together and inoculated 
 in a susceptible calf the vaccine fails "to take." 
 The serum of an unvaccinated calf has no deleterious 
 effect whatever when mixed with the vaccine. Serum 
 taken even several years after vaccination, if the case 
 is still immune, will inhibit very distinctly the action 
 of fresh vaccine virus. 
 
 The Form of Vaccine Virus Used. Vaccination is 
 now usually performed with calf virus, as this is easier 
 to obtain, is just as reliable, and practically eliminates 
 the slight possibility of the transferrence of syphilis 
 which existed in human vaccine. With active virus 
 a portion of skin only one- sixteenth of an inch in 
 diameter is scratched with the needle and the virus 
 rubbed in. If preferred it may be inserted by a 
 puncture. The vaccine is now usually mixed with 
 glycerin and water and placed in capillary tubes. So 
 
SMALLPOX AND GOWPOX. 
 
 651 
 
 prepared it is much more durable than when dried on 
 ivory points or quills. 
 
 The Appearance of the " Vaccine Organism." Williams 
 has described the appearances observed as follows : 
 
 " The epithelial cells of the skin in the infected areas 
 of vaccinated calves contain characteristic bodies. These 
 bodies are generally spheroidal, but may be quite irreg- 
 ular in outline. They vary in size from that of a 
 
 FIG. 87. 
 
 V V 
 
 Epithelial cells of sebaceous gland of hair containing vaccine bodies. 
 V. Homogeneous vaccine bodies. N. Granular nuclei. X about 600 diam. 
 
 particle of nuclear chromatin to that of the nucleus 
 iteelf, and are sometimes even larger. Usually they 
 are about one-fourth the size of the nucleus. They 
 are seen only in the body of the cell, never in the 
 nucleus, and generally only one is seen in each cell, 
 though there may be three or four. They take most 
 nuclear stains rather more faintly than the nucleus, 
 and are distinguished, moreover, by their homogeneous 
 
652 APPENDIX. 
 
 appearance. With hsematoxylin (Dela field's) and eosin 
 the nucleus of the epithelial cells take an irregularly 
 granular, dark purple stain, while the peculiar bodies 
 are a fainter, homogeneous purple, and the cell-bodies 
 pink" (Fig. 87). 
 
 Horses, rabbits, and sheep were successively vaccin- 
 ated with calf vaccine, but in none was the take any- 
 where as good as in calves, nor did it occur in every 
 instance. Guinea-pigs and dogs failed to take in a 
 few trials. The pulp and serum obtained from an 
 epidemic of cowpox took feebly in calves in a moder- 
 ate percentage of those inoculated. The characteristic 
 vaccine bodies were found practically identical with 
 those in vaccinia, except the bodies were a little larger 
 and more irregular in outline. 
 
 The Preparation of Vaccine. For the following sug- 
 gestions I am indebted to Dr. J. H. Huddleston, who 
 has had the immediate charge of the production of 
 vaccine for the New York Health Department for 
 some years : 
 
 Seed Virus. A sufficient amount of vaccine virus 
 should be on hand to vaccinate forty to fifty persons. 
 Five children in good health, and not previously vac- 
 cinated, should then be vaccinated each in a spot the 
 size of a ten-cent piece. On the fifth day after vaccin- 
 ation the top of the resulting vesicle should be removed 
 and sterilized bone slips be rubbed on the base exposed. 
 It should be possible in this manner to charge at least 
 from one to two hundred slips on each side of the slip 
 from each child. The slips should be allowed a moment 
 to dry and then placed in a sterilized box, in which, if 
 kept in cold storage, they will probably remain efficient 
 at least two or three weel^s. 
 
SMALLPOX AND COWPOX. 653 
 
 Animals. The preferable animals are female calves, 
 from two to four months of age, in good condition and 
 free from any skin disease. These can be vaccinated 
 on the posterior abdomen and inside of the thighs easily 
 by placing them on an appropriate table. It is possible 
 that on account of the character of the available supply 
 older animals may be desirable, but the calves take 
 more typically and are more easily handled. When 
 an animal is too old to be thrown and held easily it 
 may be vaccinated on the rump, each side of the spine; 
 but the skin there is tougher, and the resulting virus, 
 though efficient, is not so easily emulsified. 
 
 Vaccination. The calf should be cleaned thoroughly, 
 including the feet and the tail, and the hair should be 
 clipped from the end of the tail. The posterior abdo- 
 men and insides of the thighs are then shaved and the 
 skin beneath washed in succession with soap and water, 
 sterilized water and alcohol, and then dried with a 
 sterile towel. On this area there are then made about 
 one hundred scarifications, each from one-quarter to 
 one-half of an inch on a side. The scarification is 
 made most easily by cross-hatching with a six-bladed 
 instrument, the blades being about one-thirtieth of an 
 inch apart. The scarification is superficial, but brings 
 blood. An area as small as specified is less likely to 
 become infected than a larger one. The scarifications 
 should be separated from each other by an interval of 
 at least one-half to three-quarters of an inch. After 
 they have been made they should be dried with a sterile 
 towel or cotton and rubbed with the charged slips. One 
 to two slips, depending on the amount of virus each slip 
 contains, should be sufficient for vaccinating each ves- 
 icle. 
 
654 APPENDIX. 
 
 Collection. On the fifth or sixth day, depending upon 
 the rate of development of the vaccine vesicles, they 
 should be ready for collection. The entire shaved area 
 is washed with sterile water and sterile cotton, and the 
 crusts are picked off. The soft, pulpy, remaining mass 
 is then curetted off with an ordinary steel curette and 
 the pulp placed in a sterilized vessel. After the curet- 
 tage serum exudes from the torn base of the vesicle, 
 and ivory slips may be charged in this. The pulp 
 should be mixed with from two to three times its 
 weight of glycerin and water, equal parts, and this is 
 done most effectively by passing the mixture between 
 the rollers of a Doring mill. A watery pulp, especially 
 if it is not to be used immediately, should have the 
 smaller proportion of glycerin. The emulsion so pro- 
 duced can then be put up for issue in vials. The slips 
 charged with the serum from the calf may also be used 
 for vaccinating. Capillary tubes require especial means 
 of filling, and small vials filled and corked answer the 
 purpose admirably. 
 
 Propagation. Subsequent animals may be vaccinated 
 in any one of the three ways: (a) Slips may be charged 
 from typical vesicles on primary vaccinations, just as 
 with the first calf, and used for seed virus; (6) slips 
 charged with the serum from the calf may be used to 
 vaccinate a second calf; (c) the glycerinated emulsion 
 may be used to vaccinate succeeding calves, but in the 
 last case it is necessary to keep the emulsion a varying 
 length of time often two or three months before it 
 is fit for use to vaccinate the calf, because the use of 
 fresh glycerinated pulp on a succession of calves leads 
 to prompt degeneration of the vaccine and to the pro- 
 duction of infected vesicles. 
 
SMALLPOX AND COWPOX. 655 
 
 Laboratory. The laboratory should consist of at least 
 three rooms: (a) Stable; (6) operating-room; (c) labor- 
 atory-room. It should be possible to make and keep 
 all the rooms clean. The stable and operating-room 
 should be flushed with a hose and hot water daily. 
 Excreta should be removed immediately. The calves 
 can be kept cleaner if they stand on a raised and per- 
 forated platform, which is so short that the defecations 
 cannot fall on it, and if they have no bedding. They 
 must be fastened to keep them from licking the scari- 
 fications. If they are fed with milk the dust that 
 would be imported with other food is avoided. In the 
 health department, when a calf is removed its stall and 
 platform are scoured with a brush and sodium carbonate 
 solution. The stable should be provided with a shovel, 
 broom, hose, currycomb, mane brush, cord, halters, 
 and with buckets, scrubbing brushes, and sponges. 
 The operating-room should be well lighted and pro- 
 vided with a table and stools. 
 
 The only requisites for the table are that it should 
 be heavy and firm; that it should have holes through 
 the top so arranged that straps can be passed through 
 them to hold the calf down, and a vertical strip on one 
 side of the table to which the upper hind leg of the calf 
 can be fastened. The calf can be thrown up on the 
 table easily by two attendants. 
 
 The laboratory should also be well lighted and fur- 
 nished with tables, chairs, desk, case for instruments, 
 and refrigerator. It should also have both a steam 
 and a dry-air sterilizer, a set of scales weighing to 
 grammes or centigrammes, and a blast lamp and bel- 
 lows. In stock there should be one to two thousand 
 bone slips for seed virus, and ten to fifteen thousand 
 
65 6 APPENDIX. 
 
 smaller slips for issue, two or more scarifiers, a curette, 
 four to six razors for shaving the animals, a razor strop, 
 a pair of large scissors, curved on the flat, for clipping 
 the animals, a burette from which glycerin flows while 
 the vaccine pulp is being ground, burette holder, a 
 Dor ing vaccine grinder, clinical thermometers, to take 
 the temperature of the animals, six to twelve small 
 glass dishes with covers, a hard-rubber syringe, of four 
 ounce capacity, to make suction, absorbent cotton, glass 
 vials and corks, and several pounds of soft glass tubing, 
 three-eighths of an inch in calibre, to store virus emul- 
 sion. There should also be gowns and caps for the 
 attendants. Sodium carbonate, bichloride of mercury, 
 bromine (for a deodorizer), alcohol, and glycerin are 
 the chemicals needed. 
 
 For issue for public vaccinations there are also needed 
 packing-boxes, rubber bands, sheet wadding, needles, 
 and wooden toothpicks (for removing the virus from 
 the vials and rubbing it on the scarifications). 
 
 Yield. The material allowed from the five children 
 should vaccinate at least five calves; it may easily 
 vaccinate fifteen calves. Ten grammes of pulp and two 
 hundred charged slips would be an average yield from 
 a calf, and that, when made up, should suffice to vac- 
 cinate at least fifteen hundred persons. Calves vary 
 immensely in the yield. Of two calves vaccinated in 
 precisely the same way one may furnish material for 
 five hundred vaccinations and the other for ten thou- 
 sand vaccinations. 
 
 The Durability of G-lycerinated Virus in Sealed Tubes. 
 As a result of testing from time to time an immense 
 number of specimens of vaccine, the conclusion has 
 been reached that vaccine properly put up should 
 
SMALLPOX AND COWPOX. 657 
 
 keep at least three months. From time to time a 
 single lot of virus will fail by the end of one month. 
 Sometimes this is due to the glycerin, as when it has 
 some chemical impurity, or simply that the glycerin is 
 not diluted sufficiently with water. We find one part 
 of water to two of glycerin makes a good dilution. 
 
 Bacteria in Vaccine. It is impossible to prepare vac- 
 cine so that it is at the time of its removal free from 
 bacteria. In fact, there are usually very large numbers 
 of one or more varieties of bacteria present. When the 
 stable and animals have been kept clean the bacteria 
 comprise usually very few varieties; when dirty condi- 
 tions prevail the bacterial varieties are more numerous. 
 The number of bacteria found varies enormously. The 
 largest number found by us was 126,360 in one loopful 
 of vaccine virus, and the smallest number 523. Discrete 
 vesicles at the borders contain many less bacteria than 
 the confluent ones caused by the inoculation at the 
 scarification. The pulp has many more bacteria than 
 the contents of the vesicles. The period which elapses 
 before glycerinated virus becomes sterile is also quite 
 variable, but does not depend in any direct way upon 
 the number of bacteria originally present. A very 
 large number may disappear rapidly, and a few persist 
 for a long time. 
 
 After two or three weeks the number of living bac- 
 teria is usually greatly diminished, but seldom totally 
 destroyed. If we wait until the vaccine is surely sterile 
 it is very apt to be also useless that is, the vaccine 
 bodies are dead also. 
 
 In a very large experience we have learned that the 
 number of bacteria present has little to do with the 
 resulting vaccination. The character of the vesicles in 
 
 42 
 
(558 APPENDIX. 
 
 the calves and the trial vaccinations in young children 
 give a much more reliable basis for judging of the 
 character of the virus than any bacteriological counts 
 of colonies. 
 
 Pathogenic bacteria other than the practically non- 
 virulent skin staphylococci are not found when animals 
 are properly kept and vaccinated. The vaccine pulp 
 and serum mixture is added to two and one-half to 
 three and one-half times its bulk of a mixture consisting 
 of two parts of chemically pure glycerin and one part 
 of water. 
 
 Efficient vaccine should be inoculated in a portion of 
 skin no more than one-eighth inch in diameter. 
 
 Care of the Calves. All bedding is avoided and an 
 exclusively milk diet given; thus much of the otherwise 
 unavoidable dust is done away with. 
 
CHAPTER XL. 
 
 RABIES (HYDROPHOBIA). 
 
 ALTHOUGH neither the nature of the micro-organism 
 nor the nature of the poison of rabies has as yet been 
 determined, it is here considered because of its special 
 interest, and from the fact that it was the first infec- 
 tious disease to which a curative, or, rather, preventive, 
 method of inoculation was successfully applied. 
 
 Rabies is an acute disease of animals, dependent upon 
 a specific virus, and communicated by inoculation to 
 man. It is usually associated with an injury, such as 
 the bite of a dog, and the inoculation of the broken 
 surface with the saliva of an animal affected with the 
 disease. This is the so-called rabies of the streets. 
 Wolves, cats, foxes, and dogs; horses, cows, and deer 
 may contract the disease; monkeys, rabbits, and guinea- 
 pigs are all inoculable with it, as, indeed, are all warm- 
 blooded animals. Rabies occurs in almost all parts of 
 the world; it is most common in Russia, France, and 
 Belgium; it is not infrequent in Austria and those parts 
 of Germany bordering on Russia, and in England. It 
 is comparatively rare in this country, although it occurs 
 occasionally in various parts of the United States, also 
 in Mexico and South America. Rabies is extremely 
 rare in North Germany, Switzerland, Holland, and 
 Denmark, owing to the wise provision that all dogs 
 shall be muzzled; and in Australia it is unknown. 
 
660 APPENDIX. 
 
 Etiology and Pathogenesis. The etiology and patho- 
 genesis of rabies are still but imperfectly understood. 
 The poison, whatever may be its nature, is usually con- 
 tained in the saliva; and as early as the beginning of 
 this century experimental rabies was produced in the 
 dog by inoculation with the saliva of a hydrophobic 
 patient. The bulk of the toxic material appears to be 
 excreted in the saliva of the parotid gland, though a 
 certain small quantity may be excreted by the other 
 salivary glands, and also by the lachrymal glands, the 
 pancreas, and the mammse of rabid animals. The 
 poison may also be found in the suprarenal bodies and 
 in the fluid and substance of the cerebro-spinal nervous 
 system, especially the medulla oblongata; it is found 
 also in the peripheral nerves, though in much smaller 
 quantity than in the central nervous system. It has not 
 been found in the blood, the urine or the aqueous humor 
 of the eye; it has been reported to have been found in 
 the foetus. 
 
 That the disease is due to some form of organism 
 which has the power of multiplying in the tissues and 
 of producing a toxic substance, which appears to act 
 specifically upon the central nervous system, cannot 
 be doubted. As in other specific infectious diseases, 
 the virus is transmitted directly from animal to animal 
 through the medium of some fluid or secretion; it is 
 now very generally recognized that the disease cannot 
 arise anew, as was at one time assumed. In rabies, 
 again, as in other infectious diseases, there is a period of 
 incubation during which the poison appears to increase 
 in quantity. 
 
 The certainty with which the disease may be pro- 
 duced and its severity have been found to be deter- 
 
RABIES. 661 
 
 mined by three factors : (1) The quantity of the rabic 
 virus introduced; (2) the point of inoculation; (3) the 
 strength of the virus as determined by the kind of ani- 
 mal which affords the cultivation ground for the growth 
 of the hypothetical organism. It is a matter of com- 
 mon observation of hydrophobia in man that slight 
 wounds of the skin, of the limbs, and of the back are 
 often followed by the disease after an extremely long 
 period of incubation; while in lacerated wounds of the 
 tips of the fingers, where small nerves are numerous or 
 where the muscles and nerve-trunks are reached, or in 
 lacerated wounds of the face, where there is a similar 
 abundance of nerves, the period of incubation is usually 
 much shorter and the disease generally much more 
 rapid. Experimental infection in animals is produced 
 with the greatest certainty when the material from the 
 rabic nerve-centre (the spinal cord or bulb) of a dog, 
 or of a human being who dies of rabies, is injected into 
 the dura mater of the brain. It may be produced almost 
 as certainly when the injection is made into the anterior 
 chamber of the eye or into the greater nerve-trunks. 
 Intravenous injection is usually followed by positive 
 results in small animals, but the larger animals do not 
 succumb to this mode of inoculation. Subcutaneous 
 inoculation in animals is uncertain, because the periph- 
 eral nerves are not always injured; but injection directly 
 into a mass of muscle, especially into parts which are 
 rich in nerves, almost invariably produces the disease. 
 Absorption of the rabic poison, even from a healthy 
 mucous surface, has been said to have taken place; 
 and the conjunctiva, the nasal and genital mucous 
 membranes, and the digestive tract have been noted as 
 unabraded surfaces from which this has occurred. The 
 
662 APPENDIX. 
 
 rapidity with which the virus is diffused through the 
 body from the point of inoculation in the tissues seems 
 to vary according to the location of the wound, but it 
 is always comparatively slow. It has been found 
 that rabbits, when etherized and then presented to a mad 
 dog to be bitten on the fur, escape the disease in a very 
 large proportion of cases, although the teeth may have 
 passed well through the skin; if, on the other hand, the 
 part presented to the rabid dog be shaved before it is 
 bitten, the bitten animals contract rabies in a much 
 larger proportion of cases. So in man, in many cases the 
 rabic virus may be cleaned from the teeth by the cloth- 
 ing which covers the bitten part before they come in 
 contact with the skin. From what has been said it is 
 evident also that when the skin is thick and the nerves 
 few a small quantity of virus may find its way into a 
 wound, but riot penetrate into the nerves, and thus the 
 person bitten by a rabid animal may escape without any 
 ill effects beyond those due to a lacerated wound. This 
 will explain the fact that only about 16 per cent, of 
 the cases bitten by rabid animals appear to contract 
 hydrophobia. 
 
 Preventive Inoculation Against Rabies. The old treat- 
 ment of rabies consisted simply in encouraging bleeding 
 from the wound, or by first excising the wound and 
 then encouraging bleeding by means of ligatures, warm 
 bathing, cupping-glasses, etc. ; the raw surface was then 
 freely cauterized with caustic potash, nitric acid, or the 
 actual cautery. It is doubtful whether the disease ever 
 manifests itself after such heroic treatment if the 
 wound be small; but when the wounds were numerous 
 or extensive the mortality from it was still high. 
 As it was often impossible to apply cauterization to the 
 
RABIES. 663 
 
 wound rapidly or deeply enough to ensure complete 
 destruction of the virus, Pasteur and others were there- 
 fore led to study the disease experimentally in animals, 
 with the hope of finding some method of immunization 
 or even cure through bacteriological methods; these 
 investigations finally resulted in the discovery of 
 methods of preventive inoculation applicable to man. 
 
 Immunization against rabies may be effected in sev- 
 eral different ways. Pasteur's treatment is based upon 
 the fact that rabic virus may be attenuated or intensified 
 for any animal at will. He first observed that the tis- 
 sues and fluids taken from rabid animals varied con- 
 siderably in their virulence. Then he showed that the 
 virus taken from similar positions say the cerebro- 
 spinal fluid had always the same action in the same 
 species; but that fluid taken from an animal of different 
 species was weaker or stronger as the case might be. 
 Thus the cerebro-spinal fluid of a series of dogs is of 
 constant strength, and inoculations made from dog to dog 
 regularly produce death from rabies, the animals passing 
 through an incubation period fairly constant in length, 
 and through a series of similar symptoms up to death 
 at the same term. If, however, a series of monkeys 
 be inoculated the virus gradually becomes attenuated, 
 and this attenuation becomes more and more marked in 
 successive inoculations until eventually, after the disease 
 has run a longer and longer course in the successive 
 animals, there comes a time at which the virus is no 
 longer sufficiently active to cause death. If this atten- 
 uated fluid be now passed through a series of rabbits, 
 dogs, or guinea-pigs it comes back to such a strength 
 that it will kill, though slowly; then, however, its vir- 
 ulence gradually increases until the original intensity is 
 
664 APPENDIX. 
 
 reached. If successive inoculations be made into rab- 
 bits with fluid, either from the dog or the monkey, the 
 virulence may be so exalted beyond that of the virus 
 taken from a street dog, in which the incubation period 
 is from twelve to fourteen days, .that at the end of the 
 one hundredth passage the incubation period may be 
 reduced to about six or seven days. This, the strong- 
 est virus obtainable, was called by Pasteur the fixed 
 virus. Rabic virus appears also to become attenuated 
 under certain conditions of temperature, and if it be 
 subjected for about an hour to a temperature of 50 C. 
 its activity is completely destroyed, or in half an hour 
 if to a temperature of 60 C. A 5 per cent, solution 
 of carbolic acid, acting for the same period, exerts a 
 similar effect, as do likewise 1 : 1000 solutions of 
 bichloride of mercury, acetic acid, or potassium per- 
 manganate. The virus also rapidly loses its strength 
 by exposure to air, especially in sunlight; when pro- 
 tected from heat, light, and air it retains its virulence 
 for a long period. In his earlier experiments Pasteur 
 selected a series of rabic poisons of different strengths, 
 beginning with that obtained from the spinal cord of 
 the monkey from the very weak to the strongest that 
 he could obtain in this animal then passing through a 
 similar series obtained during the process of exaltation 
 of the virus by passage through the rabbit. By inoc- 
 ulating dogs subcutaneously with virus taken from a 
 series commencing with the weakest taken from a mon- 
 key, and gradually working up to that obtained from 
 the rabbit from the earliest to the latest in the series 
 the animals become immune not only against subcu- 
 taneous injection but against subdural injection with 
 fixed virus, and also against the bite of rabid dogs. 
 
RABIES. 665 
 
 Such a method as this, however, had several disadvan- 
 tages, and was not absolutely certain in its action, as 
 only fifteen out of twenty dogs were completely pro- 
 tected. Pasteur, therefore, assisted by Chamberland 
 and Roux, devised a more trustworthy and accurate 
 method, in which he utilized the fact that the cord of 
 a rabic animal when kept under certain conditions loses 
 its virulence in fourteen days. A series of cords cut 
 into short segments, which were held in series by the 
 dura mater, were suspended in sterile glass flasks 
 plugged with cotton stoppers, and containing a quantity 
 of some hygroscopic material, such as caustic potash; 
 and the whole was kept at a temperature of about 22 
 C. The cord when taken out at the end of the first 
 twenty-four hours was found to be almost as active as 
 the fresh untreated cord; that removed at the end of 
 forty-eight hours was slightly less active than that re- 
 moved twenty-four hours previously; and the diminu- 
 tion in virulence, though gradual, progressed regularly 
 and surely until, as already noted, at the end of the 
 fourteenth or fifteenth day the virus was inactive. An 
 emulsion of the cord of the last day was made, and a 
 certain quantity injected into a dog that had been bit- 
 ten; this was followed by an injection of an emulsion 
 of a thirteenth-day cord, and so on until the animal 
 had been injected with a perfectly fresh and, therefore, 
 extremely active cord, corresponding to the fixed virus. 
 Animals treated in this way were now found to be abso- 
 lutely protected, even against subdural inoculation with 
 considerable quantities of the most virulent virus; and 
 thus his protective inoculation against rabies became an 
 accomplished fact. As it would be impossible, how- 
 ever, or very undesirable, to inject any but persons who 
 
666 APPENDIX. 
 
 had actually been bitten by a rabid, or presumably 
 rabid, animal, Pasteur continued his experiments, in 
 order to see whether it would not be possible to cure a 
 patient already bitten. He carried on, therefore, a 
 series of experiments which led to the discovery that if 
 the process of inoculation be begun within five days of 
 the bite in animals in which the incubation period was 
 at least fourteen days, almost every animal bitten can 
 be saved; and that even if the treatment be com- 
 menced at a longer interval after the bite a certain 
 proportion of recoveries can be obtained. Thus the 
 application of this method of treatment to the human 
 subject was not tried until it had been proved in 
 animals that such protection could be obtained, and 
 that such protection would last for at least two years, 
 and probably longer. 
 
 The chance of success in the human subject appears 
 to be even greater than in the dog or rabbit, seeing that 
 on account of the resistance offered by the human tissues 
 to the virus the period of incubation is comparatively 
 prolonged; very rarely, if ever, does an outbreak of the 
 disease in man occur before an interval of at least fifteen 
 days. The first symptoms usually appear in the fifth 
 or sixth week, sometimes not until the third month ; 
 exceptionally the incubation period has lasted for a 
 year. Thus there is an opportunity of obtaining 
 immunity by beginning the process of vaccination 
 soon after the bite has been inflicted, the protection 
 being complete before the incubation period has passed. 
 In his earlier experiments Pasteur injected on each 
 succeeding day emulsions from a cord dried for one 
 day less until cords dried five days were reached; but 
 later he used those dried for only three days. This 
 
RABIES. 667 
 
 was the u simple" ten-day method. It was soon evident 
 that although this method was efficacious where the 
 wounds were not severe, and were confined to parts in 
 which the nerve-supply was not extensively interfered 
 with, it was often quite inadequate in serious cases, 
 as of wounds about the face, or of wounds inflicted 
 by a mad wolf, the virus of which is more active and 
 the lesions made more severe than that of the rabid 
 dog of the streets. In these latter cases the injec- 
 tions which, iu the simple treatment, are spread over 
 five days are made in three days; then, on the four- 
 teenth day, a fresh series of injections, or, rather, repe- 
 titions, is begun, which lasts until the twenty-first day. 
 This is the " intensive method." In the technique of 
 the treatment, which is the same in both methods, a 
 small portion (about 1 cm.) of the desiccated cord is 
 rubbed up thoroughly with about four or five times its 
 bulk of bouillon until a complete emulsion is made; 
 this, then, is injected by means of a syringe, holding 
 several cubic centimetres, first on one side of the hypo- 
 chondriac region and then on the other, the following 
 day, and so on alternately, to avoid irritation. With 
 the observance of thorough asepsis no local reaction to 
 speak of takes place, nor are abscesses ever formed. 
 The results of Pasteur's method of protective inocula- 
 tion, as recorded in the reports issued in the Annales de 
 rinstitut Pasteur and those of other antirabic institutes 
 in Italy, Russia, Roumania, etc., are very favorable. 
 Since 1886, when the treatment was first commenced at 
 the Pasteur Institute in Paris, upward of 20,000 per- 
 sons bitten by rabid, or presumably rabid, animals have 
 received preventive inoculations, with a mortality of 
 only 0.5 of 1 per cent. The mortality of those bitten 
 
668 APPENDIX. 
 
 on the face or head was 1.25 per cent, of those bitten on 
 the hand; it was 0.75 of 1 per cent, of those bitten 
 on other parts of the body, a little over 0.25 of 1 per 
 cent. As a rule, only those persons are treated who 
 have been bitten on the face or hands or whose clothes 
 have been lacerated, so that the virus has passed into 
 the wounds. Ordinarily, a certificate from a physician 
 or a veterinarian that the animal was rabid is required 
 before vaccination; but if the animal cannot be found 
 or the wounds are severe vaccination is performed with- 
 out it. Taking only the cases in which rabies has been 
 confirmed in the animal by a veterinary surgeon, the 
 mortality of the cases treated at the Pasteur Institute 
 in Paris is only 0.6 per cent. a proof, it would seem, 
 of the trustworthiness of the statistics. In view of this 
 fact there can no longer be any doubt of the value of 
 Pasteur's antirabic treatment. It has been stated by 
 some that the percentage of persons killed by the bites 
 of rabid animals is inconsiderable; but according to the 
 reliable statistics of Leblanc, from 1878 to 1883, out 
 of 515 persons bitten in Paris 83 died of hydrophobia, 
 a mortality of 16 per cent.; most authorities place the 
 mortality at a much higher figure. Extensive bites 
 on the face and head are considered to be particularly 
 dangerous ; the mortality of these is said to be at least 
 80 per cent. The bites of wolves seem to be more fatal 
 than the bites of dogs or other animals; the mortality 
 of these, in spite of the most intensive treatment, is 
 stated to be still 10 per cent., as against a previous 
 mortality, without specific treatment, of 40 to 60 
 per cent. But even Pasteur's antirabic treatment 
 appears to be unavailable when symptoms of the dis- 
 ease have manifested themselves. Our results in the 
 
RABIES. 669 
 
 New York Health Department have been very en- 
 couraging. 
 
 Other methods of immunization against rabies have 
 been proposed by different investigators. But all of 
 these methods have proved on trial to be unsatisfactory 
 and unreliable, beside being not devoid of danger. As 
 early as 1889, Babes and Lepp conceived the idea that 
 it might be possible by means of the blood to transmit 
 conferred immunity from rabies from one animal to 
 another; but although the success of these investigators 
 was not great, Tizzoni and Schwartz, and later Tizzoni 
 and Centanni, worked out a method of serum inocula- 
 tion and protection in rabies which is worthy of atten- 
 tion. In this method not the rabic poison itself but 
 the protective material formed is injected into the 
 tissues. These observers showed that the serum of 
 inoculated animals is capable of destroying the patho- 
 genic power of the rabic virus not only when mixed 
 with it before injection, but when injected simultane- 
 ously or within twenty-four hours after the intro- 
 duction of the virus into the body. This serum 
 treatment of rabies is still in the experimental stage. 
 We ourselves have had no experience with it, nor has 
 it been adopted in Paris, or, so far as we know, in other 
 places. It is quite possible that others will not obtain 
 such good results as the authors of the treatment, or 
 that it may not prove so efficacious in the treatment of 
 man as it has been found to be in experimental work. 
 
 The Cauterization of Wounds Infected with the Virus 
 of Rabies after an Interval of Twenty-four Hours. It 
 is commonly believed that unless a cautery is used 
 within an hour after infection by a suspected animal it 
 is useless to apply it. This belief is held by physi- 
 
670 APPENDIX. 
 
 cians in general, and also, apparently, so far as the 
 literature seen by me indicates, by those familiar with 
 rabies. For this reason physicians when applying a 
 cautery later than an hour after infection do so largely 
 as a matter of form, for its moral effect on the patient, 
 and so the application is not thorough, and in conse- 
 quence not effectual. There is no evidence to show 
 that this is the case at all; no systematic investigations 
 have been published, so far as we know, to prove the 
 point one way or the other. 
 
 We know that the virus of rabies is not carried into the 
 system by the blood, but through the nervous system. 
 Dr. Follen Cabot carried out an extensive series of 
 experiments in the laboratory upon guinea-pigs which 
 showed : 1. That 91 per cent, of guinea-pigs can be 
 prevented from developing rabies if the wounds be cau- 
 terized with chemically pure nitric acid at the end of 
 twenty-four hours from the time of infection, probably 
 a larger percentage if the cautery be used earlier. 2. 
 That fuming nitric acid is more effectual than the actual 
 cautery or pure nitrate of silver. 3. That some degree 
 of benefit is derived from thoroughly opening and swab- 
 bing out an infected wound within twenty four hours 
 from the time of infection when no cautery is used. 
 I believe that he demonstrated that in cases in which 
 the Pasteur treatment cannot be applied great benefit 
 may be derived from the correct use of cauterization 
 even twenty-four hours after infection, and that even 
 in cases in which the Pasteur treatment can be given, 
 an early cauterization will be of great assistance as a 
 routine practice, and should be very valuable, as the 
 Pasteur treatment is frequently delayed several days, 
 for obvious reasons, and does not always protect. In 
 
RABIES, 671 
 
 the case of small wounds all the treatment probably 
 indicated will be thorough cauterization with nitric 
 acid within twelve hours from the time of infection. 
 Our experience in dealing with those bitten by rabid 
 animals indicates that physicians do not appreciate the 
 value of thorough cauterization of the infected wounds. 
 But far more important than any treatment, curative 
 or preventive, for hydrophobia in man is the prevention 
 of rabies in dogs, through which this disease is usually 
 conveyed. Were all dogs under legislative control and 
 the compulsory wearing of muzzles rigidly enforced 
 where rabies prevails, hydrophobia would soon become 
 an almost unknown disease. This fact has been amply 
 demonstrated by the statistics of rabies in countries 
 where such laws are now in force. 
 

INDEX OF INFECTIOUS DISEASES AND 
 BACTERIA FOUND IN THEM. 
 
 Abscesses. The bacteria most commonly found in acute abscesses 
 are : Staphylococcus pyogenes aureus, 468, and albus, 470, and strep- 
 tococcus pyogenes, 481-483. The following species are also occa- 
 sionally met with: Pneumococcus, 509; colon bacillus, 449-453; 
 typhoid bacillus, 410; micrococcus tetragenus, 472, and influenza 
 bacillus, 324. In " cold abscesses " the tubercle bacillus is usually 
 the only micro-organism present. Beside these bacteria other varie- 
 ties may sometimes cause circumscribed suppurative processes. 
 
 Acne. Unna and Hodara (1894) obtained a bacillus from the 
 contents of acne pustules which they believe to be the cause of true 
 acne in man. Staphylococci are usually present in the pustules, and 
 undoubtedly exert some influence in the production of the affection. 
 
 Actinomycosis. Due to the presence of the actinomyces or ray 
 fungus, 618. 
 
 Alopecia. Although many dermatologists consider alopecia 
 areata to be of neurotic origin, others incline to the belief that 
 this affection is due to micro-organisms. Definite proof, however, 
 is still wanting of the infectiousness of the disease, or of the causal 
 relation of any specific micro-organism to it. Holborn (1895) de- 
 scribed a micra-ui' 6 ..- ' " wlvrh he named trichophyton radeus, 
 obtained it in pufre culture ^ pi^aced a similar affection in rab- 
 bits by inoculation, claiming that it was the cause of the disease in 
 man. 
 
 Angina. When not diphtheritic the pyogenic cocci, 483, includ- 
 ing the pneumococcus, are most frequently found in angina, also 
 Vincent's bacillus, 354. 
 
 Anthrax. Due to the bacillus anthracis, 554. 
 
 Appendicitis. In thirty-two put of thirty-five cases of appen- 
 dicitis bacteriologically examined by Hodenpyl (1893) the bacillus 
 
 43 
 
674 INDEX OF INFECTIOUS DISEASES. 
 
 coli communis, 451, 453, was the only micro-organism present. 
 Streptococci and other varieties of bacteria from the intestines are 
 also occasionally found. 
 
 Arthritis. The pneumococcus of Fraenkel, 509, has been fre- 
 quently fuund in arthritis following pneumonia. The gonococcus 
 of Neisser, 528, has been often met with in gonorrhceal arthritis. 
 Streptococci, 483, and staphylococci, 469, have been obtained from 
 the pus of the affected joints in suppurative arthritis following 
 scarlet fever. 
 
 Beri-beri. Various species of bacteria have been found in the 
 blood and tissues of persons affected with beri-beri, but none of these 
 have been demonstrated to be the specific cause of the disease. 
 
 Bronchitis. From the sputa of patients with putrid bronchitis 
 a spore-bearing bacillus has been obtained, the cultures of which 
 gave off the characteristic odor of fetid bronchitis. Hitzig (1895) 
 obtained two bacilli resembling the colon bacillus from a case of 
 putrid bronchitis. In ordinary acute bronchitis the pneumococcus 
 and streptococcus are most frequently found, but also small cocci like 
 the gonococci in shape, and occasionally other bacteria, especially 
 very small bacilli. In epidemics of influenza the influenza bacillus 
 is frequently found. 
 
 Bronchopneumonia. The micro-organism most frequently met 
 with in bronchopneumonia is the pneumococcus of Fraenkel, 507, 
 508, 511 ; next to this the streptococcus, 483 ; then FriedKinder's 
 bacillus, 458, and the staphylococcus alone or in combination. 
 At times the influenza bacillus, 325, is often found also. In pneu- 
 monia complicating typhoid fever the typhoid bacillus may be 
 present in almost pure culture. 
 
 Bubo. The pus from an unopened inguinal bubo following chan- 
 croid is usually sterile, though it may sometimes contain the ordinary 
 pus micrococci. 
 
 Bubonic Plague. Due to the presence of the bacillus pestis of 
 Kitasato and Yersin, in the contents of the buboes and in the blood 
 of infected animals and man, 607. 
 
 Carcinoma. No micro-organism has as yet been demonstrated 
 to bear any causal relation to cancer. Some attribute the disease to 
 protozoa. 
 
 Cerebro -spinal Meningitis. The micro-organism most fre- 
 quently found in cerebro-spinal meningitis complicating other diseases 
 is the pneumococcus, 510, 511, of Fraenkel ; while in uncomplicated 
 epidemic pases the diplococcus intracellularis raeningitidis, 516, 519, 
 
INDEX OF INFECTIOUS DISEASES. 675 
 
 of Weichselbaum is usually found. Streptococcus, 483, pyogenes has 
 also been met with in a certain number of cases, and occasionally 
 the colon and typhoid bacilli and other species of bacteria. 
 
 Chalazion. Whether stye is a specific affection or due to mixed 
 infection by the ordinary pus cocci is not known. 
 
 Chancroid. Ducrey (1890) discovered a bacillus, called by him 
 bacillus ulceris cancrosi, which he obtained from the pus of soft 
 chancres and buboes, and believed to be the cause of the disease, 
 but he and others who have found it failed to cultivate it. 
 
 Cholera Asiatica. Due to the cholera spirillum, or Koch's 
 "comma bacillus," 579. 
 
 Cholera Infantum. According to Booker and Jeffries, Bagin- 
 sky and others cholera infantum is not due to a specific micro- 
 organism, but to the action of the common putrefactive bacteria, 
 such as the colon bacillus and the proteus vulgaris and other allied 
 species, which, decomposing the food before it is digested, give rise 
 to toxic products, which are then absorbed in the alimentary canal. 
 
 Cholera Nostras. Finkler and Prior (1884) obtained from the 
 feces of patients with cholera nostras a spirillum which they be- 
 lieved to be the specific cause of this disease, but this has not been 
 corroborated by experiment It is more probable that cholera 
 nostras, summer diarrhoea, and all this class of gastro-intestinal dis- 
 orders are induced by the development of toxic products as the 
 result of the ferment action of various species of bacteria, such as the 
 colon, 452, and proteus groups. 
 
 Cholecystitis. The bacteria most commonly found are the colon 
 bacillus, 453, and less often the typhoid bacillus In the cases where 
 typhoid infection is present the bacilli may remain in the gall- 
 bladder for years. 
 
 Cholelithiasis. The colon, 453, and less often typhoid bacilli 
 are met with. Typhoid bacilli have been found at operations for 
 gallstones ten years after an attack of typhoid fever. (See Johns 
 Hopkins Hospital Bulletin, 1899.) 
 
 Conjunctivitis. The specific infectious forms of conjunctivitis 
 are undoubtedly due to the action of bacteria, as gonorrhreal, 525, 
 ophthalmia, and perhaps Egyptian catarrhal conjunctivitis (to the 
 bacillus discovered by Koch and studied by Kartulis, Weeks and 
 others), and diphtheritic conjunctivitis (to the Klebs-LofHer bacil- 
 lus, 349, when associated with diphtheria, or perhaps to the xerosis, 
 348, bacillus). The non-infectious forms of conjunctivitis, however, 
 are probably due, not to the action of specific micro-organisms, but 
 
676 INDEX OF INFECTIOUS DISEASES. 
 
 rather to some inflammation resulting from any cause aggravated 
 by the presence of pyogenic micrococci. 
 
 Coryza. It is doubtful whether this affection is due to the action 
 of any one specific micro-organism. Bacteria, however, play a part 
 in keeping up the inflammation in acute and chronic nasal catarrh ; 
 and in ozaena the offensive odor of the nasal secretions seems to be 
 due to the presence of certain bacteria, as Hajek's B. foetidus ozsense. 
 
 Cystitis. It has been shown by recent investigations that cyst- 
 itis is not caused by the mere presence of most varieties of bacteria 
 in the bladder, except, perhaps, by a few varieties, such as the gono- 
 coccus, 528, provided it be healthy ; but when the mucous membrane 
 is injured by mechanical violence, or by the presence of a foreign 
 body, cystitis is likely to result from the introduction of bacteria. 
 The micro-organisms most frequently concerned in the development 
 of chronic cystitis are the colon, 449, bacillus, the typhoid bacillus, 
 the bacillus aerogenes, and varieties of the proteus bacillus. Among 
 other bacteria which have been found in the bladder, and which may 
 influence the production of chronic inflammation, are the tubercle 
 bacillus, staphylococcus pyogenes aureus and allied species, the uro- 
 bacillus, and the urobacillus liquefaciens septicus of Krogius. 
 
 Dengue. No specific micro-organism has been found in this dis- 
 ease which would seem to bear a causal relation to it. 
 
 Dental Caries. According to Miller (1894), who has made an 
 exhaustive study of the bacteriology of dental caries, it is a mixed 
 infection due to the presence of various micro-organisms in the pulp, 
 cocci and bacilli being about equally frequent, with the occasional 
 appearance of spiral forms. The typical pyogenic cocci are seldom 
 present in the pus from the pulp, but various allied species are 
 found which cause pus formation in mice. Putrefactive processes, 
 also the result of bacterial action, greatly increase the action of the 
 pulp cocci. 
 
 Diarrhoea. The action of bacteria in the production of diarrhoea 
 has already been referred to under cholera infantum and cholera 
 nostras There is no reason to suppose that any particular micro- 
 organism is the specific cause of this class of diseases, which are due 
 probably to the toxins produced by various bacteria. Those which 
 would seem to have most to do with the production of these troubles 
 are bacilli of the colon and proteus groups. 
 
 Diphtheria. The Klebs-Loffler bacillus, 349, is now recognized 
 to be the specific cause of diphtheria. Other bacteria, however, are 
 always associated with this, producing more or less of mixed infec- 
 
INDEX OF INFECTIOUS DISEASES. 677 
 
 tion viz., the streptococcus, 352, staphylococcus, 353, and pneumo- 
 coccus, 353, mainly. Rather atypical pseudomembranous exudates 
 are also produced occasionally by the pyogenic cocci, and fairly 
 characteristic ones by the fusiform bacillus of Vincent, 354, and 
 probably by other varieties. 
 
 Distemper in Dogs. According to Schantyr (1893) the so- 
 called distemper in dogs includes three different infectious diseases : 
 
 1, abdominal typhoid, in which bacilli closely resembling those of 
 typhoid fever in man are found in the blood and various organs ; 
 
 2, dog-typhoid, in which bacilli are present which are readily cul- 
 tivated and stain by Gram's method ; and 3, genuine distemper, 
 containing bacilli which stain by Gram's method, but which do not 
 grow, or are difficult to grow, in culture media. 
 
 Dysentery. Trophic or amoebic dysentery is probably due, in 
 the majority of cases, to the presence of the amreba coli found in 
 the discharges. But this parasite has not been found in all forms 
 of dysentery and in healthy stools. Among other bacteria found 
 in the alvine discharges which may be concerned in the etiology of 
 certain cases of dysentery are : the colon bacillus, the proteus bacil- 
 lus, 542 ; the staphylococcus, the bacillus pyocyaneus, 539 ; the 
 bacillus dysenterica liquefaciens, etc. 
 
 Eczema. Various species of bacteria, micrococci and bacilli 
 have been obtained from cases of eczema seborrhoaicum by different 
 investigators, but none of these have been shown to be specific for 
 the affection. 
 
 Empyenia. The streptococcus pyogenes, 483, is the usual cause 
 of purulent inflammation of the pleura, in which it is found in 60 
 per cent, of cases. Empyema complicating pneumonia is generally 
 caused by the pneumococcus of Fraenkel, 511 ; and tubercular empy- 
 ema is due, of course, to infection by the tubercle bacillus The 
 various micro-organisms are often found together in the same case, 
 with one or the other predominating. In exceptional cases still 
 other varieties of bacteria, as the typhoid bacillus, may be met with. 
 
 Endocarditis. Numerous varieties of bacteria have been found 
 in pure culture or mixed in cases of ulcerative endocarditis, the 
 most common being pneumococci, 509, 511, streptococci, 483, and 
 staphylococci, 469 ; more rarely gonococci, 529, and other micro- 
 cocci, and occasionally bacilli of several varieties, are found. Most 
 probably the action of bacteria upon the endocardium is similar to 
 that upon the bladder, and endocarditis, like cystitis, is not usually 
 produced by them, unless some previous injury has been caused to 
 
678 INDEX OF INFECTIOUS DISEASES. 
 
 the valves, when their introduction then increases and maintains the 
 inflammatory process. 
 
 Endometritis. The healthy uterine mucous membrane is usually 
 sterile, but various species of bacteria have been observed in the 
 secretions of the cervix uteri. In inflammations of the uterus not 
 following abortion or child-birth the gonococcus is by far the most 
 frequent micro-organism found. In inflammation following child- 
 birth and operations the ordinary pus cocci and the colon bacillus 
 are also frequently met with, as well as other varieties of bacteria. 
 
 Erysipelas. Due to infection by streptococcus, 483. 
 
 Fowl-cholera. Due to infection by bacillus cholerae gallinarum 
 (Fliigge), probably identical with the bacillus of rabbit septicaemia 
 of Koch. 
 
 Furunculosis. Due to infection by the different pus cocci, and 
 more especially to the staphylococcus pyogenes aureus. 
 
 Gangrene. Etiology not positively known, but probably due to 
 the invasion of various parasitic and saprophytic bacteria into the 
 tissues when their vital resistance has become lowered by malnu- 
 trition and pressure or by a poor blood-supply. 
 
 Gas-formation. The bacillus aerogenes capsulatus, 545, has 
 been found either alone or along with pyogenic bacteria. 
 
 Glanders or Farcy. Due to infection by bacillus mallei, 600, 
 602. 
 
 Gonorrhoea. Due to "gonococcus," 528 (Neisser). 
 
 Hog-cholera. Due to infection by bacillus of hog-cholera (Sal- 
 mon and Smith). 
 
 Hog erysipelas or Swine-plague. Due to infection by bacillus 
 of swine-plague (Salmon and Smith ) . 
 
 Hydrophobia. No micro-organism has as yet been discovered 
 which is specific for this disease, 660. 
 
 Influenza. La Grippe. Due to infection by the bacillus of 
 influenza, 324. Pneumococcus inflammations often show similar 
 symptoms. 
 
 Influenza of Horses. Various micro-organisms, some resem 
 bling the pneumococcus and others the streptococcus in man, have 
 been described and claimed to be the specific cause of this epidemic 
 disease in horses. 
 
 Keratitis. According to Bach (1895) purulent keratitis is due 
 to the invasion of the cornea by micro-organisms, the pyogenic cocci, 
 pneumococci, etc., secondary to traumatism. 
 
 Leprosy. Due to the bacillus leprse, 316 
 
INDEX OF INFECTIOUS DISEASES. 679 
 
 Lupus. Due to infection by the tubercle bacillus, 276. 
 
 Lymphangitis. Usually due to streptococcus pyogenes ; occa- 
 sionally other organisms viz., staphylococcus pyogenes aureus and 
 albus and the colon bacillus, either alone or associated take part in 
 the production of this affection. 
 
 Malaria. Due to infection by the plasmodium malarise, 626. 
 
 Malignant (Edema. Due to infection by bacillus cedematis 
 maligni. 
 
 Malignant Pustule. Due to anthrax bacillus. 
 
 Mastitis. The micro-organisms commonly found in mastitis are 
 the ordinary pus cocci staphylococcus and streptococcus. Diplo- 
 cocci corresponding to the gonococcus have also been observed in 
 patients suffering at the same time from gonorrhoea. 
 
 Measles. All attempts to discover the etiology of measles, as of 
 the other specific eruptive febrile diseases except, perhaps, smallpox, 
 have thus far been futile. 
 
 Meningitis. (See Cerebro-spinal meningitis. ) 
 
 Nephritis. The urine in acute infectious diseases, and also in cases 
 of chronic nephritis, not infrequently contains various micro-organ- 
 isms, which are also found in the blood or some other of the organs. 
 Among the micro-organisms commonly found in nephritis second- 
 ary to general infection are : Streptococcus, staphylococcus, pneu- 
 mococcus, bacillus coli communis, bacillus typhi abdominalis, etc. 
 
 Ophthalmia. There can be little doubt that most acute and some 
 chronic inflammations of the eye are due to the presence of micro- 
 organisms. As is well known, the gonococcus of Neisser is the 
 cause of gonorrhoeal ophthalmia, and, according to Fuchs, a consid- 
 erable number of cases of so-called Egyptian ophthalmia are probably 
 due to the same infective agent, while other cases are perhaps caused 
 by the bacillus of Koch and Kartulis or by a combination of these 
 two micro-organisms. Many bacteria when introduced into the eye 
 give rise to inflammatory processes. The pneumococcus has been 
 found by several investigators in cases of panophthalmia and other 
 metastatic eye affections, sometimes alone or associated with the 
 streptococcus and staphylococcus pyogenes The bacillus pyocy- 
 aneus and bacillus coli communis have also been met with in these 
 affections, the inflammation being undoubtedly due to the presence 
 of the micro-organisms in the eye, which has been previously injured 
 in some way. 
 
 Osteomyelitis and Periostitis. According to most authors the 
 staphylococcus, 469, pyogenes aureus is considered to be the specific 
 
680 INDEX OF INFECTIOUS DISEASES. 
 
 cause of acute osteomyelitis ; but though present in many cases, alone 
 or associated with other bacteria, this is not the only organism found 
 in the affection. Staphylococcus pyogenes albus, streptococcus pyo- 
 genes, pneumococcus, and bacillus typhosus have also been found in 
 osteomyelitis by various observers. This disease cannot, therefore, 
 be regarded as a specific infection, but is rather a localized infectious 
 process due to various micro-organisms. Chronic osteomyelitis and 
 periostitis may also be considered in like manner as localized infec- 
 tions due to the tubercle bacillus. 
 
 Otitis Media. The micro-organisms most frequently found in 
 the purulent discharges in recent cases of otitis media are : Pneumo- 
 coccus, streptococcus, Staphylococcus pyogenes aureus and albus, and 
 Friedlander's bacillus. Occasionally found are : Bacillus pyocyaneus, 
 micrococcus tetragenus, bacillus coli com munis, and diphtheria ba- 
 cillus, etc. These bacteria are undoubtedly responsible, directly or 
 indirectly, for the inflammatory process and pus formation. 
 
 Ozsena. According to the investigations of Babes, Hajek and 
 others the micro-organisms most constantly found in the nasal 
 secretions of this affection are : Friedlander's bacillus, or a capsule 
 bacillus closely resembling this, and the bacillus ozsense of Hajek, 
 though other species of bacteria are also often present. 
 
 Parotitis. Simple uncomplicated mumps is probably due to 
 some specific micro-organism not as yet discovered, but the suppura- 
 tive inflammation is undoubtedly caused by one or other of the 
 ordinary pyogenic cocci. In parotitis occurring as a complication 
 of other infectious diseases, as pneumonia and typhoid fever, the 
 specific infective agents of these affections have been obtained in 
 pure culture from the pus of the parotid abscess. 
 
 Pericarditis. Various micro-organisms have been found in the 
 pericardial sac in pericarditis the ordinary pus cocci, pneumococci, 
 bacillus pyocyaneus, tubercle bacilli, etc. 
 
 Peritonitis. Among the bacteria found commonly in peri- 
 tonitis are : The ordinary pyogenic micrococci, the colon bacillus, 
 449, 453, the pneumococcus, gonococcus, typhoid bacillus, tubercle 
 bacillus, and proteus vulgaris. The pus cocci, especially strepto- 
 coccus and the colon bacillus, appear to be the usual cause of the 
 inflammatory process in puerperal peritonitis. In peritonitis fol- 
 lowing appendicitis and intestinal injuries the colon bacillus is 
 always present either alone or associated with other bacteria. 
 
 Pleuritis. Levy (1895), from a resume of the literature of the 
 subject, arrives at the conclusion that the pneumococcus is the usual 
 
INDEX OF INFECTIOUS DISEASES. 681 
 
 ause of pleurisy in children and of metapneumonia pleurisy, but 
 that in metastatic, pyogenic, pleuritic inflammation the strepto- 
 coccus or staphylococcus are the common infective agents. Pleurisy 
 due to streptococcus or staphylococcus infection is not in all cases 
 attended with pus formation ; the exudate in a certain proportion of 
 cases may remain serous. 
 
 In pleurisy occurring as a complication of typhoid fever the 
 bacillus typhosus has been found in the exudate. Occasionally 
 bacillus coli communis has been found. According to Flemming, 
 about 41 per cent, of the fatal cases of pleurisy are due to tubercular 
 infection. 
 
 Pleuropneumonia of Cattle. Due to infection by the pneumo- 
 bacillus liquefaciens bovis of Arloing. 
 
 Pneumonia. Characteristic lobar pneumonia is due to infection 
 by the pneumococcus, 507, 511 ; irregular cases are usually due to 
 Friedliinder's bacillus, streptococcus, staphylococcus, typhoid bacil- 
 lus, and influenza bacillus, 325. 
 
 Puerperal Fever. Due usually to infection by streptococcus, 
 482, or colon bacillus, 453. In some fatal cases staphylococcus 
 pyogenes aureus has also been found. Among other micro-organisms 
 sometimes met with, and which in these cases may have been con- 
 cerned in the production of the inflammatory process, are gonococcus 
 and proteus vulgaris. 
 
 Purpura Hsemorrhagica. No micro-organism has been shown 
 to be specific for this affection. 
 
 Pyaemia. (See Septicaemia.) 
 
 Pyelonephritis. According to Schmidt and Aschoff (1893), 
 pyelonephritis or surgical kidney is an infectious process usually due 
 to bacillus coli communis. (See also Nephritis. ) 
 
 Pyosalpinx Zweifel ( 1 892 ) has shown that a certain proportion 
 of the cases of pyosalpinx, if not all of them, are due to the pres- 
 ence of the gonococcus, 528. In some cases the infectious agent is 
 apparently streptococcus pyogenes or pneumococcus ; but Zweifel 
 believes that in the majority of cases in which the gonococcus is not 
 found it is the infectious agent, its absence being due to the fact that 
 it has died out in cases examined too late to find it. 
 
 Relapsing Fever. Due to infection by spirillum Obermeieri, 
 596. 
 
 Rheumatic Fever. The close analogy existing between true 
 rheumatism and certain of the infectious diseases, such as gonorrhoaa, 
 scarlet fever, and septic processes in general, which are frequently 
 
682 INDEX OF INFECTIOUS DISEASES. 
 
 accompanied by arthritis and endocarditis, has led to the belief that 
 acute rheumatism is an infectious disease. All investigations here- 
 tofore made, however, have failed to demonstrate the causal relation 
 of the different bacilli and cocci isolated to the disease. 
 
 Rhinitis Fibrinosa. Pseudomembranous rhinitis is often asso- 
 ciated with severe faucial diphtheria, and in these cases virulent 
 Klebs-Loffler bacilli, 349, are present. The primary form of the 
 affection, like conjunctivitis, usually runs a favorable course, and is 
 due usually to the attenuated diphtheria bacillus ; but here, too, 
 occasionally virulent diphtheria bacilli are found in the fibrinous 
 exudate. In such cases, of course, the nasal infection, however 
 mild, may give rise to severe faucial or nasal diphtheria in others. 
 In a few cases only pyogenic cocci have been found. 
 
 Bihinosderoma. A localized infectious process due, apparently, 
 to the presence of the bacillus of rhinoscleroma. 
 
 Rinderpest. The etiology of this acute exanthematous disease 
 in cattle is still obscure. Recovery from an attack, however, pro- 
 duces marked immunity, and Koch has achieved considerable 
 success in inoculating cattle against rinderpest 
 
 Scarlet Fever. Streptococci are constantly present in large 
 numbers in the pseudomembranous exudate of scarlatinal angina, 
 484, and not infrequently also in the blood and organs after death 
 from scarlet fever. The presence of these streptococci in scarlet 
 fever is probably due to the great increase in the streptococci usually 
 existing in the throat secretions, and does not indicate any specific 
 causal relation to the disease. 
 
 Septicaemia. General septicaemia in man is usually due to 
 infection by one or other of the common pyogenic cocci strepto- 
 coccus, 481, 483, pyogenes or staphylococcus, 469, aureus and albus. 
 Other micro-organisms which may sometimes be concerned in the 
 production of septicaemia are the pneurnococcus, 509, and colon ba- 
 cillus. Septicaemia in cattle, deer, swine, rabbits, and fowls is due 
 to infection by the bacillus of fowl-cholera or rabbit septicaemia 
 specifically, but various other bacteria produce septicaemia also in 
 rabbits, mice, swine, and fowls. 
 
 Stomatitis. Schimmelbusch, Lingard, Foote and others have 
 described bacilli obtained by them from the necrotic tissues in cases 
 of noma, but the etiology of the disease is by no means established. 
 
 Syphilis. The bacillus of Lustgarten, 309, is accepted by some 
 to be probably the specific cause of the disease, but this is far from 
 proven. 
 
INDEX OF INFECTIOUS DISEASES. 683 
 
 Tetanus. Due to infection by the tetanus bacillus, 388. 
 
 Texas Fever in Cattle. Due to infection by a blood parasite 
 belonging to the protozoa, described by Smith under the name of 
 pyrosoma bigeminum. 
 
 Tonsillitis. (See Angina. ) 
 
 Trachoma. Various micmcocci have been found in trachoma 
 by different investigators and claimed by them to cause the affection, 
 According to Fuchs and Hoor, trachoma is frequently, if not always, 
 due to infection by the gonococcus 
 
 Tuberculosis. All forms of tubercular infection in man and 
 animals are due to the bacillus tuberculosis. The bacillus which 
 causes tuberculosis in cattle, 299, and the one which produces it in 
 fowls, 300, though closely resembling the tubercle bacillus in man, 
 possess some slight differences. 
 
 Typhoid Fever. Due to infection by bacillus typhi abdomi- 
 nalis, 402. 
 
 Typhus Fever. The specific causative agent of this, under 
 certain circumstances, extremely infectious disease has not yet been 
 determined. 
 
 Varicella. No micro-organism has been demonstrated to bear 
 any relation to the etiology of this affection. 
 
 Variola and Vaccinia. Probably due to protozoa, 651. The 
 common pus cocci and various other micro-organisms are found in 
 the characteristic pustular eruption ; their presence is due to sec- 
 ondary infection of the pustules, and has nothing to do with the 
 cause of the disease, 657 
 
 Whooping-cough. Considered by Koplik and others to be due 
 to a small bacillus found in the nasal and bronchial secretions in 
 cases of the disease, 613. 
 
 Wool-sorter's Disease. Due to anthrax bacillus. 
 
 Yellow Fever. Sanarelli ( 1 897 ) discovered a small bacillus, 609, 
 in the blood and tissues of yellow fever cadavers, which he named 
 "bacillus icteroides," and claimed to be the specific cause of yellow 
 fever, 609. 
 
GENERAL INDEX. 
 
 CHORIONSchoenleinii (favus | Antitoxins, effect of, how pro- 
 
 fungus), 622 
 Acids, effect of, on bacteria, 158 
 formation of, from alcohol, 
 
 etc., 83 
 
 from carbohydrates, 80 
 Aerobic bacteria, 141 
 Agar, nutrient, 215 
 
 characteristic of, 225 
 Air, bacteriological examination 
 
 of, 252 
 
 Alexines, 120 
 Amoeba coli, 640 
 dysenterise, 640 
 Amoebae, occurrence of, in man, 
 
 643 
 Anaerobic bacteria, 141 
 
 culture methods for, 233 
 Aniline dyes, 166 
 
 water solution of fuchsin or 
 
 gentian violet, 202 
 Animals, use of, for diagnostic 
 
 and test purposes, 236 
 Anthrax bacillus, 547 
 
 biological characters, 551 
 growth in media, 552 
 identification of, 562 
 infection, how caused, 559 
 morphology of, 548 
 occurrence in cattle and 
 
 sheep, 556 
 in man, 557 
 pathogenesis, 554 
 spore formation, 550 
 infection, prophylaxis against, 
 
 561 
 
 Antiseptic action, 151 
 Antitoxins, absorption of, taken 
 by the mouth, 115 
 
 duced, 363 
 elimination of, by the body, 113 
 nature of, 362 
 
 results of, in treatment of diph- 
 theria, 358 
 
 stability of, in the serum, 113 
 varieties of bacteria producing, 
 
 in the body, 113 
 Apparatus, sterilization of, 222 
 Arnold steam sterilizer, 213 
 Arthrospores, 46 
 Attenuation, 151 
 Autoclave, 214 
 Auto-infection. 131 
 
 BACILLI, general characters of, 
 38 
 
 Bacillus aerogenes capsulatus, 545 
 anthracis, 547 
 
 symptomatici, 563 
 biology, 564 
 morphology, 563 
 pathogenesis, 564 
 coli communis, 444 
 of Friedlander, 458 
 icteroides (yellow fever), 609 
 of malignant oedema, 543 
 mallei (of glanders), 598 
 pestis bubonicae (plague), 606 
 proteus vulgaris, 539 
 
 biological characters, 540 
 morphology, 539 
 pathogenesis, 541 
 pyocyaneus, 535 
 biology, 535 
 morphology, 535 
 pathogenesis, 536 
 
686 
 
 GENERAL INDEX. 
 
 Bacillus of tuberculosis, 263 
 typhosus, 402 
 of whooping-cough, 613 
 Bacteria, adaptation to soil of, 91 
 attenuation of, 262 
 chemical composition of, 50 
 
 effects of, 60 
 classification of, 257 
 definition of, 34 
 elimination of, from the body, 
 
 115 
 
 entrance of, into the blood, 116 
 general characteristics of, 33 
 influence of, one species on an- 
 other, 56, 137 
 invasion of, in tissues after 
 
 death, 198 
 
 involution, forms of, 49 
 manner in which they produce 
 
 disease, 92 
 
 morphological forms of, 35, 40 
 motility of, 58 
 nutritive substances required 
 
 for growth of, 52 
 parasitic, 34, 89 
 pathogenic, presence of, on 
 
 healthy skin, 130 
 of, in respiratory tract, 130 
 presence of, in intestines, 131 
 production of light by, 59 
 products of, general symptoms 
 caused by poisons ab- 
 sorbed, 93 
 local effects, 91 
 relation of, to disease, 85 
 saprophytic, 34, 88 
 size and nature of, 33 
 spore formation of, 46 
 structure of cells of, 42 
 temperature required for growth 
 
 of, 56 
 
 their relation to oxygen, 54 
 thermic effects of, 60 
 vegetative reproduction of, 44 
 Bacterial species, permanence of, 
 
 260 ^ 
 Bactericidal substances in blood of 
 
 immunized animals, 108 
 nature of, 108 
 
 Bedding, carpets, upholstery, etc., 
 disinfection of, 187 
 
 Bichloride of mercury, 156 
 
 solution of, 170 
 
 Blood, bactericidal effect of, 119 
 cultures, how obtained, 241 
 -serum coagulator, 220 
 germicidal effect of, 119 
 as a medium, 217, 268 
 Books, disinfection of, 187 
 Bouillon nutrient, 212 
 Bovine tuberculosis, 299 
 
 relation of, to human tuber- 
 culosis, 299 
 Bromine, 160 
 Bubonic plague bacillus, 606 
 
 immunizing injections in, 
 608 
 
 P<ALCIUM compounds, 157 
 
 \J Camphor, 166 
 
 Capaldi plate medium, 436 
 
 Capsule bacteria, 43 
 
 Capsules of bacteria, staining of, 
 
 203 
 
 Carbolic acid, 165 
 solution, 170 
 Carriages, etc., disinfection of, 
 
 188 
 
 Cerebro -spinal meningitis, bac- 
 teriological diagnosis of, 520 
 Chemicals, effects of, on bacteria, 
 
 151 
 Chemotaxis, negative, 121 
 
 positive, 121 
 Chlorine, 159 
 Chloroform, 165 
 Cholera immunity, 581, 585 
 infection, how caused, 582 
 spirillum, accidental infection 
 
 in man, 579 
 biological characters of, 570 
 
 diagnosis of, 587 
 effect of chemical disinfect- 
 ants on, 576 
 
 growth in gelatin plate cul- 
 tures, 570 
 stick cultures, 572 
 immunizing injections ( Haff- 
 
 kine's), 585 
 morphology, 569 
 pathogenesis, 577 
 
GENERAL INDEX. 
 
 687 
 
 Cholera spirillum, varieties and 
 
 variations, 585 
 toxin, 580 
 Cocci, characters of, 36 
 
 staphylococcus pyogenes au- 
 
 reus, 461 
 
 streptococcus pyogenes, 476 
 Cold predisposing to infection, 
 
 130 
 
 Colon bacillus, 444 
 biology of, 445 
 differential diagnosis from 
 
 typhoid bacillus, 454 
 growth on common media, 
 
 446 
 
 immunization, 451 
 morphology of, 444 
 occurrence in man and ani- 
 mals, 451 
 pathogenesis, 448 
 Copper sulphate, 157 
 Cover-glass preparations, how 
 
 made, 198 
 how stained, 199 
 -slips, how to render free from 
 
 grease, 198 
 Cowpox, micro-organisms of, 648, 
 
 651 
 
 relation to smallpox, 648 
 Creolin, 171 
 Creosote, 166 
 Cresol, 165 
 
 Culture, contamination of, 231 
 media, reaction of, 55 
 method of making, at autopsy, 
 
 241 
 from urine and feces, 243 
 
 DENEKE'S cheese spirillum, 
 592 
 
 biology, 592 
 morphology, 592 
 Diphtheria antitoxin, actual 
 
 steps in test of, 373 
 nature of, 362 
 
 of its action on toxin, 
 
 363 
 
 persistence in the blood, 366 
 production of, for therapeutic 
 purposes, 359 
 
 Diphtheria antitoxin, technical 
 
 points in testing, 366 
 testing of, 364 
 unit, definition of, 366 
 use of, in treatment and im- 
 munization, 365 
 bacilli, exudate due to, con- 
 trasted with that due to 
 other bacteria, 376 
 persistence of, in the throat, 
 
 350 
 virulent, in healthy throats, 
 
 349 
 bacillus, 329 
 
 animal inoculation as a test 
 
 of virulence of, 383 
 biology of, 337 
 in different culture media, 
 
 336 
 
 growth on agar, 338 
 on blood serum, 338 
 in bouillon, 341 
 history of, 329 
 isolation of, 340 
 morphology of, 332 
 non-virulent forms, 347 
 pathogenesis of, 342 
 staining characteristics, 334 
 toxin of, 344 
 bacteriological diagnosis of, 
 
 377 
 characteristic appearances of, 
 
 375 
 
 direct microscopical examina- 
 tion of exudate, 3 2 
 examination of cultures, 381 
 mixed infection in, 352 
 technique of, 377 
 toxin, neutralizing value *of a 
 
 fatal dose 'of, 369 
 relation between the toxicity 
 and neutralizing value of, 
 366 
 
 transmission of, 355 
 antitoxic serum, 357 
 susceptibility and immunity 
 
 to, 356 
 value of cultures in diagnosis 
 
 of, 373 
 
 Diphtheritic inflammations, loca- 
 tion of, 347 
 
688 
 
 GENERAL INDEX. 
 
 Diplococcus intracellularis men- 
 ingitidis, 516 (See Meningo- 
 coccus^ 
 
 pneumoniae, 498 
 Disinfectants, 156-167 
 testing value of, 152 
 Disinfecting chambers, public 
 
 steam, 190 
 Disinfection, 152 
 in infectious and contagious 
 
 diseases, 171 
 
 practical (house, person, in- 
 struments, and food), 168 
 Dry-heat sterilizer, 223 
 Drying, effect of, on bacteria, 139 
 
 FHRLICH'S theory of the pro- 
 
 J-J duction of antitoxin in blood, 
 368 
 
 Electricity, influence of, on bac- 
 teria, 134 
 
 Eisner's method for cultivation 
 of typhoid bacillus, 434 
 
 Endospores, 46 
 
 Epitoxoids, Ehrlich's theory of, 
 368 
 
 Erlenmeyer flask, 221 
 
 Esmarch's tubes, 228 
 
 Essential oils, 166 
 
 l?ATS, decomposition of, 77 
 T Feces, disinfection of, 173 
 Fermentation tube, 82 
 Ferments, diastatic, 65 
 
 enzymes, 63 
 
 inverting, 65 
 
 organized and unorganized, 62 
 
 proteolytic or peptonizing, 64 
 
 rennet, 65 
 Flagella, 44 
 
 staining of, 205 
 Formaldehyde, 160 
 
 development of, from trioxy- 
 methylene pastilles, 184 
 
 forms of apparatus for develop- 
 ing, 181 
 
 gas, advantages of, over sul- 
 phur dioxide, 188 
 
 and sulphur dioxide gases, prac 
 tical employment of, 178 
 
 Formalin, 170 
 
 Friedlander's bacillus, 458 
 
 biological characters of, 458 
 morphology of, 458 
 pathogenesis of, 459 
 
 Fungi, pathogenic varieties, 619 
 
 riABBETT'S solution, 304 
 iJ Gas, formation of, from car- 
 bohydrates, 81 
 Gelatin, nutrient, 215 
 
 characteristics of, 225 
 Germination of spores, 49 
 Glanders bacillus, 598 
 isolation of, 603 
 morphology, 598 
 pathogenesis, 600 
 test for (mallein), 604 
 Gonococcus in cases of chronic 
 
 urethritis, 533 
 
 Gram's method of staining, 532 
 Neisser, 522 
 
 biological characters of, 525 
 morphological characters of, 
 
 522 
 in pus-cells, 524 
 
 affections due to, 528 
 immunizing serum for, 529 
 pathogenesis, 529 
 Gonorrhoea, bacteriological diag- 
 nosis of, 530 
 Gram's method of staining, 203 
 
 TJAFFKINE'S preventive in- 
 
 11 oculation for cholera, 585 
 for plague, 609 
 
 Hands, disinfection of, 192 
 
 Hanging drop for study of bac- 
 teria, 209 
 
 Heat, dry, effect of, on bacteria, 
 
 147 
 moist, effect of, on bacteria, 147 
 
 Hiss' media for typhoid bacillus, 
 430 
 
 Hydrogen peroxide, 159 
 
 Hydrophobia, 659 
 etiology of, 660 
 
 preventive inoculation (Pas- 
 teur), 662 
 
GENERAL INDEX. 
 
 689 
 
 Hydrophobia, preventive, cau- Koch's tuberculin, 288 
 
 terization of wounds, 669 
 
 IMMUNITY, acquired, to poi- 
 1 son, 112 
 to bacterial poisons, 123 
 decrease of, 103 
 natural, 102 
 produced by injection of serum 
 
 of immunine animals, 109 
 specific, 106 
 
 how acquired, 107 
 theories of, 119 
 Immunization by non-specific 
 
 means, 104 
 Incubators, 231 
 Infected dwellings, disinfection 
 
 of, 186 
 Infection, influence of quantity 
 
 in, 95 
 inherited and susceptibility to 
 
 or immunity from, 132 
 mixed, 98 
 
 modes of entrance of, 100 
 removal by local means, 105 
 spread of, 128 
 theories of, 123 
 Influenza bacillus, 320 
 
 biological characters, 321 
 detection of, in sputum, 322 
 immunity to, 325 
 morphology of, 321 
 pathogenesis of, 324 
 pneumonia from, 325 
 resistance of, to drying, 323 
 staining characteristics of, 
 
 321 
 
 in tuberculosis, 326 
 diagnosis of, 327 
 spread of, 327 
 
 Instruments, dressings, etc., for 
 surgical operation, disinfection 
 of, 191 
 Iodine, 160 
 
 trichloride, 160 
 lodoform, 165 
 Iron sulphate, 157 
 
 KLEBS-LOFFLER bacillus, 
 329 
 
 old tuberculin, 289 
 
 1 EPROSY bacillus, 315 
 Lj biological characters of, 316 
 differential diagnosis of, 318 
 location of, in tissues, 316 
 morphology of, 315 
 pathogenesis of, 316 
 direct inheritance of, 317 
 Ligatures, disinfection of, 192' 
 Light, influence of, on bacteria, 
 
 135 
 Lime, chloride of, 160, 170 
 
 milk of, 170 
 
 Loffler's alkaline solution of me- 
 thylene-blue for staining diph- 
 theria bacillus, 334 
 Lupus, 278 
 
 Lustgarten's bacillus, 311 
 Lysol, 171 
 
 MALARIA, diagnosis of, 635 
 infection, how acquired, 638 
 mixed infection in, 635 
 technique of blood examina- 
 tion in, 636 
 Malarial parasites. See Plasmo- 
 
 dium malarise, 626 
 Material for bacteriological ex- 
 amination, procuring of, from 
 
 those suffering from disease, 
 
 240 
 
 Mallein test for glanders, 604 
 Media to be used at autopsy, 241 
 
 sterilization of, 218 
 Meningococcus, 516 
 
 biological characters of, 517 
 
 morphology of, 516 
 
 pathogenesis, 518 
 Mercury, bichloride, 156 
 
 biniodide, 157 
 Mesophilic bacteria, 145 
 Metachromatic bodies, 42 
 Metbylene-blue solution, 202 
 Metschnikoflf's theory, 122 
 Micrococcus gonorrhoeae, 522 
 
 lanceolatus, 498 
 
 tetragenus, 472 
 Microscope, different parts of, 206 
 
 44 
 
690 
 
 GENERAL INDEX. 
 
 Microscopical examination of un- 
 stained bacteria, 198 
 Migula, classification of bacteria 
 /^ by, 258 
 
 / Milk as a culture medium, 216 
 ( sterilization of, 193 
 \. streptococci and staphylococci 
 ^ in, 117 
 
 tubercle bacilli in, 117, 283 
 Mixed infection, 137 
 
 examination for other bac- 
 teria in tuberculosis, 305 
 practical notes in the exami- 
 nation for, 307 
 
 Mosquitoes as agents of infection 
 in malaria, 638 
 
 NEISSER'S stain for diphtheria 
 bacillus, 335 
 Nitrogen combination produced 
 
 by bacteria, 79 
 Nuclein, 121 
 
 PARASITES, strict, 91 
 
 I Pasteurization, 149 
 
 Pasteur's flask, 221 
 
 Petri dish, 225 
 
 Pfeiffer's cholera reaction, 582 
 
 Phagocytosis, 122, 125 
 
 Phenolphthalein as an indicator, 
 
 218 
 Pigmented leucocytes in malaria, 
 
 632 
 
 Pigment production, 66 
 Plasmodium malaria?, 626 
 
 sestivo autumnal parasite, 
 630 
 
 effect of quinine upon, 634 
 
 inoculation experiments 
 with, 633 
 
 quartan parasite, 629 
 
 tertian parasite, 627 
 Plasmolysis, 42 
 Plate cultures, 225 
 
 study of colonies in, 228 
 of, in gelatin, 230 
 
 surface inoculation of, 228 
 
 technique of making 224 
 Pneumococcus, 498 
 
 Pneumococcus, attenuation of 
 
 virulence of, 506 
 biological characters of, 500 
 in blood, 510 
 diseases caused by, 511 
 effects of germicidal agents, 
 
 light, and drying on, 503 
 growth on ordinary media, 500 
 
 on special media, 502 
 immunity to infection by, 513 
 morphology of, 498 
 occurrence in man, 507 
 pathogenesis of, 504 
 producing mixed infection, 508 
 source of infection by, 504 
 varieties of, 511 
 
 Potatoes as medium, 216 
 
 Proteus, 71 
 
 Pseudodiphtheria bacilli, 351 
 
 Pseudomembranous inflamma- 
 tions due to bacteria other than 
 the diphtheria bacilli, 353 
 
 Pseudotuberculosis, streptothrix 
 of, 619 
 
 Psychrophilic bacteria, 145 
 
 Ptomai'ns and toxins, 68 
 
 Pure cultures, 230 
 
 Putrefaction, 78 
 
 Pyogenic cocci, 461, 476 
 
 RABIES, 659 
 cauterization of infected 
 
 wounds, 669 
 Reaction in media, correction of, 
 
 217 
 
 Reduction processes, 75 
 Rooms, disinfection of, 175 
 
 OAPROPHYTES, facultative, 
 O 90 
 
 strict, 90 
 
 Scarlet fever, streptococci in, 354 
 Sections, preparation of, 200 
 Serum, anti-pneumococcic, 514 
 treatment of pneumonia by, 
 
 514 
 
 antistreptococcic, 489 
 preparation of, 492 
 protection afforded by, 491 
 
GENERAL INDEX. 
 
 691 
 
 Serum antistreptococcic, stability 
 
 of, 492 
 standardization of. value of, 
 
 492 
 
 therapeutic results of, 493 
 protective, test of, 110 
 test, 417 
 
 Silver, nitrate, 157 
 Skin, disinfection of, 192 
 Smallpox, micro-organisms of, 
 
 648 
 
 Smegma bacillus, 313 
 Sodium compounds, 157 
 Specific immunity, nature of, 124 
 Spirilla, characters of, 39 
 Spirillum of Asiatic cholera. See 
 
 Cholera, 568 
 Finkler and Prior's, 589 
 biology, 589 
 morphology, 589 
 pathogenesis, 591 
 Metschnikovi, 593 
 
 biological characters of, 594 
 Obermeieri, 596 
 
 in relapsing fever, 596 
 Sporagenous granules, 42 
 Spores, staining of, 203 
 Spore types, 48 
 Sputum, collection of material, 
 
 301 
 
 from consumptive patients, dis- 
 infection of, 174 
 general rules in microscopical 
 
 examination of, 308 
 methods of examination for 
 
 tubercle bacilli in, 302 
 washing of, 306 
 
 Staining solutions, ordinary, 200 
 Staphylococcus epidermis albus, 
 
 471 
 
 pyogenes albus, 471 
 aureus, 461 
 biology, 461 
 growth in common media, 
 
 462 
 
 immunization, 467 
 morphology, 461 
 occurrence in man, 468 
 pathogenesis of, 464 
 production of toxic sub- 
 stances by, 466 
 
 Staphylococcus pyogenes citreus, 
 
 472 
 Sterilization, fractional, 149 
 
 incomplete, 152 
 Sternberg bulb, 251 
 Streptococci, dettction of, in pus, 
 
 etc., 496 
 of, in blood, 496 
 Streptococcus erysipelatis, 476 
 pyogenes, 476 
 
 biological characters of, 478 
 growth on media, 478 
 immunity, 487 
 morphology of, 477 
 occurrence of, in man, 483 
 pathogenesis, 481 
 toxins of, in sarcoma, 485 
 production of toxic sub- 
 stances by, 487 
 susceptibility to infection by, 
 
 487 
 use of anti-streptococcic serum, 
 
 489 
 
 Strep tothrix, 615, 619 
 actinomyces, 61 K 
 in pseudotuberculosis, 619 
 Sulphur dioxide gas, 158 
 
 in house disinfection, 189 
 Sulphuretted hydrogen, produc- 
 tion of, 74 
 
 Sunlight, effect of, 168 
 Sweat, bacteria eliminated by, 
 
 117 
 Syphilis bacillus, 311 
 
 biological and pathogenic 
 
 properties of, 312 
 morphology of, 311 
 
 TEMPERATURE, effect of, on 
 bacteria. 146 
 Tetanus antitoxin, 395 
 dosage, 400 
 
 persistence in the blood, 398 
 production of, for therapeu- 
 tic purposes, 397 
 results in treatment in teta- 
 nus, 399 
 
 technique of testing anti- 
 toxin serum, 307 
 bacillus, 385 
 
692 
 
 GENERAL INDEX. 
 
 Tetanus bacillus, biology of, 386 
 differential diagnosis of, 400 
 morphology of, 385 
 non-virulent type, 401 
 pathogenesis, 388 
 resistance of spores, 388 
 toxin, 392 
 
 action of, in the body, 393 
 varieties of, due to bacillus, 391 
 Tetragenus, micrococcus, 472 
 biology of, 474 
 growth of, on media, 474 
 morphology of, 473 
 pathogenesis of, 474 
 Thermo-regulator, 232 
 Thermophilic bacteria, 145 
 Timothy grass bacilli and differ- 
 ential diagnosis from tubercle 
 bacilli, 319 
 Toxalbumins, 71, 72 
 Toxins, 73 
 
 Trichophyton (ringworm fun- 
 gus), 620 
 
 Tricresol, 166, 171 
 Tube cultures, inoculation of, 
 
 from plate colonies, 229 
 Tubercle bacillus, 263 
 
 action of, upon tissues of 
 
 poisons, 277 
 attenuation of, 287 
 biological characters of, 265 
 distribution of, in tissues, 278 
 duration of virulence of, in 
 
 sputum, 286 
 
 effect of sunlight on, 267 
 growth on coagulated blood- 
 serum, 268 
 on peptonized veal or in 
 
 beef broth, 269 
 mode of infection, 278 
 morphological characters in, 
 
 264 
 
 pathogenesis of, 273 
 paths of infection, 278 
 practical examination for, in 
 
 sputa, 301 
 resisting power of, against 
 
 disinfectants, 266 
 smegma bacillus, and leprosy 
 bacillus, differential diag- 
 nosis of, 314 
 
 Tubercle bacillus from sputa and 
 
 infected materials, 269 
 staining of, in tissues, 310 
 a strict parasite, 267 
 transmissibilily of, to foetus, 
 
 285 
 
 Tuberculin for diagnosis of tuber- 
 culosis in cattle, 291 
 in man, 294 
 Tuberculosis, animals attacked 
 
 by, 276 
 
 antituberculous serum in, 297 
 auto-infection by swallowing 
 
 sputum, 284 
 communicability of, 263 
 different forms of, in man, 276 
 immunization in, 288 
 individual susceptibility to, 
 
 281 
 
 infection from coughing, sneez- 
 ing, etc., 283 
 by ingestion of milk and 
 
 meat, 283 
 inhalation of tuberculous dust, 
 
 279 
 
 mixed infection in, 287 
 prophylaxis of, 298 
 Turpentine, oil of, 166 
 Typhoid bacillus, 402 
 
 biological characters, 405 v ' 
 comparative value of methods 
 
 for isolation of, 439 
 detection of, in water, 443 
 differential diagnosis from 
 
 colon bacillus, 454 
 fermentation, 407 
 flagella, 404 
 growth in bouillon, 406 
 on gelatin plates, 405 
 in milk, 407 
 on potato, 406 
 indol reaction, 407 
 infection, diagnosis of, by 
 
 Widal test, 416 
 isolation of, from feces, urine, 
 
 blood, water, etc.. 429 
 length of time capable of liv- 
 ing outside the body, 413 
 location of, in body in ty- 
 phoid fever, 40'J 
 methods of infection, 412 
 
GENERAL INDEX. 
 
 693 
 
 Typhoid bacillus, morphological 
 
 characters of, 403 
 in oysters, 413 
 pathogenic properties of, 407 
 in pneumonia, 410 
 presence of, in blood, 411 
 
 in feces and urine, 440, 676 
 
 in foetus, 411 
 
 in gall-bladder, 411, 675 
 
 in sputa, 411 
 as pus producer, 410 
 staining characteristics of, 403 
 in urine, 442 
 infection of, by contaminated 
 
 drinking water, 414 
 by milk, 414 
 immunization in, 415 
 
 UREA, fermentation of, 67 
 Urine, bacteria eliminated 
 through, 117 
 
 \J ACCIN ATION , immunity 
 
 conferred by, 649, 650 
 Vaccine, bacteria in, 657 
 
 bodies, 651 
 
 durability of, 656 
 
 preparation of, 652 
 
 virus, use of, 650 
 Vincent's bacillus, 354 
 Virulence, how diminished, 96 
 
 how increased, 96 
 
 possessed by bacteria, 96 
 Vitality, increase of, strengthens 
 
 immunity, 124 
 
 WATER, bacilli of colon group 
 in, 248 
 bacteriological examination of, 
 
 245 
 
 contamination and purification 
 of drinking 253 
 
 Water, domestic purification of, 
 
 255 
 obtaining of, for examination, 
 
 249 
 
 typhoid bacilli in, 247 
 Whooping-cough, bacillus of, 613 
 Widal reaction, 421 
 test, 419 
 
 advantages and disadvan- 
 tages of serum and dried 
 blood in, 424 
 
 dilution of blood- serum to 
 
 be employed and time re 
 
 quired for development of, 
 
 reaction of, 425 
 
 mode of obtaining serum in, 
 
 423 
 persistence of reaction in, 
 
 428 
 
 proportion of cases in which 
 definite reaction occurs in, 
 427 
 
 pseudo reactions, 422 
 reaction with blood-serum 
 of healthy persons, 428 
 of those ill with diseases 
 other than typhoid fever, 
 428 
 
 use of dried blood in, 420 
 WolfFhiigel's apparatus for count- 
 ing colonies, 227 
 
 VEROSIS bacilli, 347 
 A 
 
 YEASTS, 625 
 
 1 Yellow fever, bacillus of, 609 
 
 yiEHL'S carbol-fuchsin solu- 
 L tlon for tubercle bacilli, 303 
 carbolic fuchsin solution, 202, 
 Ziehl-Neelson's method, 310 
 
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