a ^^K^Tf^FlJND-Bh:oUH?^HEgW1 A TEXT - BOOK OF BACTERIOLOGY A TEXT-BOOK OF BACTERIOLOGY A PRACTICAL TREATISE FOR STUDENTS AND PRACTITIONERS OF MEDICINE BY PHILIP HANSON HISS, Jk., M.D. PROFESSOR OF BACTERIOLOGY, COLLEGE OF PHYSICIANS AND SURGEONS, COLUMBIA UNIVERSITY, NEW YORK CITY AND HANS ZINSSER, M.D. ASSOCIATE PROFESSOR IN CHARGE OF BACTERIOLOGY, LELAND STANFORD, JR., UNIVERSITY, PALO ALTO, CALIFORNIA WITH ONE HUNDRED AND FIFTY- SIX ILLUSTRATIONS IN THE TEXT, SOME OF WHICH ARE COLORED NEW YORK AND LONDON D. APPLE TON AND COMPANY 1910 ^ Copyright, 1910, by D. APPLETON AND COMPANY PRINTED IN New York, U. S. A. PREFACE The volume here presented is primarily a treatise on the funda- mental laws and technique of Bacteriology, as illustrated by their application to the study of pathogenic bacteria. So ubiquitous are the bacteria and so manifold their activities that Bacteriology, although one of the youngest of sciences, has already been divided into special fields — Medical, Sanitary, Agricul- tural, and Industrial — having little in common, except problems of general bacterial physiology and certain fundamental technical pro- cedures. From no other point of approach, however, is such a breadth of conception attainable, as through the study of bacteria in their rela- tion to disease processes in man and animals. Through such a study one must become familiar not only with the growth character- istics and products of the bacteria apart from the animal body, thus gaining a knowledge of methods and procedures common to the study of pathogenic and non-pathogenic organisms, but also with those complicated reactions taking place between the bacteria and their products on the one hand and the cells and fluids of the animal body on the other — reactions which often manifest themselves as symptoms and lesions of disease or by visible changes in the test tube. Through a study and comprehension of the processes underlying these reactions, our knowledge of cell physiology has been broadened, and facts of inestimable value have been discovered, which have thrown light upon some of the most obscure problems of infection and immunity and have led to hitherto unsuspected methods of treatment and diagnosis. Thus, through Medical Bacteriology — that highly specialized offshoot of General Biology and Pathology — have been given back to the parent sciences and to Medicine in general methods and knowledge of the widest application. It has been our endeavor, therefore, to present this phase of our subject in as broad and critical a manner as possible in the sections vi PREFACE dealing with infection and immunity and with methods of biological diagnosis and treatment of disease, so that the student and practi- tioner of medicine, by becoming familiar with underlying laws and principles, may not only be in a position to realize the meaning and scope of some of these newer discoveries and methods, but may be in better position to decide for themselves their proper application and limitations. We have not hesitated, whenever necessary for a proper under- standing of processes of bacterial nutrition or physiology, or for breadth of view in considering problems of the relation of bacteria to our food supply and environment, to make free use of illustrations from the more special fields of agricultural and sanitary bacteriology, and some special methods of the bacteriology of sanitation are given in the last division of the book, dealing with the bacteria in relation to our food and environment. In conclusion it may be said that the scope and arrangement of subjects treated of in this book are the direct outcome of many years of experience in the instruction of students in medical and in advanced university courses in bacteriology, and that it is our hope that this volume may not only meet the needs of such students but may prove of value to the practitioner of medicine for whom it has also been written. It is a pleasure to acknowledge the courtesy of those who furnished us with illustrations for use in the text, and our indebtedness to Dr. Gardner Hopkins and Professor Francis Carter Wood for a number of the photomicrographs taken especially for this work. P. H. H., Jr., H. Z. August, 1910. CONTENTS SECTION I THE GENERAL BIOLOGY OF BACTERIA AND THE TECHNIQUE OF BACTERIOLOGICAL STUDY CHAPTER PAGE I. The Development and Scope of Bacteriology 1 II General Morphology, Reproduction, and Chemical and Physical Properties of the Bacteria 9 II. The Relation of Bacteria to Environment, and Their Classification 25 IV. The Biological Activities of Bacteria 40 V. The Destruction of Bacteria 62 VI. Methods Used in the Microscopic Study and Staining of Bacteria 93 VII. The Preparation of Culture Media 113 VIII. Methods Used in the Cultivation of Bacteria 141 IX Methods Determining Biological Activities of Bacteria . 164 X. The Bacteriological Examination of Material from Patients . . , * 174 SECTION II INFECTION AND IMMUNITY CHAPTER PAGE XL Fundamental Factors of Pathogenicity and Infection . . 181 XII. Defensive Factors of the Animal Organism 189 XIII. Toxins and Antitoxins 203 XIV. Production and Testing of Antitoxins 216 XV. Lysins, Agglutinins, Precipitins, and Other Antibodies , 224 XVI. The Technique of Serum Reactions 249 vii viii CONTENTS CHAPTER PAGE XVII. Phagocytosis 275 XVIII. Opsonins, Leucocyte Extract, and Aggressins 281 XIX. Anaphylaxis or Hypersusceptibility 295 XX. Facts and Problems of Immunity in their Bearing upon the Treatment of Infectious Diseases 305 SECTION III PATHOGENIC MICROORGANISMS CHAPTER PAGE XXI. The Staphylococci (Micrococci) 321 XXII. The Streptococci 336 XXIII. Diplococcus pneumoniae . 352 XXIV, Micrococcus intracellularis meningitidis (Meningococcus) . 371 XXV. Diplococcus gonorrhoeae (Gonococcus) , Micrococcus ca- TARRHALIS, AND OTHER GRAM-NEGATIVE COCCI 380 XXVI. Bacilli of the Colon-Typhoid-Dysentery Group — Bacillus COLI COMMUNIS, 388 XXVII. Bacilli of the Colon-Typhoid-Dysentery Group (continued) — Bacillus of Typhoid Fever 399 XXVIII. Bacilli of the Colon-Typhoid-Dysentery Group (continued) — Bacilli Intermediate between the Typhoid and Colon Organisms 428 XXIX. Bacilli of the Colon-Typhoid-Dysentery Group (continued) — The Dysentery Bacilli 433 XXX. Bacillus mucosus capsulatus 445 XXXI. Bacillus tetani 454 XXXII. Bacillus of Symptomatic Anthrax, Bacillus of Malignant Edema, Bacillus afrogenes capsulatus, Bacillus botu- linus 463 XXXIII. The Tubercle Bacillus 477 XXXIV. The Smegma Bacillus and the Bacillus of Leprosy . . . 497 XXXV. Bacillus diphtheria, Bacillus Hoffmanni, and Bacillus xerosis 504 XXXVI. Bacillus mallei 520 XXXVII. Bacillus influenzae and Closely Related Bacteria . . . 528 CONTENTS ix CHAPTER PAGE XXXVIII. Bordet-Gengou Bacillus, Morax-Axenfeld Bacillus, Zur Nedden Bacillus, Ducrey Bacillus 535 XXXIX. The Bacilli of the Hemorrhagic Septicemia Group and Bacillus pestis 543 XL. Bacillus anthracis and Anthrax 553 XLI. Bacillus pyocyaneus 567 XLII. Asiatic Cholera and the Cholera Organism 572 XLIII. Diseases Caused by Spirochetes 582 XLIV. The Higher Bacteria 606 XLV. The Yeasts 617 XL VI. Hyphomycetes 623 SECTION IV DISEASES OF UNKNOWN ETIOLOGY CHAPTER PAGE XLVII. Rabies 634 XLVIII. Smallpox 644 XLIX. Acute Anterior Poliomyelitis 651 L. Yellow Fever 654 LI. Measles, Scarlet Fever, and Foot-and-Mouth Disease . . 661 SECTION V BACTERIA IN AIR, SOIL, WATER, AND MILK CHAPTER PAGE LII. Bacteria in the Air and Soil 665 LIII. Bacteria in Water 671 LIV. Bacteria in Milk and Milk Products, Bacteria in the Industries 681 INDEX OF AUTHORS 701 INDEX OF SUBJECTS 711 LIST OF ILLUSTRATIONS FIGURE . PAGE 1. Types of bacterial morphology 10 2. Bacterial capsules .12 3. Arrangement of bacterial flagella 15 4. Various positions of spores in bacterial cell 17 5. Germination of spores 17 6. Degeneration forms of Bacillus diphtherias , . 19 7. Degeneration forms of Bacillus pestis 20 8. Hot-air sterilizer 69 9. Arnold sterilizer . 70 10. Low-temperature sterilizer 71 11. Autoclave 72 12. Lentz formalin apparatus 88 13. Breslau formaldehyde generator and section of same 89 14. Hanging drop preparation 94 15. Florence flask 114 16. Erlenmeyer flask . 114 17. Petri dish 115 18. Test tubes, showing method of stoppering 116 19. Burette for titrating media . 117 20. Tubing media 118 21. Media in tubes 119 22. Berkefeld filter 120 23. Berkefeld filter 121 24. Reichel filter . 122 25. Kitasato filter 123 26. Maassen filter, for small quantities of fluid 124 27. Platinum wires 142 28. Taking plugs from tubes before inoculation 143 29. Inoculating 144 30. Pouring inoculating medium into Petri dish 145 31. Streak plate 147 32. Deep stab cultivation of anaerobic bacteria 148 33. Deep stab cultivation of anaerobic bacteria 149 34. Cultivation of anaerobes in fluid under albolin 150 35. Wright's method of anaerobic cultivation in fluid media 151 36. Novyjar 152 37. Wright's method of anaerobic cultivation by the use of pyrogallic-acid solution 153 38. Jar for anaerobic cultivation 154 xi xii • LIST OF ILLUSTRATIONS FIGURE PAGE 39. Apparatus for combining the methods of exhaustion, hydrogen replace- ment, and oxygen absorption 155 40. Simple apparatus for plate cultivation of anaerobic bacteria 156 41. Incubator 157 42. Thermo-regulator 158 43. Thermo-regulator 158 44. Moitessier gas-pressure regulator 160 45. Variations in the conformation of the borders of bacterial colonies . . 161 46. Wolffhiigel counting-plate 162 47. Types of fermentation tubes 165 48. Types of gelatin liquefaction by bacteria 169 49. Intraperitoneal inoculation of rabbit 170 50. Intravenous inoculation of rabbit 170 51. Intraperitoneal inoculation of guinea-pig 171 52. Guinea-pig cage 171 53. Rabbit cage 172 54. Blood-culture plate showing streptococcus colonies 179 55. Toxin and body cell 206 56. Toxin and antitoxin 214 57. Ehrlich's conception of cell-receptors, giving rise to lytic immune bodies 227 58. Complement, amboceptor or immune body, and antigen or immunizing substance • . . . ' 227 59. Microscopic agglutination reaction 229 60. Macroscopic agglutination 230 61. Ehrlich's conception of the structure of agglutinins and precipitins . . 238 62. The structure of cell-receptors and immune bodies, according to Ehrlich's conception 239 63. Neisser and Wechsberg's conception of complement deviation .... 245 64. Schematic representation of complement fixation in the Bordet-Gengou reaction 247 65. Capillary pipette for removal of exudate in doing the Pfeiffer test . . 256 66. Wright's capsule for collecting blood 284 67. Pipette for opsonic work 285 68. Pipette with three substances, — corpuscles, bacteria, and serum, as first taken up 285 69. Staphylococcus pyogenes aureus . 322 70. Staphylococcus colonies 323 71. Micrococcus tetragenus 333 72. Streptococcus pyogenes 337 73. Streptococcus colonies on serum agar 340 74. Streptococcus colonies from blood culture on blood-agar plate .... 346 75. Pneumococci 354 76. Pneumococci 354 77. Meningococcus, pure culture 372 78. Meningococcus in spinal fluid 373 79. Meningococcus culture (streak plate) 375 80. Gonococcus pus from urethra 381 LIST OF ILLUSTRATIONS xiii FIGURE PAGE 81. Gonococcus 382 82. Gonococcus colony 383 83. Bacillus coli communis 390 84. Bacillus coli communis on various media 396 85. Bacillus coli communior on various media 397 86. Bacillus typhosus 400 87. Bacillus typhosus, showing flagella 401 88. Surface colony of Bacillus typhosus on gelatin . . . . „ 402 89. Bacillus coli; deep colonies on Hiss plate medium 406 90. Bacillus typhosus; deep colonies in Hiss plate medium 407 91. Bacillus typhosus; colony in Hiss plate medium, highly magnified . . . 409 92. Colon and typhoid colonies in Hiss plate medium „ 410 93. Scheme of fermentations of the dysentery- typhoid-colon-bacilli in carbo- hydrate serum-water media 443 94. Bacillus mucosus capsulatus 446 95. Bacilli of rhinoscleroma 450 96. Bacillus tetani 455 97. Young tetanus culture in glucose agar 456 98. Older tetanus culture in glucose agar 457 99. Bacillus of symptomatic anthrax 464 100. Bacillus of symptomatic anthrax, culture in glucose agar 465 101. Bacillus of malignant edema 467 102. Bacillus of malignant edema, culture in glucose agar 468 103. Tubercle bacilli in sputum 478 104. Culture of Bacillus tuberculosis in flask of glycerin bouillon 481 105. Bacillus diphtherias 505 106. Colonies of Bacillus diphtherias on glycerin agar 510 107. Bacillus Hoffmanni 515 108. Colonies of Bacillus Hoffmanni on agar • . . 516 109. Bacillus xerosis 517 110. Glanders bacillus . 521 111. Glanders bacilli in tissue 523 112. Bacillus influenzae; smear from pure culture on blood agar 529 113. Bacillus influenzae; smear from sputum . 530 114. Colonies of influenza bacillus on blood agar 531 115. Koch- Weeks bacillus 534 116. Bordet-Gengou bacillus 536 117. Morax-Axenfeld diplo-bacillus 539 118. Bacillus pestis 547 119. Bacillus pestis, involution forms .... 548 120. Bacillus anthracis; pure culture on agar 554 121. Bacillus anthracis, in kidney tissue 555 122. Bacillus anthracis, in spleen tissue 556 123. Anthrax colony on gelatin 557 124. Anthrax colony on agar 558 125. Bacillus subtilis 566 126. Cholera spirillum 573 xiv LIST OF ILLUSTRATIONS FIGURE PAGE 127. Colonies of Spirillum cholere on gelatin plate, thirty hours old .... 574 128. Cholera spirillum; stab cultures in gelatin 575 129. Spirochete pallida; smear from chancre 584 130. Spirochete pallida, in spleen of congenital syphilis 590 131. Spirochete pallida, in liver of congenital syphilis 591 132. Spirochete of relapsing fever 594 133. Spirochete of relapsing fever 595 134. Spirochete of relapsing fever „ 596 135. Spirochete of Dutton, African tick fever 598 136. Smear from the throat of a case of Vincent's angina 600 137. Throat smear, Vincent's angina 601 138. Spirochete gallinarum 604 139. Cladothrix, showing false branching 608 140. Streptothrix, showing true branching 609 141. Actinomyces granule crushed beneath a cover-glass 612 142. Actinomyces granule crushed beneath a cover-glass 613 143. Branching filaments of actinomyces 614 144. Yeast cells 618 145. Mucor mucedo . 624 146. Mucor mucedo 625 147. Mucor mucedo 626 148. Mucor ramosus ' 627 149. Penicillium glaucum 628 150. Aspergillus glaucus 629 151. Thrush 630 152. Thrush 631 153. Achorion Schoenleinii 632 154. Method of drying spinal cord of rabbit for purposes of attenuation . . 640 155. Stegomyia fasciata 657 156. Bacillus bulgaricus 696 ^°V m °y NOV 14 1910 THE GENERAL BIOLOGY OF BACTERIA AND THE TECHNIQUE OF BACTERIOLOGICAL STUDY CHAPTER I THE DEVELOPMENT AND SCOPE OF BACTERIOLOGY As we trace back to their ultimate origins the lines of development of living beings of the animal and plant kingdoms, we find them con- verging toward a common type, represented by a large group of uni- cellular organisms, so simple in structure, so unspecialized in function, that their classification in either the realm of plants or that of animals becomes little more than an academic question. However, even such microorganisms, in which the functions of nutrition, respiration, loco- motion, and reproduction are concentrated within the confines of a single cell, and in which adaptation to special conditions more readily brings about modifications leading to the production of a multitude of delicately graded transitional forms, fall into groups which, either in structure or in biological attributes show evidence of a tendency toward one or the other of the great kingdoms. Most important of these unicellular forms, for the student of medical science, are the bacteria and the protozoa. The former, by reason of their undifferentiated protoplasm, their occasional possession of cellulose membranes, their biological tendency to synthetize, as well as to break down organic compounds, and because of the transitional forms which seem to connect them directly with the lower plants, are generally placed in the plant kingdom. The latter, chiefly on the basis of metabolism, are classified with the animals. Knowledge of the existence of microorganisms as minute as the ones under discussion, was of necessity forced to await the perfection of instruments of magnification. It was not until the latter half of the seventeenth century, therefore, that the Jesuit, Kircher, in 1659, and the Dutch linen-draper, van Leeuwenhoek, in 1675, actually saw and 1 2 BIOLOGY AND TECHNIQUE described living beings too small to be seen with the naked eye. There can be no doubt that the small bodies seen by these men and their many immediate successors were, at least in part, bacteria. And indeed the descriptions and illustrations of several of the earliest workers cor- respond with many of the forms which are well known to us at the present day. During the century following the work of these pioneers, the efforts of investigators lay chiefly in the more exact morphological description of some of the forms of unicellular life, already known. Conspicuous among the work of this period is that of Otto Friedrich Miiller. In the generation following Miiller' s work, however, a marked advance in the study of these forms was made by Ehrenberg,1 who established a classification which, in some of its cardinal divisions, is retained until the present day. Meanwhile the regularity with which these " animalcula " or "in- fusion animalcula " were demonstrable in tartar from the teeth, in intes- tinal contents, in well-water, etc., had begun to arouse in the minds of the more advanced physicians of the time a suspicion as to a possible relationship of these minute forms with disease. The conception of " contagion," or transmission of a disease from one human being to another, was, however, even at this time, centuries old. The fact had been recognized by Aristotle, had been reiterated by medieval philos- ophers, and had led, in 1546, to the division of contagious diseases by Fracastor, into those transmitted "per contactum," and those con- veyed indirectly "per fomitem." It was for these mysterious facts of the transmissibility of disease, that clinicians of the eighteenth centur}^ with remarkable insight, saw an explanation in the microorganisms dis- covered by Leeuwenhoek and his followers. In fact, Plenciz of Vienna, writing in 1762, not only expressed a belief in the direct etiological connection between microorganisms and some diseases, but was the first to advance the opinion that each malady had its own specific causal agent, which multiplied enormously in the diseased body. The opinions of this author, if translated into the language of our modern knowledge of the subject, came remark- ably near to the truth, not only as regards etiology and transmission, but also in their suggestion of a specific therapy for each disease. The conception of a " contagium vivum " was thus practically es- tablished with the work of Plenciz and many others who followed in 1 " Die Infusionstierchen," etc., Leipzig, 1838. DEVELOPMENT AND SCOPE OF BACTERIOLOGY 3 his train, but the astonishingly shallow impression which the acute reasoning of these men left upon the medical thought of their day furnishes an excellent example of the futility of the most penetrating speculation when unsupported by experimental data. The real advancement in the scientific development of the subject was destined to be carried on along entirely different lines. In 1837, Schwann, a botanist, showed that the yeasts, found in fermenting sub- stances, were living beings, which bore a causal relationship to the proc- ess of fermentation. At almost the same time, similar observations were made by a French physicist, Cagniard-Latour. The opinions advanced by these men on the nature of fermentation aroused much interest and discussion, since, at that time and for a long period thereafter, fermentation was ascribed universally toproteid decomposition, a process which was entirely obscure but firmly believed to be of a purely chemical nature. Although belief in the discovery of Schwann did not completely master the field until after Pasteur had completed his classical studies upon the fermentations occurring in beer and wine, yet the conception of a " fermentum vivum " aroused much speculation, and the attention of physicians and scientists was attracted to the many analogies ex- isting between phenomena of fermentation and those of disease. The conception of such an analogy, however, was not a new thought in the philosophy of the time. Long before Schwann and Cagniard- Latour, the philosopher Robert Boyle, working in the seventeenth century, had prophesied that the mystery of infectious diseases would be solved by him who should succeed in elucidating the nature of fermenta- tion. Nevertheless, the diligent search for microorganisms in relation to various diseases which followed, led to few results, and the successes which were attained were limited to the diseases caused by some of the larger fungi, favus (1839), thrush (1839), and pityriasis versicolor (1846). During this time of ardent but often poorly controlled etiolog- ical research, it was Henle who formulated the postulates of conserva- tism, almost as rigid as the later postulates of Koch, requiring that proof of the etiological relationship of a microorganism to a disease could not be brought merely by finding it in a lesion of the disease, but that constant presence in such lesions must be proven and isolation and study of the microorganism away from the diseased body must be car- ried out. It was during this period also that one of the most fundamental 4 BIOLOGY AND TECHNIQUE questions, namely, that of the origin of these minute living beings, was being discussed with much passion by the scientific world. It was held by the conservative majority that the microorganisms described by Leeuwenhoek and others after him, were produced by spontaneous generation. The doctrine of spontaneous generation, in fact, was solidly established and sanctified by tradition, and had been applied in the past not alone to microorganisms.1 And it must not be forgotten that without the aid of our modern methods of study, satisfactory proof for or against such a process was not easily brought. Needham, who published in 1749, had spent much time in fortify- ing his opinions in favor of spontaneous generation by extensive ex- perimentation. He had placed putrefying material and vegetable in- fusions in sealed flasks, exposing them for a short time to heat, by immersing them in a vessel of boiling water, and had later shown them to be teeming with microorganisms. He was supported in his views by no less an authority than Buffon. The work of Needham, however, showed a number of experimental inaccuracies which were thoroughly sifted by the Abbe Spallanazani. This investigator repeated the ex- periments of Needham, employing, however, greater care in sealing his flasks, and subjecting them to a more thorough exposure to heat. His results did not support the views of Needham, but were answered by the latter with the argument that by excessive heating he had pro- duced chemical changes in his solutions which had made spontaneous generation impossible. The experiments of Schulze, in 1836, who failed to find living organ- isms in infusions which had been boiled, and to which air had been admitted only after passage through strongly acid solutions, and similar results obtained by Schwann, who had passed the air through highly heated tubes, were open to criticism by their opponents, who claimed that chemical alteration of the air subjected to such drastic influences, had been responsible for the absence of bacteria in the infusion. Similar experiments by Schroeder and Dusch, who had stoppered their flasks with cotton plugs, were not open to this objection, but had also failed to convince. The question was not definitely settled until the years im- 1 Valleri-Radot, in his life of Pasteur, stated that Van Helmont, in the six- teenth century, had given a celebrated prescription for the creation of mice from dirty linen and a few grains of wheat or pieces of cheese. During the centu- ries following, although, of course, such remarkable and amusing beliefs no longer held sway, nevertheless the question of spontaneous generation of minute and structureless bodies, like the bacteria, still found learned and thoughtful partisans. DEVELOPMENT AND SCOPE OF BACTERIOLOGY 5 mediately following I860, when Pasteur conducted a series of experi- ments which were not only important in incontrovertibly refuting the doctrine of spontaneous generation, but in establishing the principles of scientific investigation which have influenced bacteriological re- search since his time.1 Pasteur attacked the problem from two points of view. In the first pace he demonstrated that when air was filtered through cotton- wool, innumerable microorganisms were deposited upon the filter. A single shred of such a contaminated filter dropped into a flask of pre- viously sterilized nutritive fluid, sufficed to bring about a rapid and luxuriant growth of microorganisms. In the second place, he succeeded in showing that similar, sterilized " putrescible " liquids, if left in con- tact with air, would remain uncontaminated provided that the en- trance of dust particles were prohibited. This he succeeded in doing by devising flasks, the necks of which had been drawn out into fine tubes bent in the form of a U. The ends of these U-tubes, being left open, permitted the sedimentation of dust from the air as far as the lowest angle of the tube, but, in the absence of an air current, no dust was carried up the second arm into the liquid. In such flasks, he showed that no contamination took place but could be immediately induced by slanting the entire apparatus until the liquid was allowed to run into the bent arm of the U-tube. Finally, by exposing a series of flasks containing sterile yeast infusion, at different atmospheric levels, in places in which the air was subject to varying degrees of dust con- tamination, he showed an inverse relationship between the purity of the air and the contamination of his flasks with microorganisms. The doctrine of spontaneous generation had thus received its final refutation, except in one particular. It was not yet clear why com- plete sterility was not always obtained by the application of definite degrees of heat. This final link in the chain of evidence was supplied, some ten years later, by Cohn, who, in 1871, was the first to observe and correctly interpret bacterial spores and to demonstrate Iheir high powers of resistance against heat and other deleterious influences: 1 In a letter to his foremost opponent, at this period, Pasteur writes: "In experimental science, it is always a mistake not to doubt when facts do not compel affirmation." The critical spirit pervading the scientific thought of that time in France is also well expressed by Oliver Wendell Holmes, who said that he had learned three things in Paris: "Not to take authority when I can have facts, not to guess when I can know, and not to think that a man must take physic because he is sick." 6 BIOLOGY AND TECHNIQUE Meanwhile, Pasteur, parallel with his researches upon spontaneous generation, had been carrying on experiments upon the subject of fermentation along the lines suggested by Cagniard-Latour. As a consequence of these experiments, he not only confirmed the opinions both of this author and of Schwann concerning the fermentation of beer and wine by yeasts, but was able to show that a number of other fer- mentations, such as those of lactic and butyric acid, as well as the de- composition of organic matter by putrefaction, were directly due to the action of microorganisms. It was the discovery of the living agents underlying putrefaction, especially, which exerted the most active influence upon the medical research of the day. This is illustrated by Lister's work. The suppurative processes occurring in infected wounds had long been regarded as a species of putrefaction, and Lord Lister, working directly upon the premises supplied by Pasteur, introduced into both the active and prophylactic treatment of surgical wounds, the antiseptic principles which alone have made modern surgery possible. There now followed a period in which bacteriological investigation was concentrated upon problems of etiology. Stimulated by Pasteur's successes, the long-cherished hope of finding ,some specific microorgan- ism as the causal agent in each infectious disease was revived. Pollender, in 1855, had reported the presence of rod-shaped bodies in the blood and spleen of animals dead of anthrax. Brauell, several years later, had made similar observations and had expressed definite opinions as to the causative relationship of these rods to the disease. Convincing proof, however, had not been brought by either of these observers. Finally, in 1S63, Davaine, in a series of brilliant investi- gations, not only confirmed the observations of the two authors men- tioned above, but succeeded in demonstrating that the disease could be transmitted by means of blood containing these rods and could never be transmitted by blood from which these rods were absent. Anthrax, thus, is the first disease in which definite proof of bacterial causation was brought. Speaking before the French Academy of Medicine at this time, Davaine suggested that the manifestations of the disease might in reality represent the results of a specific fermentation produced by the bacilli he had found. This, in a crude way, expresses the modern conception of infectious disease. Within a few years after this, 1868, the adherents of the parasitic theory of infectious diseases were further encouraged by the discovery, by Obermeier, of a spirillum in the blood of patients suffering from DEVELOPMENT AND SCOPE OF BACTERIOLOGY 7 relapsing fever. It is not surprising that the successes attained in these diseases, fostering hope of analogous results in all other similar condi- tions, but without the aid of adequate experimental methods, should have led to many unjustified claims and to much fantastic theorizing. Thus Hallier, at about this time, advanced a theory as to the etiology of infectious diseases, in which he attributed all such conditions to the moulds or hyphomycetes, regarding the smaller form or bacteria as developmental stages of these more complicated forms. Extravagant conjectures of this kind, however, did not maintain themselves for any length of time in the light of the critical attitude which was already pervading bacteriological research. Progress was made during the years immediately following, chiefly in the elucidation of suppurative processes. Rindfleisch, von Reckling- hausen, and Waldeyer, almost simultaneously, described bodies which they observed in sections of tissue containing abscesses, and which they believed to be microorganisms. Notable support was given to their opinion by similar observations made upon pus by Klebs, in 1870. In view, however, of the purely morphological nature of their studies, the opinions of these observers did not entirely prevail. Satisfactory methods of cultivation and isolation had not yet been developed, and Billroth and his followers, with a conservatism entirely justified under existing conditions, while admitting the constant presence of bacteria in purulent lesions, denied their etiological significance. The contro- versy that followed was rich in suggestions which greatly facilitated the work of later investigators, but could not be definitely settled until 1880, when Koch introduced the technical methods which have made bacteriology an exact science. By the use of solid nutritive media, the isolation of bacteria and their biological study in pure culture were made possible. At about the same time the use of anilin dyes, developed by Weigert, Koch, and Ehrlich, was introduced mto morphological study and facilitated the observation of the finer structural details which had been unnoticed while only the grosser methods employed for tissue staining had been available. With the publication of Koch's work, there began an era unusually rich in results held in leash heretofore by inadequate technical methods. The discovery of the typhoid bacillus in 1880, of the bacillus of fowl cholera and the pneumococcus in the same year, and of the tubercle bacillus in 1882, initiated a series of etiological discoveries which, ex- tending over not more than fifteen years, elucidated the causation of a majority of the infectious diseases. 8 BIOLOCY AND TECHNIQUE Coincident with the elucidation of etiological facts began the inquiry into that field which is now spoken of as the science of immunity. The phenomena which accompany the development of insusceptibility to bacterial infections in man and in animals, first studied by Pasteur, have become the subject of innumerable researches and have led to results of the utmost practical value. The problems which were encountered were first studied from a purely bacteriological point of view, but their solution has shed light upon biological principles of the broadest application. Investigations into the properties of immune sera, while making bacteriology one of the most important branches of diagnostic and therapeutic medicine, have, at the same time, inseparably linked it with physiology and experimental pathology. By the revelations of etiological research, and by the study of the biological properties of pathogenic bacteria, contagion, an enemy hitherto unseen and n^sterious, was unmasked, and rational campaigns of public sanitation and personal hygiene were made possible. Upon the same elucidations has depended the development of modern surgery — a science which without asepsis and antisepsis would have been doomed to remain in its medieval condition. Apart from its importance in the purely medical sciences, the study of the bacteria has shed beneficial light, moreover, upon many other fields of human activity. In their relationship to decomposition, the conditions of the soil, and to diseases of plants, the bacteria have been found to occupy a position of great importance in agriculture. Knowl- edge of bacterial and yeast ferments, furthermore, has become the scien- tific basis of many industries, chiefly those concerned in the production of wine, beer, and dairy products. The scope of bacteriology is thus a wide one, and none of its various fields has, as yet, been fully explored. The future of the science is rich in allurement of interest, in promise of result, and in possible benefit to mankind. CHAPTER II GENERAL MORPHOLOGY, REPRODUCTION, AND CHEMICAL AND PHYSICAL PROPERTIES OF THE BACTERIA Bacteria are exceedingly minute unicellular organisms which may occur perfectly free and singular, or in larger or smaller aggregations, thus forming multicellular groups or colonies, the individuals of which are, however, physiologically independent. The cells themselves have a number of basic or ground shapes which may be roughly considered in three main classes: The cocci or spheres, the bacilli or straight rods, and the spirilla or curved rod forms. The cocci are, when fully developed and free, perfectly spherical. When two or more are in apposition, they may be slightly flattened along the tangential surfaces, giving an oval appearance. The bacilli, or rod-shaped forms, consist of elongated cells whose long diameter may be from two to ten times as great as their width, with ends squarely cut off, as in the case of bacillus anthracis, or gently rounded as in the case of the typhoid bacillus. The spirilla may vary from small comma-shaped microorganisms, containing but a single curve, to longer or more sinuous forms which may roughly be compared to a corkscrew, being made up of five, six, or more curves. The turns in the typical microorganisms of this class are always in three planes and are spiral rather than simply curved. Among the known microorganisms, the bacilli by far outnumber other forms, and are probably the most common variety of bacteria in existence. Many variations from these fundamental types may occur even under normal conditions, but contrary to earlier opinions it is now positively known that cocci regularly reproduce cocci, bacilli bacilli, and spirilla spirilla, there being, as far as we know, no mutation from one form into another. The size of bacteria is subject to considerable variation. Cocci may vary from .15 jx to 2. ll in diameter. The average size of the ordinary pus coccus varies from .8 p. to 1.2 ll in diameter. Fischer has given a graphic illustration of the size of a staphylococcus by calculating that one billion micrococci could easily be contained in a drop of water hav- 9 10 BIOLOGY AND TECHNIQUE ing a volume of one cubic millimeter. Among the bacilli the range of size is subject to even greater variations. Probably the smallest of the common bacilli is the bacillus of influenza which measures about .5 /'. in length by .2 a in thickness. The limit of the optical possibilities of the modern microscope is almost reached by some of the known micro- organisms, and it is not at all out of question that some of the diseases, for which, up to the present time, no specific microorganisms have # ® oh /d /J Y->> -V a Fig. 1. — Types of Bacterial Morphology. been found, may be caused by bacteria so small as to be invisible by any of our present methods. In fact, the virus causing the peripneumonia of cattle has been shown to pass through the pores of a Berkefeld filter, which are impenetrable to the smallest of the known bacteria.1 MORPHOLOGY OF THE BACTERIAL CELL When unstained, most bacteria are transparent, colorless, and ap- parently homogeneous bodies with a low refractive index. The cells themselves consist of a mass of protoplasm, surrounded, in most in- stances, by a delicate cell membrane. The presence of a nucleus2 in bacterial cells, though denied by the earlier writers, has been demonstrated beyond question by Zettnow, Nakanishi,3 and others. The original opinion of Zettnow was that the entire bacterial body consisted of nuclear material intimately inter- mingled with the cytoplasm. The opinion now held by most observers 1 Nocard and Roux, Ann. Past., 12, 1898. 2 A. Fischer, Jahrbi'icher f. wissen. Botanik, xxvii. 3 Nakanishi, Mi'inch. med. Woch., vi, 1900. MORPHOLOGY, REPRODUCTION, ETC. 11 who have studied this phase of the subject favors the existence of an ectoplasmic zone which includes cell membrane and flagella, but is definitely a part of the cytoplasm, and an entoplasm in which is con- centrated the nuclear material. Biitschli ] claims to have demonstrated within this entoplasmic substance a reticular meshwork, between the spaces of which lie granules of chromophilic or nuclear material. Confirmation of this opinion has been brought by Zettnow2 and others. Nakanishi, working with a special staining method, asserts that some microorganisms show within the entoplasmic zone a well-defined, minute, round or oval nucleus, which possesses a definitely charac- teristic staining reaction.3 In the bodies of a large number of bacteria, notably in those of the diphtheria group, Ernst,4 Babes,5 and others have demonstrated granular, deeply staining bodies now spoken of as metachromatic granules, or Babes-Ernst granules, or, because of their frequent position at the ends of bacilli, as polar bodies. These structures are irregular in size and number, and have a strong affinity for dyes. They are stained dis- tinctly dark in contrast to the rest of the bacterial cell with methylene blue, and may be demonstrated by the special methods of Neisser and of Roux.6 Their interpretation has been a matter of much difficulty and of varied opinion. Those who first observed them held that they were a part of the nuclear material of the cell. Others have regarded them as an earty stage in spore formation, or as arthrospores.7 Again, they have been interpreted as structures comparable to the centrosomes of other unicellular forms. As a matter of fact, the true nature of these bodies is by no means certain. They are present most regularly in microorganisms taken from young and vigorous cultures or in those taken directly from the lesions of disease. It is unlikely that they repre- 1 Biitschli, " Bau der Bakterien," Leipzig, 1890. 2 Zettnow, Zeit. f. Hyg., xxiv, 1897. 3 The method of Nakanishi is carried out as follows: Thoroughly cleansed slides are covered with a saturated aqueous solution of methylene blue. This is spread over the slide in an even film and allowed to dry. After drying, the slide should be of a transparent, sky-blue color. The microorganisms to be examined are then emulsified in warm water, or are taken from the fluid media, and dropped upon a cover slip. This is placed, face downward, upon the blue ground of the slide. In this way, bacteria are stained without fixation. Nakanishi claims that by this method the entoplasm is stained blue, while the nuclear material appears of a reddish or purplish hue. 4 Ernst, Zeit. f. Hyg., iv, 1888. 5 Babes, Zeit. f. Hyg., v, 1889. 6 See section on stains, p. 107. 7 See section on sporulation, p. 16. 12 BIOLOGY AND TECHNIQUE sent structures in any way comparable, to spores, since cultures con- taining individuals showing metachromatic granules are not more resistant to deleterious influences than are others. Their abundant presence in young and vigorous cultures, on the other hand, seems to indicate a relationship between them and the growth energy of the microorganisms. There is no evidence, however, to prove that these bodies have any bearing upon the virulence of the bacteria. Cell Membrane and Capsule. — Actual proof of the existence of a cell membrane has been brought in the cases of some of the larger forms only,1 but the presence of such envelopes may be inferred for most bacteria by their behavior during r „. _„ plasmolysis, where definite retrac- tion of the protoplasm from a - ^ well-defined cell outline has been repeatedly observed. The occur- rence, furthermore, of so-called " shadow forms" which appear as empty capsules, and of, occasion- ally, a' well-outlined cell body, after the vegetative form has en- tirely degenerated in the course of sporulation, make the assump- ^ tion of the presence of a cell membrane appear extremely well Fig. 2,-Bacterial Capsules. founded. Differing from the cell membranes of plant cells, cellulose has not, except in isolated instances, been demonstrable for bacteria, and the membrane is possibly to be regarded rather as a peripheral protoplasmic zone, which remains unstained by the usual manipula- tions. Zettnow,2 who has carefully studied the structure of some of the larger forms, takes the latter view, and regards the "ecto- plasmic" zone as a part of the cell protoplasm, which, however, is entirely devoid of nuclear material. The opinion of Zettnow is, furthermore, borne out by the greatly increased size of the bacterial cells as seen by means of special stains. Many bacteria have been shown to possess a mucoid or gelatinous envelope or capsule. According to Migula,3 such an envelope is present 1 Biitschli, loc. cit. 2 Zettnow, loc. cit. 3 Migula, " Systeme d. Bakterien," 1, p. 56. MORPHOLOGY, REPRODUCTION, ETC. 13 on all bacteria, though it is in only a few species that it is sufficiently well developed and stable to be easily demonstrable and of differential value. When stained, the capsule takes the ordinary anilin dyes less deeply than does the bacterial cell body, and varies greatly in thickness, ranging from a thin, just visible margin to dimensions four or five times exceeding the actual size of the bacterial body itself. This struc- ture is perfectly developed in a limited number of bacteria only in which it then becomes an important aid to identification. Most prominent among such bacteria are Diplococcus pneumoniae, Micrococcus tetra- genus, the bacilli of the Friedlander group, and B. aerogenes capsulatus. The development of the capsule seems to depend intimately upon the environment from which the bacteria are taken. It is most easily de- monstrable in preparations of bacteria taken directly from animal tissues and fluids, or from media containing animal serum or milk. When cultivated for any prolonged period upon artificial media, many otherwise capsulated microorganisms no longer show this characteristic structure. Capsules may be demonstrated on "bacteria taken from artificial media most successfully when albuminous substances, such as ascitic fluid or blood serum, are present in the culture media, or, as Hiss has pointed out, when the bacteria are smeared upon cover slip or slide in a drop of beef or other serum.1 It is believed by most observers that the capsule represents a swelling of the ectoplasmic zone of bacteria. By others it is regarded as an evidence of the formation of a mucoid intercellular substance, some of which remains adherent to the individual bacteria when removed from cultures. It is noticeable, indeed, that some of the capsulated bacteria, especially Streptococcus mucosus and B. mucosus capsulatus, develop such remarkably slimy and gelatinous colonies that, when these are touched with a platinum wire, mucoid threads and strings adhere to the loop. Exactly what the significance of the capsules is can not be decided as yet. A definite relation to virulence, although suggested, has not been satisfactorily demonstrated. Whether they are protective in function or whether they are evidences of degeneration is a question which must be left open until further evidence is at hand. Organs of Locomotion. — When suspended in a drop of fluid many bacteria are seen to be actively motile. It is important, however, in 1 Hiss, Jour. Exp. Med., vi, 1905. 14 BIOLOGY AND TECHNIQUE all cases to distinguish between actual motility and the so-called Brown- ian or molecular movement which takes place whenever small particles are held in suspension in a fluid. Brownian or molecular movement is a phenomenon entirely ex- plained by the physical principles of surface tension, and has absolutely no relation to independent motility. It may be seen when particles of carmine or any other insoluble substance are suspended in water, and consists in a rapid to and fro vacillation during which there is actually no permanent change in position of the moving particle except inas- much as this is influenced by currents in the drop. The true motility of bacteria, on the other hand, is active motion due to impulses originating in the bacteria themselves, where the actual position of the bacterium in the field is permanently changed. The ability to move in this way is, so far as we know, limited almost entirely to the bacilli and spirilla, there being but few instances where members of the coccus group show active motility. In all cases, with the exception of some of the spirochetes, where motility may occasionally be due to an undulating membrane marginally placed along the body, bacterial motility is due to hair-like organs known as flagella. These flagella have rarely been seen during life, and their recognition and study has been made possible only by special staining methods, such as those devised by Loeffler, van Ermengem, Pitt, and others. In such stained preparations, the bacterial cell bodies often appear thicker than when ordinary dyes are used, and the flagella apparently are seen to arise from the thickened ectoplasmic zone. The flagella are long filaments, averaging in thickness from one-tenth to one-thirtieth that of the bacterial body, which often are delicately waved and undulating, and, judging from the positions in which they become fixed in preparations, move by a wavy or screw-like motion. In length they are subject to much variation, but are supposed to be generally longer in old than in young cultures. Very short flagella have been described only on nitrosomonas, one of the nitrifying bacteria discovered by Winogradsky.1 As to the finer structures of flagella, little can be made out except that they possess a higher refractive index than the cell body itself, and that they can be stained only with those dyes which bring- clearly into view the supposedly true cytoplasm of the cell. Whether they penetrate this cytoplasmic membrane or whether they 1 Winogradsky, Arch, des sci. biologiques, St. Petersburg, 1892, I, 1 and 2. MORPHOLOGY, REPRODUCTION, ETC. „ 15 are a direct continuation of this peripheral zone of the bacterial body, can not be decided. The manner in which bacteria move is naturally subject to some var- iation depending upon the number and position of the flagella possessed by them. Whether bacteria exercise or not the power of motility de- pends to a large extent upon their present or previous environment. They are usually most motile in vigorous young cultures of from twenty- four to forty-eight hours' growth in favorable media. In old cultures motility may be diminished or even inhibited by acid formation or by other deleterious products of the bacterial metabolism. At the optimum growth-temperature motility is most active, and a diminution or increase of the temperature to any considerable degree diminishes or inhibits it. Thus actively motile organisms, in the fluid drop, may be seen to diminish distinctly in activity when left for any prolonged time in a cold room, or when ^ Q A ^ & ' riG. 6. —Arrangement of the preparation is chilled. Any influence, Bacterial Flagella. in other words, chemical or physical, which tends to injure or depress physiologically the bacteria in any way, at the same time tends to inhibit their motility. Messea * has proposed a classification of bacteria ^ which is based upon the arrangement of their organs of motility, as follows: I. Gymnobacteria, possessing no flagella. II. Trichobacteria, with flagella. 1. Monotricha, having a single flagellum at one pole. 2. Lophotricha, having a tuft of flagella at one pole. 3. Amphitricha, with flagella at both poles. 4. Peritricha, with flagella completely surrounding the bac- terial body. Bacterial Spores. — A large number of bacteria possesses the power of developing into a sort of encysted or resting stage by a process commonly spoken of as sporulation or spore formation. The formation of spores by bacteria depends largely upon environmental conditions, and the optimum environment for spore formation differs greatly for various species. It is usually necessary that a temperature of over 20° C. exist in order that spores may be formed. Unfavorable factors, like acid formation, accumulation of bacterial products in old cultures, or 1 Messea, Cent. f. Bakt., I, Ref, ix, 1891. 16 BIOLOGY AXD TECHNIQUE lack of nutrition, frequently seem to constitute the stimuli which lead to sporulation. In the case of some species, notably the anthrax bacillus, spores are formed only in the presence of free oxygen and are therefore never formed within the tissues of infected animals. It is claimed that some of the pathogenic anaerobes, like B. tetani and the bacillus of malignant edema, may form spores anaerobic ally. Nevertheless it has been observed that when an absolute exclusion of oxygen is practiced in the cultivation of these bacteria, vegetative forms only are seen in the cultures.1 The process of sporulation is by no means to be regarded as a method of multiplication, since it rarely occurs that a single bacil- lus produces more than one spore. In some species of bacteria the formation of several spores in one individual has occasionally been observed, but there can be no question about the fact that such a condition is exceptional. Varieties of spores are often recognized, the so-called arthrospores and the true spores or endospores. It is seriously in doubt whether the structures once spoken of as arthrospores should be considered as in any way comparable to true spores. They are represented by the granular and globular appearances occasionally observed in old cultures of some bacteria, notably streptococcus, cholera spirillum, diphtheria bacillus, and others. It was believed that they were due to a transformation of certain individuals of the cultures into more resistant forms. It is probable, however, that such structures are merely to be regarded as evidences of involution or degeneration, since it has never been demon- strated that cultures containing them are more resistant either to dis- infectants or to heat, than cultures showing no evidences of such forms. The true spores or endospores are most common among bacilli, and are rarely observed among the spherical bacteria. They arise within the body of the individual bacterium as a small granule which probably represents a concentration of the protoplasmic substance. Xakanishi2 claims that there is a definite relation between these sporogenic globules and the nuclear material of the bacterial cell. At the time at which sporulation occurs there is usually a slight and gradual thickening of the bacillary body. After the formation of this thickening, a spore mem- brane appears about the same thickened area. The completed spore is usually round or oval, has an extremely high refractive index, and a 1 Zinsser, Jour. Exp. Med., viii, 1906. p. 542. 2 Xakanishi, Munch, rued. Woch., 1900. p. 680. MORPHOLOGY, REPRODUCTION, ETC. 17 Fig. 4. Various Positions of Spores in Bacterial Cell. a I c d e f g 00QD<2 cP on membrane which is very resistant. Muhlschlegel * believes that the spore membrane is a double structure, and, as stated before, Nakanishi believes that the spore contains nuclear material. The position of the spore in the mother cell is of some differential importance in that it is usually con- stant for one and the same species. Thus, the spores of the tetanus bacillus are regularly situated at the extreme ends of the bacillary bodies, while those of anthrax are situated at or near the middle. Physiologically, sporulation is probably to be regarded as a method of encystment for the purpose of resisting unfavorable environment, A and it is indeed true that species of bacteria the vegetative forms of which are rather easily injured by heat, light, drying, and chemicals /? have a comparatively enormous re- sistance to these agents after the formation of spores. Thus, while a 10-per-cent solution of carbolic acid will kill the vegetative forms of anthrax bacilli within twenty min- utes, anthrax spores are able to resist the same disinfectant for a long- period in a concentration of over 50 per cent; and while the vegetative forms of the same bacillus show little more resistance against moist heat than other vegetative forms, the spores will withstand the action of live steam for as long as ten to twelve minutes and more. Whenever the spores of any mi- ^ a I C 000 i Z? A&ff V B SO i.%. C aQ &0 c( Fig. 5. — Germination of Spores. A, Bacillus subtilis, equatorial spore germination; B, Bacillus anthracis, germination by simple transition; C, Clostridium butyricum, polar germi- nation. croorganism are brought into an en- vironment suitable for bacterial growth as to temperature, moisture, and nutrition, the spores develop into vegetative forms. This process differs according to species. In general it consists of an elongation of Muhlschlegel, Cent. f. Bakt., II Abt., vi, 1900, p. 65. 18 BIOLOGY AND TECHNIQUE the spore body with a loss of its highly refractile character and resist- ance to staining fluids. The developing vegetative cell may now rupture and slip out of the spore membrane at one of its poles, leav- ing the empty spore capsule still visible and attached to the bacillary body. Again, a similar process may take place equatorially instead of at the pole. In other species again, there may be no rupture of the spore-membrane at all, the vegetative form arising by gradual elongation of the spore and an absorption or solution of the mem- brane which is indicated by change in staining reaction. Division by fission in the ordinary way then ensues. REPRODUCTION OF BACTERIA Bacteria multiply by cell division or fission. A young individual increases in size up to the limits of the adult form, when, by simple cleavage, at right angles to the long axis, without any discoverable process of mitosis or nuclear changes, it divides into two individuals. In spite of the claims of various bacteriologists, notably Nakanishi,1 traceable analogy to the karyokinesis of other cells has not been definitely established. In the case of the spherical bacteria a slight change to the elliptical form takes place just before cleavage and this cleavage may occur in one plane only, in two planes, or in three planes. According to the limitations of cleavage direction, the cocci assume a chained appearance (streptococci), a grape-like appearance (staphylococci), or an arrangement in packets or cubes having three dimensions (sarcinae). In the cases of bacilli and spirilla, cleavage takes place in the direction of the short axis. The individuals, after cleavage, may separate from each other, or may remain mutually coherent. The cohesion after cleavage is pronounced in some species of bacteria and slight in others, and, together with the plane of cleavage, determines the morphology of the cell-groups. Thus among the cocci diplo- or double forms, long chains and short chains may arise and fur- nish a characteristic of great aid in differentiation. Similarly among the bacilli there are forms which appear characteristically as single individuals and others which form chains of varying length. The rate of growth varies to a certain extent with the species, and also with the favorable or unfavorable character of the environment. A generation, that is, the time elapsing in the interval between one 1 Nakanishi, Cent. f. Bakt., I, xxx, 1901. MORPHOLOGY, REPRODUCTION, ETC. 19 cleavage and the next, has been estimated by A. Fischer1 as being about twenty minutes for the cholera spirillum and 16-20 minutes for bacillus coli communis, under the most favorable conditions. The same author has calculated that under these conditions a single cholera spirillum would yield 1600 trillions in a single day. Such a multiplica- tion rate, however, is probably not usual under natural or even artificial conditions, both on account of lack of nutritive material and because of inhibition of the growth of the bacteria by their own products. VARIATIONS OF BACTERIAL FORMS Variations from the basic forms considered in the preceding sec- tion may occur, but are not common among bacteria under normal conditions. Thus the formation of club shapes by a thickening of the X^^^^MTW® Fig. 6. — Degeneration Forms of Bacillus Diphtheria. (After Zettnow.) bacillary body at one or both ends has been frequently observed among bacteria of the diphtheria group, and in the glanders bacillus, and an irregular beading is not infrequently observed in tubercle bacilli under normal conditions. Such pictures can not, in these cases, be regarded as degeneration or involution forms, since they are visible in young, actively growing cultures under ordinary conditions. It is a well-known 1 A. Fischer, " Vorlesungen tiber Bakt.," Jena, 1903. 20 BIOLOGY AND TECHNIQUE fact, furthermore, that the sizes and contours of bacteria may vary to some extent according to the medium on which they are grown. This may, to a certain degree, be due to osmotic relations. On fluid media for instance, many bacteria may appear larger and of a less dense con- sistency than do members of the same species cultivated upon solid media. Degeneration Forms. — When bacteria are grown under conditions which are not entirely favorable for their development, or when they are grown for a prolonged period upon artificial culture media without transplantation, there may occur variations which often depart consider- ably from the ground type, known as degeneration or involution forms. A Fig. 7. — Degeneration Forms of Bacillus Pestis. (After Zettnow.) Thus, in the case of the diphtheria bacillus, old cultures may contain long, irregularly beaded forms with broad expansions at the ends. In the case of bacillus pestis the formation of oval, vacuolated bodies in old cultures occurs so regularly and in such numbers that the fact has become of differential value.1 Among the cocci, marked evidences of involution are often seen in cultures of the meningococcus in the form of large, swollen poorly- staining spheres, and in the case of the pneumococcus in the so-called shadow forms which have the appearance of empty capsules. There are 1 Harikin and Leumann, Cent. f. Bakt., I, xxii, 1897. MORPHOLOGY, REPRODUCTION, ETC. 21 few microorganisms indeed, in which prolonged cultivation on artificial media or other unfavorable influences do not produce variations from the ground type which may often make the cultures morphologically unrecognizable. In the case of many of the spirilla (spirillum Milleri, spirillum Metchnikovi, etc.) the degeneration forms may appear within so short a time as two or three days after transplantation. CHEMICAL AND PHYSICAL PROPERTIES OF THE BACTERIAL CELL Chemical Constituents. — The quantitative chemical composition of bacteria is subject to wide variations, dependent upon the nutritive materials furnished them. Approximately 80 to 85 per cent of the bacterial body is water. The remainder consists chiefly of proteids which constitute roughly from 50 to 80 per cent of the dry substances. Remaining, after extrac- tion of these, are fats, and in some cases true wax (fatty acid combina- tions with higher alcohols), traces of cellulose (in some bacteria only), and the ash which makes up usually about 1 to 2 per cent of the dry substances. The extensive researches of Cramer1 have shown how widely at va- riance quantitative analyses may be when made of cultures of the same species of bacteria grown upon different media. Thus the dry sub- stances of the cholera vibrio were found to be made up of 65 per cent of proteids when the microorganisms were grown upon nutrient broth as against 45 per cent when the same bacteria had been grown upon the proteid-free medium of Uschinsky. Analyses made by Kappes2 of B. prodigiosus and by Nencki3 and Scheffer of some of the putrefactive bacteria, may serve to illustrate the approximate proportions of the substances making up the bacterial body. B. prodigiosus Water 85 . 45 per cent. Proteids 10.33 " " Fats 0.7 " Ash 1.75 " " Residue 1.77 " " Putrefactive Bacteria 83 42 per cent. 13 96 u U 1 a a 0 78 u a 0 84 11 ic 1 Cramer, Arch. f. Hyg., xii, xiii, xvi, xxii, xxviii. 2 Kappes, " Analyse der Massenkulturen," etc. Diss., Leipzig, 1889. 3 Nencki und Scheffer, Jour. f. prakt. Chemie, new ser. xix, 1880. 22 BIOLOGY AND TECHNIQUE Analyses of the tubercle bacillus by Ruppel,1 Hammerschlag,2 Weyl,3 and others, have yielded the following approximate results (calculated from results of above-mentioned authors). Tubercle bacillus Water 85 to 86 per cent. Proteids 8 . 5 to 9 " " Fat and waxes 3 . 5 to 4 Ash and carbohydrates 1 . 2 to 1.4 " " The proteids which are contained in the bacterial dry substances consist partly of nucleoproteids, globulins, and proteids differing ma- terially from those ordinarily met with. Ruppel, in an analysis of the tubercle bacillus, obtained the following values, for 100 grams of dried tubercle bacilli: Nucleic acid 8.5 grams. (Tuberculinic acid) Nucleoprotamin 25 . 5 " Nucleoproteid 23 Albuminoids , 8.3 " (Keratin, etc.) Fat and wax 26 . 5 " Ash 9.2 " A true globulin has been isolated from bacteria by Hellmich,4 and true proteids, coagulable by heat, have been demonstrated by Buchner,5 in the " Presssaft " or juice obtained by subjecting bacteria to mechanical pressure. In this connection, too, we should not fail to consider the thermolabile toxic substances contained in many bacteria, the endo- toxins, which though of uncertain chemical nature, are probably pro- teid in composition.6 The fats which are demonstrable both by micro-chemical methods, staining with Sudan III., Scharlach R., Osmic acid, and by alcohol- ether extraction, consist of fatty acids, true fats, and, in the case of the tubercle bacillus at least, of waxy substances.7 1 Ruppel, Zeit. f. physiol. Chemie, xxvi, 1898. 2 Hammerschlag , Zeit. f. klin. Med., 1891. 3 Weyl, Deut. med. Woch., 1891. 4 Hellmich, Arch. f. exper. Pathol., etc., xxvi. 5 Buchner, Munch, med. Woch., 1897. 6 Shattock, Lancet, May, 1898. * De Schweinitz and Dorset, Cent. f. Bakt., I, xxii, 1897. MORPHOLOGY, REPRODUCTION, ETC. 23 The carbohydrates isolated from various bacteria consist chiefly of small quantities of cellulose and allied bodies, presumably concerned in the formation of the bacterial cell membrane. The demonstration of these substances has been successful only in isolated cases and has not found universal confirmation. Glycogen-like substances have been demonstrated, according to A. Fischer,1 in B. subtilis and B. coli. These bacteria stained a reddish brown color when stained with iodin, and after treatment with weak acids were shown to contain dextrose. The bacterial ash, remaining after removal of other substances, con- sists largely of phosphates and chlorides of potassium, sodium, cal- cium, and magnesium. Osmotic Properties of the Bacterial Cell. — Like all other animal and vegetable cells, the bacterial cell forms in itself a small osmotic unit which reacts delicately to differences of pressure existing between its own protoplasm and the surrounding medium. The perfect and normal morphology of a microorganism, therefore, can exist only when the osmotic pressure within the protoplasm of the cell is isotonic or equal to that of its own environment. The changes produced in the morpho- logical relations of a cell when transferred from one environment into another of varying osmotic pressure, depend intimately upon the " permeability " of the cell membrane for different substances. When such a membrane is permeable for water and not for substances in solu- tion, it is technically spoken of as "semi-permeable." Now, as a matter of fact, the bacterial cell membrane is easily permeable for water, but its permeability differs greatly in various species of bacteria for other substances. Thus, for instance, the cholera vibrio shows great perme- ability for common salt and B. fluorescens liquefaciens shows a lower permeability for potassium nitrate than do many other bacteria.2 When a microorganism is suddenly removed from an environment of low osmotic pressure into one showing a high pressure, say, from a dilute to a concentrated solution of NaCl, an abstraction of water from the cell occurs, with a consequent shrinkage of the protoplasm away from the cell membrane. This process is spoken of as "plasmolysis." Cell death does not usually occur with plasmolysis, but by slow diffusion of the salt itself into the protoplasm, the equilibrium may eventually be restored and the normal morphology of the cell resumed. In all cases 1 A. Fischer, " Vorlesungen fiber die Bakt./' Jena, 1903. 2 Gottschlich, in Fli'igge, ': Mikroorganismen," i, p. 91. 24 BIOLOGY AND TECHNIQUE the speed and completeness of the return to normal depends upon the permeability of the cell membrane for the dissolved substances. There is no evidence to support the view that the internal pressure of a cell may be in any way increased by an inherent power of the protoplasm independently of the laws of diffusion. As a general rule, old cultures are more susceptible to plasmolysis than are young and vigorous strains. Spores and, according to A. Fischer,1 flagella are much less susceptible to osmotic changes than are the vegetative bodies. When, on the other hand, bacteria are suddenly removed from a medium possessing a high osmotic pressure to one comparatively low, say, from a concentrated salt solution to distilled water, a bursting of the cell may occur, a process spoken of as "plasmoptysis." Plasmoptysis leads to cell death, and is probably the cause of the death of micro- organisms so often observed in distilled-water emulsions of bacteria. Other Physical Properties of Bacteria. — The refractive index of the vegetative bacterial body is low, in contrast to the highly refractive character of the spores and flagella. According to Fischer, the ectoplasm or cell membrane shows a higher index than does the endoplasm. The specific gravity of various microorganisms has been investi- gated by Bolton,2 Rubner,3 and others. Some of Rubner' s results are the following : Gelatin fluidifiers Sp. gr. 1 .0651 Gas formers " " 1 .0465 Cultures from potato " " 1 . 038 M. prodigiosus " " 1 . 054 1 A. Fischer, quoted from Gottschlich in Flugge, " Mikroorganismen," I, p. 91. 2 Bolton, Zeit. f. Hyg., i. 1886. 3 Rubner, Arch. f. Hyg., xi, 1890. CHAPTER III THE RELATION OF BACTERIA TO ENVIRONMENT, AND THEIR CLASSIFICATION NUTRITION OF BACTERIA Like all protoplasmic bodies, bacteria consist of carbon, oxygen, hydrogen, and nitrogen, to which are added inorganic salts and varying quantities of phosphorus and sulphur. In order that bacteria may develop and multiply, therefore, they must be supplied with these sub- stances in proper quantity and in forms suitable for assimilation. To formulate definite laws based on chemical structure as to the compounds suitable, and those unsuitable for use by the bacteria, is obviously im- possible owing to the great metabolic variations existing within the bacterial kingdom, and notable attempts to do so, such as those by Loew,1 have not successfully withstood critical inquiry. Carbon. — The carbon necessary |or bacterial nourishment or ana- bolism may be obtained either directly from proteids, carbohydrates, and fats, or from the simpler derivatives of these substances. Thus, the amido-acids, leucin and tyrosin, ketons, and organic acids, like tartaric, citric, and acetic acids, glycerin, and even some of the alcohols, may furnish carbon in a form suitable for bacterial assimilation. A limited number of bacterial species, furthermore, notably the nitrobacteria of Winogradsky, are capable of obtaining their required carbon from atmospheric C02, and possibly from other simple carbon compounds added to culture media.2 Oxygen. — Oxygen is obtained, by the large majority of bacteria, directly from the atmosphere in the form of free 02. For many micro- organisms, moreover, the presence of free oxygen is a necessary condi- tion for growth. These are spoken of as the " obligatory aerobes." Among the pathogenic bacteria proper, many, like the gonococcus, bacillus influenzae, and bacillus pestis, show a marked preference for a well-oxygenated environment. Probably there is no pathogenic micro- *Loew, Cent. f. Bakt., I, xii, 1892. 2 Muntz, Compt. rend, de Pacad. des sciences, t. iii, 25 26 BIOLOGY AND TECHNIQUE organism which, under certain conditions of nutrition, is entirely unable to exist and multiply in the complete absence of this gas. The conditions existing within the infected animal organism cause it to seem likely that all incitants of infection may, at times, thrive in the complete absence of free oxygen. There is another class of organisms, on the other hand, for whose development the presence of free oxygen is directly injurious. These microorganisms, known as "obligatory anearobes," obtain their supply of oxygen indirectly, by enzymatic processes of fermentative and pro- teolytic cleavage, from carbohydrates and proteids, or by reduction from reducible bodies. Among the pathogenic microorganisms the class of " obligatory anaerobes " is represented chiefly by Bacillus tetani, the bacillus of malignant edema, the bacillus of symptomatic anthrax, Bacillus aerogenes capsulatus, and Bacillus botulinus. Intermediate between these two classes is a large group of bacteria which thrive well both under aerobic and anaerobic conditions. Some of these, which have a preference for free oxygen but nevertheless possess the power of thriving under anaerobic conditions, are spoken of as "facultative anaerobes." In others the reverse of this is true; these are spoken of as "facultative aerobes." These varieties of bacteria are by far the most numerous and comprise most of our parasitic and saprophytic bacteria. The relation of microorganisms to oxygen is extremely subtle, there- fore, and not to be biologically dismissed by a rigid classification into aerobes, facultative anaerobes, and obligatory anaerobes. Both Engel- mann,1 by a method of observing motile bacteria in the hanging drop as to their behavior in relation to the oxygen given off by a chlorophyll- bearing alga, and Beijerinck,2 by a macroscopic method of observing similar bacteria as to their motion away from or toward an oxygenated area, were able to demonstrate delicately graded variations between species, favoring various degrees of oxygen pressure. The discovery by Pasteur that certain bacteria develop only in the absence of free oxj^gen, produced a revolution in our conceptions of metabolic processes, since up to that time it was believed that life could be supported only when a free supply of 02 was obtainable. Pasteur's original explanation for this phenomenon was that anaerobic conditions of life were always associated with some form of carbohydrate fermenta- 1 Engelmann, Botanische Zeitiing,-i881. 2 Beijerinck, Cent, f. Bakt., I, xiv, 1893. RELATION TO ENVIRONMENT— CLASSIFICATION 27 tion and that oxygen was obtained by these microorganisms by a split- ting of carbohydrates. As a matter of fact, for a large number of micro- organisms, this is actually true, and the presence of readily fermentable carbohydrates not only increases the growth energy of a large number of anaerobic bacteria, but in many cases permits otherwise purely aerobic bacteria to thrive under anaerobic conditions.1 On the other hand, the basis of anaerobic growth can not always be found in the fermentation of carbohydrates or in the simple process of reduction. The favorable influence of certain actively reducing bodies, like sodium formate or sodium-indigo-sulphate, upon anaerobic cultivation is probably referable to their ability to remove free oxygen from the media and thus perfect the anaerobiosis.2 A number of strictly anae- robic bacteria, however, may develop in the entire absence of carbohy- drates or reducing substances, obtaining their oxygen supply from other suitable sources, some of which may be the complex proteids. Thus the tetanus bacillus may 3 thrive when the nutritive substances in the media are entirely proteid in nature. (See p. 28.) As Hesse 4 has shown, the respiratory processes of aerobic bacteria consist in the taking in of oxygen and the excretion of C02. The C02 excretion has been shown, in these cases, to be markedly less than is represented in the intake of oxygen. Anaerobes, likewise, show an excretion of C02 which must, in these cases, be a result of bacterial katabolism. Certain bacteria, like the red sulphur bacteria, have the power of utilizing atmospheric oxygen in the same way in which this process takes place in the chlorophyll-bearing plants. While a profuse supply of oxygen absolutely inhibits the growth of most anaerobes, a number of these may, nevertheless, develop when only small quantities of oxygen are present. Minute quantities of free oxy- gen in culture media have been shown by Beijerinck5 and others not to inhibit the growth of Bacillus tetani and Theobald Smith 6 has recently demonstrated that when suitable nutritive material in the form of fresh liver tissue is added to bouillon, a number of anaerobic bacteria may be 1 Theobald Smith, Cent. f. Bakt., I, xviii, 1895. 2 Kitasato and Weyl, Zeit. f. Hyg., viii, 1890. 3 Chudiakow, Cent. f. Bakt., Ref., II, iv, 1898. * Hesse, Zeit. f. Hyg., xv, 1897. 5 Beijerinck, Cent. f. Bakt., II, vi, 1900. 6 Th. Smith, Brown, and Walker, Jour. Med. Res., ix, 1906. 28 BIOLOGY AND TECHNIQUE induced to grow in indifferently anaerobic environment. Ferran,1 moreover, succeeded in gradually adapting the tetanus bacillus to an aerobic environment. In this case, however, the virulence of the bacil- lus was lost. Nitrogen. — The nitrogen required by bacteria is taken, in most cases, from proteids. Most important in this respect, of course, are the dif- fusible proteicls; but many of the non-diffusible albumins may be rendered assimilable by the proteolyzing enzymes possessed by many microorganisms. Among the pathogenic, more strictly parasitic bac- teria, moreover, a delicate specialization may be observed as to the particular varieties of animal albumin which may be utilized by them. Thus the gonococcus grows more readily only upon uncoagulated human blood serum; the Pfeiffer bacillus requires hemoglobin, and the diph- theria bacillus outgrows other bacteria upon a medium composed for the greater part of coagulated beef serum. For bacteria that do not absolutely require native animal proteid for their development, the most common nitrogenous ingredient of culture media is pepton, added in solutions of varied concentration. A large number of bacteria (pathogenic ' and saprophytic) , on the other hand, may thrive on media containing absolutely no proteid, in which case, of course, a synthetic proteid production by the micro- organisms must be assumed. A medium which has been extensively used to demonstrate this phenomenon is that devised by Uschinski,2 containing ammonium lactate, glycerin, asparagin (the amide of amido- succine acid), and inorganic salts. Creatin, creatinin, urea and urates, and even ammonia compounds and nitrates, may serve as adequate sources of nitrogen for many of the less parasitic bacteria. A limited number of species, moreover, the bacilli in the root tubercles of the leguminosse and the nitrogen-fixing organ- isms of the soil, possess the power of obtaining their supply of nitrogen directly from the free N2 of the atmosphere. Although the sources of carbonaceous and of nitrogenous food supply have been separately treated in the preceding paragraphs, it should not be forgotten that, in many instances, both elements are taken up within the same compound, and that separate supplies are a necessity in isolated cases only. Hydrogen. — Hydrogen is obtained by bacteria largely in combina- » Ferran, Cent. f. Bakt., I, xxiv, 1898. 2 Uschinski, Cent. f. Bakt., I, xiv, 1893. RELATION TO ENVIRONMENT— CLASSIFICATION 29 tion as water and together with the carbon and nitrogen containing substances. Salts. — The phosphatic constituents of the bacterial body are taken in, chiefly, as phosphates of magnesium, calcium, sodium, or potassium. The phosphates seem to be necessary constituents of culture media, while chlorides, on the other hand, according to Proskauer 1 and Beck are not absolutely essential. Sodium salts, as a rule, seem to be more advantageous for purposes of bacterial cultivation than potassium salts. The uncombined sulphur, which is a constituent of the bacterial body in many cases, is usually supplied by soluble sulphates. In the case of the thiobacteria of Winogradsky, however, the presence of free H2S is necessary for its formation.2 The iron contained in some of the higher bacteria is taken in in the form of ferrous compounds, and is oxidized in the bacterial body into ferric compounds. The relative quantities of the various nutritive substances in culture media are of importance only in so far as too high concentrations may have a distinctly inhibitory influence. In this respect, however, separate species may show widely divergent tastes. The development of bacteria in any given medium, it may be noted, is far oftener arrested by the accumulation of waste products than by an exhaustion of nutrient materials. PARASITISM AND SAPROPHYTISM When we speak of bacteria as parasites or as saprophytes, we classify them, primarily, according to their relationship to the bodies of higher animals. " Parasites " are those bacteria which are capable of living and multiplying within the human or animal body, whereas the term " sapro- phytes " refers to the multitude of microorganisms which are unable to hold their own under the environmental conditions found in the tis- sues of higher animals, but are found, almost ubiquitously, in air, soil, manure, and water. The separation is by no means a sharp one and carries with it other implications, which the use of these terms always conveys. While parasites are usually very fastidious as to nutritional and temperature requirements, most saprophytes are easily cultivated upon the simplest media. Thus certain parasitic bacteria, such as the 1 Proskauer and Beck, Zeit. f. Hyg., xviii, 1895. 2 Voges, Cent. f. Bakt., I, xviii, 1893. 30 BIOLOGY AND TECHNIQUE bacillus of influenza, the -gone-coccus, and others, are dependent upon specific forms of animal proteids for their food supply, while typical saprophytes, like Bacillus proteus, may thrive and multiply upon even the simplest organic proteid derivatives. Between the strict parasites and the saprophytes, however, there is a large class of bacteria, to which the majority of our pathogenic varieties belong, the members of which are capable of developing luxuriantly under both conditions. These bacteria are often spoken of as facultative parasites. More recently the question of parasitism and saprophytism has become closely interwoven with our conceptions of virulence. Bail (see section on Aggressins) has classified parasites into strict parasites and half parasites. By the first term he designates bacteria like Bacillus anthracis, which actually invade all the tissues of their host, while, by the term "half parasites," he refers to microorganisms like the spiril- lum of cholera which gain a foothold upon some part of the body of the host, but do not actually penetrate into the general circulation. All pathogenic bacteria, therefore, must be grouped as parasites, strict or facultative, while the saproprrytes, as a class, perform the far more thankful task of breaking up organic matter outside of the animal body, by putrefaction and fermentation. Absolute separation between the two classes, however, can not be maintained, since many ordinarily saprophytic bacteria may display parasitic qualities if administered in large numbers to animals or man in whom resistance to bacterial invasion is at a low ebb. ANTAGONISM AND SYMBIOSIS OF BACTERIA The ubiquity of bacteria in nature naturally carries with it the simul- taneous presence of many species in all places where special conditions have provided a favorable environment for growth. Thus bacteriological investigation of water, milk, manure, soil, or organic infusions, always reveals the presence of a large number of different varieties within the same substance. If the food supply in such a natural culture is at all limited in quantity, or the removal of waste products is prohibited, it will usually be found that gradually the numbers of varieties will dimin- ish and a few species, or even only one, will prevail. In the case of milk, for instance, after standing for three or four da}rs at a suitable temper- ature, two or three varieties will be found to have taken the place of the twenty or thirty, which may have been present originally. RELATION TO ENVIRONMENT— CLASSIFICATION 31 This behavior is due to the influences which various microorganisms exert upon each other and is known as antagonism. Such antagonism probably depends upon the fact that the metabolic products of the pre- dominant species (the one or ones for whom the special cultural condi- tions are most favorable) inhibit the growth of the less vigorous varieties. Many examples, experimentally supported, of such antagonism, can be given. Thus, the gonococcus is distinctly inhibited by the soluble pro- ducts of Bacillus pyocyaneus,1 while in the presence of pyogenic cocci it develops luxuriantly, and the bacillus of plague is completely inhibited when streptococci are present in the culture.2 Mutual inhibition may also be clue to the monopolizing of the nutri- tion in the medium by the predominating species or to the change in re- action produced by its growth. This last consideration is probably the secret of the frequently noticed inhibitory effect exerted by acid-pro- ducers upon bacteria of putrefaction, and has received practical thera- peutic application in MetchnikofFs lactic-acid bacillus therapy, which see. When the simultaneous presence of two bacterial species within the same environment favors the development of both species, the condi- tion is spoken of as symbiosis. Such dependence is not so frequent as antagonism, but it does occur. Examples of such a condition have been observed in cultures containing diphtheria bacilli and streptococci 3 and have been frequently observed in cultures containing both aerobic and anaerobic bacteria, where the former favor the development of the latter by monopolizing the supply of free oxygen. Symbiosis may also take place in cultures in which complex food products are split up by one species, furnishing substances for ingestion by species with a lesser digestive ability. RELATIONS OF BACTERIA TO PHYSICAL ENVIRONMENT Relation to Temperature. — Like all other living beings, bacteria develop and multiply by virtue of a series of chemical and physical processes, by means of which growth energy is obtained by destruction or catabolism, and the lost tissues resupplied by absorption of nutritive materials. It is natural, therefore, that the conditions of external 1 Schafer, Fortschr. d. Med., 5, 1896. 2 Bitter, Rep. Egypt Plague Com., Cairo, 1897, 3 Hilbert, Zeit. f. Hyg., xxix, 1895. 32 BIOLOGY AND TECHNIQUE temperature should intimately affect the metabolic processes. The range of temperature at which bacteria may grow is subject to wide variations among different species. Each species, on the other hand, may thrive within a more or less elastic range of temperature, each one having an optimum, a minimum, and a definite maximum tempera- ture. When the optimum temperature is present in the environment, the functions of absorption and excretion keep pace with each other, and the chemical balance is well preserved. When the temperature is lower than the optimum, all metabolic processes take place more slowly, and the bacterium gradually enters into a resting or latent stage, at which ac- tual growth ma}r be exceedingly slow7 or entirely inhibited. . When the temperature is higher than the optimum, the destructive processes are carried on more rapidly than the substitution of wTaste products by ab- sorption, and a gradual weakening of vital energy, or even a gradual death of the bacterium, may take place. When certain bacteria form spores, they become very much more resistant against both high and low temperatures, probably because a true resting stage has been reached, during wmich metabolism has been reduced to a minimum, there being practically no nutritive material taken in and corresponding- ly little destruction taking place wTithin the body of the microorganism. The optimum temperature for various bacteria depends upon the habitual environment, in which the particular species is accustomed to exist. Thus, for the large majority of bacteria pathogenic for human beings, the optimum temperature is at or about 37.5° C. There are a large number of bacteria common in water, however, which grow hardly at all at the body temperature, but thrive most luxuriantly at temperatures of about 20° C. F. Forster,1 moreover, described certain phosphorescent bacteria, isolated from sea-water, which grow readily at 0° C, or a little above. On the other hand, Miquel2 has described, non- motile bacilli, which he isolated from the wrater of the Seine, which grow rapidly at temperatures ranging about 70° C, and the so-called "muce- dinees thermophiles," described by Tsiklinski,3 develop most readily at temperatures very little above this. It is thus plain that the tempera- tures favored by various bacteria depend to a large extent upon an adaptation of these bacteria through many generations to specific en- vironmental conditions. A good illustration of this is furnished by the bacillus of avian tuberculosis, a microorganism differing essentially 1 F. Forster, Cent. f. Bakt,, ii, 1887. 2 Miquel, Bull, de la Stat. Mimic, de Paris, 1879. 3 Tsiklinski, Ann. Past,, 1889. RELATION TO ENVIRONMENT— CLASSIFICATION 33 from the bacillus of human tuberculosis in that its optimum growth temperature lies at 41°-42° C, a temperature which exceeds the op- timum temperature for the human type by as much as the normal tem- perature of birds exceeds that of man. The same principle is illustrated by the facts that the bacteria which have a very low optimum tem- perature are usually those isolated from water, and the so-called ther- mophile or high-temperature bacteria are obtained from hot springs and from the upper layers of the soil, where, according to Globig,1 occasion- ally temperatures ranging from about 55° C. occur. As stated before, one and the same species may develop within a wide temperature range, and it may be possible, by persistent cultiva- tion at special temperatures, to adapt certain bacteria to grow luxu- riantly at temperatures removed by several degrees from their normal optimum. In such cases it may often occur that special characteristics of the given species may be lost. An example of this is the loss of viru- lence and of spore-formation which takes place when anthrax bacilli are cultivated at 42° C, or the loss of the power to produce pigment when bacillus prodigiosus is grown at temperatures above 30° C. The vegetative forms of most of the pathogenic bacteria may grow at temperatures ranging between 20° C. and 40° C. This can, however, by no means be regarded as applicable to all of the pathogenic bacteria, as some of these, like the gonococcus, the pneumococcus, the tubercle bacillus, and others, are delicately susceptible to temperature changes and have the power of growing only within limits varying but a few degrees from their optimum. Others, on the other hand, like bacilli of the colon group, Bacillus anthracis, Spirillum cholerse asiaticse, etc., may develop at temperatures as low as 10° C. and as high as 40° C, or over. The range of temperature at which saprophytic bacteria may develop is usually a far wider one. When temperatures exceed in any considerable degree the maximum growth temperature, the vegetative forms of bacteria perish. Thus, ten minutes' exposure to a temperature of between 55° and 60° C. causes death of the vegetative forms of most microorganisms. Death in such cases is due probably to a coagulation of the protoplasm, and since all such processes of coagulation take place best in the presence of water, the thermal death point of most bacteria is lower when heat is applied in the form of boiling water or steam, than when employed as dry heat. (See section on Sterilization.) When spores are present in cultures, the resistance to heat is enor- * Globig, Zeit. f. Hyg., iii. 34 BIOLOGY AND TECHNIQUE mously increased. Exactly what the explanation of this is can not be at present stated. It may be that the high concentration in which the protoplasmic mass is found in the spores renders it less easily coagulable than is the protoplasm of the vegetative body. A more detailed discus- sionof these relations will be found in the section on Heat sterilization. The thermal death points of a large number of bacteria have been very carefully studied by Sternberg/ by a special technique described elsewhere. The thermal death points ascertained by him in this way, with an exposure of ten minutes in a fluid medium, for some of the more common non-sporogenic bacteria are as follows: Spirillum cholerse asiaticae 52° C. Diplococcus pneumonia? 52° C. Streptococcus pyogenes 54° C. Bacillus typhosus 56° C. Bacillus pyocyaneus 56° C. Bacillus mucosus capsulatus 56° C. Bacillus prodigiosus 58° C. Staphylococcus pyogenes aureus 58° C. Gonococcus : 60° C. Staphylococcus pyogenes albus 62° C. The bacillus tuberculosis, though not a spore bearer, seems to be slightly more resistant to heat than other purely vegetative microorganisms. Thus, according to the researches of Smith 2 and others, ten and twenty minutes' exposure to a temperature of 70° C. is necessary to destroy tubercle bacilli in a fluid medium. For the effectual destruction of spores by moist heat, a temperature of 100° C, or boiling point, is usually necessary. Low temperatures are much less destructive than the high ones, and are even in a number of cases useful in keeping bacteria alive for long periods, inasmuch as metabolic processes are inhibited and life is maintained without actual development in a sort of resting state. Actual destruction by low temperatures rarely takes place. The exposure of diphtheria, typhoid, and other bacilli to temperatures as low as 200° C. below zero has been carried out without destruction of the microorganisms, a fact which is of great importance in considering the possibility of infection by the vehicle of ice. Meningococci and gonococci, on the other hand, die out rapidly when exposed to 0° C. 1 Sternberg, "Textbook of Bacteriology," New York, 1901. 2 Th. Smith, Jour, of Experimental Med., No. 3, 1899. RELATION TO ENVIRONMENT— CLASSIFICATION 35 Relation to Pressure. — High pressure does not exert any noticeable effects upon bacteria. In the experiments of Certes,1 a pressure of two atmospheres seemed to have no influence upon the growth and motility of anthrax bacilli suspended in blood. Relation to Moisture. — For the growth and development of all bac- teria, the presence of water in the culture medium is necessary. It is self-evident that nutritive materials can not be absorbed by an osmotic process unless in a state of solution. While complete dryness does not permit growth, its destructive action upon various bacteria is subject to great differences. The effect of complete drying upon bacteria will be found more fully discussed in the section upon the destruction of bacteria by physical agents. (See page 62.) In the same section may be found a discussion of the effects of light, electricity, x-ray, and radium rays upon bacteria. THE CLASSIFICATION OF BACTERIA Too simple in structure, too varied in biological properties to be definitely identified with either the vegetable or animal kingdom, the bacteria are placed at the bottom of the scale of all living beings. Closely linked on the one hand to the plant kingdom by the yeasts and the molds, and on the other to the animal kingdom by the protozoa, they themselves combine, within one and the same division, attributes so widely divergent as to structure, metabolism, and biological activity that their grouping is more a matter of working convenience than of actual scientific classification. Thus, for instance, all stages of metabolic ac- tivity fill in the gap between the synthetizing sulphur and nitrifying bacteria and the purely katabolic activities of some of the aerobic and anaerobic microorganisms which cause putrefaction. Growth takes place within the limits of a wide temperature range, and the specific modes of life and cultural conditions are subject to the widest varia- tions, from those of an indisputably useful saprophytism to those of the most exquisite parasitism. Although, therefore, strictly speaking, the bacteria can be classified as a whole neither in the animal nor in the vegetable realms, being non chlorophyll-bearing, they are for conve- nience classified with the fungi or colorless plants. The relationship of the bacteria to other simple plants may be graphically represented by the following scheme: 1 Certes, Compt. rend, de l'acad. d. sc, 99, Paris, 1884. 36 BIOLOGY AND TECHNIQUE Cryptogam ia. I Thallophyta. Alg-«. SCHYZO.MYCETES (Bacteria). Lichens. Blastomycetes (Yeasts). Fungi. Hyphomycetes (Moulds — Oidia). Coccacea. Bacteriacese. Spirillacese. Chlamydobacteria. (Higher bacteria.) Streptothrix. Cladothrix. Leptothrix. Actinomyces. The special classification of the bacteria has offered still greater difficulties, for the lower we proceed in the phylogenetic scale of living beings, the less specialized the morphological and biological charac- teristics of any group become, and the more difficult it is to establish a classification which can in any way be regarded as final. It is, there- fore, quite impossible to classify the bacterial varieties or species on any basis which can hope to satisfy all the demands of scientific accuracy and it is necessary to resort to the expedient of utilizing some one characteristic which remains constant for the individual genus and to base upon this an attempt at grouping. When bacteria were first dis- covered, and for many years following, numerous observers contended that the form of the microorganism observed was not a constant one for each genus, but that cocci could be converted into bacilli or spirilla according to environmental conditions. It was Cohn 1 who, in 1872, first recognized the constancy of the morphology of bacteria and es- tablished, upon morphological basis, a classification which, with minor changes, has been retained until the present day. Such classifications can not, however, be regarded as anything more than a convenient make-shift pending the day when the finer structure and true biological relations of the various bacteria shall have been more accurately inves- tigated. The scheme most commonly accepted at present is the one given below, proposed by Migula2: i Cohn, "Beitrage zur Biol. d. Pflanzen," Heft 1 u. 2, 1872. 2 Migula, "System d. Bakt.," Jena, 1897. RELATION TO ENVIRONMENT— CLASSIFICATION 37 Bacteria (Schizomycetes) . — Fission fungi (chlorophyll free) with cell division in one, two, or three directions of space. Many varieties possess the power of forming endospores. Whenever motility is present, it is carried on by means of flagella, or, more rarely, by undulating membranes. Family I. Coccace^e. — Cells in free state perfectly spherical. Division in one, two, or three directions of space, by which each spherical cell divides into two, four, or eight segments, each of which again develops into a perfect sphere. Endospore formation rare. Genus I. Streptococcus. — Cells divide in one direction of space only, for which reason, if they remain connected after fission, bead- like chains may be formed. No organs of locomotion. Genus II. Micrococcus (Staphylococcus). — -Cells divide in two directions of space, whereby, if the cells remain connected after fission, tetrad and grape-like clusters may be formed. No organs of locomotion. Genus III. Sarcina. — Cells divide in three directions of space, whereby, if they remain connected after fission, bale-like packets are formed. No organs of locomotion. Genus IV. Planococcus. — Cells divide in two directions of space, as in micrococcus, but possess flagella. Genus V. Planosarcina. — Cells divide in three directions of space as in sarcina, but possess flagella. Family II. Bacteriace^e.— Cells long or short, cylindrical, straight, never spiral. Division in one direction of space only, after pre- liminary elongation of the rods. Genus I. Bacterium. — Cells without flagella, often with endospores. Genus II. Bacillus. — Cells with peritrichal flagella, often with endospores. Genus III. Pseudomonas. — Cells with polar flagella. Endospores occur in a few species, but are rare. Family III. Spirillace^e. — Cells spirally curved or representing a part of a spiral curve. Division in one direction of space only, after preceding elongation of cell. Genus I. Spirosoma. — Cells without organs of locomotion. Rigid. Genus II. Microspira. — Cells rigid, with one or, more rarely, two or three polar undulated flagella. 38 BIOLOGY AND TECHNIQUE Genus III. Spirillum. — Cells rigid, with polar tufts of five to twenty flagella usually curved in semicircular or flatly undulating curves. Genus IV. Spirochete. — Cells sinously flexible. Organs of locomo- tion unknown, perhaps a marginal undulating membrane. Family IV. Chlamydobacteriace^e. — Forms of very varying stages of evolution, but all distinguished by a rigid sheath (Hiille) or covering, which surrounds the cells. The cells are united in branched or unbranched threads. Genus I. Streptothrix. — Cells united in simple, unbranched threads. Division in one direction of space only. Reproduction by non- motile coniclia. Genus II. Cladothrix. — Cells united or pseudoclichotomously branch- ing threads. Division in one direction of space only. Vegeta- tive multiplication by separation of entire branches. Repro- duction by swarming forms with polar flagella. Genus III. Crenothrix. — Cells united in unbranched threads, at first with division in one direction of space only. Later the cells divide in all three directions of space. The daughter cells be- come rounded and develop into reproductive cells. Genus TV. Phragmidiothrix. — Cells at first united in unbranched threads, dividing in three directions of space, thus forming a rope of cells. Later some of the cells may penetrate through the delicate sheath, and thus give rise to branches. Genus V. Thiotkrix. — Unbranched, non-motile threads, inclosed in fine sheaths. Division of cells in one direction only. Cells contain sulphur granules. Family V. Beggiatoace^e. — Cells united in sheathless threads. Division in one direction of space only. Motility by undulating membrane as in Oscillaria. Genus Beggiatoa. — Cells with sulphur granules. It will be seen in reviewing the classification just given that the sub- divisions are based upon questions of form, motility, and situation of flagella. "While these characteristics, so far as we know, are constant, there are, nevertheless, many instances in which types entirely similar in these respects must be differentiated. This can be done only by care- ful stud}' of staining reactions, finer structure, cultural characteristics, and biological activities. RELATION TO ENVIRONMENT— CLASSIFICATION 39 As a matter of fact, while the botanical classification of the bacteria offers almost insurmountable difficulties, actual identification is not so complicated a task as this would indicate. Identification, once roughly made on a morphological basis, is further carried on by the aid of cul- tural characteristics, such as the conditions favorable and unfavor- able for growth, appearance of growth on different media, and pigment formation, by biochemical reactions and by pathogenic properties. The bacteria occupy so important a place in agriculture, in medicine, and in hygiene, that it rarely becomes necessary for a worker in any par- ticular field to survey the entire group. The habitat of a large number of species is so well known that this consideration alone often gives a clew invaluable for actual identification. CHAPTER IV THE BIOLOGICAL ACTIVITIES OF BACTERIA While the bacteria pathogenic to man and animals largely usurp the attention of those interested in disease processes, this group of micro- organisms is after all but a small specialized off-shoot of the realm of bacteria, and, broadly speaking, actually of minor importance. Sur- veying the existing scheme of nature, as a whole, it is not an extrava- gant statement to say that without the bacterial processes which are constantly active in the reduction of complex organic substances to their simple compounds, the chemical interchange between the animal and vegetable kingdoms would fail, and all life on earth would of necessity cease. To understand the full significance of this, it is neces- sary to consider for a moment the method of the interchange of matter between the animal and vegetable kingdoms. All animals require for their sustenance Organic compounds. They are unable to build up the complex protoplasmic substances which form their body cells from chemical elements or from the simple inorganic salts. They are dependent for the manufacture of their food-stuffs, therefore, directly or indirectly, upon the synthetic or anabolic activi- ties of the green plants. These plants, by virtue of the chlorophyll contained within the cells of their leaves and stems, and under the influence of sunlight, possess the power of utilizing the carbon of the carbonic acid gas of the atmos- phere, and of combining it with water and the nitrogenous salts ab- sorbed by their roots, building up from these simple radicles the highly complex substances required for animal sustenance. These products of the synthetic activity of the green plants, then, are ingested by members of the animal kingdom, either directly, in the form of vegetable food, or indirectly, as animal matter. They are utilized in the complex laboratory of the animal body and are again broken down into simpler compounds, which leave the body as excreta and secreta. The excreta and secreta of animals, however, are, in a small part only, made up of substances simple enough to be directly utilized by plants. The dead bodies, moreover, of both animals and plants would 40 THE BIOLOGICAL ACTIVITIES OF BACTERIA 41 be of little further value as stores of matter unless new factors inter- vened to reduce them to that simple form in which they may again enter into the synthetic laboratory of the green plant. Agents for further cleavage of these compounds are required, and these are supplied by the varied activities of the bacteria. On the other hand, bacteria are also important in the process of synthesis. The main supply of nitrogen available for plant life is found in the elementary state in the atmosphere — a condition in which it can not be utilized as a raw product by the plant. This gap again is bridged by the bacteria found in the root bulbs of the leguminous plants — bacteria which possess the power of assimilating or aiding in the as- similation of atmospheric nitrogen and its preparation for further use by the plant itself. Another bacterial activity which may be classified as an anabolic process is the oxidation of the ammonia, released by decomposi- tion, into nitrites and nitrates. This is carried on by certain bacteria of the soil. These are to be treated of in greater detail in another section. There is a constant circulation, therefore, of nitrogen and carbon compounds, between the plant and the animal kingdoms, by virtue of an anabolic or constructive process in the one, and a katabolic or destruc- tive process in the other, rendering them mutually interdependent and indispensable. The circuit, however, is not by any means a closed one; there are important gaps, both in the process of cleavage and in that of synthesis, which, if left unbridged by the bacteria, would effectually arrest all life-activity of plants and eventually of animals. Far from being scourges, therefore, these minute microorganisms are paramount factors in the great cycle of living matter, supplying necessary links in the circulation of both nitrogenous and carbon com- pounds. KATABOLIC ACTIVITIES OF BACTERIA The katabolic activities of bacteria, then, consist in the fermentation of carbohydrates and in the cleavage of proteids and fats. Fermentation is carried out to a large extent by the yeasts, but also to no inconsiderable degree by bacteria. Proteid decomposition and the cleavage of fats are carried out almost exclusively by bacteria. For our knowledge of the fundamental laws underlying these phe- nomena of fermentation and proteid decomposition, we are indebted to the genius of Pasteur,1 who was the first to prove experimentally the 1 Pasteur, " Etude sur la bi're," Paris, 1876. 42 BIOLOGY AND TECHNIQUE exclusive and specific parts played by various microorganisms in these processes. While the observations and deductions made by Pasteur have not been greatly modified, a large store of information has been gained since his time, which has thrown additional light upon the chemical de- tails and the more exact manner of action of the factors involved. The actual work of cleavage in both fermentation and proteid cleav- age is carried out by substances known as enzymes or ferments, the nature of which we must further discuss before their manner of action can be fully comprehended. Bacterial Enzymes or Ferments. — A ferment or enzyme is a substance produced by a living cell, which brings about a chemical reaction with- out entering into the reaction itself. The enzyme itself is not bound to any of the end products and is not appreciably diminished in quantity after the reaction is over, although its activity may be finally inhibited by one or another of the new products. The action of bacterial enzymes is thus seen to be closely similar to that of the chemical agents technically spoken of as "katalyzers," represented chiefly by dilute acids. Thus, if an aqueous solution of saccharose is brought into contact with a dilute solution of sulphuric acid, the disaccharid is hydrolyzed and is decomposed into levulose and dextrose. Thus: C12H22011 + H2 O = C6H1206 + C6H1206 In contact with Dextrose Levulose dilute H2S04 During this process, which is known as "inversion," the concentration of the sulphuric acid remains entirely unchanged. While theoretically the changes brought about by enzymes and katalyzers are usually such as would occur spontaneously, the time for the spontaneous oc- currence would be, at ordinary temperatures, infinitely long. The defini- tion for enzymes and katalyzers is given by Ostwald, therefore, as " substances which hasten a chemical reaction without themselves taking part in it." Exactly the same result which is obtained by the use of dilute sulphuric acid is caused by the ferment "invertase" produced, for instance, by B. megatherium. Were a solution of saccharose sub- jected to heat, without katalyzer or ferment, a similar change would occur, but by the mediation of these substances the inversion is pro- duced without other chemical or physical reinforcement. This analogy between enzymes and katalyzing agents is very striking. Thus, as stated, both katalyezrs and enzymes bring about THE BIOLOGICAL ACTIVITIES OF BACTERIA 43 changes without themselves being used up in the process, both act without the aid of heat, and the reactions brought about by both have occasionally been shown to be reversible. While this last phe- nomenon has been variously shown for katalyzers, the process of re- versibility has been demonstrated for bacterial enzyme action only in isolated cases. Thus, it has been found that by the action of the yeast enzyme maltase upon concentrated dextrose solutions, a re-formation of maltose may occur. In both cases, moreover, the quantity of enzyme or katalyzer is infinitely small in proportion to the amount of material converted by their action. There is a close similarity, furthermore, between the bacterial en- zymes and the ferments produced by specialized cells of the higher ani- mals and plants. For instance, the action of the ptyalin of the saliva or of the diastase obtained from plants is entirely analogous to the starch- splitting action of the amylase produced by many bacteria. The action of all enzymes depends most intimately upon environ- mental conditions. For all of them the presence of moisture is essential. All of them depend for the development of their activity upon the exist- ence of a specifically suitable reaction. Strong acids or alkalies always inhibit, often destroy them. Temperatures of over 70° C. permanently destroy most enzymes, whereas freezing, while temporarily inhibiting their action, causes no permanent injury, so that upon thawing, their activity may be found almost unimpaired. Direct sunlight may injure, but rarely destroys, ferments. Against the weaker disinfectants in com- mon use, enzymes often show a higher resistance than do the bacteria which give rise to them. The optimum conditions for enzyme action, then, consist in the presence of moisture, the existence of a favorable reaction, weakly acid or alkaline, as the case may be, and a temperature ranging from 35°- 45° C.1 Proteolytic Enzymes. — In nature, the decomposition of dead animal and vegetable matter occurs only when the conditions are favorable for bacterial development. Thus, as is well known, freezing, sterilizing by heat, or the addition of disinfectants will prevent the rotting of organic material. In the laboratory, the presence of proteolytic enzymes is determined chiefly by the power of bacteria to liquefy gelatin, fibrin, or coagulated blood serum. These ferments are not always secretions from the bac- 1 Oppenheimer, " Die Fermente," etc. Leipzig, 1900. 44 BIOLOGY AND TECHNIQUE terial cell, but in some cases may be closely bound to the cell-body and separable only by extraction after death. In such cases they are spoken of as endoenzymes. Whenever they are true secretory products, however, they can be obtained separate from the microorganisms which form them by filtration through a Berkefelcl candle. From such filtrates they may, in some cases, be obtained in the dry state by precipitation with alcohol. When obtained in this way the precipitated enzyme is usually much more thermostable than when in solution, for while soluble enzymes in filtrates are usually destroyed by 70° C, and even less, the dried powder may occasionally withstand 140° C. for as long as ten minutes.1 Apart from the general conditions of temperature and moisture, the development of these enzymes seems to depend directly upon the presence of proteids in the culture media. The number of bacterial species which produce proteolytic enzymes is legion. Among those more com- monly met with are staphylococci, B. subtilis, B. proteus, B. faecalis liquefaciens, Spirillum cholerse asiaticse, B. anthracis, B. tetani, B. pyo- cyaneus, and a large number of others. The inability of any given micro- organism to liquefy gelatin or fibrin by no means entirely excludes the formation by it of proteolytic enzymes, since these ferments may often be active for one particular class of proteid only. In order to study the qualitative and quantitative powers of any given bacterial proteolyzing enzyme or protease, it is, of course, neces- sary to study these processes in pure culture in the test tube with media of known composition. In the refuse heap, in sewage, or in rotting excreta, the process is an extremely complicated one, for besides the bacteria which attack the proteid molecule itself, there are many other species supplementing these and each other, one species attacking the more or less complex end-products left by the action of the others. Exactly what the chemical reactions are which take place in these cleavages is not entirely clear. It is believed, however, that most of the cleavages are of an hydrolytic nature. In general, the action of the proteid-splitting ferments is comparable to that of the pancreatic ferment trypsin, and they are most often active in an alkaline environment. They differ, among themselves, chiefly in the form of proteid which they are competent to attack, and in the extent to which they are able to reduce it toward its simple radicles. A distinction is occasionally made between the terms putrefaction and decay, the former being used to refer to the decomposition taking 1 Fuhrmann, " Die Bakterienzyme/' p. 45. THE BIOLOGICAL ACTIVITIES OF BACTERIA 45 place under anaerobic conditions, that is, in the absence of oxygen, a process usually resulting in incomplete cleavage of the proteid medium; the latter being used to signify decompositions under aerobic conditions and leading to a more complete splitting, the end-products often being represented by such simple compounds as carbon dioxide, water, and ammonia. In general, the products of putrefaction are largely repre- sented by the amino-acids, leucin and tyrosin, fatty acids, mercaptan, indol, and skatol. The gases generated in such decomposition are largely made up of C02, hydrogen, NH4 and H2 S. The coincident presence, furthermore, of the carbohydrate-splitting bacteria and of denitrifying microorganisms renders the actual process of putrefaction a chaos of many activities in which the end-products and by-products are qualita- tively determinable only with much inexactitude, and which com- pletely defies any attempt at quantitative analysis. Ptomains. — There are certain products, however, resulting from the proteolytic action of bacterial enzymes upon proteids which claim more than a purely chemical interest because of their toxic action upon the animal organism, and their consequent importance as incitants of dis- ease. Pre-eminent among these are the ptomains. The word ptomain (from r,T(bij.al a dead body) is used to designate organic chemical compounds produced by the action of bacteria, which are basic in char- acter; that is, are able to combine with an acid to form a salt. They should be definitely distinguished from the so-called leucomains, a term employed to designate similar substances formed in the course of proteid metabolism within the animal body, and not bacterial in origin. Both in their basic characters and in their nitrogenous constitu- tion, the ptomains resemble the vegetable alkaloids, and for this reason are sometimes spoken of as " animal alkaloids." The ptomains must be sharply distinguished from the bacterial toxins, which are products of the bacterial growth irrespective of the medium in which they are grown, except in so far as this hinders or abets the development of the microorganisms. Thus, toxins may be developed by diphtheria organisms, for instance, in proteid-free media. As will be seen in a subsequent section, the true toxins are comparable to the enzymes themselves, rather than to their cleavage products, rep- resented in this instance by the ptomains. A great number of ptomains are chemically known. Many of these possess little or no toxicity. Others, however, like putrescin (tetramethylenediamin, C4H12N2) and cadaverin (C5H14N2) are very highly poisonous. It is to one or another of these ptomains that most 46 BIOLOGY AND TECHNIQUE cases of so-called meat poisoning (kreatoxismus) , cheese poisoning (tyrotoxismus) , or vegetable poisoning (sitotoxismus) are clue. In each individual case the variety of ptomain resulting from a bac- terial decomposition varies with the individual species of microorganism takmg part in the process and with the nature of the proteid upon which its development takes place. In breaking down animal excreta, the task of the bacteria is rather a simpler one than when dealing with the cadavers themselves, for here a part of the cleavage has already been carried out either by the destruc- tive processes accompanying metabolism, or by partial decomposition by bacteria begun within the digestive tract. This material outside of the body is further reduced by bacterial enzymes into still simpler sub- stances, the nitrogen usually being liberated in the form of ammonia. One example of such an ammoniacal fermentation may be found in the case of the urea fermentation by Micrococcus urea?, in which the cleavage of the urea takes place by hydrolysis according to the follow- ing formula: (XH2)2 CO + 2H2 O = C02 + 2NH3 + H2 O Similar ammoniacal fermentations are carried out, though perhaps according to less simple formulae, by a large number of microorganisms. Perhaps the most common species which possesses the power is the group represented by B. proteus vulgaris (Hauser). From what has been said it follows naturally that, so far, the decom- position of the proteid molecule from its complex structure to ammonia or simple ammonia compounds is an indispensably important function, not only for agriculture, but for the maintenance of all life processes. It is clear, on the other hand, that a further decomposition of ammonia compounds into forms too simple to be utilized by the green plants wotdd be a decidedly harmful activity. And yet this is brought about by the so-called denitrifying bacteria which will be considered in a subsequent section. Lab Enzymes. — There are a number of ferments produced by bacteria which, although affecting proteids, can not properly be classified with the proteolytic enzymes. These are the so-called coagulases or lab enzymes, which have the power of producing coagulation in liquid pro- teids. Just what the chemical process underlying this coagulation is, is not known. If Hammarsten's 1 conclusions as to the hydrolytic 1 Hammarsten, "Textbook of Physiol. Chemistry," Translation by Mandel. THE BIOLOGICAL ACTIVITIES OF BACTERIA 47 nature of the changes produced by them are true, these enzymes are brought into close relationship to the proteolyzers, although a coagula- tion can hardly be regarded as a true katabolic process. In milk where the lab-action becomes evident by precipitation of casein7 a strict dif- ferentiation must be made between this coagulation and that brought about by acids or alkalies. In the former case, casein is not only pre- cipitated and converted into paracasein, but is actually changed so that when redissolved it is no longer precipitated by lab.1 Coagulating enzymes for milk proteids, blood, and other proteid solutions are produced by a large variety of bacteria. They have been observed in cultures of the cholera vibrio, B. prodigiosus, B. pyocyaneus, and several others.2 The lab enzymes are easily destroyed by temperatures of 70° C. and over, and are very susceptible to excessive acidity or alkalinity. Fat-Splitting Enzymes {Lipase). — The fat-splitting powers of bac- teria have been less studied than some of the other bacterial func- tions and are correspondingly more obscure. It is known, nevertheless, that the process is due to an enzyme and that it is probably hydrolytic in nature. The following formula represents the simplest method in which some of the molds and bacteria produce cleavage of fats into glycerin and fatty acid. C3 H5 (Cn H1M 02)3 + 3H2 O = C3 H5 (OHs) + 3Cn H2n 02 Glycerin Fatty acid Some of the bacteria endowed with the power of producing lipase are the spirillum of cholera, B. fluorescens liquefaciens, B. prodigiosus, B. pyocyaneus, Staphylococcus pyogenes aureus, and some members of the streptothrix family. The methods of investigating this function of bacteria, originated by Ejkmann, 3 consists in covering the bottom of a Petri dish with tallow and pouring over this a thin layer of agar. Upon this, the bacteria are planted. Any diffusion of lipase from the bacterial colonies becomes evident by a formation of white, opaque spots in the tallow. Carriere4 was able to demonstrate a fat-splitting ferment for the tubercle bacillus. Apart from the importance of these enzymes in nature for the destruction of fats, they are industrially important be- 1 Oppenheimer, "Die Fermente u. ihre Wirkung/' Leipzig, 1903. 2 Torini, Atti dei laborat. d. sanita, Rome, 1890. 3 Ejkmann, Cent. f. Bakt., I, xxix, 1901. 4 Carriere, Comptes rend, de la soc. de biol., 53, 1901. 48 BIOLOGY AND TECHNIQUE cause of their action in rendering butter, milk, tallow, and allied prod- ucts rancid, and are medically of interest for their action upon fats in the intestinal canal. Enzymes of Fermentation (The Cleavage of Carbohydrates by Bacteria). — The power to assimilate carbon dioxide from the atmosphere is possessed only by the green plants and some of the colored algae, and the sulphur or Thiobacteria. All other living beings are thus dependent for their supply of carbon upon the synthetic activities carried on by these plants to the same degree in which they are de- pendent upon similar processes for their nitrogen supply. The return of this carbon to the atmosphere is, of course, brought about to a large ex- tent by the respiratory processes of the higher animals. The carbon, which, together with nitrogen, forms a part of proteid combinations, is freed, as we have seen in a previous section, by the processes of proteid cleavage. That, however, which is inclosed in the carbohydrate mole- cule, is set free by the action of yeasts, molds, or bacteria, by an enzy- matic process similar in every respect to that described above for the process of proteid cleavage. Fermentation. — The power of carbohydrate cleavage is possessed by a large number of the yeasts and bacteria. The process, as has been indicated, is of great importance in the cycle of carbon compounds for the return of carbon to its simplest forms, and is. furthermore, as will be seen in a later section, of great utility in the industries. In each case the power to split a particular carbohydrate is a more or less specific characteristic of a given species of microorganism, and for this reason has been extensively used as a method for the biological differen- tiation of bacteria. In the course of much careful work upon this question it has been ascertained that the specific carbohydrate-splitting powers of any given species are constant and unchanged through many generations of artificial cultivation. Thus, differentiation of the Gram-negative bacteria, the members of the pneumococcus-streptococ- cus group, and the diphtheria group, can now largely be made by a study of their sugar fermentations. In most of these cases, as far as we know, the cleavage is produced by a process of hydrolysis. A convenient nomenclature which has been adopted for the designation of these ferments is that which employs the name of the converted carbohydrate adding the suffix " ase " to indicate the enzyme. There are thus ferments known as amylase, cellulase, lac- tase, etc. Amylase (Diastase or Amylolytic Ferment). — Amylases or starch- THE BIOLOGICAL ACTIVITIES OF BACTERIA 49 splitting enzymes are formed by many plants (malt) and by animal organs (pancreas, saliva, liver). Among microorganisms amylase is produced by many of the streptothrix group, by the spirilla of Asiatic cholera and of Finkler-Prior, by B. anthracis, and many other bacteria. A large number of the bacteria found in the soil, furthermore, have been shown to produce amylases. By cultivating bacteria upon starch- agar plates, amylase can be readily demonstrated by a clearing of the medium immediately surrounding the colonies.1 Since, of course, there are several varieties of starches, it follows that the exact chemical action of amylase differs in individual cases. The determination of the structural disintegration of starch by these fer- ments is fraught with much difficulty, owing to the polymeric constitu- tion of the starches. Primarily, however, a cleavage takes place into a disaccharid such as maltose (hexobiose) , and the non-reducing sugars and dextrin. Beyond this point, however, the further cleavages are subject to much variation and are not entirely clear. The dextrins upon further reduction yield eventually dextrose. Cellulase. — Cellulose is fermented by a limited number of bacteria, most of them anaerobes. The chemical process by which this takes place is but poorly understood.2 Gelase. — An agar-splitting ferment has been found by Gran.3 Invertase. — The enzymes which hydrolytically cause cleavage of saccharose into dextrose and levulose are numerous. The chemical process takes place according to the following formula: C12 H22 On + H2 0 = C6 H12 06 + C6 H1206 Saccharose Dextrose Levulose Invertase is produced by many of the yeasts. It is one of the most common of the enzymes produced by bacteria, and has been found in cultures of B. megatherium, B. subtilis, pneumococcus, some strepto- cocci, B. coli, and many others. Invertase is usually very susceptible to heat, being destroyed by temperatures of 70° C. and over. A slightly acid reaction of media abets the inverting action of these enzymes. Strong acids and alkalies inhibit them. Inverting enzymes may be precipitated out of solution by alcohol. Antiseptics even in weak con- centrations will inhibit their action. 1 Ejkmann, Cent. f. Bakt., xxix, 1901, and xxxv, 1904. 2 Omelianski, Lafar's " Handb. d. techn. Mykologie," Bd. iii, Chap. 9. 3 Gran, Bergens Museum Aarbog, 1902, Hft. I. 4 50 BIOLOGY AXD TECHNIQUE Lactase. — Lactose-splitting ferments are extremely common both among bacteria and among the yeasts. The process is here again a hydrolytic cleavage resulting in the formation of the monosaccharids as dextrose and galactose. Maltose. — A. maltose-splitting ferment has also been found in the cultures of many bacteria, leading to the formation of dextrose. Lactic Acid Fermentation. — Lactic acid (oxyproprionic acid, C3H6 03) is one of the most common substances to appear among the prod- ucts of bacterial activity, both in media containing carbohydrates and in those consisting entirely of albuminous substances. In most of these cases, the lactic acid is formed merely as a by-product accom- panying many other more complicated chemical cleavages. In some instances, however, lactic acid is produced from carbohydrates, both clisaccharids and monosaccharids. as an almost pure product due to a specific bio-chemicai^j)>^ss:/ir?h£>)e^ions taking place in this phenom- enon may be brj^^^xjDre^e^Uaeeorcliri^to the following formulae: 4C3 H6 03 or Lactic acid 2C,H,0, Dextrose Lactic acid In the same way lactic acid may be produced by bacteria from levu- lose. Examples of lactic acid formation are furnished by the streptococcus lacticus, and B. lactis aerogenes. In the case of the former, the fer- mentation may indeed proceed by the simple chemical process indi- cated in the formula?, since the action of the bacillus is entirely unac- companied by the evolution of gas. Numerous other bacteria produce large amounts of lactic acid from lactose, possibly by chemical processes less simply formulated. Among these are bacilli of the colon group, B. prodigiosus, B. proteus vulgaris, and many others. Although lactic acid is usually the chief product in the bacterial fermentation of the simpler carbohydrates, acetic, formic, and butyric acids may often be found as by-products in variable amounts.1 Oxydases (Oxydizing Enzymes). — The most common example of oxidation by means of bacterial ferments is the production of acetic acid 1 Buchner und Meisenheimer, Ber. d Deut. chem. Gesellsch., xxxvi, 1903. THE BIOLOGICAL ACTIVITIES OF BACTERIA 51 from weak solutions of ethyl alcohol. This process, which is the basis of vinegar production, is universally carried out by bacterial ferments. While possessed to some extent by a considerable number of microorgan- isms, acetic acid formation is a function pre-eminently of the bacterial groups described by Hansen, including "Bacterium aceti" and "Bac- terium pasteurianum." To these two original groups a number of others have since been added. The organisms are short, plump bacilli, with a tendency to chain- formation, and occasionally showing characteristically swollen centers and many irregular involution forms. In the production of vinegar, as generally practiced by the farmer with cider or wine, these bacteria accumulate on the surface of the fluid as a pellicle or scum which is popularly known as the "mother of vinegar. " Destruction of these bacteria by disinfectants or by sterilization with heat promptly arrests the process of vinegar formation. Chemically, the conversion of the alcohol consists in a double oxidation through ethyl aldehyde into acetic acid as shown in the following formulae : 1. C2H5 (OH) + O = CH3 (COH) Alcohol Ethyl aldehyde 2. CH3 (COH) + O = CH3 (COOH) Acetic acid Alcoholic Fermentation (Zymase). — The formation of alcohol as an end product of fermentation is of great importance in a number of the industries, primarily in the production of wine and beer. While accom- plished by a number of bacteria, this form of fermentation is carried out chiefly by the yeasts. Expressed in formulae the simplest varieties of alcoholic fermenta- tion, from mono- and disaccharids, may be represented as follows: C6H1206 = 2C2H5 (OH) + 2 C02 Dextrose Ethyl alcohol or C12H22On + H20 = 4C2H5(OH) + 4C02 Saccharose Ethyl alcohol In all cases the process may not be so simple as indicated by the equa- tions, since by-products, such as higher alcohols, glycerin, succinic and acetic acids, may often be found in small traces among the end- products of such fermentations. The conditions which favor alcoholic 52 BIOLOGY AND TECHNIQUE fermentation by the yeasts are extremely important, since, upon obser- servance of these, depends much of the uniformity of result which is so desirable in the industries mentioned above. The optimum concentra- tion of sugar for the production of the highest quantity of alcohol is at or about 25 per cent. The temperature favoring the process ranges about 30° C. Under such conditions fermentation may continue until the alcohol forms almost a 20-per-cent solution. Most of the fermenta- tions important in the wine, beer, and spirit industries, take place under anaerobic conditions, since the carbon dioxide which is formed soon shuts out any excess of air. In the industrial employment of yeasts for fermentative purposes, it is necessary to work with specific strains, and in scientifically conducted vineyards, breweries, and distilleries the study and pure cultivation of the yeasts form no unimportant part of the work. Certain races of yeasts are more uniform in their fermentative powers than others, and the by- products formed by some races differ sufficiently from those of other races to cause material differences in the resulting substances. In the wine industries, the yeasts differ much from one another according to climatic and other environmental conditions. In vineyards, natural inoculation of the grapes' occurs by transportation of the yeast from the soil to the surface of the grapes by wasps, bees, or other insects, through whose alimentary canals the microorganisms pass uninjured. In the autumn the yeast is returned to the soil by falling berries and remains alive in the upper layers of the ground throughout the winter months. In actual practice this natural yeast inoculation is not de- pended upon, but pure cultures of artificially cultivated yeasts are employed for inoculation. In some of the wine-growing countries these are supplied by special government experiment stations. Denitrifying Bacteria. — Nitrogen is most readily absorbed by plants in the form of nitrates. These are furnished to the soil chiefly by the proteid decomposition induced by the proteolytic bacterial enzymes. It is self-evident, therefore, that any cleavage which reduces nitrog- enous matter beyond the stage of nitrates, to nitrites and ammonia, detracts from the value of the nitrogen as a food stuff for plants, and the eventual setting free of nitrogen in the elementary state ren- ders it entirely valueless for any but the leguminous plants. Nevertheless, this process of nitrogen waste or denitrification is constantly going on in nature. In the course of ordinary decomposition, there is a constant reduction of nitrogenous matter to nitrites and salts of ammonia, actively taken part in by a host of bacteria, as many as THE BIOLOGICAL ACTIVITIES OF BACTERIA 53 S5 out of 109 investigated by Maassen 1 being found to possess this power. This, however, is not nearly so harmful a source of nitrogen waste as the process technically spoken of as true denitrification, in which nitrates are reduced, through nitric and nitrous oxides, to elementary nitrogen. This phenomenon, more widely spread among bacteria than at first believed, depends essentially upon simple oxygen extraction from the nitrates by the bacteria, and for this reason goes on most actively when the supply of atmospheric oxygen is low. The first bacteria described as possessing this power of denitrification were the so-called B. denitri- ficans I and II, the first an obligatory anaerobe, the other a facultative aerobe. Since then numerous other bacteria, among them B. coli and B. pyocyaneus, have been shown to exhibit similar activities. It is important agriculturally, therefore, to know that many species which are able to utilize atmospheric oxygen when supplied with it, will get their oxygen by the reduction of nitrates and nitrites when free oxygen is withheld. It is thus clear that a loss of nitrogen is much more apt to proceed rapidly in manure heaps which are piled high and poorly aerated. There are other factors, however, in regard to the physi- ology of these microorganisms, which must be considered for practical purposes. In order that these bacteria may develop their denitrifying powers to the best advantage, it is necessary to supply them with some carbon compound which is easily absorbed by them. This, in decomposing material, is furnished by the products of the carbohydrate cleavage going on side by side with the proteolytic processes. It is still more or less an open question whether the facilitation of denitrification brought about in manure heaps by the presence of hay and straw is due to the carbon furnished by these materials, or whether it is due to the fact that bacilli of this group are apt to adhere to the straw which acts in that case as a means of inoculation. The actual danger of nitrogen depletion of the soil by denitrifying processes is probably much less threatening than was formerly supposed ; for, in the first place, the conditions for complete denitrification are much more perfect in the experiment than they ever can be in nature, and the nitrifying processes going on side by side with denitrification make up for much of the loss sustained. 1 Maassen, Arb. a. d. kais. Gesundheitsamt, 1, xxviii, 1901. 54 BIOLOGY AND TECHNIQUE ANABOLIC OR SYNTHETIC ACTIVITIES OF BACTERIA Nitrogen Fixation by Bacteria. — The constant withdrawal of nitroge- nous substances from the soil by innumerable plants would soon lead to total depletion were it not for certain forces continually at work re- plenishing the supply out of the large store of free nitrogen in the atmos- phere. This important function of returning nitrogen to the soil in suitable form for consumption by the plants is performed largely by bacteria. It is well known that specimens of agricultural soil when allowed to stand for any length of time without further interference will increase in nitrogenous content, but that similar specimens, if sterilized, will show no such increase.1 The obvious conclusion to be drawn from this phenomenon is that some living factor in the unsterilized soil has aided in increasing the nitrogen supply. Light was thrown upon this problem when Winogradsky,2 in 1893, discovered a microorganism in soil which possessed the power of assimilating large quantities of nitrogen from the air. This bacterium, which he named " Clostridium Pasteurianum," is an obligatory anaerobe which in nature always occurs in symbiosis with two other facultatively anaerobic microorganisms. In sym- biosis with these, it can be cultivated under aerobic conditions and thus grows readily in the upper well-aerated layers of the soil. Although, until now, no other bacteria with equally well-developed nitrogen-fixing powers have been discovered, yet it is more than likely that Clostridium Pasteurianum is not the only microorganism endowed with this function. In fact, Penicillium glaucum and Aspergillus niger, two molds, and two other bacteria described by Winogradsky, have been shown to possess this power slightly, but in an incomparably less marked degree than Clostridium Pasteurianum.3 According to the calculations of Sachse,4 unsterilized soil may, under experimental conditions, gain as much as 25 milligrams of nitrogen in a season, a statement which permits the calculation of a gain of twelve kilograms of nitrogen per acre annually.5 It is very unlikely, however, that such gains actually occur in nature, where nitrogen-fixation and nitrogen-loss usually occur side by side. 1 Berthelot, Compt. rend, de la soc. de biol., cxvi, 1893. 2 Winogradsky, Compt. rend, de la soc. de biol., cxvi, 1893, ibid., t. cxviii, 1894. 3 Tacke, Landwirtsch. Jahresber., xviii, 1889. * Sachse, "Agr. Chem.," 1883. 6 Pfeffer, Pflugers Physiologie, p. 395. THE BIOLOGICAL ACTIVITIES OF BACTERIA 55 Agriculturally of even greater importance than the free nitrogen- fixing bacteria of the soil are the bacteria found in the root tubercles of a class of plants known as "leguminosse." It has long been known that this class of plants, including clover, peas, beans, vetch, etc., not only does not withdraw nitrogen from the soil, but rather tends to enrich it. Upon this knowledge has depended the well-known method of alternation of crops employed by farmers the world over. The actual reason for the beneficial influence of the leguminosse, however, was not known until 1887, when Hellriegel and Wilfarth 1 succeeded in demonstrating that the nitrogen-accumulation was directly related to the root tubercles of the plants, and to the bacteria contained within them. These tubercles, which are extremely numerous — as many as a thousand sometimes occurring upon one and the same plant — are formed by the infection of the roots with bacteria which probably enter through the delicate root-hairs. They vary in size, are usually situated near the main root-stem, and, in appearance, are not unlike fungus growths. Their development is in many respects comparable to the develop- ment of inflammatory granulations in animals after infection, inas- much as the formation of the tubercle is largely due to a reactionary hyperplasia of the plant tissues themselves. They appear upon the seedlings within the first few weeks of their growth as small pink nodules, and enlarge rapidly as the plant grows. At the same time, later in the season, when the plants bear fruit, the root tubercles begin to shrink and crack. When the crops are harvested, the tubercles with the root remain, rot in the ground, and re-infect the soil. Histologically the tubercles are seen to consist of large root cells which are densely crowded with microorganisms. The microorganism itself, " Bacillus radicicola," was first observed within the tubercles by Woronin 2 in 1866. The bacilli are large, slender, and actively motile during the early development of the tubercles, but in the later stages assume a number of characteristic involution forms, commonly spoken of as "bacteroids." They become swollen, T and Y shaped, or branching and threadlike. Their isolation from the root tubercles usually presents little difficulty, since they grow readily upon gelatin and agar under strictly aerobic conditions. On the artificial media the bacillary form is usually well retained, involution forms appearing only upon old cultures. 1 Hellriegel und Wilfarth, Cent. f. Bakt., 18S7. 2 Woronin, Bot. Zeit., xxiv, 1866. 56 BIOLOGY AND TECHNIQUE The classical experiments of Hellriegel and Wilfarth conclusively demonstrated the important relation of these tubercle-bacteria to nitro- gen assimilation by the leguminosse. These observers cultivated various members of this group of plants upon nitrogen-free soil — sand — and prevented the formation of root tubercles in some, by sterilization of the sand, while in others they encouraged tubercle formation by inoculation. An example of their results may be given as follows : * Lupinus luteus was cultivated upon sterilized sand. Some of the pots were inoculated with B. radicicola, others were kept sterile. Com- parative analyses were made of the plants grown in the different pots with the following striking result: Harvested _, , , , dry weight Root tubercles ^^ present j (b) 33.755 ., .-.. , . I (c) 0.989 No root tubercles j ( Carbolic acid < ' 1 in 30 1 in 40 1 in 100 1 in 110 growth growth growth growth growth growth growth In the above table, formalin 1 in 30 killed in the same time as carbolic acid 1 in 110. Thus the carbolic-acid coefficient of formalin in this test = yW = .27. The Rideal-Walker method has been much used and is recommended by many workers.4 Its value, however, is doubtful. Determination of Antiseptic Values. — The antiseptic or in- hibitive strength of a chemical substance, sometimes spoken of as the " coefficient of inhibition," is determined by adding to definite quantities of a given culture medium, graded percent- 1 Kronig und Paul, loc. cit. 2 Rideal and Walker, Jour, of the Sanitary Tns. London, xxiv. 3 Simpson and Hewlett, Lancet, ii, 1904. * Sommerville, Brit. Med. Jour., 1904. THE DESTRUCTION OF BACTERIA 81 INHIBITION STRENGTHS OF VARIOUS ANTISEPTICS. Adapted from Flugge, Leipzig, 1902. Acids Sulphuric. . . . Hydrochloric Sulphurous Arsenous. . Boric Alkalies Potass, hydrox . Amnion, hydrox. Calcium hydrox. Salts Copper sulphate Ferric sulphate Mercuric chlorid . Silver nitrate . . . Potass, perman. Halogens and Compounds Chlorin Bromin Iodin Potass, iodid Sodium chlor Anthrax Bacilli. 1 : 3,000 1 : 3,000 1 :800 1 :700 1 :700 1 : 100,000 1 : 60,000 1 : 1,000 1 : 1,500 1 : 1,500 1 : 5,000 Other Bacteria. Choi. spir. 1 : 6,000 B. diph. 1 : 3,000 B. mallei 1 : 700 B. typh. 1 : 500 Choi. spir. 1 : 1,000 B. diphth. 1 : 600 Choi. spir. 1 : 400 B. typh. 1 : 400 Choi. spir. 1 : 500 B. typh. 1 : 500 Choi. spir. 1 : 1,100 B.typh. 1 : 1,100 B. typhosus 1 : 60,000 Choi. spir. B. typhosus 1 : 50,000 Organic Compounds Ethyl alcohol Acetic and oxalic acids Carbolic acid Benzoic acid . . Salicylic acid . . Formalin (4% hyde) formalde- Camphor Thymol Oil mentha pip Oil of terebinth Peroxide of hydrogen 6 1 :60 1: 12 1 :800 1 : 1,000 1 : 1,500 1 : 1,000 1 : 10,000 1 : 3,000 1 : 8,000 B. diph. 1 : 500 B. typh. 1 : 400 Choi. spir. 1 : 600 Choi. spir. 1 : 20,000 Staphylo. 1 : 5,000 Putrefactive Bac- teria in Bouillon. 1 : 6,000 1 :200 1 :100 1 : 1,000 1:90 1 : 20,000 1 :500 1 : 4,000 1 : 2,000 1 : 5,000 1:7 1:10 1 :400 1 : 1,000 1 : 3,500 1 : 2,000 82 BIOLOGY AND TECHNIQUE ages of the chemical substance which is being investigated and planting in these mixtures equal quantities of the bacteria in question. The medium used for the tests may be nutrient broth or melted gelatin or agar. If broth is used, growth is estimated by turbidity of the medium and by morphological examination; if the agar or gelatin are employed plates may be poured and actual growth observed. Thus, in the case of carbolic acid, for instance, a five per cent or ten per cent solution of the antiseptic is prepared and this is added in gradually decreasing quantities to tubes of the medium in the follow- ing way: Tube 1 contains 5% carbolic 2 c.c. + broth 8 c.c. = 1 : 100 carbolic acid. " 2 " 5 " 1 c.c. + broth 9 c.c. = 1 : 200 " "3 " 5 " .5 c.c. + broth 9.5 c.c. = 1 : 400 " " "4 " 5 " 7 c.c. + broth 9.8 c.c. = 1 : 1.000 " "5 " 5 " .lcc. + broth 9.9 c.c. = 1 : 5,000 " To each of these tubes a definite quantity of the bacteria is added either by means of a standard loopful of a fresh agar culture, or better by a measured volume of an even emulsion in sterile salt solution. In prac- tice, it is often regarded as sufficiently accurate to add the graded quan- tities of the disinfectant to a constant 10 c.c. of the medium. This, however, is naturally not so accurate as the method given above. The inoculated tubes are then incubated at a temperature corresponding to the optimum growth temperature for the microorganism in question. The tubes are examined for growth from day to day. From the tubes containing the higher dilutions, in which no growth is visible, trans- plants are made to determine the presence of living bacteria. This serves to distinguish between simple inhibition or antisepsis and bac- terial death or disinfection. Determination of Disinfectant Values. — The determination of the bactericidal or disinfectant value of a chemical substance may be carried out by a variety of methods. Koch,1 using anthrax spores as the indicator, dried the spores upon previously sterilized threads of silk. These were exposed to the disinfectant at a definite temper- ature for varying times, the disinfectant was then removed by wash- ing in sterile water, and the threads planted upon gelatin or blood- serum media and incubated. A serious objection to this method was pointed out by Geppert,2 who maintains that it is impossible by simple 1 Koch, Arb. a, d. kais. Gesimdheitsamt. 1, 1881. 2 Geppert, Berl. klin. Woch., xxvi, 1889. THE DESTRUCTION OF BACTERIA 83 BACTERICIDAL STRENGTHS OF COMMON DISINFECTANTS. Adapted from Flugge, Leipzig, 1902. Acids Sulphuric . . . Hydrochloric Sulphurous . Sulphurous Boric Alkalies Potass, hydrox. . Amnion, hydrox. Calcium Salts Copper sulphate Mercuric chlor. Silver nitrate . . Potass, permang. " Calc. chlorid " Halogens and Com- pounds Chlorin Tri chlorid of iodin . . . Organic Compounds Ethyl alcohol Acetic and oxalic acids Carbolic acid Lysol Creolin Salicylic acid Formalin (40% for- maldehyde) Peroxide of hydrogen . Strepto- and Staphylo- 5 Minutes. 1:10 1 :10 1:5 1 : 10,000 to 1,000 1 : 1,000 1 :200 1 per cent. 1 :200 70%-15 minutes 1 :60 1 :300 1 : 1,000 1 :10 Cone. Anthrax and Typhoid Bacilli. Cholera Spirillum. 5 Minutes. 1:100 1 :100 1 :300 1 :300 1 : 1,000 1 : 2,000 1 :500 .1 per cent. 1 : 1,000 70%-10miris Cholera 1:200 Typh. 1 : 50 1 :300 1 :100 1 :20 1 :200 2-24 Hours. 1 : 1,500 1 : 1,500 (Typhoid 1 : 700) 1 : 300 (Gas 10 vol. %) 1 :30 1 : 10,000 1 : 4,000 1 : 2-300 1 .300 1 : 3,000 1 : 1,000 1 : 500 Anthrax Spores. 1 : 50 in 10 days 1 : 50 in 10 days Cone. sol. incomplete disinfection 1 : 20 (5 days) 1:2,000 (26 hours) 1 : 20 (1 day) 1 : 20 (1 hour) 2 per cent (in 1 hr.) 1 : 1,000 (in 12 hrs.) Alcol. 50% for 4 months without killing spores. Koch.1 1 : 20 (4-45 days) (at 40° in 3 hrs.) (10% in 5 hrs.) 1 :20 (in 6 hrs.) 1 : 100 (in 1 hr.) 3 : 100 (in 1 hr.) 1 Koch, Arb. a. d. kais. Gesundheitsamt, 1, 1881. 84 BIOLOGY AND TECHNIQUE washing to remove completely the disinfectant in which the thread has been soaked. This author suggests that, whenever possible, the disin- fectant, at the end of the time of exposure, should be removed by chem- ical means. In the case of bichloride of mercury Geppert exposes emul- sions of the bacteria to aqueous solutions of the disinfectant, and at the end of exposure precipitates out the bichloride of mercury with ammo- nium sulphid. In the case of a large number of disinfectants, however, this is not possible, and, when the thread method is used, removal of the chemical agent by washing must be practiced. Complete removal of the disinfectant is especially desirable, since spores previously exposed to these substances are more easily inhibited by dilute solutions than are normal spores. To do away with the source of error introduced by the difficulty of washing a disinfectant out of a silk thread, the spores may be dried upon the end of a glass rod, which, after exposure, is washed in distilled water or salt solution and then immersed in sterile broth.1 A simple method often practiced is that in which graded percentages of the disinfectant are added to the menstruum, such as blood, blood serum, broth, etc., in which the disinfectant is to be tested, and equal quantities of bacteria thoroughly emulsified in water or salt solution are added. Such an emulsion may be obtained by filtering through cotton, glass wool, or filter paper. Loopfuls of these mixtures are then planted from time to time in agar or gelatin plates upon which colony counts can afterward be made. In all such tests it is of primary importance to remember that the presence of organic fluids, blood serum, mucus, etc., considerably alters the efficiency of germicides, and whenever practical deductions are made, experimental imitation of the actual conditions should be at- tempted. Practical Disinfection. — In practical disinfection with chemical agents, the disinfectant must be chosen to a certain extent in accordance with the material to be disinfected. Sputum is a substance extremely difficult to disinfect because the bacteria present are surrounded by dense envelopes of mucus, through which disinfectants do not easily diffuse. For sputum disinfection, es- pecially tuberculous sputum, carbolic acid — 5 per cent solution — or any of the phenol derivatives in similar concentration, may be used. Bichloride of mercury is of very little use in sputum disinfection be- 1 Hill, Rep. Am. Pub. Health Assn., xxiv, 1898. THE DESTRUCTION OF BACTERIA 85 cause of the dense protective layers of albuminated mercury which form about the microorganisms. Sputum should always be received into cups containing the disinfectant, and contaminated handkerchiefs should be soaked in the solution. Feces from typhoid, dysentery, and cholera patients should be steril- ized by burning, if possible, or by thoroughly mixing with large quan- tities of boiling water; but if chemical disinfectants are to be used, five per cent carbolic acid or dilute formalin are convenient. Milk of lime and chlorid of lime are useful, though somewhat inconvenient. Bichlo- rid of merurcy is of little value in this case for the same reason that it is valueless in sputum disinfection. In all cases of feces disinfection it is extremely important that the chemical agent should be added in large quantities and thoroughly mixed with the discharge. Linen, napkins, and other cloth materials which have come into con- tact with patients should be soaked for one or two hours in one per cent formaldehyde, five per cent carbolic acid, or 1 : 5,000 or 1 : 10,000 bichlorid of mercury. After this, the material may be taken from the sick-room and boiled. It is extremely important that cloth material should never be removed from the sick-room in a dry state. Urine may be easily disinfected by the addition in proper con- centration of any of the disinfectants named above. The methods for sterilization of surgical instruments and the prepara- tion of the skin of the patient for operation are subject to so many local variations that it is hardly within the scope of a textbook on bacteriology to mention them. Metal instruments are usually sterilized by boiling in soda solution and may be subsequently immersed in five per cent car- bolic acid solution. Catgut may be sterilized by boiling in alcohol or by subjecting it to temperatures of 140° C. and over, for several hours in oils (albolin). The disinfection of the hands is also a matter of much variation. Two methods which are frequently quoted are that of Welch and that of Fiirbringer. In Welch's method the hands are brushed with green soap in water as hot as it can be borne for at least five minutes. They are then rinsed and immersed for two minutes in a warm saturated solution of perman- ganate of potash in which they are rubbed with a sponge or sterile cotton. They are then transferred to a saturated solution of oxalic acid, until the red color has entirely disappeared. Following this, they are rinsed in sterile water and then immersed in a 1 : 500 bichlorid of mercury solution for ome to two minutes. 86 BIOLOGY AND TECHNIQUE According to Fi'irbringer's method, the finger nails are carefully cleaned with an orange-wood stick or nail file; the hands are then thor- oughly brushed with a nail brush in green soap and hot water for five minutes. Following this they are immersed in 60 per cent alcohol for one minute, then in 3 per cent carbolic acid solution for one minute; after which they are rinsed in sterile water and dried. Rooms, closets, and other closed spaces which are contaminated, must be disinfected largely by gaseous disinfectants. After such disinfection in the case of cellars, privies, and other places where feasible, walls and ceilings should be whitewashed. Gaseous Disinfectants for Purposes of Fumigation. — There are a large number of gaseous agents which are harmful to bacteria. Only a few, however, are of sufficient bactericidal strength to be of practical im- portance. Oxygen, especially in the nascent state, may exert distinct bacteri- cidal action upon some bacteria. That strictly anaerobic strains are inhibited by its presence, has already been mentioned. Ozone was shown by Ransome and Fullerton x to exert considerable germicidal power when passed through a liquid medium in which bac- teria were suspended, but was absolutely without activity when em- ployed in the dry state. Chlorin because of its powerful germicidal action was once looked upon with favor, but has been found quite inadequate from a practical point of view because of its injurious action upon materials, and its irregularity of action. Chlorin, too, is but weakly efficient unless in the presence of moisture.2 Sulphur dioxide or sulphurous anhydrid (S02), which was formerly much used for room disinfection, is no longer regarded as uniformly ef- ficient for general use. The gas is produced by burning ordinary roll sulphur. In order that it shall be at all effective, water should be vaporized at the same time, since the disinfectant action is dependent upon the formation of suphurous acid. The concentration of the gas should be at least 8 per cent of the volume of air in the room. For this purpose about three pounds of sulphur should be burned for every thousand cubic feet of space. It should be allowed to act for not less than twenty-four hours. The researches both of Wolff hugel 3 and of 1 Ransome and Fullerton, Rep. Public Health, July, 1901. 2 Fischer und Proskauer, Mitt. a. d. kais. Gesundheitsamt, x, 11, 1882. 3 Wolffhiigel, Mitt. a. d. kais. Gesundheitsamt, i, 1881. THE DESTRUCTION OF BACTERIA 87 Koch 1 have shown that the gas is not sufficient for the destruction of spores, under the best circumstances, probably because of its lack of pen- etrating power. Park 2 believes that sulphur dioxide used in quantities of four pounds of sulphur to 1,000 cubic feet is of practical value for fumigating purposes in cases of diphtheria and the exanthemata. Of all known gaseous disinfectants by far the most reliable is form- aldehyde. There are many methods of generating this gas, and many devices for its practical use have been introduced. In all cases where formaldehyde fumigation is intended, clothing, bed-linen, and fabrics should be spread out, cupboards and drawers freely opened. The cracks of windows and doors should be hermetically sealed with paper strips or by calking with cotton. The generation of gas may be carried out in an apparatus left within the room or it may be generated outside and the gas introduced by a tube passed within the keyhole. In all cases moisture should be provided for, either in the generating appa- ratus or by a separate boiler. The first of the methods of generating formaldehyde for fumigation purposes was that of Trillat,3 who devised a lamp in which formaldehyde was produced by the incomplete combustion of methyl alcohol. This method has proved expensive because of the complete oxidation of a large percentage of the alcohol. Direct evaporation of formaldehyde from formalin solutions has been the principle underlying some other devices. If such evaporation is attempted from an open vessel, however, polymerization of formal- dehyde to the solid trioxymethylene occurs. To prevent this, Trillat4 and others have constructed special autoclaves in which 20 per cent of calcium chloride is added to formalin which is then vaporized under pressure. By this means polymerization is practically eliminated. The Trillat autoclave, as well as others constructed on the same principle, consists of a strong copper chamber of a capacity of about a gallon, fitted with a cover which can be tightly screwed into place. The cover is perforated by an outlet vent, a pressure gauge, and a thermometer. The whole apparatus is adjusted upon a stand and set over a kerosene lamp. Into the chamber is put about one-half to three- quarters its capacity of 40 per cent formaldehyde (commercial formalin) 1 Koch, Mitt. a. d. kais. Gesundheitsamt, i, 1881. 2 Park, "Pathogen. Bact.," N. Y.. 1908. 3 Trillat, Compt. rend, de Facad. des sc., 1892. 4 Trillat, Compt. rend, de 1'acad. des sc, 1896. 88 BIOLOGY AND TECHNIQUE containing 15-20 per cent calcium chloride. The solution used should be free from methyl alcohol, since this leads to the formation, with formaldehyde, of methylal, which is absolutely without germicidal action. The name is lighted and the exit tube kept closed until the pressure gauge indicates a pressure of three atmospheres. Then the vapor is allowed to escape through the tube. For a room of about 3,000 Fig. 12. — Lentz Formalin Apparatus. cubic feet Trillat advises the continuance of the gas flow for about an hour. The method is not uniformly reliable. A method which has found much favor is that in which glycerin — usually in a concentration of 10 per cent — is added to formalin. Ac- cording to Schlossmann x the presence of glycerin hinders polymeriza- tion. An apparatus in which this mixture is conveniently utilized is 1 Schlossmann, Munch, med. Woch., 45, 1898. THE DESTRUCTION OF BACTERIA 89 that of Lentz (see Fig. 12). Formalin with 10 per cent glycerin is placed in the copper tank and heated by a burner. Formaldehyde leaves the nozzle (which can be introduced through the keyhole) mixed in a fine spray with steam. This apparatus has been favorably endorsed by the War Department of the United States. The so-called Breslau method of generating formaldehyde depends Fig. 13. — Breslau Formaldehyde Generator and Section of Same. (After v. Brunn.) a, Formalin tank; b, Inlet; c, Exit vent; d, Alcohol lamp. upon the evaporation of formaldehyde from dilute solutions, v. Brunn i claims that where formalin in 30 to 40 per cent strength is evaporated, water vapor is generated more rapidly than formaldehyde is liberated , and a concentration leading to polymerization occurs. If, however, 1 v. Brunn, Zeit. f. Hyg., xxx, 1899. 90 BIOLOGY AND TECHNIQUE dilution As carried out until the formaldehyde in the solution is not more than 8 per cent, the generation of water vapor and formaldehyde takes place at about equal speed and no concentration occurs. Schloss- mann 1 furthermore claims that polymerization in the vaporized formal- dehyde does not occur if sufficient water vapor is present — a principle which may also contribute to the efficiency of the Breslau method. In practice, the apparatus devised by v. Brunn (Fig. 13) consists of a strong- copper kettle of about 34 cm. diameter by 7.5 cm. height. This is com- pletely closed except for two openings in the slightly domed top, one of which is the exit vent, the other, laterally placed, is for purposes of filling and is closed by a screw stopper. The kettle is set up on a metal stand over an alcohol lamp, so arranged with a double circle of burners that heating may be carried out rapidly. The tank is filled with a solu- tion of formalin of a strength of from 8 to 10 per cent (commercial form- alin 1 : 4) . The apparatus permits the evaporation of large quantities of fluid in a short time (3 liters in one hour) . When the lamp is left in a closed room care should be taken to fill it with a quantity of alcohol about proportionate to the amount of fluid to be evaporated. This, according to v. Brunn, is about one-quarter of the volume of formalin solution used. By using 1.5 liters of 8 per cent formalin for each 1,000 cubic feet of space, this apparatus is said to yield a concentration of formaldehyde of about 25 grams to the cubic meter, a strength suffi- ficient to complete surface disinfection within seven hours. To do away with the use of liquid formalin solutions, a method has been devised which depends in principle upon the breaking up by heat of the solid polymer of formaldehyde (trioxymethylene) . The apparatus (trade name, "Schering's Paraform Lamp") as described by Aronson2 consists of a cylindrical mantle of sheet-iron placed upon a stand and supplied below with an alcohol lamp. Set into the top of the mantle is a small chamber, into which 1 gram tablets of trioxymetlYylene are placed. The alcohol lamp, so placed that the wicks project but slightly — to avoid overheating — is lighted, and the formalin generated passes out through slits in the upper case, mingling with the water vapor and other gases liberated by the alcohol flame. The more modern devices have water-boiler attachments to insure sufficient moisture. Two tablets are sufficient for the fumigation of about thirty-five cubic feet, and 2 c.c. of alcohol are filled into the lamp for each tablet. One hundred to one hundred and fifty tablets are usually enough for the ordinary 1 Schlossmann, loc. cit. 2 Aronson, Zeit. f. Hyg., xxv, 1897. THE DESTRUCTION OF BACTERIA 91 room. Modifications of this method are in common use, some well- known firms preparing so-called "paraform candles/' in which para- form, in the powdered state, is volatilized by heat. A simple method of generating formaldehyde is that which is known as the "lime method." In a wide shallow pan 40 per cent formalde- hyde solution (commercial formalin) is poured over quicklime (CaO). According to Park, the previous addition of concentrated sulphuric acid to the formalin, in proportions of one to ten, increases the speed of formalin liberation, and aids in limiting polymerization. About one and one-half to two pounds (one-half to one kilogram) of quicklime are used for every 500 c.c. of the formalin solution. The heat generated in the slaking of the lime produces volatilization of the formalin. A modification of this method is that of Schering * in which tablets of paraform and unslaked lime are together laid into a pan and warm water is poured over them. A highly efficient method, which has universal approval because of its simplicity, is the potassium permanganate method of Evans and Russell.2 This method depends upon the active reaction occurring when formalin and potassium permanganate are mixed. In practice, about 300s gram of small crystals of potassium permanganate are poured into a half liter of 40 per cent formalin. The mixture results in an active evolution of heat and the evaporation of formalin together with water vapor. Because of the active foaming which takes place, high cylin- drical vessels should be used about one foot in height, preferably with a funnel-like flare at the top. The yield of gas by this method is said to be about 80 per cent of the amount present in the solution, and within the first five minutes most of this is liberated. In gauging the amount of formalin to be used for definite spaces by the various methods, it is safe to follow, as a general rule, the calcula- tions made by Harrington,3 who states that the equivalent of 110 c.c. of formalin suffices to produce sterility within two and a half hours in a space of one thousand cubic feet. In practice, whenever the formalin generators are left in a closed room, it is a point of great importance to place the apparatus upon a large piece of sheet-iron in order to avoid accidents from fire. 1 Schering, Hyg. Rundschau, 1900. 2 Evans and Russell, Rep. State Bd. Health, Maine. 1904. 3 Harrington, "Practical Hygiene," Phila., 1905. 92 BIOLOGY AND TECHNIQUE The room in which formaldehyde has been liberated is kept sealed, in the manner already described, for at least twelve hours, after which the windows and doors are opened and thorough airing practiced. The odor which remains after formaldehyde fumigation may be removed by sprinkling with ammonia, or by the use of some one or another of the various sorts of apparatus devised for the liberation of ammonia. CHAPTER VI METHODS USED IN THE MICROSCOPIC STUDY AND STAINING OF BACTERIA MICROSCOPIC STUDY OF BACTERIA Bacteria may be studied microscopically, in the living and un- stained state, and, after the application of dyes, in colored preparations. For the manipulation of bacteria for such study, glass slides and cover- slips of various design are used. These must be perfectly clean if the preparations are to be of any value.1 The Study of Bacteria in the Living State. — Living bacteria may be studied in what is spoken of as the "hanging-drop" preparation. For this purpose a so-called hollow slide is employed, in the center of which there is a circular concavity about three-quarters of a centimeter to one centimeter in diameter. The preparation is manipulated as follows: If the bacteria are growing in a fluid medium a drop of the culture fluid is transferred to the center of a cover-slip. If taken from solid media, an emulsion may be made in broth or in physiological salt solution, and a drop of this transferred to the cover-slip, or the bac- teria may be emulsified in a drop of salt solution, or broth, directly upon the cover-slip. The concavity on the slide, having first been rimmed with vaseline, by means of a small cameTs-hair brush, the cover-slip is inverted over the slide in such a way that the drop hangs freely within the hollow space. The preparation is then ready for examination under the microscope. 1 Although the silicates of which glass is composed are extremely stable, never- theless alkaline silicates which are said to separate out on the surface, together with grease and dirt left upon the glass by handling, during blowing and cutting, neces- sitate cleansing before use. This may be accomplished by a variety of methods. A simple one suitable for general application is as follows: (1) The slides and cover- slips are thrown singly into boiling water and left there for half an hour; (2) wash in twenty-five per cent sulphuric acid; (3) rinse in distilled water; (4) wash in alcohol; (5) wipe with a clean cloth and keep dry under a bell-jar. Another method convenient for routine use is to immerse, after thorough washing in soap-suds and acid, in ninety-five per cent alcohol and to leave in this until the time of use. 93 94 BIOLOGY AND TECHNIQUE Another method, known as the " hanging block method/' devised by Hill,1 for the study of living bacteria in solid media is carried out as fol- lows : Nutrient agar is poured into a Petri dish and allowed to solidify. Out of this layer a piece about a quarter of an inch square is cut. This is placed on a sterile slide. The upper surface of the agar block is then inoculated with bacteria by surface smearing, and the preparation covered with a sterile dish and allowed to dry for a few minutes in the incubator. A sterile cover-slip is then dropped upon the surface of the Fig. 14. — Hanging Drop Preparation. block and sealed about the edges with agar. Block and cover-slip are then taken from the slide and fastened over a moist chamber with paraf- fin. The entire preparation can be placed upon the stage of a microsocpe. This method is especially designed for the study of cell-division. Living bacteria may also be studied in stained preparations by the so-called " intravital " method of Nakanishi. Thoroughly cleaned slides are covered with a saturated aqueous solution of methylene-blue. This is spread over the slide in an even film and allowed to dry. After drying the slide should appear of a transparent sky-blue color. The micro- organisms which are to be examined are then emulsified in water, or are taken from a fluid medium and placed upon a cover-slip. This is dropped, face downward, upon the blue ground of the slide. In this way bacteria may be stained without being subjected to the often destructive proc- esses of heat or chemical fixation. According to Nakanishi, cytoplasm is stained blue, while nuclear material assumes a reddish or purplish hue. The Study of Bacteria in Fixed Preparations. — Stained preparations of bacteria are best prepared upon cover-slips, the process consisting of the following steps : (1) Spreading on cover-slip; (2) drying in air; (3) fixing; (4) staining; (5) washing in water; (6) blotting; (7) mounting. (1) Smearing. — Bacteria from a fluid medium are transferred in a small drop of the fluid, with a platinum loop, to a cover-slip and care- fully spread over the surface in a thin film. If taken from a solid medium a small drop of sterile water is first placed upon the cover-slip and the bacteria are then in very small quantity carefully emulsified in this drop with the platinum needle or loop and spread in an extremely thin film. 1 Hill, Jour, of Med. Research, vii, 1902. MICROSCOPIC STUDY AND STAINING 95 (2) The film is allowed to dry in the air. (3) When thoroughly dried, fixation is carried out by passing the preparation, film side up, three times through a Bunsen flame, at about the rate of a pendulum swing. Fixation by heat in this manner is most convenient for routine work, but is not the most delicate method, in- asmuch as the degree of heat applied can not be accurately controlled. The other methods which may be employed are immersion in methyl alcohol, formalin, saturated aqueous bichloride of mercury, Zenker's fluid, or acetic acid. If chemical fixatives are used, they must be re- moved by washing in water before the stain is applied. If a prepara- tion is made upon a slide instead of a cover-slip, passage through the flame should be repeated eight or nine times. (4) Staining. — The dyes used for the staining of bacteria are, for the greater part, basic anilin dyes, such as methylene-blue, gentian- violet, and fuchsin. These may be applied for simple staining in 5 per cent aqueous solutions made up from filtered saturated alcoholic solutions, or directly by weight. They are conveniently kept in the laboratory as saturated alcoholic solutions. The strengths of some saturated solutions are as follows: Saturated Solutions 1 (Stains Gruebler or Merck). Fuchsin (aqueous), 1.5 per cent. Fuchsin (alcohol 96 per cent), 3 per cent. Gentian- violet (aqueous), 1.5 per cent. Gentian-violet (alcohol 96 per cent), 4.8 per cent. Methylene-blue (aqueous), 6.7 per cent. Methylene-blue (alcohol 96 per cent), 7 per cent. The staining solution, in simple routine staining, is left upon the fixed bacterial film for from one-half to one and one-half minutes according to the efficiency of the stain used. Methylene-blue is the weakest of the three stains mentioned; gentian-violet the strongest. (5) The excess stain is removed by washing with water. (6) The preparation is thoroughly dried by a blotter or between layers of absorbent paper. (7) A small drop of Canada balsam is placed upon the film side of the dry cover-slip, which is then inverted upon a slide. The prepara- tion is now ready for microscopical examination. 1 After Wood, " Chemical and Microscopical Diagnosis," Appendix. N. Y., 1909. 96 BIOLOGY AND TECHNIQUE The chemical principles which underlie the staining process-are still more or less in doubt.1 Suffice it to say here that most of the dyes in common use by bacteriologists and pathologists are coal-tar derivatives belonging to the aromatic series, all of them containing at least one "benzolring" combined with what Michaelis terms a " chromophore group," chief among which are the nitro-group (N02), the nitroso-group (NO), and the azo-group (N = N). Just what the actual process of stain- ing consists in, is a question about which various opinions are held, some believing that the phenomenon is purely chemical, in which a salt is formed by the combination of the dye and the protoplasm of the cells, others that there is no such salt formation, and that the process takes place by purely physical means. To support the latter view it is argued that certain substances like cellulose are stainable without possessing the property of salt formation, and that staining may often be accom- plished without there being a chemical disruption of the dye itself. Michaelis sums up his views by stating that probably both processes actually take place. A dye stuff, as a whole, may enter into and be de- posited upon a tissue or cell by a process which he speaks of as " insorp- tion." In such a case the coloring matter may be subsequently ex- tracted by any chemically indifferent solvent. On the other hand, a dye after being thus deposited upon or within a cell, may become chemically united to the protoplasm by the formation of a salt, and in such a case the color can be removed only by agents which are capable of decom- posing salts, such as free acids. The staining power of any solution may be intensified either by heating while staining, by prolonging the staining process, or by the addition of alkalies, acids, anilin oil, and other substances which will be mentioned in the detailed descriptions of special staining methods. One of the most common examples of such an intensified stain is the so-called Loeffler's alkaline methylene-blue. This is made up in the following way: Saturated alcoholic solution of methylene-blue, 30 c.c. 1 : 10,000 solution potassium hydrate in water, 100 c.c. Another solution designed with a similar purpose is the Koch-Ehrlich anilin-water solution. Anilin oil, one part, is shaken up with dis- tilled water, nine parts; after thorough shaking, the mixture is filtered 1 For comprehensive reviews of the subject, the reader is referred to dissertations such as those of Mann (" Physiol. Hist. Methods and Theory," Oxford, 1902) and of Michaelis (" Einfiihrung in die FarbstorTchemie," etc., Berlin, 1902). MICROSCOPIC STUDY AND STAINING 97 through a- moist filter paper until perfectly clear. A saturated alco- holic solution of either fuchsin or gentian-violet is added to this anilin water in proportions of about one to ten or until a slightly iridescent pellicle appears upon the surface of the solution. An extremely useful and very strong staining solution is the Ziehl carbol-fuchsin solution, made up as follows: 1 Fuchsin (basic) 1 gm. Alcohol (absolute) 10 c.e. Five per cent carbolic acid 100 c.c. To make up this staining solution, mix 90 c.c. of a five per cent aque- ous solution of carbolic acid with 10 c.c. of saturated alcoholic basic fuchsin. It may also be made up as follows : Weigh out Basic fuchsin 1 gram Carbolic acid 5 grams Dissolve in Distilled water 100 c.c. Filter and add Absolute alcohol 10 c.c. SPECIAL STAINING METHODS Spore Stains. — Abbott's Method.2 — Cover-slips are smeared and fixed by heat in the usual manner. Cover with Loeffler's alkaline methylene-blue and heat the stain until it boils, repeat the heating at intervals but do not boil continuously. Keep this up for one minute. Rinse in water. Decolorize with a mixture of alcohol eighty per cent 98 c.c. and nitric acid 2 c.c, until all blue has disappeared. Rinse in water. Dip from three to five seconds in saturated alcoholic solution of eosin 10 c.c, and water 90 c.c Rinse in water, blot, and mount in balsam. By this method the spores are stained blue, the bodies of the bacilli are stained pink. » Ziehl, Deut. med, Woch., 1882, 2 Abbott, "Prin, of Bact.," Phila., 1905. 7 98 BIOLOGY AND TECHNIQUE Moeller's Method.1 — Cover-slips are prepared as usual and fixed in the flame. Wash in chloroform for two minutes. Wash in water. Cover with five per cent chromic acid one-half to two minutes. Wash in water. Invert and float cover-slip on carbol-fuchsin solu- tion in a small porcelain dish and heat gently with a flame until it steams; continue this for three to five minutes. (This step can also be done by covering the cover-glass with carbol-fuchsin and holding over flame.) Decolorize with five per cent sulphuric acid five to ten seconds. Wash in water. Stain writh aqueous methylene-blue one-half to one minute. By this method spores will be stained red, the body blue. Capsule Stains. — Welch's Method.2 — Cover-slips are prepared as usual and fixed by heat. Cover with glacial acetic acid for a few seconds. Pour off acetic acid and cover with anilin water gentian-violet, renewing stain repeatedly until all acid is removed. This is done by pouring the stain on and off three or four times and then finally leaving it on for about three minutes. W^ash in two per cent salt solution and examine in this solution. Hiss' Methods.3 — (1) Copper Sulphate Method. — Cover-slip prepara- tions are made by smearing the organisms in a drop of animal serum, preferably beef-blood serum. Dry in air and fix by heat. Stain for a few seconds with — Saturated alcoholic solution of fuchsin or gentian-violet 5 c.c, in distilled water 95 c.c. The cover-slip is flooded with the d}re and the preparation held for a second over a free flame until it steams. Wash off dye with twenty per cent aqueous copper sulphate solution. Blot (do not wash). Dry and mount. By this method permanent preparations are obtained, the capsule appearing as a faint blue halo around a dark purple cell body. (2) Potassium Carbonate Method. — This method consists in using as a dye a half -saturated solution of gentian-violet. Gentian-violet in » Moeller, Cent. f. Bakt., I, x, 1891. 2 Welch, Johns Hopkins Hosp. Bull., 1892. s Hiss, Cent. f. Bakt., xxxi, 1902; Jour. Exper. Med., vi, 1905. MICROSCOPIC STUDY AND STAINING 99 substance is added in excess to distilled water and allowed to dissolve to its full extent. The solution is then filtered and diluted to twice its volume. Cover-glass preparations are made by spreading the bacteria on a cover-slip in a drop of animal serum as in preceding method. They are allowed to dry in the air and fixed by heat as usual. The dye is then poured upon the preparation and allowed to remain for a few seconds. It is then washed off with a twenty-five-hundredth per cent solution of potassium carbonate in water, and studied in this solution. The cover-slip inverted on a slide may be rimmed with vaseline to prevent evaporation. Buerger's Method.1 — Cover-slip preparations are made by smear- ing in serum as in Hiss' method. As the edges of the smear begin to dry, pour over it Mueller's fluid and warm in flame for three seconds. (Mueller's fluid is composed of potassium bichromate 2.5 gm., sodium sulphate 1 gm., water 100 c.c, saturated with bichloride of mercury.) Wash in water. Flush with ninety-five per cent alcohol. Cover with tincture of iodin, U. S. P., one to three minutes. Wash with ninety-five per cent alcohol. Dry in the air. Stain with anilin water gentian-violet two to five seconds. Wash with two per cent salt solution. Mount and examine in salt solution. Wads worth's Method.2 — W^adsworth has devised a method of staining capsules which depends upon the fixation of smears with forma- lin. After such fixation capsules may be demonstrated both with simple stains and by Gram's method. The technique is as follows: Smear preparations, made as usual, are treated as follows: 1. Formalin, 40 per cent, two to five minutes. 2. Wash in water, five seconds. Simple Stain. Differential Stain (Gram's Method). 3. Ten per cent aqueous gentian-violet. 3. Anilin gentian-violet, two minutes. 4. Wash water, five seconds. 4. Iodin solution, two minutes. 5. Dry, mount in balsam. 5. Alcohol, 95 per cent, decolorize. 6. Fuchsin, dilute aqueous solution. 7. Wash water, two seconds. 8. Dry, mount in balsam. 1 Buerger, Med. News, Dec, 1904 2 Wadsworth, Jour. Inf. Dis., 1906. 100 BIOLOGY AND TECHNIQUE It is important that the formalin be fresh and the exposure to water momentary. When decolorizing in the Gram method, strong alcohol only should be used. Wadsworth also found that encapsulated pneumococci could be demonstrated in celloidin sections of pneumonic lesions hardened in strong formalin. The lungs should be distended with the formalin or the lesions cut in very thin bits, hardened, dehydrated, embedded, and cut in the usual way. The celloidin sections may be fixed on the slides by partially dissolving the celloidin in alcohol and ether and setting the celloidin quickly in water before staining. Failure to obtain pneumococci encapsulated in such sections is usually due to improper or inadequate fixation in the formalin. The differential method employed by Wadsworth for tissue staining is as follows: 1. Fix in formalin forty per cent, two to five minutes. 2. Wash in water. 3. Anilin gentian-violet, two minutes. 4. Iodin solution, two minutes. 5. Alcohol, ninety-five per cent, decolorize. 6. Eosin alcohol, counterstain. 7. Clear in oil of origanum. 8. Mount in balsam. Flagella Stains. — All flagella stains, in order to be successful, neces- sitate particularly clean cover-slip preparations, best made from young agar cultures emulsified in sterile salt solution. Scrupulous care should be exercised in cleaning the glassware used. Loeffler's Method.1 — The preparation is dried in the air and fixed by heat. It is then treated with the following mordant solution: Twenty per cent aqueous tannic acid 10 parts. Ferrous sulphate aq. sol. saturated at room temperature . 5 parts. Saturated alcoholic f uchsin solution 1 part. This solution, which should be freshly filtered before using, is poured over the cover-glass and allowed to remain there for one-half to one minute, during which time it should be gently heated, but not allowed to boil. Wash thoroughly in water. Stain with five per cent anilin water fuchsin or anilin water gen- ^Loeffler, Cent. f. Bakt., I, vi, 1889. MICROSCOPIC STUDY AND STAINING 101 tian-violet made slightly alkaline by the addition of one-tenth per cent sodium hydrate. The stain should be filtered directly upon the cover-slip. Warm gently and leave on for one to two minutes. Wash in water. Mount in balsam. Van Ermengem's Method.1 — This method requires the preparation of three solutions. (1) Twenty per cent tannic acid solution 60 c.c. Two per cent osmic acid solution 30 c.c. Glacial acetic acid 4-5 drops. The cover-slip carrying the fixed preparation is placed in this solu- tion for one hour at room temperature, or for five minutes at 100° C. (boiling) . Wash in water. Wash in absolute alcohol. Immerse the cover-slip for one to three seconds in (2) Silver nitrate, 0.25-0.5 per cent solution. Without washing, transfer to (3) Gallic acid 5 gm. Tannic acid 3 " Fused potassium acetate 10 " Distilled water 350 c.c. Immerse in this for a few minutes, moving the cover-slip about. Return to the silver nitrate solution until the preparation turns black. Wash thoroughly in water. Blot and mount. Smith's Modification of Pitfield's Method.2 — A saturated solu- tion of bichloride of mercury is boiled and is poured while still hot into a bottle in which crystals of ammonia alum have been placed in quantitiy more than sufficient to saturate the fluid. The bottle is then shaken and allowed to cool. Ten c.c. of this solution are added to 10 c.c. of freshly prepared ten per cent tannic acid solution. To this add 5 c.c. carbol- fuchsin solution. Mix and filter. To stain, filter the above mordant directly upon the fixed cover-slip 1 Van Ermengem, Cent. f. Bakt., I, xv, 1894. 2 Smith, Brit. Med. Jour., I, 1901, p. 205. 102 BIOLOGY AND TECHNIQUE preparation. Heat gently for three minutes, but do not allow to boil. Wash in water and stain with the following solution: Saturated alcoholic solution gentian-violet 1 c.c. Saturated solution ammonia alum 10 c.c. Filter the stain directly upon the preparation and heat for three or four minutes. Wash in water, dry, and mount in balsam. Differential Stains. — Gram's Method.1 — By this method of staining, which is extremely important in bacterial differentiation, bacteria are divided into those which retain the initial stain and those which are subsequently decolorized and take the counterstain. The former are often spoken of as the Gram-positive, the latter as Gram-negative bacteria. Preparations are made on cover-slips or slides in the usual way. The preparation is then covered with an anilin gentian-violet solu- tion which is best made up freshly before use. The staining fluid is made up, according to Gram's original direc- tions,2 as follows: Five c.c. of anilin oil are shaken up thoroughly with 125 c.c. of dis- tilled water. This solution is then filtered through a moist filter paper. To 108 c.c. of this anilin water, add 12 c.c. of a saturated alcoholic- solution of gentian-violet. The stain acts best when twelve to twenty- four hours old, but may be used at once. It lasts, if well stoppered, for three to five days. A more convenient and simple method of making up the stain is as follows: To 10 c.c. of distilled water in a test tube add anilin oil until on shaking the emulsion is opaque; roughly, one to ten. Filter this through a wet paper until the filtrate is clear. To this acid saturated alcoholic solution. of gentian-violet until the mixture is no longer transparent, and a metallic film on the surface indicates saturation. One part of alcoholic saturated gentian-violet to nine parts of the anilin water will give this result. This mixture may be used immediately and lasts two to five days if kept in a stoppered bottle. Cover the preparation with this; leave on for five minutes. Pour off excess stain and cover with Gram's ioclin solution, Iodin 1 gm. Potassium iodid 2 gm. Distilled water 300 c.c. 1 Gram, Fortschr. d. Med., ii, 1884. 2 Gram, loc. cit. MICROSCOPIC STUDY AND STAINING 103 Decolorize with ninety-seven per cent alcohol until no further traces of the stain can be washed out of the preparation. This takes usually thirty seconds to two minutes, according to thinness of prepara- tion. Wash in water. Counterstain with an aqueous contrast stain, preferably Bismarck brown.1 Paltauf's Modification of Gram's Stain.2 — The staining fluid as prepared by this modification possesses the advantage of retaining its staining power for a longer period than does the anilin-water-gentian- violet described in the original method. The staining fluid is prepared as follows: 3-5 c.c. anilin oil are added to 90 c.c. distilled water and 7 c.c. absolute alcohol. This mixture is thoroughly shaken and filtered through a moist filter paper until clear. Then add: Gruebler's gentian- violet 2 gm. The fluid should stand twenty-four hours, during which a precipi- tate forms. This is filtered before use. This gentian-violet solution retains its staining power for from four to six weeks. It is good only when a metallic luster develops on the surface. It is used in the following way: Spreads on cover-slips or slides are dried and fixed as usual. Then apply: — Anilin water gentian-violet (as above), three minutes. Gram's iodin solution, two minutes. Absolute alcohol (with stirring), thirty seconds. Counterstain, without washing in water, in aqueous fuchsin or in weak carbol-fuchsin. 1 To make up Bismarck brown solution, prepare a saturated aqueous solution of the powdered dye by heating. Allow it to cool, and filter. Dilute one to ten with distilled water. 2 Sharnosky, Proc. N. Y. Pathol. Soc, Oct., 1909, n. s., ix, 5. 104 BIOLOGY AND TECHNIQUE Classification of the Most Important Pathogenic Bacteria According to Gram's Stain. Gram-positive. {Retain the Gentian-violet.) Micrococcus pyogenes aureus Micrococcus pyogenes albus Streptococcus pyogenes Micrococcus tetragenus Pneumococcus Bacillus subtilis Bacillus anthracis Bacillus diphtheria? Bacillus tetanus Bacillus tuberculosis and other acid -fast bacilli Bacillus aerogenes capsulatus Bacillus botulinus Gram-negative. {Take Counterstain.) Meningococcus Gonococcus Micrococcus catarrhalis Bacillus coli Bacillus dysenterise Bacillus typhosus Bacillus paratyphosus Bacillus fecalis alkaligenes Bacillus enteritidis Bacillus proteus (proteus) Bacillus mallei Bacillus pyocyaneus Bacillus influenzae Bacillus mucosus capsulatus Bacillus pestis Bacillus maligni cedematis Spirillum cholera? Bacillus Koch-Weeks Bacillus Morax-Axenfeld Stains for Acid-Fast Bacteria. — These methods of staining are chiefly useful in the demonstration of tubercle bacilli. These bacteria because of their waxy cell membranes are not easily stained by any but the most intensified dyes, but when once stained, retain the color in spite of ener- getic decolorization with acid. For this reason they are known as acid- fast bacilli. The first method devised for the staining of tubercle and allied bacilli was that of Ehrlich. Ehrlich Method.1 — This method is now rarely used. Cover-slip preparations are prepared as usual and fixed by heat. Stain with anilin water gentian- violet, hot, three to five minutes, or twenty-four hours at room temperature. i Ehrlich, Deut. med. Woch., 1882. MICROSCOPIC STUDY AND STAINING 105 Decolorize with thirty-three per cent nitric acid one-half to one minute. Treat with sixty per cent alcohol, until no color can be seen to come off. Counterstain with aqueous methylene-blue. Rinse in water, dry, and mount. Ziehl-Neelson Method.1 — Thin smears are made upon cover- slips or slides. Fix by heat. Stain in carbol-fuchsin solution as given on page 97. The slide or cover-slip may be flooded with the stain, and this gently heated with the flame until it steams, or else the cover-slip may be inverted upon the surface of the staining fluid, in a porcelain dish or watch-glass, and this heated until it steams. This is continued for three to five min- utes. Decolorize with either five per cent nitric acid, five per cent sulphuric acid, or one per cent hydrochloric acid for three to five seconds. The treatment with the acid is continued until subsequent washing with water will give only a faint pink color to the preparation. Wash with ninety per cent alcohol until no further color can be re- moved. If, after prolonged washing with alcohol, a red color still re- mains in very thick places upon the smear, while the thin areas appear entirefe^Lecolorized, this may be disregarded. mlsh in water and counterstain in aqueous methylene-blue for one-half to one minute. Rinse 'n water, dry, and mount. By this method the tubercle bacilli are colored red, other bacteria and cellular elements which may be present are stained blue. Gabbet's Method.2 — Gabbet has devised a rapid method in which the decolorization and counterstain ing are accomplished by one solu- tion. The specimen is prepared and stained with carbol-fuchsin as in the preceding method. It is then immersed for one minute directly in the following solution: Methylene-blue 2 gms. Sulphuric acid 25 per cent (sp. gr. 1018) 100 c.c. Then rinse in water, dry, and mount. This method, while rapid and very convenient, is not so reliable as the Ziehl-Neelson method. 1 Ziehl, Deut. med. Woch., 1882; Neelson, Deut. med. Woch., 1883. 2 Gabbet, Lancet, 1887. 106 BIOLOGY AND TECHNIQUE Pafpenheim's Method.1 — The method of Pappenheim is devised for the purpose of differentiating between the tubercle bacillus and the smegma bacillus. Confusion may occasionally arise between these two microorganisms, especially in the examination of urine where smegma bacilli are derived from the genitals, and less frequently in the examina- tion of sputum where smegma bacilli may occasionally be mixed with the secretions of the pharynx and throat. Preparations are smeared and fixed by heat in the usual way. Stain with hot carbol-fuchsin solution for two minutes. Pour off dye without washing and cover with the following mixture : Corallin (rosolic acid) 1 gm. Absolute alcohol 100 c.c. Methylene-blue added to saturation Add glycerin 2 20 c.c. This mixture is poured on and drained off slowly, the procedure being repeated four or five times, and finally the preparation is washed in water. The combination of alcohol and rosolic acid decolorizes the smegma bacilli, but leaves the tubercle bacilli stained bright red. Bunge and Trautenroth Method.3— This method is designed to differentiate between the tubercle and smegma bacilli. Smear and fix by heat in the usual way. Wash with absolute alcohol to remove fat. Treat with five per cent chromic acid for fifteen minutes. Wash in several changes of water. Stain with hot carbol-fuchsin for five minutes. Decolorize with sixteen per cent sulphuric acid for three minutes. Counterstain with alcoholic methylene-blue for five minutes. Wash in water, dry, and mount. By this method the tubercle bacillus remains red, the smegma bacil- lus is decolorized. Baumgarten's Method.4 — This method is recommended by the author for differentiation between the bacillus of tuberculosis and the bacillus of leprosy and depends upon the fact that the tubercle bacillus is less easily stained than Bacillus leprae. Smears are prepared and fixed by heat in the usual way. 1 Pappenheim, Berl. klin. Woch., 1898. 2 The glycerin is added after the other constituents have been mixed. 3 Bunge und Trautenroth, Fortschr. d. Med., xiv, 1896. 6 Baumgarten, Zeit. f. wissensch. Mikrosk., 1, 1884. MICROSCOPIC STUDY AND STAINING 107 Stain in dilute alcoholic fuchsin for five minutes. Decolorize for twenty seconds in alcohol, ninety-five per cent, ten parts, nitric acid one part. Wash in water. Counterstain in methylene-blue. Wash in water, dry, and mount. The tubercle bacillus should be blue and the bacillus of leprosy red. Special Stains for Polar Bodies. — These staining methods are designed to bring into view polar bodies as found, for instance, in the bacilli of diphtheria and plague. Neisser's Method.1 — Smear and fix in the usual manner. Stain for two to five seconds in the following solution: Methylene-blue 1 gm. Absolute alcohol 20 c.c. Glacial acetic acid 50 c.c. Distilled water 1,000 c.c. Wash in water. Counterstain in two per cent aqueous Bismarck brown solution for five seconds. B3r this method polar bodies are stained blue, while the bacillary bodies are stained brown. Roux's Method.2 — Two solutions are necessary. (1) Dahlia violet 1 gm. Alcohol 90 per cent 10 c.c. Aqua destillata ad 100 c.c. (2) Methyl-green 1 gm. Alcohol 90 per cent 10 c.c. Aqua destillata ad 100 c.c. Before use, one part of solution No. 1 is mixed with three parts of solution No. 2. The preparation is stained with the mixture for two minutes in the cold. Polychrome Stains. — The various polychrome stains are of value to the bacteriologist chiefly for the staining of pus and exudates where the relation of bacteria to cellular elements is to be demonstrated. They are also extremely useful in the study of fixed specimens of protozoan parasites. There is a large number of these stains in use; a few only, 1 Neisser, Zeit. f. Hyg., xxiv, 1897. 2 Roux and Yersin, Annal. de l'inst. Past., 1890. 108 BIOLOGY AND TECHNIQUE however, can be given here. In principle, all these stains depend upon a combination of eosin and methylene-blue, these elements staining not only as units, but acting together in combination. One and the same solution, therefore, contains at least three elements which color the various structures of the preparation selectively. Jenner's Method.1 — This stain, because of its simplicity, is useful for routine use. It is made up as follows: Equal parts of eosin (Gruebler, "W. G.") one and two-tenths per cent aqueous solution, and methylene- blue (medicinal, Gruebler) one per cent aqueous solution, are mixed and allowed to stand for twenty-four hours. A coarse granular precipitate is formed which appears dark, with a metallic luster on its surface. This is separated by filtration and washed with distilled water until the fil- trate appears almost clear. To make up the stain 0.5 gram of the dry precipitate is dissolved in 100 c.c. of methyl alcohol. In using the stain, preparations are not fixed, but simply dried in the air and immersed in the stain for one to two minutes. After this, wash in distilled water and examine. Wright's Modification of Leishman's Method.2 — A one per cent solution of methylene-blue (Gruebler) in five-tenths per cent solution of sodium bicarbonate in distilled water is steamed in a sterilizer at 100° C. for one hour. After this has cooled, a one-tenth per cent aqueous solution of eosin (Gruebler, W. G.) is added until a metallic scum ap- pears on the surface of the mixture. (About five parts of eosin solution to one of methylene-blue is necessary.) The precipitate which forms is collected by filtration, dried, and a saturated solution then made in methyl alcohol. This is filtered and diluted with one-quarter its bulk of methyl alcohol. To stain, cover the dried preparation with the stain for one to one and one-half minutes. Dilute by dropping upon the stain distilled water from a pipette until a metallic film appears upon the top. Leave this on for three to fifteen minutes. Wash in distilled water. Giemsa's Method.3 — The method of Giemsa is really a modification of the Romanowsky method. It is widely applicable, being of great value in the staining of the Spirochete pallida, Vincent's spirilla, pro- tozoa, and Negri bodies. The stain has been modified several times by 1 Jenner, Lancet, i, 1889. 2 Wright, Jour. Med. Research, ii, 1902. 3 Giemsa, Cent. f. Bakt., I, xxxvii, 1904. MICROSCOPIC STUDY AND STAINING 109 its originator, the following being the formula given by him in 1904: The substance referred to as azur II and purchasable under that name, consists of pure methylenazur chloralhydrate combined with an equal quantity of methylene-blue chloralhydrate. The substance referred to as azur II-eosin is a combination of this substance with eosin. The staining fluid is made up as follows:1 Azur II-eosin 3 gms. Azur II 8 gms. This mixture is thoroughly dried over sulphuric acid in a desiccator, finely powdered, and rubbed through a fine sieve. It is then dissolved in 250 gms. of C. P. glycerin (Merck), at 60° C. To this ic added methyl alcohol (Kahlbaum) 250 c.c, previously warmed to 60° C. This mix- ture is well shaken and allowed to stand at room temperature for twenty-four hours. The mixture is now ready for use. For use 10 c.c. of distilled water are poured into a test tube and one to two drops of a one per cent potassium carbonate solution are added. Ten drops of the staining solution described above (one drop to the c.c.) are mixed with this slightly alkaline water. The preparation which is to be stained is fixed in methyl alcohol, dried, and covered with the diluted staining solution. For the staining of protozoa and ex- udates containing bacteria, ten to fifteen minutes are sufficient. For the staining of Negri bodies or Spirochete pallida, one or more hours of staining should be employed. After staining, wash in running tap water and blot. Wood's Method.2 — Wood has devised a simple staining method based on the principles of the Giemsa stain, in which azur II and eosin may be used in separate solutions. Preparations are fixed in strong methyl alcohol for five minutes and are then stained in a 0.1 per cent aqueous solution of eosin until the preparation is pink. The eosin is then poured off and the preparation is covered with a 0.25 per cent aqueous solution of azur II for one-half to two minutes. Following this, it is washed in tap water and dried by blotting. When an intense stain is desired, the solution of eosin and azur II may be flooded over the preparation together, using an excess of azur II. They are then left on from five to ten minutes. At the end of this time washing and drying as before completes the process. 1 It is best not to attempt to make up the undiluted staining fluid, since this is purchasable under the name of "Giemsa Losung fur Romanowsky Farbung." 2 Wood, Med. News, 83, 1903. 110 BIOLOGY AND TECHNIQUE The Staining of Bacteria in Tissues. — The preparation of tissue for bacterial staining is, in general, the same as that employed for purposes of cellular studies, in histology. For bacteriological studies the most useful fixative is alcohol; other fixations, such as that by formalin, Zenker's fluid, or Mueller's fluid, give less satisfaction. In other respects the details of dehydration and embedding are the same as those used in histological studies, except that it is desirable that the tissues should be handled rather more carefully than is necessary for ordinary patholog- ical work, and the changes from the weaker to the stronger alcohols should be made less abruptly.1 Embedding in paraffin is preferable to celloidin, although the latter method is not unsuccessful if carefully carried out. The chief disadvan- tages of celloidin are the retention of color by the celloidin itself and the consequent unclearness of differentiation. It is also easier to cut thin sections from paraffin blocks than from those prepared with celloidin. When staining tissue sections for bacteria, it is most convenient to carry out the process with the section attached to a slide. For cel- loidin sections this may be accomplished by means of ether vapor. For paraffin sections it is necessary to cover the slide with an extremely thin layer of a filtered mixture of equal quantities of egg albumin and glycerin, to which a small crystal of camphor or a drop or two of carbolic acid has been added. The sections are then floated upon a slide so prepared, and set away in the thermostat for four or five hours. Loeffler's Method.2 — Stain in alcoholic methylene-blue solution five to fifteen minutes, or in Loeffler's alkaline methylene-blue solution one to twenty-four hours. Wash in one to one-thousand acetic acid solution for about ten seconds. Treat with absolute alcohol by pouring the alcohol over the prepara- tion for ten to twenty seconds. Clear with xylol. Mount in balsam. When celloidin sections are stained in this way ninety-five per cent alcohol should be substituted for the absolute. A number of other staining solutions may be used in the same way, aqueous fuchsin or aqueous gentian-violet yielding good result. 1 For details of such work reference should be had to the standard textbooks on pathological technique, notably the very excellent one of Mallory and Wright. 2 Loeffler, Mitt. a. d. kais. Gesundheitsamt, ii, 1884. MICROSCOPIC STUDY AND STAINING 111 Nicolle advises the use of a ten per cent aqueous solution of tannic acid for a few seconds after washing with the acetic acid. This fixes the stain and prevents a too vigorous decolorization during the process of dehydration. Method of Staining Gram-Positive Bacteria in Tissue Sections. — Celloidin Sections. — After fixing section to the slide by pressure with a filter paper or by ether vapor, cover with anilin-water gentian-violet five minutes. Pour off excess of stain and cover with Gram's iodin solution for two minutes. Decolorize with ninety-five per cent alcohol until no more color comes out. Stain quickly with eosin-alcohol (ninety-five per cent alcohol to which enough eosin has been added to give a transparent pink color; about 1 : 15). Clear in eosin-oil of origanum (oil of origanum, 25 c.c. and eosin alcohol, as above, about 3 c.c). Blot and mount in balsam. Paraffin Sections. — Stain with anilin-water gentian-violet five to ten minutes. Wash in water. Cover with Gram's iodin solution one minute. Wash in water. Decolorize with absolute alcohol until no more color comes out. Clear in xylol. Mount in balsam. Gram-Weigert Method.1 — (For celloidin sections.) — Stain for one-half hour in the following freshly filtered solution: Carmine 3-5 grams. Saturated aqueous solution of lithium carbonate. . . .100 c.c. Dehydrate in ninety-five per cent alcohol. Stick section to slide with ether vapor. Stain in anilin-water gentian-violet for five to fifteen minutes (or in a saturated solution of aqueous crystal violet diluted with water one to ten, five to fifteen minutes). Wash in physiological salt solution. Cover with Gram's iodin solution one to two minutes. Wash in water and blot. 1 Weigert, Fortschr. d. Med., v, 1887. 112 BIOLOGY AND TECHNIQUE Decolorize with anilin oil until no more color comes off. This both decolorizes and dehydrates. Treat with xylol. Mount in balsam. Method of Staining for Tubercle Bacilli in Sections.1 — Paraffin Sections. — Stain in carbol-fuchsin solution hot for five minutes (or better cold, for twenty-four hours) . Wash in water. Decolorize and counterstain in Gabbet's methylene-blue sulphuric acid mixture for one minute. Wash in water. Dehydrate in absolute alcohol. Clear in xylol. Mount in balsam. Celloidin Sections.2 — Stain lightly in alum hematoxylin. Wash in water. Dehydrate in ninety-five per cent alcohol. Attach the slide by ether vapor. Stain with steaming carbol-fuchsin two to five minutes. Wash in water. Wash with Orth's acid alcohol (alcohol ninety per cent., 99 c.c; cone. HC1, 1 c.c.) one-half to one minute. Wash in water several changes. Treat with ninety-five per cent alcohol until red color is entirely gone. Blot and cover with xylol until clear. Mount in balsam. Method of Staining Actinomyces in Sections. — Mallory1 s Method3. — 1. Stain deeply in saturated aqueous eosin ten minutes. 2. Wash in water. 3. Anilin gentian-violet two to five minutes. 4. Wash in normal saline solution. 5. Weigert's iodin solution (iodin 1, KI 2, and water 100 parts) one minute. 6. W^ash in water and blot. 7. Clear in anilin oil. 8. Xylol several changes. 9. Mount in balsam. i Mallory and Wright, " Pathol. Tech.," p. 413. 2 After Mallory and Wright. 3 Mallory and Wright, " Pathol. Tech.," 1904. CHAPTER VII THE PREPARATION OF CULTURE MEDIA GENERAL TECHNIQUE The successful cultivation of bacteria upon artificial media requires the establishment of an environment which shall be suitable in regard to the presence of assimilable nutritive material, moisture, and osmotic relations. These requirements are fulfilled in the composition of the nutrient media described in another section, media which are to some extent varied according to the special requirements of the bacteria which are to be cultivated. If cultivation, furthermore, is to have any value for scientific study of individual species, it is necessary to ob- tain these species free from other varieties of microorganisms, that is, in pure culture, and to protect such cultures continuously from con- tamination with the other innumerable species which are everywhere present. The technique which is employed for these purposes has been gradu- ally evolved from the methods originally devised by Pasteur; Koch. Cohn, and others. Bacterial cultivation is carried out in glassware of varied construc- tion, the forms most commonly employed being test tubes of various sizes, Erlenmeyer flasks, the common Florence flasks, and Petri dishes. All glassware, of course, must be thoroughly cleansed before being used. Preparation of Glassware. — The cleansing of glassware may be ac- complished by any one of a number of methods. New glassware may be immersed in a one per cent solution of hydrochloric or nitric acid in order to remove the free alkali which is occasionally present on such glass. It is then transferred to a one per cent sodium hydrate solution for a few hours, and following this is washed in hot running water. In the case of old glassware which has contained culture media, sterilization in the autoclave is first carried out, then the glassware is boiled in five per cent soda solution or in soapsuds. After this, thorough mechanical cleansing is practiced, and the glassware may be treated by acid and alkali followed by running water, as given above. These last 8 113 114 BIOLOGY AND TECHNIQUE steps, however, are not essential, thorough washing in hot water after the soapsuds or soda solution being usually sufficient to yield good results. Other workers have recommended immersion of the glassware after mechanical cleansing in five per cent to ten per cent potassium bichromate solution in twenty-five per cent sulphuric acid. This is followed by thorough washing in hot running water, and drying. Clean flasks and test tubes are then stoppered with cotton, which has been found to be a convenient and efficient seal against the bacteria of the air, catching them in the meshes of the fibers as in a filter. The technique of the stoppering or plugging of glass receptacles is important, Fig. 15. — Florence Flask. Fig. 16. — Erlenmeyer Flask. in that, when poorly plugged, sterility is not safeguarded, and the pur- pose of culture study is defeated. In almost all laboratories in this country non-absorbent cotton or " cotton batting " is used for the plug. In a few of the German labora- tories the absorbent variety is employed. The disadvantages of the latter, especially in the case of fluid media, are obvious. The plugs should fit snugly, but not so tightly that force is necessary to remove them. Care should be taken, furthermore, that no creases are left be- tween the surface of the glass and the periphery of the plug; for these, if present, may serve as channels for the entrance of bacteria. Fig. 18, accompanying, will illustrate some of the more common and un- desirable defects in poorly made plugs. The plugging itself is carried out by tearing a small piece of cotton, about 2X2 inches, from the roll, THE PREPARATION OF CULTURE MEDIA 115 folding over one of its corners, and, applying the smooth end of a glass rod to the folded portion, gently pushing it into the mouth of the tube. After plugging and before media are introduced into the tubes and flasks, these should be sterilized. This is best done in one of the "hot- air sterilizers " (see Fig. 8, p. 69), by exposing the tubes for one hour to a temperature of 150° C. If greater speed is desired exposure to 180° to 190° C. for half an hour is usually safe. If by mistake, however, the temperature is allowed to rise above 200° C, a browning of the cotton plugs occurs and the glassware is apt to be stained by the burning of the fat and other organic material derived from the cotton. Petri dishes Fig. 17. — Petri Dish. after cleansing are fitted together in the manner shown in Fig. 17, and are sterilized in the hot-air chamber at 150° C. for one hour. Glassware so prepared is ready for the reception of media. Ingredients of Culture Media. — The food requirements of bacteria have been discussed in another section. From what has there been said, it is apparent that artificial culture media must, to a certain extent, be adjusted to the peculiarities of individual bacteria. In the cases of the more strictly parasitic microorganisms growth can be obtained only by the most rigid observance of special requirements. For the large majority of pathogenic bacteria, however, routine or standard media may be employed, which, while slightly more favorable for one species than for another, are sufficiently general in their composition to per- mit the growth of all but the most fastidious varieties. The basis of many of our common media is formed by the soluble constituents of meat. These substances are best obtained by macerating 500 grams of lean beef in 1,000 c.c. of distilled water. The mixture is 116 BIOLOGY AND TECHNIQUE Kfc. II $ \\\^ allowed to infuse in the ice chest over night, and then strained through cheese-cloth. To this infusion are added the other required constituents in the manner given in the detailed instructions below. The soluble constituents of meat, however, may also be procured in a simpler way by the use of the commercial meat extracts, such as that of Liebig. These extracts are dissolved in quantities of five grams to the liter, and other constituents are added to this nutrient basis. Though simpler to make, the meat-ex- tract media are less favorable for the culti- vation of the more delicate organisms than are the media made directly from fresh meat. Nevertheless, they suffice for the cultivation of the large majority of the more saprophytic pathogenic microorganisms and hold an im- portant place in laboratory technique. The ingredients and methods used in va- rious laboratories in the preparation of such standard media should be, as much as pos- sible, uniform, in order that confusion in re- sults may be avoided ; for, as is well known, the biological characteristics of one and the same bacterial species may vary considerably if grown on media differing in their compo- sition. A committee of the American Public Health Association,1 appointed in 1897 for the sake of standardizing the methods of preparation of media, recom- mended that the following rules should govern the choice of ingredients: 1. Distilled water should be used in all cases. 2. The meat used should be fresh, lean beef (when veal or chicken is substituted the change should be stated). 3. The pepton used should be Witte's pepton, dry, made from meat. 4. Only C. P. NaCl should be used. 5. For alkalinizing C. P. sodium hydrate should be used in normal solutions. W b Fig. 18.— Test Tube (a) incorrectly stoppered; (6) correctly stoppered. 1 Rep. Com. of Amer. Bact. to Com. of Amer, Pub. Health Assn. Meeting, Philadelphia, Sept., 1897, THE PREPARATION OF CULTURE MEDIA 117 6. For acidification C. P. hydrochloric acid in normal solution should be used. 7. When glycerin is used, this should be of the redistilled variety. 8. The agar-agar employed should be of the finest grade of commer- cial thread agar. 9. The gelatin should be the commercial sheet gelatin washed as free as possible of acid and impurities. 10. Chemicals and carbohydrates which are used should be as nearly chemically pure as possible. Titration of Media. — Next in importance to the actual composi- tion of media is the adjustment of their reaction. Bacteria are highly susceptible to variations in the acidity and alkalinity of media, excessive degress of either may completely inhibit development or moderate variations may lead to marked modifications of cultural characteristics. It is necessary, therefore, to adjust the reaction both for the sake of favoring growth and in order to insure uni- formity of growth characters. This is accomplished by titration which is best carried out according to the recommendations of the committee mentioned above. The color indicator employed for the titration is a five-tenths per cent solution of phenolphthalein in fifty per cent alcohol. The chief advan- tage of this indicator over others is due to the fact that it indicates the presence of organic acid and acid compounds in its reaction. For actual titration -^ (^ normal) solutions of sodium hydrate or of hy- drochloric acid are used. Since media in the process of preparation are usually acid, the NaOH solution is the one most frequently needed. Five c.c. of the medium to be tested is measured accurately in a care- 1 1 • ■ ■■' h :; f ■■;«,:■ I ■ . ■ . ■ ■ ''..'. Fig. 19. — Burette for Titrating Media. 118 BIOLOGY AND TECHNIQUE fully washed pipette and transferred into a porcelain evaporating dish. To this are added 45 c.c. of distilled water. The mixture is thoroughly boiled for three minutes over a free flame. The boiling drives off C02, giving the true neutral point, and approximates the conditions prevailing during the further sterilization of the medium from which the 5 c.c. have been taken. After boiling, 1 c.c. of the phenolphthalein is added. If the medium is acid, no color is present; if alkaline, a pink or red color appears. The ■£$ alkali or acid solution is allowed to drop into the dish from a graduated burette. When the neutral point is approached in an acid solution, each drop of sodium hydrate added brings forth at first a deep red, which, however, upon slight stir- ring with a clean rod, completely disappears.1 The end reaction is reached when a faint but clear and distinct pink color remains in the fluid after stirring. When titrating alkaline media, the addition of the phenolphthalein produces a red color in the hot medium which gradually fades upon the addition of |^ HC1, becoming- colorless at the end point of titration. Titration should be done quickly and in a hot solution. From the result of the titration the computation for the neutralization of the entire bulk of the medium can be made by a simple arithmetical process as illustrated in the following example: Let us suppose that we have used : 2.5 c.c. of -i^- NaOH to neutralize 5 c.c. of the medium, then 2.5 c.c. of f NaOH will neutralize 100 c.c. " and 25 c.c. of ^ NaOH will neutralize 1,000 c.c, or one liter. Fig. 20. — Tubing Media. 1 See standard textbooks on volumetric analysis. THE PREPARATION OF CULTURE MEDIA 119 The adjustment of the reaction of media is largely determined 03' the particular uses for which the media are designed. For examinations in the practice of sanitation, such as analyses of water, ice, and milk, etc., the American Public Health Association recommends a standard reac- tion of + 1 per cent (the plus sign is used to indicate acidity, the minus alkalinity; + 1 per cent is the expression used to indicate that one per per cent of y sodium hydrate solution would be required to neutralize the medium or 10 c.c. to the liter). For general work with pathogenic bacteria, the most favorable reaction for routine media is slight alka- linity, neutrality, or an acidity not exceeding + 1 per cent. Methods of Clearing Media. — Clearing with Eggs. — When culture media are prepared from substances containing no coagulable proteid, it is often necessary, for purposes of clearing, to add the whites of eggs, and then to heat for forty-five min- utes in the Arnold sterilizer. In the following detailed descriptions, the direction " clear with egg" has been given whenever such a step is deemed necessary. The exact tech- nique of such a procedure is as follows : In a small pot or pan, the whites of several eggs (one or two eggs to each liter of medium) are beaten up thoroughly with a little water (20 c.c). This egg white is then poured into the medium, which, if hot, as in the case of melted agar or gelatin, must first ■"% ir 1/ V' ?$*a be cooled to about 50° to 55° C. The mixture is then thoroughly shaken and steamed in the Arnold sterilizer for thirty minutes. At the end of this time the flask con- taining the medium is removed from the sterilizer and thoroughly shaken so as completely to break up the coagulum which has formed. It is then replaced and allowed to steam for another fifteen minutes. At the end of this time the medium between the coagula should be clear. It is now ready for filtration through cotton. Fig. 21. — Media in Tubes: a, broth; b, agar slant; c, potato. 120 BIOLOGY AND TECHNIQUE Filtering Media through Cotton. — The filtration of media after clearing, either by the addition of eggs or by the coagulation of the pro- teicls originally contained in it, is best done through absorbent cotton. A small spiral, improvised of copper wire, is placed as a support in the bottom of a large glass funnel. A square piece of absorbent cotton is Fig. 22. — Berkefeld Filter. then split horizontally, giving two squares of equal size. Ragged edges and incisures should be avoided. These two layers of cotton are then placed in the funnel, one piece above the other in such a way that the direction of the fibers of the two layers is at right angles one to the other. They are then gently depressed into the filter with the closed fist. The THE PREPARATION OF CULTURE MEDIA 121 edges of the cotton are made to adhere to the sides of the funnel by allowing a thin stream of tap water to run over them, while smoothing them against the glass with the hand. The medium, when poured into such a filter, should be poured along a glass rod at first, to avoid running down the sides or bursting the filter. After filtration has begun, the filter should be kept as full as possible. The first liter or so which comes through may not be clear, but the filter gains in efficiency as the coag- ulum settles into the fibers of the cotton, and the first yield may be sent through a second time. Filtration of agar or gelatin is best done in a warm room with windows and doors closed, and the filter covered with a lid, to avoid too rapid cooling. The funnel and filter should be warmed just before use. Filtering through Paper. — Many media may be efficiently cleared by filtration through close filter paper without the aid of coagula. The Tubing of Media. — Most of the media described in the foregoing section are used in test tubes. In order to fill these tubes, the media are best poured into a large glass funnel to which a glass discharging tube has been fitted by means of a short piece of rubber tubing (see Fig. 20) . Upon this is placed a thumb cock. The plug is then re- moved from the test tube by catching it be- tween the small and ring fingers of the right hand and the glass outlet is thrust deeply into the test tube, in order to prevent the medium from touching the upper portion of the test tube where the cotton plug will be lodged. About 7 to 8 c.c. is put in each test tube. Sterilization of Media. — By Heat. — Media which contain neither sugars, gelatin, glycerin, nor animal serum may be sterilized in the auto- clave at fifteen pounds pressure for fifteen minutes to half an hour. Media which contain these or other substances subject to injury from the high temperature, must be sterilized by the fractional method, Fig. 23. — Berkefkld Filter. 122 BIOLOGY AND TECHNIQUE i.e., by twenty minutes' exposure in the live steam sterilizer (Arnold, Fig. 9, p. 70) on each of three consecutive days. During the intervals between sterilizations, they should be kept at room temperature or in the incubator, to permit the germination of spores which may be present. Media containing animal serum or other albuminous solutions which are to be sterilized without coagulation, may be sterilized in wate baths, or in hot-air chambers (Fig. 10, p. 71), at temperatures varying ^♦^^i I JtL d Fig. 24. — Reichel Filter. from 60° to 70° C, by the fractional method. In such cases five or six exposures of one hour on succeeding days should be employed. By Filtration. — It is often desirable in bacteriological work to free fluid from bacteria. This is frequently necessary for the sterilization of blood-serum or exudate fluids, or for obtaining toxins free from bac- teria. For these purposes a large variety of filters are in use. Those most commonly employed are of the Chamberland1 or Berkefeld type, which consist of hollow candles made of unglazed porcelain or dia- tomaceous earth. Both these types are made in various grades of fine- ness, upon which depend both the speed of filtration and the efficiency. They are made in various forms and models, some of which are shown 1 Pasteur and Chamberland, Compt. rend, de Pacad. des sci., 1884. THE PREPARATION OF CULTURE MEDIA 123 in the accompanying figures. In most of the methods of filtration commonly employed the fluid which is to be filtered is sucked through the walls of the filter, either by a hand suction-pump or by some form of vacuum-pump attached to an ordinary water-tap. The hollow candle-filter may either be firmly fitted into a cylin- drical glass chimney and surrounded by the fluid which is to be filtered, or else the candle may be connected to the collecting flask with sterile rubber tubing and suspended freely in the fluid. Perfect filters of these types will hold back any of the bacteria known to us at present. Filters before use must be sterilized. The candles themselves are subjected to 150° C. in the hot-air sterilizer for one hour. The glassware and washers necessary for setting up the apparatus may be sterilized by boiling. In order that filters may be re- peatedly used with good result, it is neces- sary that they should be carefully cleaned from time to time. This is best done in the following way: Filters through which fluids from living cultures have passed are first sterilized in the Arnold steam sterilizer. Their exterior is then carefully cleaned with a fine brush. Following this a five-tenths per cent solu- tion of potassium permanganate is passed through them and this again removed by sucking through a five per cent solution of bisulphite of soda. This last is washed out by sending a considerable quantity of dis- tilled water through the filter, which is then dried and sterilized by heat. The suction necessary for filtration through these filters is usually applied by means of the ordinary suction-pump attached to a running faucet. Slanting of Media. — Solid media which are to be used in slanted form in test tubes should be inclined on a ledge (easily improvised of glass tubing) at the proper slant, after the last sterilization. Agar, the medium Fig. 25. — Kitasato Filter. 124 BIOLOGY AND TECHNIQUE most frequently employed in this way, should be left in this position for two or three days. (See Fig. 21, b.) ACTUAL STEPS IN THE PREPARATION OF NUTRIENT MEDIA Broth.— Meat Extract Broth.— 1. To 1,000 c.c. of distilled or clear tap water add 5 gms. Liebig's meat extract, 10 gms. Witte's pepton, and 5 gms. common salt (NaCl). 2. Weigh solution with containing vessel (any suitable agate-ware vessel or glass flask will do). 3. Heat over free flame until thor- oughly dissolved, stirring constantly. 4. Weigh again and make up loss by evaporation. 5. Determine volume. 6. Titrate and adjust to required reaction, heating over free flame for five minutes. 7. Filter through paper until clear. 8. Sterilize. If medium can not be cleared by filtering through paper, clearing by white of egg Yaay be resorted to and the medium filtered through cotton. Meat Infusion Broth. — 1. Infuse 500 gms.1 of lean meat, twelve to twenty-four hours, with 1,000 c.c. of distilled water in refrigerator. 2. Strain through wet cotton flan- nel or wet cheese-cloth and make up volume to 1,000 c.c. 3. Add 5 gms. common salt and 10 gms. Witte's pepton. 4. Weigh with containing vessel. 5. Warm over flame or water bath, stirring until pepton is dissolved, not allowing temperature to rise above 50° C. 6. Determine volume. Fig. 26. — Maassen Filter, for small Quantities of Fluid. 1 Roughly, 1 pound (1± lb.). THE PREPARATION OF CULTURE MEDIA 125 7. Titrate and adjust reaction to neutral. 8. Heat in Arnold sterilizer for thirty minutes; shake or stir well and heat again for fifteen minutes. 9. Determine weight and restore loss by evaporation. 10. Determine volume, titrate, and adjust reaction to desired point (usually one per cent acid). 11. Heat again for five minutes if adjustment of reaction has been necessary.1 12. Filter through absorbent cotton, passing the filtrate through the same filter until clear. 13. Titrate and record the final reaction. Place in cotton-plugged sterile flasks or plugged sterile test tubes, and sterilize for thirty minutes in the Arnold sterilizer on three suc- cessive days, leaving at room temperature in the intervals. Sugar-Free Broth. — 1. Make 1 liter of meat infusion broth, following steps 1, 2, 3, 4, 5, 6, 7, and 82; then filter through thin cotton filter to remove gross particles — total clearing is not necessary. 2. Put the broth in a flask and cool. Then add 10 c.c. of a twenty- four-hour broth culture of B. coli communis. 3. Place the flask, stoppered with cotton, in the incubator at 37° C. for eighteen hours. (The bacteria will ferment and thus destroy any sugar [monosaccharid] which may be present in the broth, and thus render the broth sugar-free and acid.) 4. Heat thoroughly to kill the bacteria. 5. Determine weight and bring to 1,015 gms. Then determine volume and titrate, and adjust to neutral. Heat thoroughly again. 6. Filter through filter paper until clear. 7. The pure sugars, dextrose, lactose, saccharose, etc., are then added to separate portions (250 c.c.) of the broth in the proportion of one per cent. 8. When the sugars are dissolved, tube the broth immediately in fermentation tubes, and sterilize by discontinuous sterilization, never heating over twenty minutes at a time, as heat tends to destroy or change the sugars. Glycerin Broth. — To ordinary, slightly acid or neutral meat infusion broth, add six per cent of C. P. glycerin. Sterilize by fractional method. 1 Media become more acid on boiling, probably because of a driving out of C02, and a second titration therefore becomes necessary. 2 These steps refer to the regular directions for making infusion broth. One liter of previously made infusion broth may be used instead. 126 BIOLOGY AND TECHNIQUE Calcium Carbonate Broth. — -This medium is designed for obtaining mass cultures of pneumococcus or streptococcus for purposes of im- munization or agglutination. To 100 c.c. of meat infusion broth in small flasks, add one per cent of powdered calcium carbonate, and one per cent of glucose. It is a wise precaution to sterilize the dried calcium carbonate in the hot-air chamber before using. Small pieces of marble may be used as sug- gested by Bolduan. Pepton-Salt Solution (Dunham's solution) : 1. Distilled water 1,000 c.c. Pepton (Witte) 10 gms. NaCl 5 " 2. Heat until ingredients are thoroughly dissolved. 3. Filter through filter paper until perfectly clear. 4. Tube twenty-five tubes, and store remainder in 250 c.c. flasks. Sterilize by discontinuous method. Nitrate Solution. — 1. Distilled water 1,000 c.c. Pepton , 1 gm. Potassium nitrate 0.2 " 2. Heat until ingredients are thoroughly dissolved. 3. Filter through filter paper until perfectly clear. 4. Tube twenty-five tubes, and store remainder in 250 c.c. flasks. Sterilize by discontinuous sterilization. Uschinsky'sProteid-Free Medium.1 — To one liter of distilled water add : Asparagin 3.4 grams. Ammonium lactate 10 Sodium chloride 5 Magnesium sulphate 0.2 " Calcium chloride 0.1 " Potassium phosphate 1.0 " When these substances are thoroughly dissolved, add 40 c.c. of glycerin Tube and sterilize. Gelatin. — Meat-Extract Gelatin. — 1. To 1,000 c.c. of distilled water add Liebig's extract 5 gms., pepton 10 gms., NaCl 5 gms., and 120 gms. of the finest French sheet gelatin.2 1 Uschinsky, Cent. f. Bakt., 1, xiv, 1893. 2 The acidity and consistence of the different commercial gelatins vary con- siderably and care should be taken in selecting a uniform and suitable brand, such as Hesterberg's gold label gelatin. It is advisable, when working during the summer or in hot climates, to add 130 instead of 120 grams. THE PREPARATION OF CULTURE MEDIA 127 2. Weigh with containing vessel. 3. Dissolve by warming. 4. Adjust weight, determine volume, titrate, and adjust reaction. 5. Cool to 60° C, add whites of two eggs, and stir thoroughly. G. Heat for thirty minutes, stir thoroughly, and heat for fifteen minutes. 7. Adjust weight. 8. Filter through cotton. 9. Sterilize. Meat-Infusion Gelatin. — 1. Infuse 500 gms. lean meat twelve to twenty-four hours with 1,000 c.c. of distilled water in refrigerator. 2. Strain through wet cotton flannel or wet cheese-cloth and make up volume to 1,000 c.c. 3. Add 5 gms. common salt, 10 gms. Witte's pepton, and 120 gms. of the finest French sheet gelatin. 4. Weigh with containing vessel. 5. Warm over flame or water bath, stirring till pepton and gelatin are dissolved and not allowing temperature to rise above 50° C. 6. Determine volume. 7. Titrate and adjust reaction to neutral. 8. Heat in Arnold sterilizer for thirty minutes; shake or stir well and heat again for fifteen minutes. 9. Determine weight and restore loss by evaporation. 10. Determine volume, titrate, and adjust reaction to desired point, if necessary (one per cent acid). 11. Heat five minutes over free flame, constantly stirring, if ad- justment of reaction has been necessary. 12. Filter through absorbent cotton, passing the filtrate through the same filter until clear. 13. Titrate and record the final reaction. Place gelatin in cotton-plugged sterile 250 c.c. flasks or about 8 c.c. in plugged sterile test tubes and sterilize for thirty minutes in the Arnold sterilizer on three successive days, leaving at room temperature in the in- tervals. Never heat the gelatin for longer than is necessary to comply with directions, or it may not be solid enough for use. With some brands of gelatin it may be necessary to add thirteen per cent in order to obtain sufficient stiffness. Agar. — Meat-Extract Agar. — 1. To 1,000 c.c. of distilled water (or tap water) add 15 gms. of thread agar, 10 gms. of Witte's pepton, and 5 gms. of Liebig's meat extract, and 5 gms. of common salt. 128 BIOLOGY AND TECHNIQUE 2. Weigh with containing vessel. 3. Heat over free flame until agar is dissolved, thirty to forty-five minutes. (Great care should be exercised in determining that agar is completely in solution.) 4. Determine weight and make up loss by evaporation. 5. Determine volume, titrate, and adjust to desired reaction. 6. Cool to 60° C. 7. Add whites of two eggs and stir thoroughly. 8. Heat in Arnold sterilizer thirty minutes, stir, and reheat fifteen minutes. 9. Weigh and make up loss by evaporation. 10. Determine volume, titrate, and correct reaction if necessary.1 11. Heat for five minutes, if reaction is corrected. 12. Filter through cotton, tube, and sterilize. Meat-Infusion Agar.2 — (A) 1. Infuse 500 gms. lean meat twelve to twenty-four hours in 500 c.c. of distilled water in refrigerator. 2. Strain through wet cotton flannel or wet cheese-cloth, and make up volume to 500 c.c. 3. Add 10 gms. of Witte's pepton and 5, gms. of common salt. 4. Weigh solution and containing vessel. 5. Warm over free flame or water bath till pepton and salt are dis- solved, not allowing temperature to rise above 50° C. 6. Determine volume, titrate, and neutralize. (B) 7. Add 15 gms. of thread agar to 600 c.c. of distilled water and boil over free flame for thirty to forty-five minutes, watching and stirring constantly till agar is completely dissolved. This will lose weight by evaporation; final weight should be 515 gms. 8. Cool this to about 60° C. (C) 9. Then to the solution A of meat infusion (at 50° C.) add the solution B of agar (at 60° C). 10. Heat for thirty minutes in Arnold sterilizer. Shake or stir thoroughly, and heat fifteen minutes more. Adjust weight by adding water. 11. Determine volume, titrate, and adjust reaction to plus one per cent acid or any desired reaction. 12. Boil for two minutes over free flame, constantly stirring. 1 While titrating, care should be taken that medium does not solidify along sides of vessel. Agar may be made more quickly and successfully in autoclave. 2 Glycerin agar is made by adding 6 per cent of C. P. glycerin to meat-extract or meat-infusion agar. THE PREPARATION OF CULTURE MEDIA 129 13. Filter through absorbent cotton, passing the filtrate through the same filter until clear. 14. Titrate and record final reaction. Place agar in cotton-plugged sterile flasks or plugged sterile test tubes and sterilize for thirty minutes on three successive days. Lactose-Litmus-Agar (Wurtz). — 1. Put 1,500 c.c. distilled water in previously weighed agate-ware vessel. 2. Add 15 gms. thread agar and boil over free flame for thirty to forty-five minutes, watching and stirring constantly till the agar is completely dissolved. 3. Add 5 gms. Liebig's extract of meat, 5 gms. NaCl, 10 gms. Witte's pepton, and dissolve completely. 4. Restore loss by evaporation to 1,035 gms. 5. Determine volume, titrate, and adjust reaction to one per cent acid. 6. Place in a flask and cool to 60° C. 7. Add the whites of two eggs beaten up in 50 c.c. of water and mix thoroughly. 8. Heat for thirty minutes in Arnold sterilizer, shake thoroughly, and heat again for fifteen minutes. 9. Adjust weight. 10. Filter through absorbent cotton to clear. 11. Add two per cent pure lactose (milk sugar).1 12. Add enough pure five per cent litmus solution 2 to bring to purple color when cold. 13. Tube and sterilize. Welch's Modification of Gnarnieri's Medium.3 — This medium is made on a meat-infusion basis, according to the directions given for the prep- aration of meat-infusion agar. It contains 5 grams of agar, 80 grams of gelatin, 5 grams of NaCl, and 10 grams of pepton to one liter. It should 1 Add lactose and litmus to 250 c.c. for 25 tubes; keep the remainder, with- out lactose, stored in small sterile flasks for further use. 2 The litmus solutions used in the preparation of media are best made up as fol- lows: Litmus in substance — Merck's purified, or Kaulbaum's — is dissolved in water to the extent of 5 per cent. The solution is made by heating in an Arnold sterilizer for about one to two hours, shaking occasionally. The solution is then filtered through paper and sterilized. It should be kept sterile, as molds will grow in it otherwise. A standard litmus solution, which is marketed for laboratory purposes, known as " Kubel and Thiemann's " solution, may be used. 3 Welch, Bull. Johns Hopkins Hosp. 9 130 BIOLOGY AND TECHNIQUE be adjusted to a neutral reaction. It is used for stab cultures and is designed chiefly for pneumococcus cultivation and storage. Dorsett Egg Medium. — This medium is chiefly useful for the culti- vation of tubercle bacilli. 1. Carefully break eggs and drop the contents into a wide-mouthed flask. Break up the yolk with a sterile platinum wire, and shake up the flask until the whites and yolks are thoroughly mixed. 2. Add 25 c.c. of distilled water to every four eggs; strain through sterile cloth. 3. Pour 10 c.c. each into sterile test tubes and slant in an inspissa- tor and expose to 73° C. for four to five hours on two days. 4. On the third day, raise the temperature to 76° C. 5. The sterilization may be finished by a single exposure to 100° C. in the Arnold sterilizer for fifteen minutes. Before inoculation, add two or three drops of sterile water to each tube. Potato Media. — Large potatoes are selected, carefully washed in hot water, and scrubbed with a nail brush. They are then peeled, considerably more than the cuticle being removed. The peeled potatoes are again washed in running water for a short time, following which cylindrical pieces are removed from them with a large apple corer. The cylinders are cut into wedges by oblique cuts. Since the reaction of the potato is normally acid, this should be cor- rected by washing the pieces in running water over night, or, better, by immersing them in a one per cent solution of sodium carbonate for half an hour. The pieces are then inserted into the large variety of test tubes known as " potato tubes." (See Fig. 21, c.) In the bottom of the tubes a small amout of water (about 1 c.c.) or a small quantity of moist absorbent cotton should be placed in order to retard drying out of the potato. The tubes are sterilized by fractional sterilization, twenty minutes to half an hour in the Arnold sterilizer on three successive days. Glycerin Potato. — In preparing glycerin potato the potato wedges are treated as above, and are then soaked in a ten to twenty-five per cent aqueous glycerin solution for one to three hours. A small quantity of a ten per cent glycerin solution should be left in the tubes. In steril- izing these tubes, thirty minutes a day in the Arnold after heating of the sterilizer should be regarded as sufficient, to avoid changes in the glycerin. Milk Media. — Fresh milk is procured and is heated in a flask for THE PREPARATION OF CULTUR EMEDIA 131 fifteen minutes in an Arnold sterilizer. It is then set away in the ice chest for about twelve hours in order to allow the cream to rise. Milk and cream are then separated by siphoning the milk into another flask. It is rarely necessary to adjust the reaction of milk prepared in this way, since, if acid at all, it is usually but slightly so. If, however, it should prove more than 1.5 per cent acid, it should be discarded or neutralized with sodium hydrate. The milk may then be poured into test tubes without further additions, or litmus solution may be added in a quantity sufficient to give a purplish blue color. The tubes are sterilized by fractional sterilization in the Arnold sterilizer for thirty minutes on three successive days. Serum Media. — Loeffler's Medium. — Beef blood is collected at the slaughter house in high cylindrical jars holding two quarts or more. While absolute sterility in getting such blood can not usually be as- sured, it is desirable that attempts should be made to avoid contamination as much as is feasible by previously sterilizing the jars, keeping them covered, and exercising care in the collection of the blood. The blood is allowed to coagulate in the jars, and should not be moved from the slaughter house until coagulated. All unnecessary shaking of jars should be avoided. As soon as the coagulum is fully formed, adhesions between the clot and the sides of the jar should be carefully separated with a sterile glass rod or wire. The jars are then set away in the ice chest for twenty-four to thirty-six hours, to allow the serum to separate from the clot. At the end of this time clear serum will be found over the top of the clot, and between the clot and the jar, and this should be pipetted off, preferably with a large pipette of 50 to 100 c.c. capacity, or siphoned off with sterile glass tubing, and trans- ferred to sterile flasks. If, by accident or through carelessness, many red cells have been taken off with the serum, the flasks may again be set away in the ice chest to permit settling out of the corpuscles, when the serum is again siphoned off. To three parts of the clear serum is then added one part of a one per cent glucose beef infusion or veal infusion bouillon. The mixture is filled into tubes, preferably the short test tubes commonly used for diagnostic diphtheria cultures. The tubes are then placed in a slanting position in the apparatus known as an inspissator (seep. 71). This is a double-walled copper box covered by a glass lid, cased in asbestos, and surrounded by a water jacket. It is heated below by a Bunsen flame. Together with the tubes a small open vessel containing water 132 BIOLOGY AND TECHNIQUE should be placed in the inspissator to insure sufficient moisture. The temperature of the inspissator is now raised to 70°-75° C, care being taken that the rise of temperature takes place slowly. The temperature is maintained at this point for two hours, and the process is repeated, for the same length of time, at the same temperature, on six successive days, preferably without removing the tubes from the inspissator at any time. It is also possible, though less regularly yielding good results, to sterilize in the inspissator for one day, following this on the second and third days by exposure for thirty minutes to 100° C. in the Arnold steam sterilizer. In doing this, the Arnold should be very gradually heated, at first without outer jacket, this being lowered only after thorough heating has taken place. If such precautions are not taken, the medium will be spoiled. Serum-Water Media for Fermentation Tests. — For the deter- mination of the fermentative powers of various microorganisms for purposes of differentiation, Hiss has devised the following media in which the cleavage of any given carbohydrate is indicated, not only by the production of an acid reaction, but by the coagulation of the serum proteids. Obtain clear beef serum by pipetting from clotted blood in the same way as this is obtained for the preparation of Loeffler's blood-serum medium. Add to this, two or three times its bulk of distilled water, making a mixture of serum and water in proportions of one to two or three. Heat the mixture for fifteen minutes in an Arnold sterilizer at 100° C. to destroy any diastatic ferments present in the serum. Add one per cent of a five per cent aqueous litmus solution (the varia- tion in the different litmus preparations as obtained in laboratories necessitates a careful addition of an aqueous litmus solution until the proper color, a deep transparent blue, is obtained, rather than rigid adherence to any quantitative directions). Add to the various fractions of the medium thus made, one per cent respectively of the sugars which are to be used for the tests. For the preparation of inulin medium, made in this way for pneu- mococcus-streptococcus differentiation, it is necessary to sterilize the inulin dissolved in the water to be added to the serum in an autoclave at high temperature (fifteen pounds for fifteen minutes) in order to kill spores before mixing with the serum. Before the serum is added, the inulin solution should be cooled. The serum-water media are sterilized by the fractional method at 109° C, at which temperature they remain fluid. , THE PREPARATION OF CULTURE MEDIA 133 Special Media for Colon -Typhoid Differentiation.1 — Hiss' Plating Me- dium.2— The composition of this medium is as follows: Agar 15 gms. Gelatin 15 " Liebig's meat extract 5 " Sodium chloride 5 " Dextrose 10 " Distilled water 1,000 c.c. The agar is thoroughly dissolved in 1,000 c.c. of distilled water , either over a free flame or in the autoclave. When the agar is melted, the gela- tin, meat extract, and salt are added and dissolved by further heating. Any loss in weight is then adjusted by the addition of water. No titra- tion or adjustment of reaction is necessary. The medium should be cleared, in the usual way, with the whites of two eggs, and filtered through cotton. To the cleared medium is added one per cent of dextrose, and the medium tubed, about 8 c.c. to each tube, and sterilized. Hiss' Tube Medium. — The composition is as follows: Agar 5 gms. Gelatin 80 " Liebig's meat extract 5 " Sodium chloride 5 " Dextrose 10 " Distilled water 1,000 c.c. The method of preparation is the same as for the plating medium. The agar is thoroughly dissolved, and then the gelatin, meat extract, and salt are added and dissolved. After adjusting the loss in weight, the volume should be determined, a careful titration made, and the re- action adjusted to one and five-tenths per cent acid by the addition of Y HC1 solution. The medium is then cleared with the whites of eggs, filtered, and one per cent dextrose added. It is then tubed and sterilized. Hesse's Medium,.3 — The medium devised by Hesse for typhoid-colon differentiation depends for its usefulness, as does the Hiss tube medium, upon the great motility of the typhoid bacillus. It may be used directly for the examination of feces or, as suggested by Jackson and Melia,4 after preliminary enrichment of the material to be examined by the use of the lactose-bile medium of Jackson. (See p. 138.) 1 For details of use of these special media see also chapter on Bacillus typhosus. 2 Hiss, Jour. Exp. Med., ii, 1897; Jour. Med. Research, viii, 1902. 3 Hesse, Zeit. f. Hyg., lviii, 1908. 4 Jackson and Melia, Jour, of Inf. Dis., vi, 1909. 134 BIOLOGY AND TECHNIQUE The Hesse medium is made up as follows: Agar 5 gms. (4.5 gms. absolutely dry.) Pepton (Witte) 10 " Liebig's beef extract .... 5 " Sodium chloride 8.5 " Distilled water 1.000 c.c. Jackson and Melia, in studying this medium, have found that complete drying of the agar and the use of 4.5 gms. of this dried prepara- tion give more uniform results, since the amount of moisture in com- mercial agar varies considerably. The preparation of the medium is as follows: Dissolve 4.5 gms. of dry agar in 500 c.c. of distilled water over a free flame, making up for loss by evaporation. In another vessel 10 gms. of pepton, 5 gms. of beef extract, and 8.5 gms. of salt are dis- solved in 500 c.c. of distilled water. This may be heated until com- plete solution has taken place and the loss by evaporation made up. The two solutions are then mixed and heated for thirty minutes; loss by evaporation is then made up with distilled water and the solu- tion is filtered through cotton until clear. The reaction is then adjusted to one per cent acidity and the medium tubed — 10 c.c. to each tube. Sterilize in autoclave, cool, and store in ice chest. The typhoid bacillus is characteristic on the Hesse medium only when the dilution poured in the plates is so high that only a few colonies appear. The typhoid colonies are much larger than are the colon colonies and may often be several centimeters in diameter. They show a small opaque center and a delicately opalescent body. Their form is perfectly circular. Piorkowski's Urine Gelatin.1 — Normal urine of a specific gravity of about 1.020 is collected for several days. At the end of this time, when its reaction has become alkaline, pepton five per cent and gelatin three and three-tenths per cent are added. This mixture is heated upon a water bath for one hour, filtered, and tubed. The tubes are sterilized by the fractional method. This medium allows the differentiation of typhoid-bacillus colonies from those of the colon bacillus. In using it for the isolation of typhoid bacilli from the feces, two loopfuls of feces are placed into a tube of the melted urine gelatin, and from this, dilutions are made into other tubes, taking four loopfuls from the first into the second and six to eight from i Piorkowski, Deut. med. Woch., vol. 25, 1899. THE PREPARATION OF CULTURE MEDIA 135 the second to the third. Plates are then poured and kept at about 20° C. The typhoid colonies show fine processes or filaments, while the colon colonies are quite compact. Capaldi's Medium} — The composition of this medium is as follows: Distilled water 1,000 c.c. Pepton (Witte) 20 gms. Gelatin 10 Agar 20 Dextrose (or preferably mannit) 10 NaCl 5 KC1 5 It is advisable to dissolve the agar in 500 c.c. of water, making up the loss by evaporation with distilled water, and to dissolve the other ingredients in a similar quantity of water, finally mixing the solutions. The mixture is rendered alkaline by the addition of 10 c.c. of normal NaOH, and is cleared, filtered, and filled into test tubes. Plates are made with this medium and surface smears made of the suspected material. B. typhosus grows in small, glistening, bluish translucent colonies. Colonies of B. coli are larger, more opaque, and show a brownish tinge. Conradi-Drigalski Medium.2 — The following are the directions given by the originators of this medium for its preparation, (a) Three pounds of meat are infused in two liters of water for twelve hours or more. After straining, boil for one hour and add 20 gms. of Witte's pepton, 20 gms. of nutrose, 10 gms. of NaCl; boil one hour and filter. To the filtrate add 60 gms. of agar. Boil for three hours (or one hour in an autoclave) until agar is dissolved. Render weakly alkaline to litmus paper, filter, and boil for half an hour more. (b) Litmus solution: Two hundred and sixty c.c. of litmus solution are boiled for ten minutes. (The litmus solution used by Conradi and Drigalski is the very sensitive aqueous litmus recommended by Kubel and Thiemann, and purchasable under the name.) After boiling, 30 grams of chemically pure lactose are added to the litmus solution. The mixture is then boiled for fifteen minutes and, if a sediment has formed, is carefully decanted. (c) Add the hot lactose mixture to the hot fluid agar solution ; mix well and, if necessary, again adjust to a weakly alkaline reaction, litmus 1 Capaldi, Zeit. f. Hyg., xxiii, 1896. 2 Conradi-Drigalski, Zeit* f. Hyg., xxxix, 1902. 136 BIOLOGY AND TECHNIQUE paper being used as an indicator. To this mixture add 4 c.c. of a hot, sterile, ten per cent solution of sodium carbonate, in order to render it alkaline, and 20 c.c. of a freshly made solution of violet crystal (c. p. Hochst), 0.1 gram in 100 c.c. of sterile distilled water. The medium contains thirteen per cent of litmus solution, and one one-thousandth per cent of crystal violet. Large plates are poured. (The plates used by Conradi and Drigalski are large plates 15 to 20 cm. in diameter.) Surface smears are made upon the medium after solidification. These are incubated twenty-four hours. Typhoid colonies are small, blue, and transparent. Colon colonies are large, red, and opaque. Endo's Medium} — 1. Prepare one liter of meat infusion three per cent agar, containing 10 grams of pepton and 5 grams of NaCl. 2. Neutralize and clear by filtration. 3. Add 10 c.c. of ten per cent sodium hydrate solution in order to render it alkaline. 4. Add 10 grams of chemically pure lactose. 5. Add 5 c.c. of alcoholic fuchsin solution, filtered before using. (Endo in his original contribution does not mention the strength of this fuchsin solution, which, however, should be saturated.) This colors the medium red. 6. Add 25 c.c. of a ten per cent sodium sulphite solution. This again decolorizes the medium, the color not entirely disappearing, how- ever, until the agar is cooled. 7. Put into test tubes, 15 c.c. each, and sterilize. The medium should be kept in the dark. For use, plates are poured and surface smears of stools made. Endo claims that upon this medium the typhoid bacillus outgrows the colon bacillus and its colonies remain colorless, while those of bacilli coli become red. Malachite-Green Media.2 — The principle of these media is that mal- achite green inhibits the growth of the colon bacillus without exerting any such influence upon the typhoid bacillus. To make one liter: 1. Prepare a neutral, one-half strength, meat-infusion bouillon (500 grams of meat to 2 liters of water) by the usual technique. 2. Acidify this with 7.5 c.c. of normal hydrochloric acid (to facilitate the solution of agar). 1 Endo, Cent. f. Bakt., xxxv, 1904. 2Loeffler, Deut. med. Woch., 32, 1906. THE PREPARATION OF CULTURE MEDIA 137 3. Dissolve in this 30 grams of agar (three per cent) by boiling. 4. Neutralize with 7 c.c. normal KOH or NaOH (until neutral to litmus) . 5. Add 5 c.c. of normal sodium carbonate solution to make it alka- line and heat in Arnold sterilizer for several hours. 6. Add 100 c.c. of a ten per cent nutrose solution (one per cent). This agar may be sterilized and stored in quantities of 100 c.c. without further manipulation. 7. Before use, redissolve, and to 100 c.c. add 2 to 2.9 c.c. of a two per cent solution of malachite green (trade mark, "Hochst 120"). This solution is made in sterilized water but is not boiled. 8. Fifteen to twenty c.c. of this medium are poured into Petri dishes, allowed to cool, and inoculated by surface smears. It is extremely important to obtain the proper malachite green. Leuchs' Modification of Loeffler's Malachite-Green Medium.1 — 1. To 100 c.c. of one-half strength bouillon made as for Loeffler's malachite- green medium, add — 0 . 5 grams NaCl 1 . " dextrin 3 . " agar 2. Neutralize to litmus. 3. Add 0.5 c.c. normal sodium carbonate. 4. Add 10 c.c. of ten per cent nutrose solution. 5. Boil, filter, and sterilize. 6. Add 1.6 c.c. of a one-tenth per cent solution of malachite green. The malachite green solution is prepared by dissolving 0.1 of a gram in 100 c.c. of hot sterile water. Leuchs does not use the "Hochst 120 " malachite green recommended by Loeffler, but prefers the purified malachite green in crystallized form, which is not the zinc double salt, but the oxalate, and is sent out by various firms marked " Malachite green, pure, crystalline, oxalate." Malachite-Green Bouillon (Peabody and Pratt).2 — To 100 c.c. of beef infusion broth, add 10 c.c. of one per cent solution of malachite green, Hochst 120, made with sterile water. This is tubed, 10 to 15 c.c. being put in each tube. This medium is used merely as an enriching fluid. One drop of the suspected material (emulsified stool) is added to each tube and after 1 Leuchs, Deut. med. Woch., 32, 1906. 2 Peabody and Pratt, Boston Med. and Surg. Jour., clviii, 7, 1908. 138 BIOLOGY AND TECHNIQUE incubation for eighteen to twenty-four hours inoculations may be made upon Conradi-Drigalski plates or other media. According to Peabody and Pratt, the usefulness of the medium is largely dependent upon the reaction. In their work, they found a reaction of five-tenths per cent acidity to phenolphthalein most favor- able. Bile Medium} — (Recommended for blood cultures by Buxton and Coleman.) The medium is prepared as follows: 900 c.c of ox bile 100 c.c glycerin and 20 grams of pepton are mixed. Put into small flasks containing quantities of about 100 c.c. each, and sterilized by fractional sterilization. Jackson's Lactose-Bile Medium.2 — Jackson has devised a medium, now in general use by water-analysts, which is of great use in isolating B. typhosus and B. coli from water, and serves as a valuable enriching medium in isolating these organisms from other sources, such as feces. Jackson and Melia 3 have found that in this medium B. typhosus and B. coli outgrow all other microorganisms and that eventually B. typhosus will even outgrow B. coli. The medium consists of sterilized undiluted ox-bile (or an eleven per cent solution of dry, fresh ox-bile) to which has been added one per cent of pepton and one per cent of lactose. This is filled into fer- mentation tubes holding 40 c.c. of the liquid, and these are sterilized by the fractional method. MacConkey's Bile-Salt Agar. — Sodium glycocholate 5 per cent. Pepton 1.5 " Lactose 3.5 " " Agar 1.5 " Tap water q.s. The agar and pepton are dissolved and cleared in the usual manner and the lactose and sodium glycocholate added before tubing. In this medium the typhoid bacilli produce no change, while the bacillus coli, by producing acid from the lactose, causes precipitation of the bile salts. i Conradi, Deut. med. Woch., 32, 1906. 2 Jackson, " Biol. Studies of Pupils of W. T. Sedgwick," 1906, Univ. Chicago Press. 3 Jackson and Melia, Jour. Inf. Dis., vi, 1909. THE PREPARATION OF CULTURE MEDIA 139 Neutral-Bed Medium. — To 100 c.c. of a one per cent or two per cent glucose agar, add 1 c.c. of a saturated aqueous solution of a neutral- red. The medium is used in tubes, stab or shake cultures being made. The typhoid bacillus produces no change, while members of the colon group render the medium colorless by reduction of the neutral-red and produce gas by fermentation of the sugar. Barsiekoiv's Medium.1 — To 200 c.c. of cold water, add 10 grams of nutrose and allow to soak for one-half to one hour. Pour this into 800 c.c. of boiling water, and heat for twenty minutes in an Arnold sterilizer at 100° C. Filter through cotton and to the opalescent solution of nutrose add 5 grams of NaCl, 10 grams of lactose, and sufficient aqueous litmus solution to give a pale blue color.2 Enriching Substances Used in Media. — For the cultivation of microor- ganisms which are sensitive to their food environment, it is often neces- sary or advisable to add to the ordinary media enriching substances, which empirical study has shown to favor the growth of the organism in question. The substances most commonly used for such enrichment are glucose, nutrose (sodium casemate), glycerin, sodium formate, and unsoliclified animal proteids. As animal and blood serum and whole blood must frequently be used in this way, an understanding of the methods emploj^ed in obtaining these substances is necessary. Method of Obtaining Blood and Blood Media. — Blood serum from beef and sheep may be collected in the manner recommended for the collection of such serum in the preparation of Loeffler's medium, pipetted into test tubes, and sterilized in the fluid state by exposure to tempera- tures ranging from 60° to 65° C, for one hour upon six consecutive days. It is not a simple matter to sterilize serum in this way and requires much time and care. The method most commonly employed, in laboratories which have access to hospitals, for obtaining clear serum depends upon the collection of exudate or transudate fluids by sterile methods directly from the pleural cavity, the abdominal cavity, or the hydrocele cavity. Sterile flasks or test tubes are prepared and the fluid is allowed to flow directly out of the cannula into these. Whenever this is done, it is necessary to eliminate the use of carbolic acid or other disinfectants from the sterili- zation of the instruments and rubber tubing which are used during the 1 Barsiekow, Wien. klin. Rund., xliv, 1901. 2 Filtration may be done through paper, but takes a long time. 140 BIOLOGY AND TECHNIQUE operation. These should be brought into the ward in the water in which they have been boiled and not in strong antiseptic solutions as is fre- quently done. The fluid so obtained may be incubated and the contam- inated tubes discarded. The serum may then be added with sterile pipettes or by direct pouring from tube to tube, in proportions of one to three, to sterile broth or melted agar or gelatin. Agar, if used in this way, is melted and cooled to 60° C, or below. One-third of its volume of warmed exudate fluid is added, and the plates are poured. Serum may be rendered free of bacteria by nitration through a Berkefeld or Pasteur-Chamberland filter. This is an effectual method, but requires much time and care, and for practical work the direct col- lecting of the fluid from the patient into sterile glassware is preferable. Whole blood may be obtained for cultural purposes by bleeding- rabbits or dogs or other animals directly from a blood-vessel into tubes of melted agar. In the case of a rabbit, after the administration of an anes- thetic (ether), an incision is made directly over the trachea, and, by careful section, the carotid artery is isolated, lying close to the side of the trachea. The artery is then closed off with a weak clamp and an incision made into its wall with a sharp-pointed pair of scissors. A small glass cannula to which a piece of rubber tubing is attached is inserted into the vessel and held in place by a thread ligature. The clamp is then removed and the blood, 1 to 2 c.c. at a time, is allowed to flow into melted agar tubes cooled to 60° C. and immediately mixed and slanted. After twenty-four hours the tubes are incubated and the contaminated ones are discarded. This method requires but little prac- tice and yields anywhere from thirty to forty tubes per rabbit. If carefully done, very few of the tubes will be contaminated.1 1 At times in hospital work, the writers have found it convenient to utilize the blood agar plates obtained in sterile blood cultures in which no growth has taken place after three or four days of incubation. These may be kept on ice, best sur- rounded by a broad rubber band to prevent evaporation, and used as required. CHAPTER VIII METHODS USED IN THE CULTIVATION OF BACTERIA INOCULATION OF MEDIA The transference of bacteria from pathological material to media, or from medium to medium, for purposes of cultivation, is usually ac- complished by means of a platinum wire or loop. The platinum wire used should be thin and yet possess a certain amount of stiffness and be about two to three inches in length. This is fused into the end of a glass rod six to eight inches long. It is an advantage, though not necessary, to use rods of so-called " sealing-in " glass which, having the same co- efficient of expansion as platinum, does not crack during sterilization. For work with fluid media, the wire should be bent at its free end so as to form a small loop which will pick up a drop of the liquid. For the inoculation of solid media and the making of stab cultures, a straight " needle " or wire should be used. Other shapes of these wires and spat- ulse from heavy wire have been devised for various purposes and are easily improvised as occasion demands. (See Fig. 27.) When making a transfer from one test tube to another, the tubes should be held between the thumb and first and second fingers of the left hand, as shown in Fig. 28. The plugs are then removed by grasping them between the small and ring fingers and ring and middle fingers of the right hand, first loosening any possible adhesions between glass and plugs b)^ a slight twisting motion. The platinum wire is held meanwhile by the thumb and index fingers of the right hand in the manner of a pen. The wire is heated red hot in a Bunsen flame, and is then passed into the culture tube without being allowed to touch the glass. It is held sus- pended within the tube for a few seconds to permit of cooling before touching the bacterial growth. The wire is then allowed to touch lightly the surface of the growth and a small amount is picked up. (See Fig. 29.) It is then removed from the tube without allowing it to touch the sides of the glass, and is passed into the tube which is to be inooulated. If the tube contains a slanted medium, such as agar, a light stroking motion from the bottom of the slant to its apex will deposit the bacteria 141 142 BIOLOGY AND TECHNIQUE f£^ upon the medium evenly along a central line. The needle may also be plunged downward into the substance of the nutritive material so that in the same tube both surface growth and deep growth may be observed. If a stab culture is to be made in unslanted agar or in gelatin, the needle is simply plunged straight downward as nearly as possible along the axis of the medium. If a fluid medium is being inoculated, the wire should be introduced only into the upper part of the liquid and the bac- teria gently rubbed into emulsion against the side of the glass. The needle is then removed from the tube, the stopper carefully replaced, and the platinum wire immediately sterilized in the flame. This sterilization of plati- num needles after they have been in contact with bacteria should become second nature to those working wTith bacteria, since an infraction against this rule may give rise to serious and widespread consequences. In burning off platinum needles it is wTell to re- member that a part of the glass rod, as well as the wire itself, is introduced into the tubes and may become con- taminated, and for this reason the rod itself, at least in its lower two or three inches, should be passed through the flame as wrell as the wire. As an extra precaution against contamina- tion, the lips of test tubes and flasks and the protruding edges of cotton plugs may be passed through the flame and singed. Fig. 27. — Platinum Wires. THE ISOLATION OF BACTERIA IN PURE CULTURE It is obvious that in many cases where bacteria are cultivated from water, milk, pathological material, or other sources, many species may be present in the same specimen. It is likewise obvious that scientific bacteriological study of any bacterium can be made only if we obtain METHODS USED IN CULTIVATION OF BACTERIA 143 this particular species entirely apart from others, in what is known as "pure culture." The earliest methods for accomplishing this were the methods of Pasteur and of Cohn who depended upon the power of one species to outgrow all others, if cultivated for a sufficient length of time in fluid media. This method, of course, was inadequate in that it was often purely a matter of chance which one of the mixture of species was finally obtained by itself. A later method, by Klebs, depends upon serial dilution, in test tubes of fluid media, by which the eventual transference of one germ only, to the last tube was attempted. Such methods, none of them of great practical value, have been entirely dis- Fig. 28. — Taking Plugs from Tubes before Inoculation. placed by those made possible by Koch's introduction of solid media which may be rendered fluid by heat. The methods now employed for the isolation of bacteria depend upon the inoculation of gelatin or agar, when in the melted state, the thorough distribution of the bacteria in these liquids by mixing, and the sub- sequent congealing of these media in thin layers. By this means the in- dividual bacteria, distributed in the medium when liquid, are held apart and separate when the medium becomes stiff. The masses of growth or " colonies" which develop from these single isolated microorganisms are discrete and are descendants of a single organism, and can be trans- ferred, by means of a process known as " colony-fishing," to fresh sterile culture media. 144 BIOLOGY AND TECHNIQUE Plaitng. — The first method employed by Koch for bacterial isolations was one that consisted in the use of simple plates of glass, about 3x4 inches in size, mounted upon a leveling stand. A wooden triangle, supported upon three adjustable screw-feet, formed the base of this apparatus. Upon this was set a covered crystallizing dish which could be filled with ice water. Upon the top of this rested the sterilized plates under a bell j ar. By screwing up or down upon the supports of the triangle, leveling of the plate could be achieved and controlled by a spirit- level placed at its side. The inoculated gelatin was poured upon the Fig. 29. — Inoculating. plate and spread and rapidly cooled and hardened. by the cold water contained in the crystallizing dish. The original method of Koch has been modified considerably and the method universally employed at present depends upon the use of circular covered dishes, the so-called Petri dishes. These obviate the necessity of a leveling stand and prevent contamination of the plate when once poured. Each Petri dish plate consists of two circular glass dishes; the smaller and bottom dish has an area of 63.6 square centimeters; the larger is used as a cover for the smaller, and forms a loosely fitting lid. METHODS USED IN CULTIVATION OF BACTERIA 145 The plates when fitted together are sterilized and thus form a closed cell which, if properly handled, may remain sterile indefinitely. The technique for making a pour plate for the purpose of isolating bacteria from mixed culture is as follows : The actual "pouring" of plates is preceded by the preparation of usually three graded dilutions of the material to be examined. For this purpose three agar or gelatin tubes are melted and, in the case of the agar, are cooled to a temperature of about 42° C. in a water bath. A platinum loopful of the material to be examined is transferred to one of these tubes. The bacteria are then thoroughly distributed throughout the melted Fig. 30. — Pouring Inoculated Medium into Petri Plate. gelatin or agar by alternately depressing and raising the plugged end of the tube, giving it a rotary motion at the same time. This thoroughly distributes the bacteria throughout the medium without allowing the formation of air-bubbles. Two loopfuls of this mixture are then trans- ferred to the second tube and a similar mixing process is repeated. This second tube contains the bacteria in much greater dilution than the first and the colonies which will form in the plate poured from this tube will be farther apart. A third dilution is then made by transferring five loopfuls of the mixture in the second tube to the third. This again is mixed as before. The contents of the tubes are then poured into three 10 146 BIOLOGY AND TECHNIQUE sterile Petri dishes. The pouring should be done with great care. The cover of the dish is raised along one margin simply far enough to permit the insertion of the end of the test tube, the plug of which has been removed and the lips passed, with a rotary movement, through the name. The medium is poured into the dish without the lips of the tube being allowed to touch either the bottom or the cover of the dish. The cover is then replaced and the medium allowed to harden. When agar has been used, the dishes may be placed in an incubator at 37° C. It is well to place the plates upside down in the incubator. This prevents the condensation water, squeezed out of the agar dur- ing hardening, from collecting on its surface, and forming channels for the diffuse spreading of bacteria. The same end may be attained by the use of Petri plates provided with porous earthenware lids, as suggested by Hill. Simple inversion of the plates, however, usually suffices. When gelatin has been used, the plates are allowed to remain in a dark place at room temperature or in a special thermostat kept at 22°-25° C. Colonies in agar, kept at 37.5° C. , usually develop in eighteen to twenty- four hours ; those in gelatin or agar at room temperature in from twenty- four to forty-eight hours, depending upon the species of bacteria which are being studied. Often in the second dilution, more frequently in the third, the colonies will be found well apart and can then be " fished." The process of " colony-fishing " is one which requires practice and should always be done with care, for upon its success depends the purity of the sub-culture obtained. Colonies should never be fished under the naked eye, no matter how far apart and discrete they may appear, since not infrequently close to the edge of or just beneath a larger colony there may be a minute colony of another species which may be too small to be visible to the naked eye, but which, nevertheless, if touched by accident will contaminate the sub-culture. For proper "fishing," the Petri plate with cover removed, should be placed upon the stage of the microscope and examined with a low power objective, such as Leitz No. 2 or Zeiss AA. The sterilized platinum needle, held in the right hand, is then carefully directed into the line of focus of the lens, while the small finger of the hand is steadied upon the edge of the microscope stage. When the point of the needle is clearly visible through the microscope, it is gently depressed until it is seen to touch the colony and to carry awa}^ a portion of it. The needle is then withdrawn without again touching the nutrient medium or the edges of the glass or the lens, and transferred to a tube of what- ever medium is desired. In this way, individuals of one colony, de- METHODS USED IN CULTIVATION OF BACTERIA 147 scendants of a single bacterium of the original mixture, — are carried over to the fresh medium. Esmarch Roll Tubes.1 — A simple method of obtaining separate colo- nics is that devised by von Esmarch and known as " roll-tube " cultiva- tion. Tubes of melted gelatin are inoculated with various dilutions of the bacterial mixture and, while still liquid, are laid in an almost horizon- Fig. 31. — Streak Plate. tal position upon a block of ice, which has been grooved slightly by means of a test tube filled with hot water. The test tube containing the gelatin, after being placed in this groove, is rapidly revolved by passing the fingers of the right hand across it, while its base is carefully steadied with the left hand. If the revolving is carried out with Esmarch, Zeit. f. Hyg., i, 1886. 148 BIOLOGY AND TECHNIQUE sufficient speed, the gelatin will harden in a thin layer on the inner surface of the tube. The colonies will develop in this layer and may be " fished " by means of a platinum wire with bent point introduced into the tube. This method is useful for certain purposes, but is too inconvenient for routine work. It is now rarely used. Separation of Bacteria by Surface Streaking. — When it is necessary to isolate bacteria like the gonococcus, Bacillus influ- enzae, the pneumococcus, and others, which, because of great sensitiveness to environment and possibly a preference for free oxygen, are not readily grown in pour plates, it is often advantageous first to pour plates of suitable media, allow them to harden, and then gently smear over their surfaces dilutions of the infectious material, usually in three or four parallel streaks. (See Fig. 31.) Upon such plates, if dilutions have been prop- erly made, and this is only a question of judgment based upon an estimation of the numbers of bac- teria in the original material, discrete colonies of the microorganisms sought for may develop, and can be " fished " in the usual manner. The media most favorable for the cultivation of various microorganisms will be discussed in the sections dealing with the individual species. ANAEROBIC METHODS We have seen in a preceding chapter (p. 26) pIG> 32 Deep ^na^ many bacteria, the so-called anaerobes, will Stab Cultivation develop only in an environment from which free of Anaerobic oxygen has been excluded. Bacteria. ^p^g exclusion of oxygen for purposes of anaero- bic cultivation may be accomplished by a variety of methods, depending upon a few simple principles which have been applied, either separately or in combination, by many workers. The earliest methods depended upon the simple exclusion of air by mechanical devices. In other methods, the oxygen of the air is displaced by inert gases (hydrogen), and others again depend upon the oxygen- absorbing qualities of alkaline solutions of pyrogallol. Cultivation by the Mechanical Exclusion of Air. — Koch succeeded in METHODS USED IN CULTIVATION OF BACTERIA 149 growing anaerobic bacteria upon plates by simply dropping upon the surface of the inoculated agar or gelatin, a flat piece of sterile mica. This method, however, rarely succeeds in sufficiently excluding the air and is hardly to be recommended. Liborius' Method.1 — This method consists in the use of deeply filled tubes of agar or gelatin, from which all oxygen has been removed by boiling for fifteen minutes or more. It is advan- tageous, as has been pointed out in the section on anaerobiosis, that media used for this purpose should contain carbohydrates in some form, pref- erably glucose. After boiling, the tubes are rapidly transferred to ice water in order to has- ten the cooling process so that as little oxygen as possible may be absorbed during the harden- ing of the medium. The tubes are then inoc- ulated by deep stabs. After inoculation, the top of the medium may be covered with a thin layer of agar, gelatin, or oil (albolin), and then further sealed with sealing-wax to prevent oxygen- absorption. This method may be utilized for the isolation of anaerobes (as in the original method of Liborius) by inoculating the medium just before it solidifies. The tubes may be gently shaken in order to distrib- ute the bacteria throughout the medium and then rapidly cooled. In this case colonies which de- velop may be scattered throughout the deeper layers of the agar or gelatin, and may be " fished" after breaking the tube. Esmarch' s Method.2 — von Esmarch has applied Fig. 33. — Deep the principles of his roll-tube to the cultivation of Stab Cultiva- anaerobic bacteria. Gelatin tubes are inoculated as TION OF Anaer°- above and roll-tubes prepared. The tubes are then set into cold water to prevent melting of the thin gelatin layer and the interior of the tube is filled with melted gelatin. Roux's Method.3 — Roux recommends the cultivation of anaerobic bacteria by sucking the inoculated gelatin or agar into narrow tubes, 1 Liborius, Zeit. f. Hyg., i, 1886. 2 Von Esmarch, loc. cit 3 Roux, Ann. Past., i, 1887. 150 BIOLOGY AND TECHNIQUE which are then closed at both ends by fusing in the flame. After growth has taken place the tubes may be broken at any desired place and the organism recovered by "fishing." Fluid Media Covered with Oil. — Erlenmeyer flasks orother vessels are partially filled with glucose-bouillon over which a thin layer of al- bolin or other oil is allowed to flow. The oxygen is driven out of the liquid by vigorous boiling for fifteen minutes or more. The simple exclusion of air, also, is the principle underlying the cultivation of anaerobic bacteria in the closed arm of a Smith fermentation tube. Wright's Method.1 — Wright has described a simple and excellent method for the cultivation of anaero- bic bacteria in fluid media. The ap- paratus necessary is easily improvised with the materials at hand in any laboratory. A short piece of glass tubing, constricted at both ends and fitted at each end with a small piece of soft-rubber tubing, is inserted into a test tube containing nutrient broth. The upper end of the inserted glass tubing is connected by the rubber with a pipette passed through the cot- ton plug in the tube. The entire ap- paratus, plus broth, may be sterilized after being put together. When a cul- tivation is made, the fluid in the test tube is inoculated as usual. The fluid is then sucked up into the glass tub- ing until this is completely filled. A downflow of the fluid is then prevented by placing the finger over the pipette through which the suction has been made or by constricting a small piece of rubber tubing attached to the upper end of the pipette. The entire system of tubes is then pushed downward in such a way that both pieces of rubber tubing, attached to the ends of the little glass Fig. 34. — Cultivation of Anaerobes in Fluid under Albolin. 1 Wright, J. H. 1904. Quoted from Mallory and Wright, "Path. Technique," Phila., METHODS USED IN CULTIVATION OF BACTERIA 151 chamber, are kinked. The entire apparatus may then be incubated. Growth of anaerobic bacteria takes place within the air-tight chamber formed by the short glass tubing within the test tube. The fluid in the test tube, outside of this chamber, usually remains clear. When cultivation has been successful, the bacteria may be obtained either for morphological study or for further cultivation, by simply allowing the fluid to flow out of the little air-tight chamber back into the test tube. The method is simple and usually successful. Methods Based upon the Displacement of Air by Hydrogen. — The principle of air-displace- ment by hydrogen, first utilized by Hauser,1 has been widely applied to the cultivation of anaerobic bacteria. In substance it consists of the conduction of a stream of hydrogen through an air-tight chamber in which plates or tubes containing inoculated media have been placed. For the production of the hydrogen, the most convenient apparatus is the Kipp hydro- gen generator or some modification of it. Hydrogen is generated from zinc and sul- phuric acid and this may be passed through a series of Woulfe-bottles, containing solu- tions of lead acetate and of pyrogallic acid, to remove traces of sulphuretted hydrogen and of oxygen, respectively. Some authors recommend also the interpolation of a bottle containing LugoPs solution to absorb traces of acid vapor, and of one with a silver- nitrate solution to take up any hydrogen arsenide which may be derived from impurities contained in the zinc. For anaerobic cultivation upon solid media, the inoculated tubes or plates are placed in an apparatus such as the Novy jar. This is connected with the hydrogen apparatus and hydrogen allowed to flow through it for five or ten minutes, and the stop-cocks then closed. Fig. 35.- Method of Cultivation Media. - Wright's Anaerobic in Fluid 1 Hauser, " Ueber Faulnissbakterien," 1885. 152 BIOLOGY AND TECHNIQUE In applying the hydrogen method to fluid media, flasks containing the broth are fitted with sterile, tightly fitting rubber stoppers per- forated by two holes, through which glass tubes are passed. One of these tubes, the inlet, passes below the surface of the liquid. The other one, the outlet, extends only a short distance below the stopper and is always kept above the surface of the medium. The flasks are inoculated and hydrogen is passed through the medium so that it enters the long tube, bubbles up through the fluid, and leaves by the short tube. The broth may be covered with a thin layer of liquid paraffin or albolin. The Use of Pyrogallic Acid Dissolved in Alkaline Solutions for Oxygen Absorption. — Buchner1 has applied the principle of chemical absorption for the re- moval of oxygen to the cultiva- tion of anaerobic bacteria. This has been made use of in a number of different ways. The method is based upon the fact that alkaline solutions of pyro- gallol possess the power of ab- sorbing large quantities of free oxygen. At first such solutions are of a light straw-color, which becomes dark brown as oxygen is absorbed. The absorption of all the oxygen in the environ- ment may be assumed when there is no further deepening of the brown color. Buchner first utilized this principle by placing a small wire or glass holder within a large test tube, dropping dry pyrogallol (pyrogallic acid) into the bottom of this tube, then running a five per cent sodium hydrate solution into it, and inserting within this large tube a smaller test tube containing the inoculated culture medium. The large tube was then tightly closed FiG' 36. — Novy Jar. i Buchner, Cent, f. Bakt., I, iv, 1888. METHODS USED IN CULTIVATION OF BACTERIA 153 with a rubber stopper. In this way, the air space surrounding the smaller tube was rendered oxygen free. A simple modification of the preceding method of Buchner has been devised by Wright.1 Stab-cultures of gelatin or agar in test tubes are made in the usual way. The cotton stopper closing the tube is then thrust into the tube to such a depth that its upper end lies at least 1 cm. below the mouth of the tube. A small quantity of sodium or potassium hydrate solution in which some pyrogallic acid has been dissolved, is then allowed to flow on to the cotton of the plug and the mouth of the tube is immediately sealed by a tightly fitting rubber stopper. The cotton stopper in these cases must be made of ab- sorbent cotton; 1.5 to 2.5 c.c. of the pyro- gallic acid solution is usually sufficient for test tubes of ordinary size. For cultivation of anaerobic bacteria upon agar slants, a simple technique may be applied and easily improvised in the labora- tory as follows : The tube of slant agar is inoculated with the infectious material in the usual way. It is then, with stopper removed, inverted into a tumbler or beaker containing about a gram of dry pyrogallic acid. A small quantity of a five per cent or three per cent sodium hydrate solution is then run into the tumbler and this is covered with a thin layer of liquid paraffin or albolin before the pyrogallic acid has been completely dis- solved. In this way, completely anaerobic conditions are obtained in the tube and the growth of anaerobes takes place upon the surface of the slant. For the cultivation of anaerobes in Petri dishes, for purposes of separation, a combination of the pyrogallic acid method and the hydrogen displacement methods is often em- ployed. For this purpose the jars devised by Novy and by Bulloch are extremely convenient. 1 Wright, Jour, of the Boston Soc. of Med. Sci., Dec, 1900. Fig. 37. — Wright's Meth- od of Anaerobic Cultiva- tion by the Use or Pyro- gallic Acid Solution. 154 BIOLOGY AND TECHNIQUE In using the Novy jar, the inoculated plates are set upon a wire frame resting about an inch above the bottom of the jar. The cover is then tightly set in place and the air in the jar exhausted by means of a suction pump. The arrangement of the double stop-cock in the top renders it possible now, by simply turning this, to admit hydrogen from a Kipp generator into the jar. The process of alternate exhaustion and admission of hydrogen may be several times repeated. A combination of air exhaus- tion, oxygen absorption, and hy- drogen replacement ma}^ be prac- ticed in jars such as that shown in Fig. 39. Tubes or plates after inoculation are placed in this jar, on a raised wire frame. Dry py- rogallic acid is placed in the bottom of the jar and the cover tightly fitted. An opening in the side of the jar connects its interior with a bottle containing sodium or potassium hydrate so- lution. Through the stopper of this bottle pass two glass tubes, one of them of such length that it can be pushed down into the alkaline solution, or pulled up- ward above the level of the fluid. This tube connects the jar with the bottle. The other glass tube is short, passing just through the stopper and at the top made in the form of a T, one arm of the T being connected with a Kipp hydrogen generator, the other with a suction-pump. After the jar has been sealed, the glass tube connecting the jar and the bottle is raised above the level of the fluid in the bottle and, the con- nection to the hydrogen generator being closed, the air in the jar is exhausted with the suction-pump. Connection to the suction may then be closed, and the other arm of the T being open, hydrogen is allowed to flow into the jar. Alternate suction and hydrogen replacement may be Fig. 38. — Jar for Anaerobic Cul- tivation. METHODS USED IN CULTIVATION OF BACTERIA 155 carried out two or three times. After the last exhaustion, the glass tube in the bottle connecting it with the jar is pushed down into the fluid, and the vacuum will draw the sodium hydrate solution into the bottom of the jar, dissolving the pyrogallol, which will then absorb any traces of free oxygen remaining in the j ar. Hydrogen is again introduced and the jar closed. If exhaustion of oxygen has been sufficiently thorough, the pyrogallic solution in the bottom of the j ar will remain light brown. A simple method for the separation of anaerobes in plates without the use of hydrogen or of specially constructed jars, may be carried out as follows l: The apparatus used consists of two circular glass dishes, fitting one into the other as do the halves of a Petri dish, and similar to these in every respect except that they are higher, and that a slightly greater space is left between their sides when they are placed together. The From hydrogen generator To exhaustion pump Fig. 39. — Apparatus for Combining the Methods of Exhaustion, Hydrogen, Replacement, and Oxygen Absorption. dishes should be about three-fourths to one inch in height, they need be of no particular diameter, although those of about the same size as the usual Petri dish are most convenient. An important requirement necessary for the success of the method is that the trough left between the two plates, when put together, shall not be too broad, a quarter of an inch being the most favorable. Into the smaller of these plates the inoculated agar is poured exactly as this is done into a Petri dish in the ordinary aerobic work. Pro- longed boiling of the agar before plating is not essential. When the agar film has become sufficiently hard on the bottom of the smaller dish, the entire apparatus is inverted. The smaller dish is now lifted out of the 1 Zinsser, Jour. Exp. Med., viii, 1906. 156 BIOLOGY AND TECHNIQUE larger, and placed, still inverted, over a moist surface — a towel or the wet surface of the table — to prevent contamination. Into the bottom of the larger dish, which now stands open, there is placed a quantity (about 3 grams) of dry pyrogallic acid. Into this, over the pyrogallic acid, the smaller dish, still inverted, is then placed. A five per cent solu- tion of sodium hydrate is poured into the space left between the sides of the two dishes, in quantity sufficient to fill the receiving dish one-half full. While this is gradually dissolving the pyrogallic acid, albolin, or any other oil (and this is the only step that requires speed), is dropped from a pipette, previously filled and placed in readiness, into Fig. 40. — Simple Apparatus for Plate Cultivatioii of Anaerobic Bacteria. (Zinsser.) the same space, thus completely sealing the chamber formed by the two dishes. If these steps have been performed successfully, the pyrogallic solu- tion will at this time appear of a light brown color, and the smaller plate, with its agar film, will float unsteadily above the other. Very rapidly, as the pyrogallic acid absorbs the free oxygen in the chamber, this plate is drawn down close to the other, and the acid assumes a darker hue, which remains without further deepening even after three or four days' incubation. THE INCUBATION OF CULTURES After bacteria have been transferred to suitable culture media, it is necessary to expose them to a temperature favorable to their develop- ment. In the case of many saprophytes, the ordinary room temperature METHODS USED IN CULTIVATION OF BACTERIA 157 is sufficiently near the optimum to obviate the use of any special appara- tus for maintaining a suitable temperature; in the case of most patho- genic bacteria, however, the body temperature of man, 37.5° C, is Fig. 41. — Incubator^ either a necessary requirement, or at any rate favors speedy and char- acteristic development. For the purpose of obtaining a uniform temperature of any required degree; the apparatus in general use is the so-called incubator or ther- 158 BIOLOGY AND TECHNIQUE mostat. This may be adjusted for gelatin cultivation at 20 to 22° C, or for agar, broth, or other media at 37.5° C. Incubators, while varying in detail, are all constructed upon the same principles. They consist of double-walled copper chambers, which are fitted with a set of double doors, the outer being made of as- bestos-covered metal, the inner of glass. (See Fig. 41.) The space be- Fig. 42. — Thermo-regulator. (Lautenschlager . ) Fig. 43. — Thermo-regulator. (Reichert.) tween the two walls is filled with water, which, being a poor heat conduc- tor, tends to prevent rapid changes of temperature within the chamber as the result of changes in the external environment. Both walls are perforated above by openings to admit thermometers into the in- terior and one wall is perforated so that a thermo-regulator may be inserted into the water jacket. The under surface of the chamber is METHODS USED IN CULTIVATION OF BACTERIA 159 heated by a gas flame, the size of which is automatically regulated by the thermo-regulator. A number of thermo-regulators are on the market, all of them con- structed upon modifications of the same principle. One of the most efficient of those in common use is that shown in Fig. 42. This con- sists of a long tube of glass fitted with a metal cap through which an in- let tube (A) projects into the interior. Slightly below the middle of the tube there is a glass diaphragm separating its interior into two com- partments. In the middle of the diaphragm an aperture leads into a spiral of glass which projects into the lower compartment. The lower compartment is filled with ether and mercury. The lower end of the inlet tube (A) has a wedge-shaped slit. The gas from the supply pipe passing through the tube (A) is conducted through the slit-like opening in its lower end into the inner chamber and passes out to the burner through the elbow (B). When the temperature is raised, the ether and mercury in the lower chamber expand and the mercury rises in the upper chamber, gradually restric ting the opening through the V-shaped slit in the inlet tube. Thus the gas supplied to the burner is diminished, the flame reduced, and the temperature again falls. The temperature can be arbitrarily adjusted by raising or lowering the inlet tube. A scale at the upper end of the inlet tube allows exact adjustment. Complete shutting off of the gas is prevented by a small circular opening placed in the inlet tube just above the slit. Another cheaper and simpler ther no-regulator is shown in Fig. 43. This consists of a long tube open at the top and fitted about 1 J inches from the top with two hollow glass elbows. One of these elbows remains open, the other, situated on a slightly lower level, is closed by a brass screw-cap. The tube is filled with mercury to a point slightly above the level of the elbow containing the screw-cap. The height of the mercury can thus be increased or decreased by screwing in or out upon the cap. Into the upper end of the tube there is fitted another device which consists of a T-shaped system of glass tubes, one arm of the T being open and the other closed, the perpendicular leg of the T tapering to a minute opening at the bottom. The gas passes into one arm of the T down through the tapering leg and into the space immediately above the mercury. It then passes out through the open elbow of the main tube. As the mercury rises, it gradually diminishes the space between its surface and the small opening in the tapering tube above it, finally completely shutting off the gas from this source. Gas can now pass only through a minute hole perforating the vertical leg of the 160 BIOLOGY AND TECHNIQUE T an inch above its end. The flame decreases and the temperature again sinks. Since gas pressure in laboratories is apt to vary, it is convenient to interpose between the gas supply and thermo-regulator some one of the various forms of gas-pressure regulators. The use of these is not ab- solutely necessary but aids considerably in the maintenance of a con- stant temperature. The one most commonly employed is the so-called Moitessier apparatus. This consists of a cylindrical metal chamber within which there is fitted an inverted metal bell. Glycerin is poured into the cylinder to the depth of about two inches. An inlet pipe con- Fig. 44. — Moitessier Gas-Pressure Regulator. ducts gas into the open space between the top of the glycerin and the bell. From the top of the bell is suspended a conical piece of metal which hangs free in the outlet pipe. As the gas pressure under the bell increases, this is raised and the opening of the outlet pipe is gradually diminished by the cone. Thus the relation between the pressure in the inlet pipe and the actual quantity of gas passing through is equalized. A cup con- nected to the top of the bell through the roof of the cylinder by a bar can be filled with birdshot and the pressure against the gas can thus be modified to conform with existing conditions. METHODS USED IN CULTIVATION OF BACTERIA 161 Colony Study. — Cultures are usually incubated for from twelve to forty-eight hours. Considerable aid to the recognition of species is derived from the observation of both the speed of growth and the ap- pearance of the colonies. It is therefore necessary to proceed in the study of developed co onies in a systematic way. The development of colonies should be observed in all cases both upon gelatin and upon agar. In forming any judgment about colonies, the acidity or alkalinity, and the special constitution of the media should be taken into account. The colonies are carefully examined with a hand lens and with the low power (Leitz No. 2, Zeiss AA, Ocular No. 2) of the microscope. The colonies should be observed as to size, outline, transparency, texture, color, and elevation from the surface of the media. Much information, V % Fig. 45. — Variations in the Conformation of the Borders of Bacterial Colonies. (After Chester.) also, can be obtained by observing whether a colony appears dry, mucoid, or glistening, like a drop of moisture. By a careful obser- vation of these points, definite differentiation, of course, can not usu- ally be made, but much corroborative evidence can be obtained which may guide us in the methods to be adopted for further identification and for a final summing up of species characteristic as a whole. The Counting of Bacteria. — It is often necessary to determine the number of bacteria per c.c. contained in water, milk, or other substances. For this purpose definite quantities of the material to be analyzed are mixed with gelatin or agar and poured into Petri plates. The exact dilutions of the suspected material must largely depend upon the number of germs which one expects to find in it. The plates, if prepared with gelatin, are allowed to develop at room temperature for twenty-four to 11 162 BIOLOGY AND TECHNIQUE forty-eight hours. If agar has been used, they are usually placed in the incubator at 37.5° C. At the end of this time, the colonies which have developed are enumerated. For this purpose, a Petri dish is placed upon a Wolffhugcl plate. This plate consists of a disk or square of glass which is divided into small squares of one square centimeter each. Diagonal lines of these squares running at right angles to each other are subdivided into nine divisions each in order to facilitate counting when the colonies are unusually abundant. The Petri dish is placed upon the plate in such a way that the center of the dish corresponds to the center of the plate. Fig. 46. — Wolffhugel Counting Plate. The colonies in a definite number of squares are then counted. The greater the number of squares that are counted the more accurate the estimation will be. When the growth is so abundant that only a limited number of squares can be counted, these should be chosen as much as possible from different parts of the plate, and in practice one counts usually six squares in one direction and six at right angles to these, so as to preclude errors arising from unequal distribution. The final calcu- lation is then made by ascertaining the average number of colonies con- tained in each square centimeter. If standard Petri dishes have been METHODS USED IN CULTIVATION OF BACTERIA 163 used, this is multiplied by 63.6, the number of squares in the area of the dish, and then by the dilution originally used. • Thus if twelve squares have been counted with a total number of one hundred and forty-four colonies — the average for each square is twelve. Twelve times 63.6 is 763.2, which represents the total number of colonies in the plate. Now if 0.1 c.c. of the original material (water or milk) has been plated, this material may be assumed to have contained 10 X 763.2, or 7,632 bacteria to each cubic centimeter. If dishes of an unusual size are employed, the square area must be ascertained by measuring the diameter and multiplying it by n (x X diameter = area) (x = 3.141592). CHAPTER IX METHODS OF DETERMINING BIOLOGICAL ACTIVITIES OF BACTERIA ANIMAL EXPERIMENTATION Gas Formation. — Bacteria of many varieties produce gas from the proteid and the carbobiydrate constituents of their environment. Gas formation can be observed in a very simple manner by making stab cultures in gelatin or agar containing the fermentable nutrient substances. In such cultures bubbles of gas will form along the track of the inoculation, or, in the case of such semisolid media as the tube medium of Hiss, will spread throughout the tube. In the case of some anaerobes gas formation in stab cultures will occur to such an extent that the medium will split and break. It should be borne in mind in carrying out such methods that air is readily carried into the medium with the inoculating needle or loop by splitting of the medium, also that media which have been stored in the cold may absorb air. Expansion of the air in such tubes may simulate small amounts of gas formation and lead to error. It is advisable, therefore, whenever making stab inoculations with the above purpose, to heat the media and rapidly cool them before use. A more accurate method of gas determination is by the use of fer- mentation tubes, such as those devised by Smith. The gas which is formed collects in the closed arm of the fermentation tube and may be quantitatively estimated. The fermentation, with gas production, of certain substances such as carbohydrates, may be determined by adding these materials in a pure state to the media before inoculation with organisms. In the case of carbohydrates this method has proved of great differ- ential value, since the power of splitting specific carbohydrates with gas production is a species characteristic of great constancy for many forms of bacteria. Analysis of Gas Formed by Bacteria. — Carbon Dioxide. — For the estimation both qualitatively and roughly quantitatively of carbon dioxide produced by bacteria, cultures are grown in fermentation tubes containing sugar-free broth (see page 125) to which one per cent of pure dextrose, lactose, saccharose, or other sugars has been added. The tubes are incubated until the column of gas formed in the closed arm no longer 164 DETERMINING BIOLOGICAL ACTIVITIES OF BACTERIA 165 increases (twenty-four to forty-eight hours). The level of the fluid in the closed arm is then accurately marked and the column of gas measured. The bulb of the fermentation tube is then completely filled with f NaOH solution, the mouth closed with a clean rubber stopper, and the bulb inverted several times in order to mix the gas with the soda solution. The tube is then again placed in the upright position, allow- ing the gas remaining to collect in the closed arm. The gas lost may be roughly estimated as consisting of C02. Hydrogen.— -The gas remaining, after removal of the C02 in the pre- ceding experiment, at least when working with carbohydrate solutions, {Sl\ Fig. 47. — Types of Fermentation Tubes. may be estimated as hydrogen. When allowed to collect near the mouth, further evidence of its being hydrogen may be gained by exploding it with a lighted match. Hydrogen Sulphide (H2 S, Sulphuretted hydrogen). — In alkaline media sulphuretted hydrogen, if formed, will not collect as gas, but will form a sulphide with any alkali in the solution. For the estimation of the formation of hydrogen sulphide, bacteria are cultivated in a strong pepton solution to which 0.1 c.c. of a one per cent solution of ferric tartrate or lead acetate has been added. The addition of these substances gives rise to a yellowish precipitate in the bottom of the tubes. If, on 166 BIOLOGY AND TECHNIQUE subsequent inoculation, the bacteria produce H2 S, this precipitate will turn black. The solution recommended by Pake for this test is prepared as follows: 1. Weigh out 30 grams of pepton and emulsify in 200 c.c. of tap water at 60° C. 2. Wash into a liter flask with 80 c.c. tap water. 3. Add sodium chloride 5 grams and sodium phosphate 3 grams. 4. Heat at 100° C. for 30 minutes, to dissolve pepton. 5. Filter through paper. 6. Fill into tubes, 10 c.c. each, and to each tube add 0.1 c.c. of a one per cent solution of ferric tartrate or lead acetate. These solutions should be neutral. 7. Sterilize.1 Accurate quantitative gas axalyses of bacterial cultures can be made only b}^ the more complicated methods used in chemical labora- tories for quantitative gas analysis. The gas; in such cases, is collected in a bell jar mounted over mercury, and subjected to analysis by the usual method described in works on analytical chemistry. Acid and Alkali Formation by Bacteria. — Many bacteria produce acid or alkaline reactions in culture media, their activity in this respect depending to a large extent upon the nature of the nutrient material. Many organisms which on carbohydrate media produce acid will give rise to alkali if cultivated upon media containing only proteids. Information as to the production of acid or alkali can be obtained by the addition of one of a variety of indicators to neutral media. The indicators most often employed for this purpose are litmus or neutral red. Changes in the color of these indicators show whether acids or alkalis have been produced. Great help in differentiation is obtained by adding chemically pure carbohydrates to media to which litmus has been added and then de- termining whether or not acid is formed from the substances by the microorganisms. These tests have been of special importance in the differentiation of the typhoid-colon groups of bacilli. Quantitative estimation of the degree of acidity or alkalinity pro- duced by bacteria may be made by careful titration of definite volumes of the medium before and after bacterial growth has taken place. The variety of acid formed by bacteria depends largely upon the nature of the nutrient medium. The acids most commonly resulting from bacterial growth are lactic, acetic, oxalic, formic, and hippuric acids. Qualitative and quantitative estimation of these acids may be made by any of the methods employed by analytical chemists. Quoted from Eyre, " Bact. Technique," Phila., 1903. DETERMINING BIOLOGICAL ACTIVITIES OF RACTERIA 167 Indol Production by Bacteria. — Many bacteria possess the power of producing inclol. Though formerly regarded as a regular accompani- ment of protcid decomposition, later researches have shown that indol production is not always coexistent with putrefaction processes and occurs only when pepton is present in the pabulum. Indol formation by bacteria is determined by the so-called nitroso- indol reaction. Organisms are grown in sugar-free pepton broth or in the pepton-salt bouillon of Dunham. (See page 126.) Media containing fermentable substances are not favorable for indol production since acids interfere with its formation. The cultures are usually incubated for three or four days at 37° C. At the end of this time, ten drops of con- centrated sulphuric acid are run into each tube. If a pink color appears, indol is present, and we gather the additional information that the microorganism in question has been able to form nitrites by reduction (e.g., cholera spirillum). If the pink color does not appear after the addition of the sulphuric acid alone, nitrites must be supplied. This is done by adding to the fluid about 1 c.c. of a 0.01 per cent aqueous solution of sodium nitrite. The sodium nitrite solu- tion does not keep for any length of time and should be freshly made up at short intervals. Phenol Production by Bacteria. — Phenol is often a by-product in the course of protei'd cleavage by bacteria. To determine its presence in cultures, bacteria are cultivated in flasks containing about 50-100 c.c. of nutrient broth. After three to four days' growth at 37° C, 5 c.c. of concentrated HC1 are added to the culture, the flask is connected with a condenser, and about 10-20 c.c. are distilled over. To the distillate may be added 0.5 c.c. of Millon's reagent (solution of mercurous nitrate in nitric acid) , when a reel color will indicate phenol ; or 0.5 c.c. of a ferric chloride solution, which will give a violet color if phenol is present. Reducing Powers of Bacteria. — The power of reduction, possessed by many bacteria, is shown by their ability to form nitrites from nitrates. This is easily demonstrated by growing bacteria upon nitrate broth (see page 126). Bacteria are transferred to test tubes containing this solution and allowed to grow in the incubator for four or five days. The presence of nitrites is then chemically determined as follows: Two test solutions are necessar}^. I. Naphthylamin 0.1 gram. Aq. dest 20. c.c. Acetic acid (25 per cent) 150. c.c. 16S BIOLOGY AND TECHNIQUE The naphthylamin is dissolved in the water by boiling, is then cooled and filtered through paper, and to the clear filtrate is added the acetic acid. II. Sulphanilic acid 0.5 grams Acetic acid (dil. of 1 to 16) 150 c.c. The solutions are kept separate and mixed in equal parts just before use. To make this test about 4 c.c. of the culture fluid is poured into a clean test tube, and to it are gradually added 2 c.c. of the mixed test solutions. A pink color indicates the presence of nitrites, the intensity of the color being proportionate to the amount of nitrite present. The reducing powers of bacteria may also be shown by their ability to decolorize litmus, methylene-blue, and some other anilin dyes, which on abstraction of oxygen form colorless leukobases. Enzyme Action. — The action of the enzymes produced by bacteria may be demonstrated by bringing the bacteria, or their isolated fer- ments, into contact with the proper substances and observing both the physical and chemical changes produced. In obtaining enzymes free from living bacteria, it is convenient to kill the cultures by the addition either of toluol or of chloroform. Both of these substances will destroy the bacteria without injuring the enzymes. Enzymes may also be obtained separate from the bodies of the bacteria by filtration. Proteolytic Enzymes. — The most common evidences of proteolytic enzyme action observed in bacteriology are the liquefaction of gelatin, fibrin or coagulated blood-serum, and the peptonization of milk. This may be observed both by allowing the proper bacteria to grow upon these media, or by mixing sterilized cultures with small quantities of these substances.1 The products of such a reaction may be separated from the bacteria by filtration and then tested for pepton by the biuret reaction. Proteolytic 2 enzymes may also be determined by growing the bac- teria upon fluid media containing albumin solutions, blood serum, or milk serum, then precipitating the proteids by the addition of ammonium sulphate (about 30 grams to 20 c.c. of the culture fluid) and warming between 50 to 60° C. for thirty minutes. The precipitate is then filtered off, the filtrate made strongly alkaline with NaOH, and a few drops of copper sulphate solution added. A violet color indicates the pres- ence of pepton — proving proteolysis of the original albumin. 1 Bitter, Archiv f. Hyg., v, 1886. 2 Hankin and Wesbrook, Ann. Past., vi, 1892, DETERMINING BIOLOGICAL ACTIVITIES OF BACTERIA 169 O* P^B. Di astatic Enzymes. — The presence of diastatic ferments may be determined by mixing broth cultures of the bacteria with thin starch paste. It is necessary that both the cultures and the starch paste be absolutely free from sugar. After remaining in the incubator for five or six hours, the fluid is filtered and the filtrate tested by methods used for determining the presence of sugars. Inverting Ferments. — Inverting ferments are determined by a pro- cedure similar to the above in principle. Dilute solutions of cane sugar are mixed with old cultures or culture filtrates of the respective J^)} bacteria and the mixture allowed %3^M to stand. It is then filtered, and the filtrate tested for glucose, preferably by Fehling's solution. ANIMAL EXPERIMENTATION In the study of pathogenic microorganisms, animal experi- mentation is essential in many instances. The virulence of any given organism for a definite ani- mal species and the nature of the lesions produced are character- istics often of great value in differentiation. Isolation, more- over, of many bacteria is greatly facilitated by the inoculation of susceptible animals and recovery of the pathogenic organism from the heart's blood or from the lesions produced in various organs. That investigations into the phenomena of immunity would be absolutely impossible without the use of animal inoculation is, of course, self- evident, for by this method only can the action of bacteria in relation to living tissues, cells, and body-fluids be observed . The animals most commonly employed for such observations are guinea-pigs, white mice, white rats, and rabbits. The method of inoculation may be either subcutaneous, intrapleural, intraperi- toneal, intravenous, or subdural, etc. It must be borne in mind always that the mode of inoculation may influence the course of an \J Fig. 48. — Types of Gelatin Liquefac- tion by Bacteria. 170 BIOLOGY AND TECHNIQUE infection no less than does the virulence of the microorganism or the size of the dose. Inoculations are made with some form of hypodermic needle fitted to Fig. 49. — Intraperitoneal Inoculation of Rabbit. Fig. 50. — Intravenous Inoculation of Rabbit. a syringe. The most convenient syringes are the all-glass Luer or the Debove syringes, which, however, are expensive. Any form of steriliz- able syringe may be used. In making inoculations the hair of the DETERMINING BIOLOGICAL ACTIVITIES OF BACTERIA 171 animal should be clipped and the skin disinfected with carbolic acid or alcohol. Subcutaneous inoculations are most conveniently made in the abdom- Fig. 51. — Intraperitoneal Inoculation of Guinea-pig. Fig. 52. — Guinea-pig Cage, inal wall, where the skin is thin. After clipping and sterilizing, the skin is raised between the fingers of the left hand and the needle plunged 172 BIOLOGY AND TECHNIQUE in obliquely so as to avoid penetrating the abdominal wall and entering the peritoneum. In making intraperitoneal inoculations, great care must be exercised not to puncture the gut. This can be avoided by passing the needle first through the skin in an oblique direction, then turning it into a posi- tion more vertical to the abdomen and perforating the muscles and perito- neum by a very short and carefully executed stab. Intravenous inoculations in rabbits are made into the veins running along the outer margins of the ears. The hair over the ear is clipped and the animal held for a short time head downward so that the vessels of the head may fill with blood. An assistant holds the animal firmly in Fig. 53. — Rabbit Cage. a horizontal position, the operator grasps the tip of the ears with the left hand, and carefully passes his needle into the vein in the direction as nearly as possible parallel to its course. (See Fig. 50.) Mice are usually inoculated under the skin near the base of the tail. They may be placed in a j ar over which a cover of stiff wire-gauze is held. They are then grasped by the tail, by which they are drawn up between the side of the jar and the edge of the wire cover, so that the lower end of the back shall be easily accessible. The skin is then wiped with a piece of cotton dipped in carbolic solution and the needle is in- serted. Great care must be exercised to avoid passing the needle too close to the vertebral column. Mice are extremely delicate, and any injury to the spine usually causes immediate death. DETERMINING BIOLOGICAL ACTIVITIES OF BACTERIA 173 The various forms of animal holders which have been devised are rarely necessary in bacteriological work unless working unassisted, im- mobilization of the animals being easily accomplished by the hands of a skilled assistant. Autopsies upon infected animals must be carefully made. The ani- mals are tied, back down, upon pans fitted in the corners with clamps for the strings. They are then moistened either with hot water or with a weak solution of carbolic acid, so that contamination by hair may be avoided. A median cut is made, the skin is carefully dissected back, and the body cavities are opened with sterile instruments. Cultures may then be taken from exudates, blood, or organs under precautions similar to those recommended below for similar procedures at autopsy upon man. Inoculated animals should be, if possible, kept separate from healthy animals. Rabbits and guinea-pigs are best kept in galvanized iron-wire cages, which are fitted with floor-pans that can be taken out and cleaned and sterilized. Mice ma}^ be kept in battery jars fitted with perforated metal covers. The mice should be supplied with large pieces of cotton upon batting since they are delicately susceptible to cold. CHAPTER X THE BACTERIOLOGICAL EXAMINATION OF MATERIAL FROM PATIENTS In making bacteriological examinations of material taken from living patients, or at autopsy, the validity of result is as fully dependent upon the technique by which the material is collected, as upon proper manipulation in the later stages of examination. Material taken at autopsy should be, if possible, directly transferred from the cadaver to the proper culture media. If cultures are to be taken from the liver, spleen, or other organs, the surface of the organ should first be seared with a hot scalpel and an incision made through the cap- sule of the organ in the seared area, with the same instrument. The platinum needle can then be plunged through this incision and material for cultivation be taken with little chance of surface contamination. When blood is to be transferred from the heart, the heart muscle may be incised with a hot knife, or else the needle of a hypodermic syringe may be plunged through the previously seared heart muscle and the blood aspirated. The same end can be accomplished by means of a pointed, freshly prepared Pasteur pipette. In taking specimens of blood at au- topsy it is safer to take them from the arm or leg, by allowing the blood to flow into a broad, deep cut made through the sterilized skin, than from the heart, since it has been found that post-mortem contamination of the heart's blood takes place rapidly, probably through the large veins from the lungs. Exudates from the pleural cavities, the pericardium, or the peritoneum may be taken with a sterilized syringe or pipette. Materials collected at the bedside or in the operating-room should be transferred directly to the proper media or else into sterile test tubes and so sent to the laborator}^ When the material is scanty, it may be collected upon a sterile cotton swab, which should be immediately re- placed in the sterilized containing tube and sent to the laboratory. Syringes, when used for the collection of exudates or blood, should be of some variety which is easily sterilizable by dry heat, or boiling. Most convenient of the forms in common use are the all-glass "Luer" syringe, or the cheaper "Sub-Q" model. Instruments which can be sterilized only by chemical disinfectants should not be used. When 174 EXAMINATION OF MATERIAL FROM PATIENTS 175 fluids are collected for bacteriological examination, such as spinal fluid, paracentesis fluid, or pleural exudate, it is convenient to have them taken directly into sterilized centrifuge tubes, since it is often necessary to concentrate cellular elements by centrifugalization. By immediate col- lection in these tubes, the danger of contamination from a subsequent transference is avoided. Examination of Exudates. — Pus. — Pus should first be examined morphologically by some simple stain, such as gentian-violet, and by the Gram stain. It is convenient, also, to stain a specimen by Jenner's stain, in order to show clearly the relation of bacteria to the cells. Such morphological examination not only furnishes a guide to future manipulation, but supplies a control for the results obtained by cultural methods. Specimens of the pus are then transferred to the proper media, and pour-plates made or streaks made upon the surface of previously prepared agar or serum-agar plates. A guide to the choice of media is often found in the result of the morphological examination. In most cases, it is well also to make anaerobic cultures by some one of the simpler methods. (See page 148 et seq.) The colonies which develop upon the plates should be studied under the microscope, and specimens from the colonies transferred to cover-glasses and slides for morphological examination and to the various media for further growth and identification. Animal inoculation and agglutination tests must often also be resorted to. A knowledge of the source of the material may furnish considerable aid in making a bacteriological diag- nosis, though great caution in depending upon such aid is recommended. In the examination of peritoneal, pericardial, or pleural exudates it is often advantageous to use the sediment obtained by centrifugalization. A differential count of the cells present may be of aid in confirming the bacteriological findings. Morphological examination and cultural exam- ination are made as in the case of pus. Specimens should also in these cases be stained for tubercle bacilli. Whenever morphological exami- nations of such fluids are negative, no bacteria being found, and especially when among the cellular elements the lymphocytes preponderate, the search for tubercle bacilli should be continued by means of animal inocu- lation. Guinea-pigs should be inoculated intraperitoneally from speci- mens of the fluid. The chances for a positive result are considerably increased if the fluid is set away in the ice chest until a clot has formed and the animals are inoculated with the material from the broken-up clot. 176 BIOLOGY AND TECHNIQUE The routine examination of spinal fluid is best made upon the sedi- ment of centrifugalized specimens. The microorganisms with which we deal most frequently in this fluid are the meningococcus, the pneumococ- cus, the streptococcus, and the tubercle bacillus. If morphological ex- amination reveals bacteria resembling the first three of these in appear- ance and staining-reaction, surface smears should preferably be made upon plates of serum agar, blood agar, or upon tubes of Loeffler's co- agulated blood-serum. Failure to find organisms morphologically does not exclude their presence and careful cultivation should be done in all cases. When organisms are not found by simple morphological examina- tion and the fluid and sediment are scanty, specimens should be stained by the Ziehl-Neelsen method for tubercle bacilli. In such cases it is often of advantage to set away the specimen until a thin threacl-like clot of fibrin has formed in the bottom of the tube. In smears of such a clot, tubercle bacilli are found with far greater ease than they are found in centrifugalized specimens. If these examinations are without result, inoculation of guinea-pigs should be resorted to. Examination of Urine. — Bacteriological examination of the urine is of value only when specimens have been taken Avith sterile catheters, and care has been exercised in the disinfection of the external genitals. Many of the numerous finds of bacillus coli in urine are unquestionably due to defective methods of collecting material. Urine should be cen- trifugalized and the sediment examined morphologically and pour- plates made and surface smears made upon the proper media. If necessary, animal inoculation may be done. In examining urine for tubercle bacilli, special care should be taken in staining methods so as to differentiate from Bacillus smegmatis. Examination of Feces. — Human feces contain an enormous num- ber of bacteria of many varieties. Klein,1 by special methods, es- timated that there were about 75,000,000 bacteria in one milligram of feces. It has been a noticeable result of all the investigations upon the feces, that although enormous numbers can be counted in morpho- logical specimens, only a disproportionately smaller number can be cultivated from the same specimen. This is explicable upon the ground that special culture media are necessary for many of the species found in intestinal contents and upon the consideration that many of the bacteria which are present in the morphological specimen are dead, show- ing that there are bactericidal processes going on in some parts of the 1 Klein, Ref. Cent, f, Bakt., I, xxx, 1901. EXAMINATION OF MATERIAL FROM PATIENTS 177 intestinal tract, possibly through the agency of intestinal secretions, bile, and the action of the products of metabolism of the hardier species present. By far the greater part of the intestinal flora consists of mem- bers of the colon group, bacilli of the lactis aerogenes group, Bacillus fsecalis alkaligenes, Bacillus mesentericus, and relatively smaller num- bers of streptococci, staphylococci, and Gram-positive anaerobes. Many other species, however, may be present without being necessarily con- sidered of pathological significance. Certain writers have recently laid much stress upon a preponderance of Gram-positive bacteria in speci- mens of feces, claiming that such preponderance signifies some form of intestinal disturbance. Herter * has recently advanced the opinion that the presence of Bacillus aerogenes capsulatus in the intestinal canal is definitely associated with pernicious anemia. The determination of these bacilli in the stools is made both by morphological examination by means of Gram stain and by isolation of the bacteria. Such isola- tion is easily done by the method of Welch and Nuttal.2 A suspension of small quantities of the feces in salt solution is made and 1 c.c. of the filtered suspension is injected into the ear vein of a rabbit. After a few minutes the rabbit is killed and placed in the incubator. After five hours of incubation, the rabbit is dissected, and if the Welch bacillus has been present in the feces, small bubbles of gas will have appeared in the liver from which the bacilli may be cultivated in anaerobic stab- cultures. Bacteriological examination of feces is most often undertaken for the isolation of Bacillus typhosus. This is accomplished with a great deal of difficulty because of the overwhelming numbers of colon bacilli which easily outgrow the typhoid germs, and because of the similarity of their colonies in most media. Many methods have been devised for this purpose, all of which depend upon the use of special media aimed at the inhibition of colon and other bacilli and the production of recog- nizable differences in the colonies of typhoid and colon bacilli. Such media are those of Eisner, Hiss, Conradi-Drigalski, Loeffler, Hesse, and others, which are described in the section upon special media. (See page 133.) The methods of using these media will be found described in the chapter on Bacillus typhosus (p. 399.) Cholera spirilla may be recognized in and isolated from the stools of patients by morphological examination, and by cultivation. (See section on Sp. cholerae.) 1 Herter, " Common Bacterial Infections of the Digestive Tract/' N. Y., 1907. 2 Welch and Nuttal. See ref. p. 469. 12 178 BIOLOGY AND TECHNIQUE For the isolation of dysentery bacilli from feces, no satisfactory special methods have as yet been devised. Here we can depend only up- on careful plating upon agar and gelatin and extended colony "fishing," and the study of pure cultures. The complete absence of motility of these bacteria is of much aid in such identification. The determination of tubercle bacilli in stools is difficult and of questionable significance, in that they may be present in people suffer- ing from pulmonary tuberculosis as a consequence of swallowing sputum or infected food, and in that there may be other acid-fast bacilli, such as the timothy bacillus, present. Blood Cultures. — The diagnosis of septicemia can be positively made during life only by the isolation of bacteria from the blood. Such exam- inations are of much value and are usually successful if the technique is properly carried out. A large number of methods are recommended, the writers giving, however, only the one which they have found successful and simple for general use. The blood is taken by preference from the median basilic vein of the arm. If, for some reason (both forearms having been used for saline infusion), these veins are unavailable, blood may be taken from the internal saphenous vein as it turns over the internal malleolus of the ankle joint. The skin over the vein should be prepared, preferably an hour before the specimen is taken, with green soap, alcohol, and bichlo- rid of mercury, as for a surgical operation. The syringe which is used should be of some sterilizable variety (the most convenient the Luer model), which is easily manipulated and does not draw with a jerky, irregular motion. Its capacity sheulcl be at least 10 c.c. It may be sterilized by boiling for half an hour, or preferably, when all-glass syringes are used, they may be inserted into potato-tubes and sterilized at high temperature in the hot-air chamber. Before drawing the blood, a linen bandage is wound tightly about the upper arm of the patient in order to cause the veins to stand out prominently. When the veins are plainly in view, the needle may be plunged through the skin into the vein in a direction parallel to the vessel and in the direction of the blood-stream. After perforation of the skin, while the needle is groping for the vein, gentle suction may be exerted with the piston. Great care should be ex- ercised, however, that the piston is not allowed to slip back, and air be, by accident, forced into the vessel. In most cases no suction is necessary, the pressure of the blood being sufficient to push up the piston. After the blood has been drawn, it should be immediately transferred to the proper media. Epstein has recently recommended the mixture of the blood EXAMINATION OF MATERIAL FROM PATIENTS 179 with sterile two per cent ammonium oxalate solution in test tubes, by which means the clotting is prevented, and transfers can be made more leisurely to culture media. While this method is convenient in cases where blood must be taken at some distance from the laboratory, it is Fig. 54. — Blood-Culture Plate Showing Streptococcus Colonies. halo of hemolysis about each colony. Note preferable, whenever possible, to make cultures from the blood im- mediately at the bedside. The choice of culture media for blood cultures should, to a certain extent, be adapted to each individual case. For routine work, it is best to employ glucose-meat-infusion agar and glucose-meat-infusion broth. At least six glucose-agar tubes should be melted and immersed in water at 45° C. Before the blood is mixed with the medium, the agar should be 180 BIOLOGY AND TECHNIQUE cooled to 41° in order that bacteria, if present, may not be injured by the heat. The blood is added to the tubes in varying quantities, ranging from 0.25 to 1 c.c. each, in order that different degrees of concentration may be obtained. Mixing is accomplished by the usual dipping and rotary motion, the formation of air-bubbles being thus avoided. The mouth of each test tube should be passed through the flame before pour- ing the contents into the plates. Three flasks of glucose broth, contain- ing 100 to 150 c.c. of fluid each, should be inoculated with varying quantities of blood — at least one of the flasks containing the blood in high dilution. The most stringent care in the withdrawal and replace- ment of the cotton stoppers should be exercised.1 The. writers have found it convenient to use, in place of one of these flasks, one containing, in addition to the glucose, 1 gm. of powdered calcium carbonate. This insures neutrality, permitting pneumococci or streptococci, which are sensitive to acid, to develop and retain their vitality. In making blood cultures from typhoid patients, various methods have been recommended. Buxton and Coleman 2 have obtained excel- lent results by the use of pure ox-bile containing ten per cent of glycerin and two per cent of peptone in flasks. The writers have had no difficulty in obtaining typhoid cultures by the use of slightly acid meat-extract broth in flasks containing 200 or more c.c. to which comparatively little blood has been transferred in order to insure high dilution. In estimating the results of a blood culture, the exclusion of contami- nation usually offers little difficulty. If the same microorganism ap- pears in several of the plates and flasks, if colonies upon the plates arc well distributed within the center and under the surface of the medium, and if the microorganisms themselves belong to species which commonly cause septicemia, such as streptococcus and pneumococcus, it is usually safe to assume that they have emanated from the patient's circulation. When colonies are present in one plate or in one flask only, when they are situated only near the edges of a plate or upon the surface of the medium, and when they belong to varieties which are often found sapro- phytic upon the skin or in the air, they must be looked upon with ex- treme suspicion. It is a good rule to look upon all staphylococcus blood cultures skeptically, discarding Staphylococcus albus as a contamina- tion, and taking, if possible, another corroborative culture when the organism is Staphylococcus pyogenes aureus. 1 Small Florence flasks are preferable to the Erlenmeyer flasks' usually employed. 2 Buxton and Coleman, Am.. Jour, of Med. Sci., 1907. SECTION II INFECTION AND IMMUNITY CHAPTER XI FUNDAMENTAL FACTORS OF PATHOGENICITY AND INFECTION When microorganisms gain entrance to the animal or human body, and give rise to disease, the process is spoken of as infection. Bacteria are ever present in the environment of animals and human beings and some find constant lodgment on various parts of the body. The mouth , the nasal passages, the skin, the upper respiratory tract, the conjunctivae, the ducts of the genital system, and the intestines are invariably inhabited by numerous species of bacteria, which, while sub- ject to no absolute constancy, conform to more or less definite charac- teristics of species distribution for each locality. Thus the colon organ- isms are invariably present in the normal bowel, Doderlein's bacillus in the vagina, Bacillus xerosis in many normal conjunctivae, and staphy- lococcus, streptococcus, various spirilla, and pneumococcus in the mouth. In contact, therefore, with the bodies of animals and man, there is a large flora of microorganisms, some as constant parasites, others as transient invaders; some harmless saprophytes and others capable of becoming pathogenic. It is evident, therefore, that the production of an infection must depend upon other influences than the mere presence of the micro- organisms and their contact with the body, and that the occurrence of the reaction — for the phenomena of infection are in truth reactions be- tween the germ and the body defenses — is governed by a number of important secondary factors. In order to cause infection, it is necessary that the bacteria shall gain entrance to the body by a path adapted to their own respective cultural requirements, and shall be permitted to proliferate after gaining a foot- hold. Some of the bacteria then cause disease by rapid multiplication, progressively invading more and more extensive areas of the animal tissues, while others may remain localized at the point of invasion and 181 182 INFECTION AND IMMUNITY exert their harmful action chiefly by local growth and the elaboration of specific poisons. The inciting or inhibiting factors which permit or prohibit an in- fection are dependent in part upon the nature of the invading germ and in part upon the conditions of the defensive mechanism of the subject attacked. Bacteria arc roughly divided into two classes, saprophytes and parasites. The saprophytes are those bacteria which thrive best on dead organic matter and fulfill the enormously important function in nature of reducing by their physiological activities the excreta and dead bodies of more highly organized forms into those simple chemical substances which may again be utilized by the plants in their con- structive processes. The saprophytes, thus, are of extreme importance in maintaining the chemical balance between the animal and plant kingdoms. Parasites, on the other hand, find the most favorable conditions for their development upon the living bodies of higher forms. While a strict separation of the twro divisions can not be made, nu- merous species forming transitions betAveen the two, it may be said that the latter class comprises most of the so-called pathogenic or disease-producing bacteria. Strict saprophytes may cause disease, but only in cases where other factors have brought about the death of some part of the tissues, and the bacteria invade the necrotic areas and break down the proteids into poisonous chemical sub- stances such as ptomains, or through their own destruction give rise to the liberation of toxic constituents of their bodies. It is necessary, therefore, that bacteria, in order to incite disease, should belong strictly or facultatively to the class known as para- sitic. It must not be forgotten, however, that the terms are relative, and that bacteria ordinarily saprophytic may develop parasitic and pathogenic powers when the resisting forces of the invaded subject are reduced to a minimum by chronic constitutional disease or other causes. Organisms that are parasitic, however, are not necessarily pathogenic, and there are certain more or less fundamental requirements which experience has taught us must be met by an organism in order that it may be infectious (or pathogenic) for any given animal; and by infec- tiousness is meant the ability of an organism to live and multiply in the animal fluids and tissues. For instance, an organism which is shown not to grow at the body temperature of warm-blooded animals may safely be assumed not to be infectious for such animals ; and experience is FACTORS OF PATHOGENICITY AND INFECTION 183 gradually teaching us that strictly aerobic organisms, those thriving only in the presence of free oxygen and not able to obtain this gas in available combination from carbohydrates, can also be safely excluded from the infectious class. We have also learned that anaerobic organ- isms, although infectious when gaining entrance to tissues not abun- dantly supplied with blood, are practically unable to multiply in the blood stream and give rise to generalized infection. The pathogenic microorganisms differ very much among themselves in the degree of their disease-inciting power. Such power is known as virulence. Variations in virulence occur, not only among different species of pathogenic bacteria, but may occur within the same species. Pneumococci, for instance, which have been kept upon artificial media or in other unfavorable environment for some time, exhibit less viru- lence than when freshly isolated from the bodies of man or ani- mals. It is necessary, therefore, in order to produce infection, that the particular bacterium involved shall possess sufficient virulence. Whether or not infection occurs depends also upon the number of bacteria which gain entrance to the animal tissues. A small number of bacteria, even though of proper species and of sufficient virulence, may easily be overcome by the first onslaught of the defensive forces of the body. Bacteria, therefore, must be in sufficient number to overcome local defenses and to gain a definite foothold and carry on their life processes, before they can give rise to an infection. The more virulent the germ, other conditions being equal, the smaller the number necessary for the production of disease. The introduction of a single individual of the anthrax species, it is claimed, is often sufficient to cause fatal infection; while forms less well adapted to the parasitic mode of life will gain a foothold in the animal body only after the introduction of large numbers. The Path of Infection. — The portal by which bacteria gain entrance to the human body is of great importance in determining whether or not disease shall occur. Typhoid bacilli rubbed into the abraded skin may give rise to no reaction of importance, while the same microorganism, if swallowed, may cause fatal infection. Conversely, virulent strepto- cocci, when swallowed, may cause no harmful effects, while the same bacteria rubbed into the skin may give rise to a severe reaction. Animals and man are protected against invasion by bacteria in various ways. Externally the body is guarded by its coverings of skin and mucous membranes. When these are healthy and undisturbed, microorganisms are usually held at bay. While this is true in a gen- eral way bacteria may in occasional cases pass through uninjured 184 INFECTION AND IMMUNITY skin and mucosa. Thus the Austrian Plague Commission found that guinea-pigs could be infected when plague bacilli were rubbed into the shaven skin, and there can hardly be much doubt of the fact that tubercle bacilli may occasionally pass through the intestinal mucosa into the lymphatics without causing local lesions. Even after bacteria of a pathogenic species, in large numbers and of adequate virulence, have passed through a locally undefended area in the skin or mucosa of an animal or a human being by a path most favor- ably adapted to them, it is by no means certain that an infection will take place. The bodies of animals and of man have, as we shall see, at their disposal certain general, systemic weapons of defense, both in the blood serum and the cellular elements of blood and tissues which, if normally vigorous and active, will usually overcome a certain number of the invading bacteria. If these defenses are abnormally depressed r or the invading microorganisms are disproportionately virulent or plen- tiful, infection takes place. Bacteria, after gaining an entrance to the body, may give rise merely to local inflammation, necrosis, and abscess formation. They may, on the other hand, from the local lesion, gain entrance into the lymphatics and blood-vessels and be carried freely into the circulation, where, if they survive, the resulting condition is known as bacteriemia or septicemia. Carried by the blood to other parts of the body, they may, under favor- able circumstances, gain foothold in various organs and give rise to secondary foci of inflammation, necrosis, and abscess formation. Such a condition is known as pi/emia. The disease processes arising as the result of bacterial invasion may depend wholly or in part upon the mechanical injury produced by the process of inflammation, the dis- turbance of function caused by the presence of the bacteria in the capil- laries and tissue spaces, and the absorption of the necrotic products resulting from the reaction between the body cells and the micro- organisms. To a large extent, however, infectious diseases are char- acterized by the symptoms resulting from the absorption or diffusion of the poisons produced by the bacteria themselves. Bacterial Poisons. — It was plain, even to the earliest students of this subject, that mere mechanical capillary obstruction or the absorption of the products of a local inflammation were insufficient to explain the profound systemic disturbances which accompany many bacterial in- fections. The very nature of bacterial disease, therefore, suggested the presence of poisons. It was in his investigations into the nature of these poisons that FACTORS OF PATHOGENICITY AND INFECTION 185 Brieger * was led to the discovery of the ptomains. These bodies, first isolated by him from decomposing beef, fish, and human cadavers, have found more extended discussion in another section. Accurately classified, they are not true bacterial poisons in the sense in which the term is now employed. Although it is true that they are produced from pro- teid material by bacterial action, they are cleavage products derived from the culture medium upon the composition of which their nature intimately depends. The bacterial poisons proper, on the other hand, are specific products of the bacteria themselves, dependent upon the nature of the medium only as it favors or retards the full development of the physiological functions of the microorganisms. The poisons, pro- duced to a greater or lesser extent by all pathogenic microorganisms, may be of several kinds. The true toxins, in the specialized meaning which the term has acquired, are soluble, truly secretory products of the bacterial cells, passing from them into the culture medium during their life. They may be obtained free from the bacteria by filtration and in a purer state from the filtrates by chemical precipitation and a vari- ety of other methods. The most important examples of such poisons are those elaborated by Bacillus diphtheria? and Bacillus tetani. If cultures of these bacteria or of others of this class are grown in fluid media for several days and the medium is then filtered through porce- lain candles, the filtrate will be found toxic often to a high degree, while the residue will be either inactive or comparatively weak. Moreover, if the residue possesses any toxicity at all, the symptoms evidencing this will be different from those produced by the filtrate. There are other microorganisms, however, notably the cholera spirillum and the typhoid bacillus, which act in an almost diametrically opposed manner. If these bacteria are cultivated and separated from the culture fluid by filtration in the method described above, the fluid filtrate will be toxic to only a very slight degree, whereas the residue may prove very poisonous. In these cases, we are dealing, evidently, with poisons not secreted into the medium by the bacteria, but rather at- tached more or less firmly to the bacterial body. Such poisons, separable from the bacteria only after death by some method of extraction, or by autolysis, are termed endotoxins. The greater number of the patho- genic bacteria seem to act chiefly by means of poisons of this class. The" first to call attention to the existence of such intracellular poisons was Buchner, who formulated his conclusions from the results of ex- 1 Brieger, "Die Ptomaine," Berlin, 1885 and 1886. 186 INFECTION AND IMMUNITY periments made with a number of microorganisms, notably the Fried- lancler bacillus and Staphylococcus pyogenes aureus, with dead cultures of which he induced the formation of sterile abcesses in animals and symptoms of toxemia. The conception of "endotoxins," subsequently, however, received its clearest and most definite expression in the work of Pfeiffer * on cholera poison. Some clarity of conception, based on visual perception, may possibly be gained by comparing some of the products of pathogenic bacteria with bacterial pigments and with insoluble interstitial or intercellular substance, which may be seen accompanying bacteria in cover-glass preparations. Soluble toxic secretions are to be compared to such pig- ments as the pyocyanin of Bacillus pyocyaneus, which is so readily soluble in culture media; endotoxins proper, to pigments confined to the bacterial cell, or at least, when secreted, being insoluble in culture media, such for instance as the well-known red pigment of Bacillus pro- digiosus, which may often be seen free among the bacteria in irregular red granules like carmine powder. That bodies such as this latter might be extruded from pathogenic bacteria and not be soluble in the usual cul- ture fluids, is not improbable, and the fact that more or less insoluble interstitial substances are not infrequent among bacteria is well known. In all bacterial bodies, after removal of toxins and endotoxins, a certain proteid residue remains which, if injected into animals, may give rise to localized lesions such as abscesses or merely slight temporary inflammations. The nature of this residue has been carefully studied, especially by Buchner, who has named it bacterial protein and he believes the substance to be approximately the same in all bacteria, without specific toxic action, but with a general ability to exert a positive chemotactic effect on the white blood cells, thereby causing the forma- tion of pus. The nature of the bacterial proteins is by no means clear, and it is still in doubt whether the separation of these substances from the endotoxins can be upheld. A number of bacteria may give rise to both varieties of poisons. Thus, recently, Kraus has claimed the discovery of a soluble toxin for the cholera spirillum and Doerr for the dysentery bacillus, both of which microorganisms were regarded as being purely of the endotoxin-pro- ducing type. It is plain, moreover, that occasionally it may be very difficult to distinguish between a soluble toxin and an endotoxin. In the filtration i Pfeiffer, Zeit. f. Hyg., xl, 1892. FACTORS OF PATHOGENICITY AND INFECTION 187 experiment recorded above, it might well be claimed that the toxicity of the filtrate, when not very strong, may depend upon an extraction of endotoxins from the bodies of the bacteria by the medium. The final test, in such instances, lies in the power of true toxins to stimu- late in animals the production of antitoxins; for, as we shall see later, the injection of true soluble toxins into animals gives rise to antitoxins, whereas the formation of such neutralizing bodies in the serum or plasma does not, it is claimed, follow the injection of endotoxins. This distinc- tion will become clearer as we proceed in the discussion of immunity. It must not be forgotten, however, that our knowledge of bacterial poisons is by no means complete, and that sharp distinctions as those given above must be regarded to a certain extent as tentative. In resistance to chemical action and heat, the various poisons show widely divergent properties. As a general rule, most true soluble toxins are delicately thermolabile, they are destroyed by moderate heating, and deteriorate easily on standing. Their chemical nature is by no means clear, but, on precipitation of toxic solutions with mag- nesium sulphate, these poisons come down together with the globulins. The nature of the endotoxins is still less clearly understood. Most of them, while less labile than the extracellular poisons, are, nevertheless, destroyed by exposure to 70° C. On the other hand, certain specific and powerful intracellular poisons, like those of the Gartner bacillus of meat poisoning, may undergo exposure to even 100° C. and still retain their toxic properties. The nature of each individual poison will be discussed in connection with its microorganism. The Mode of Action of Bacterial Poisons. — Close study of the toxic products of various microorganisms has shown that many of the bac- terial poisons possess a more or less definite selective action upon special tissues and organs. Thus, certain soluble toxins of the tetanus bacillus and Bacillus botulinus attack specifically the nervous system. Again, certain poisons elaborated by the staphylococci, the tetanus bacillus, the streptococci, and other germs, the so-called "hemolysins," attack primarily the red blood corpuscles. Other poisons again act on the white blood corpuscles; in short, the characteristic affinity of specific bacterial poisons for certain organs is a widely recognized fact. In explanation of this behavior, much aid has been given by the researches of Meyer,1 Overton,2 Ehrlich,3 and others upon the causes for 'Meyer, Arch. f. exper. Pathol., 1899, 1901. 2 Overton, "Studien iib. d. Narkose," Jena, 1901. 3 Ehrlich, "Sauerstoffs-Bediirfniss des Organismus," Berlin, 1885. 188 IMMUNITY AND INFECTION the analogous selective behavior of various narcotics and alkaloids. It seems probable, from the researches of these men, that the selective action of poisons depends upon the ability, chemical or physical or both, of the poisons to enter into combination with the specifically affected cells. From the nature of the combinations formed, it seems not unlikely fhat the physical factors, such as solubility in the cell plasma, may also play an important part. Observations of a more purely bacteriological nature have tended to^bear out these conclusions. Wassermann and Takaki,1 for instance, have shown that tetanus toxin, which specifically attacks the nervous system, may be removed from solution by the addition of brain sub- stance. Removal of the brain tissue by centrifugation leaves the solu- tion free from toxin. In the same way it has been shown that hemo- lytic poisons can be removed from solutions by contact with red blood cells, but only when the red blood cells of susceptible species are employed. Similar observations have been made in the case of leukocidin, a bacterial poison acting upon the white blood cells specifically.2 That bacterial poisons injected into susceptible animals rapidly disappear from the circulation is a fact which bears out the view that a combination between affected tissue and toxin must take place. Donitz,3 for instance, has shown that within four to eight minutes after the injections of certain toxins, considerable quantities will have dis- appeared from the circulation. Conversely, Metchnikoff 4 has ob- served that tetanus toxin injected into insusceptible animals (lizards) may be detected in the blood stream for as long as two months after administration. 1 Wassermann und Takaki, Berl. klin. Woch., 1898. 2 Sachs, Hofmeister's Beitrage, 11, 1902. 3 Donitz, Deut. med. Woch., 1897. * Metchnikoff , "h 'immunity dans les malad. infect." CHAPTER XII DEFENSIVE FACTORS OF THE ANIMAL ORGANISM GENERAL CONSIDERATIONS We have seen that the mere entrance of a pathogenic microorganism into the human or animal body through a breach in the continuity of the mechanical defenses of skin or mucosa does not necessarily lead to the development of an infection. The opportunities for such an invasion are so numerous, and the contact of members of the animal kingdom with the germs of disease is so constant, that if this were the case, sooner or later all would succumb. It is plain, therefore, that the animal body must possess mor ^ subtle means of defense, by virtue of which pathogenic germs are, even after their entrance into the tissues and fluids, dis- posed of, or at least prevented from proliferating and elaborating their poisons. The power which enables the body to accomplish this is spoken of as resistance. When this resistance, which in some degree is com- mon to all members of the animal kingdom, is especially marked, it is spoken of as "immunity." From this it follows naturally that the terms resistance and immunity, as well as their converse, susceptibility, are relative and not absolute terms. Degrees of resistance exist, which are determined to a certain extent by individual, racial, or species peculiarities; and persons or animals are spoken of as immune when they are unaffected by an ex- posure or an inoculation to which the normal average individual of the same species would ordinarily succumb. The word does not imply, however, that these individuals could not be infected with unusually virulent or large doses, or under particularly unfavorable circumstances. Thus, birds, while immune against the ordinary dangers of tetanus bacilli, may be killed by experimental inoculations with very large doses of tetanus toxin.1 Similarly, Pasteur rendered naturally immune hens susceptible to anthrax by cooling them to a subnormal temperature, and Canalis and Morpurgo did the same with doves by subjecting them to starvation. 1 Quoted from Abel, Kolle und Wassermann, "Handbuch," etc. 189 190 INFECTION AND IMMUNITY Absolute immunity is exceedingly rare. The entire insusceptibility of cold-blooded animals (frogs and turtles) under normal conditions to inoculation with even the largest doses of many of the bacteria patho- genic for warm-blooded animals, and the immunity of all the lower animals against leprosy, are among the few instances of absolute immu- nity known.1 Apart from such exceptional cases, however, resistance, immunity, and susceptibility must be regarded as purely relative terms. The power of resisting any specific infection may be the natural heritage of a race or species, and is then spoken of as natural immunity. It may, on the other hand, be acquired either accidentally or artificially by a member of an ordinarily susceptible species, and is then called acquired immunity. Natural Immunity. — Species Immunity. — It is well known that many of the infectious diseases which commonly affect man, do not, so far as we know, occur spontaneously in animals. Thus, infection with B. typhosus, the vibrio of cholera, or the meningococcus occurs in ani- mals only after experimental inoculation. Gonorrheal and syphilitic infection, furthermore, not only does not occur spontaneously, but is produced experimentally in animals with the greatest difficulty — the consequent diseases being incomparably milder than those occurring in man. Other diseases, like leprosy, influenza, and the exanthemata,2 have never been successfully transmitted to animals. Conversely, there are diseases among animals which do not spon- taneously attack man. Thus, human beings enjoy immunity against Rinderpest, and, to a lesser degree, against chicken cholera. Among animal species themselves great differences in susceptibility and resistance toward the various infections exist. Often-quoted ex- amples of this are the remarkable resistance to anthrax of rats and dogs, and the immunity of the common fowl against tetanus. The factors which determine these differences of susceptibility and resistance among the various species are not clearly understood. It has been suggested that diet in some instances may influence these re- lations, inasmuch as carnivorous animals are often highly resistant to glanders, anthrax, and even tuberculous infections, to which herbiv- orous animals are markedly susceptible.3 It is likely, too, that the great differences between animals of various species in their metabolism, temperature, etc., may call for special cultural adaptation on the part Lubarsch, Zeit. f. klin. Mediz., xix. With the possible exception of smallpox. 3 Hahn, in Kolle und Wassermann, vol. iv. DEFENSIVE FACTORS OF THE ANIMAL ORGANISM 191 of the bacteria. The fact that the bacillus of avian tuberculosis — whose natural host has a normal body temperature of 40° C. and above — will grow on culture media at 40 to 50° C, whereas B. tuberculosis of man can not be cultivated at a temperature above 40° C, would seem to lend some support to this view. The difference between warm- and cold-blooded animals has already been noted. The necessity for cultural adaptation, too, would seem to be borne out by the great enhancement observed in the virulence of certain microorganisms for a given species after repeated passage through individuals of this species. Racial Immunity. — Just as differences in susceptibility and im- munity exist among the various animal species, so the separate races or varieties within the same species may display differences in their reac- tions toward pathogenic germs. Algerian sheep, for instance, show a much higher resistance to anthrax than do our own domestic sheep, and the various races of mice differ in their susceptibility to anthrax and to glanders. Similar racial differences are common among human beings. As a general rule, it may be said that a race among whom a certain disease has been endemic for many ages is less susceptible to this disease than are other races among whom it has been more recently introduced. The appalling ravages of tuberculosis among negroes, American Indians, and Esquimaux, bear striking witness to this fact. Conversely, the compar- ative immunity of the negro from yellow fever, a disease of the greatest virulence for Caucasians, furnishes further evidence in favor of this opinion. It must not be forgotten, however, in judging of these rela- tions, that the great differences in the customs of personal and social hygiene existing among the various races may considerably affect the transmission of disease and lead to false conclusions. In so far as the statement made above is true, however, it seems to indicate that the endemic diseases have carried in their train a certain degree of inherited immunity. In other cases l— as in the instance of the malaria-immunity of negroes — the resistance seems to be acquired rather than inherited, for, as Hirsch was first to note, death from this disease occurred frequently among the children, while adult negroes were rarely attacked. Differences in Individual Resistance. — In bacteriological ex- perimentation with smaller test animals, a direct ratio may often exist between body weight and dosage in determining the outcome of an : Hahn, in Kolle und Wassermann, loc. cit. 192 INFECTION AND IMMUNITY infection, provided the mode of inoculation has been the same and the virulence of the germ not excessive. It would seem, therefore, that among these animals the difference in resistance in the face of an arti- ficial infection between individuals of the same race is very slight. In higher animals, however, especially in the case of man, the ex- istence of such apparent individual differences is a well-established fact, although in judging of them we must not forget that the conditions of infection are not subject to the uniformity and control which animal experimentation permits. Of a number of persons exposed to any given infection there are always some who are entirely unaffected and there are great variations in the severity of the disease in those who are attacked. In the absence of positive evidence in support of the direct inheritance of this individual immunity, the most reasonable explanation for such differences in resistance seems to lie in attrib- uting them to individual variations in metabolism or body chem- istry. Depressions, for instance, in the acidity of the gastric secretion would predispose to certain infections of gastro-intestinal origin. Ana- tomical differences, too, may possibly influence resistance. Thus, Birch-Hirschfeld believed that certain anomalous arrangements of the bronchial tubes predisposed to tuberculosis. Instances of transient susceptibility induced by physical or mental overwork, starvation, etc., should hardly be classified under this head- ing, since the conditions in such cases correspond simply to experi- mental depression of natural species or race resistance. Acquired Immunity. — It is a matter of common experience that many of the infectious diseases occur but once in the same individual. This is notably the case with typhoid fever, j^ellow fever, and most of the exanthemata, and is too general an observation to require extensive illustration. A single attack of any of the diseases of this class alters in some way the resistance of the individual so that further exposure to the infective agent is usually without menace, either for a limited period after the attack, or for life. Resistance acquired in this way is often spoken of as acquired immunity. The protection conferred by certain diseases against further attack was recognized many centuries ago, and there are records which show that attempts were made in ancient China and India to inoculate healthy individuals with pus from small-pox pustules in the hope of producing by this process a mild form of the disease and its consequent immunity. Pasteur, before all others, thought philosophic ally about the phenom- ena of acquired immunity, and, wTith adequate knowledge, realized the DEFENSIVE FACTORS OF THE ANIMAL ORGANISM 193 possibility of artifically bestowing immunity without inflicting the dangers of the fully potent infective agent. The first observation which, made by him purely accidentally, inspired the hope of the achievement of such a result, occurred during his experiments with chicken cholera. The failure of animals to die after inoculation with an old culture of the bacilli of chicken cholera, fully potent but a few weeks previously, pointed to the attenuation of these bacilli by their prolonged cultivation without transplantation. With this observation as a point of departure he carried out a series of investigations with the purpose of discovering a method of so weakening or attenuating various incitants of disease that they could be introduced into susceptible individuals without en- dangering life and yet without losing their property of conferring pro- tection. The brilliant results achieved by Jenner, many years before, in protecting against smallpox by inoculating with the entirely innocu- ous products of the pustules of cowpox furnished an analogy which gave much encouraging support to this prospect. The experimental work which Pasteur carried out to solve this prob- lem not only reaped a rich harvest of facts, but gave to science the first and brilliant examples of the application of exact laboratory methods to problems of immunity. ACTIVE IMMUNITY Active Artificial Immunity. — The process of conferring protection by treatment with either an attenuated form or a sublethal quantity of the infectious agent of a disease, or its products, is spoken of as " active immunization." Whatever the method employed, the immunized individuals gain their power of resistance by the unaided reactions of their own tissues. They themselves take an active physiological part in the acquisition of this new property of immunity. For this reason, Ehrlich has aptly termed these processes " active immunization." There are various methods by which this can be accomplished, all of which were, in actual application or in principle, discovered by Pasteur and his associates, and can be best reviewed by a study of their work. Active Immunization with Attenuated Cultures. — In the course of his experiments upon chicken cholera, as mentioned above, Pasteur 1 1 Pasteur, Compt. rend, de l'acad. des sci., 1880, t. xc. 13 194 INFECTION AND IMMUNITY accidentally discovered that the virulence of the bacilli of this disease was greatly reduced by prolonged cultivation upon artificial media. This was especially noticeable in broth cultures which had been stored for long periods without transplantation. By repeated injections of such cultures into fowl, he succeeded in rendering the animals immune against subsequent inoculations with lethal doses of fully virulent strains. During the same year, 1880, in which Pasteur published his observa- tions on chicken cholera, Toussaint * succeeded in immunizing sheep against anthrax by inoculating them with blood from infected animals, defibrinated and heated to 55° C. for ten minutes. Toussaint wrongly believed, however, that the blood which had been used in his immuniza- tions was free from living bacteria. In repeating this work Pasteur showed that the protection in Toussaint's cases was conferred by living bacteria, the virulence of which had been reduced by their subjection to heat. In following out the suggestions offered by these experiments, Pasteur2 discovered that he could reduce the virulence of anthrax bacilli much more reliably than by Toussaint's method, by cultivating the organisms at increased temperatures (42 Q to 43° C.) . By this process of attenuation he was able to produce " vaccines " of roughly measurable strength, with which he succeeded in immunizing sheep and cattle. A successful demonstration of his discovery was made by him at Pouilly- le-Fort, soon after, upon a large number of animals and before a commis- sion of professional men. It is a fact well known to bacteriologists that certain of the pathogenic microorganisms, when passed through several individuals of the same animal species, become gradually more virulent for this species. In his studies on the bacillus of hog cholera, Pasteur observed that when this microorganism was passed through the bodies of several rabbits it gained in virulence for rabbits, but became less potent against hogs. He suc- ceeded, subsequently, in protecting hogs against fully virulent cultures by treating them with strains which had been attenuated by their passage through rabbits. A further principle of attenuation for purposes of immunization was, at about this time, contributed by Chamberland and Roux,4 who re- 1 Toussaint, Compt. rend, de l'acad. des sci., 1880, t. xci. 2 Pasteur, Chamberland et Roux, Compt. rend, de l'acad. des sci., 1881, t. xcii. 3 Pasteur, Compt. rend, de l'acad. des sci., 1882, t. xcv. 4 Chamberland et Roux, Compt. rend, de l'acad. des sci., 1882, t. xcvi. DEFENSIVE FACTORS OF THE ANIMAL ORGANISM 195 duced the virulence of anthrax cultures by growing them in the presence of weak antiseptics (carbolic acid 1 : 600, potassium bichromate 1 : 5,000, or sulphuric acid 1 : 200) . Cultivated under such conditions the bacilli lost their ability to form spores and became entirely avirulent for sheep within ten days. A similar result was later obtained by Behring * when attenuating B. diphtherias cultures by the addition of terchlorid of iodin. Active Immunization with Sublethal Doses of Fully Virulent Bacteria. — The use of fully virulent microorganisms in minute quantities for purposes of immunization was first suggested by Chau- veau,2 and is naturally inapplicable to extremely virulent organisms like B. anthracis. The principle, however, is perfectly valid, and has been experimentally applied by many observers, notably by Ferran 3 in the case of cholera. A similar method proved of practical value in the hands of Theobald Smith and Kilborne 4 in prophylaxis against the protozoan disease, Texas fever. Active Immunization with Dead Bacteria. — Suggested by Chau- veau, the method of active immunization with gradually increasing doses of dead microorganisms has been successfully employed by various ob- servers, chief among whom are PfeifTer, Brieger, Wright, and Wasser- mann. The method is especially useful against that class of bacteria in which the cell bodies (endotoxins) have been found to be incomparably more poisonous than their extracellular products (toxins). From a practical point of view, the method is of the greatest importance in routine laboratory immunization against B. typhosus, Vibrio cholerse asiaticse, B. pestis, and a number of other bacteria. In the therapy of human disease, this method has recently come into great prominence, chiefly through the work of Wright, whose investigations will be more fully discussed in a subsequent section. Active Immunization with Bacterial Products. — Many bacteria when grown in fluid media produce extracellular, soluble poisons which remain in the medium after the microorganisms have been removed by filtration or centrifugalization. Since the diseases caused by such microorganisms are, to a large extent, due to the soluble poisons excreted by them, animals can be actively immunized against this class of bac- 1 Behring, Zeit. f. Hyg., xii, 1892. 2 Chauveau, Compt. rend, de l'acad. des sci., 1881, t. xcii. 3 Ferran, Compt. rend, de l'acad. des sci., 1895, t. ci. 4 Th. Smith and Kilborne, U. S. Dept. of Agri., Bureau of Ani. Indust., Wash., 1893. 196 INFECTION AND IMMUNITY teria by the inoculation of gradually increasing doses of the specific poison or toxin. This method is naturally most successful against those microorganisms which possess the power of toxin formation to a highly developed degree. Most important among these are B. diphtheria and B. tetani. The first successful application of this principle of active immunization, however, was made by Salmon and Smith1 in the case of hog cholera. PASSIVE IMMUNITY In Pasteur's basic experiments, as in those of the other scientists who followed in his footsteps, the methods of immunization were based upon the development of a high resistance in the treated subject by virtue of its own physiological activities. This process we have spoken of as "active immunization" and it is self-evident that a method of this kind can, in the treatment of disease, be employed prophylactically only against possible infection, or in localized acute infections, or at the beginning of a long period of incubation before actual symptoms have appeared, as in rabies or in chronic conditions in which the infection is not of a severe or acute nature. A new and therapeutically more hopeful direction was given to the study of immunity when, in 1890 and 1892, v. Behring and his collabora- tors discovered that the sera of animals immunized against the toxins of tetanus 2 and of diphtheria 3 bacilli would protect normal animals against the harmful action of these poisons. The animals thus pro- tected obviously had taken no active part in their own defense, but were protected from the action of the poison by the substances trans- ferred to them in the sera of the actively immunized animals. Such immunity or protection, therefore, is a purely passive phenomenon so far as the treated animal is concerned, and the process is for this reason spoken of as "passive immunization.'' Passive immunization of this description is practically applicable chiefly against diseases caused by bacteria which produce powerful toxins, and the sera of animals actively immunized against such toxins are called antitoxic sera. In the treatment of the two diseases men- tioned above, diphtheria and tetanus, the respective antitoxic sera have 1 Salmon and Smith, Rep. of Com. of Agri., Wash., 1885 and 1886. 2 v. Behring and Kitasato, Deut. med. Woch., 49, 1890. 3 v. Behring and Wernicke, Zeit. f. Hyg., 1892. DEFENSIVE FACTORS OF THE ANIMAL ORGANISM 197 reached broad and beneficial therapeutic application, and innumerable lives have been saved by their use. Passive immunization against microorganisms not characterized by marked toxin formation was attempted, even before Behring's dis- covery, by Richet and Hericourt,1 experimenting with cocci, and by Babes,2 in the case of rabies; and the underlying thought had been the basis of Toussaint's work upon anthrax. Microorganisms, however, which exert their harmful action rather by the contents of the bacterial cells than by secreted, soluble toxins, do not, so far as is known, pro- duce antitoxins in the sera of immunized animals. The substances which they call forth in the process are directed against the invading organisms themselves in that they possess the power of destroying or of causing dissolution of the specific germs used in their production. Such antibacterial sera are extensively used in the laboratory in the passive immunization of animals against a large number of germs, and are fairly effectual when used before, at the same time with, or soon after, infection. Their therapeutic use in human disease, however, has, up to the present time, been disappointing and their prophylactic and cura- tive action has been almost invariably ineffectual or feeble at best, ex- cept when the antibacterial sera could be brought in direct contact with the germs, in closed cavities or localized lesions. Thus, in epidemic meningitis, such sera have proved extremely useful in the hands of Flexner, when injected directly into the spinal canal. ANTIBODIES AND THE SUBSTANCES GIVING RISE TO THEM In the foregoing sections we have seen that the process of active immunization so changes the animal body that it becomes highly resistant against an infection to which it had formerly in many in- stances been delicately susceptible. In the absence of visible anatomical or histological changes accompanying the acquisition of this new power, investigators, in order to account for it, were led to examine the physio- logical properties of the body cells and fluids of immunized subjects. While it was reasonable to suppose that all the cells and tissues were affected by, or might have taken part in, a physiological change so profoundly influencing the individual, the blood, because of its unques- tionably close relation to inflammatory reactions, and because of the J Richet et Hericourt, Compt. rend, de l'acad. des sci., 1888. 2 Babes et Lepp, Ann. de l'inst. Pasteur, 1889. 198 INFECTION AND IMMUNITY ease with which it could be obtained and studied, claimed the first and closest attention. The bactericidal properties of normal blood serum noted in 1886 by Nuttall,1 v. Fodor,2 and Fliigge, moreover, aided in pointing to this tissue as primarily the seat of the immunizing agents. It is an interesting historical fact, that, long before this time, the English physician Hunter had noted that blood did not decompose so rapidly as other animal tissues. The study of the blood serum of immunized animals as to simple changes in chemical composition or physical properties has shed little light upon the subject. Beljaeff 3 in a recent investigation found little or no alteration from the normal in the blood sera of immunized animals as to index of refraction, specific gravity, and alkalinity. Joachim 4 and Moll agree in stating that immune blood serum is comparatively richer in globulin than normal serum. Similar observations had been made by Hiss and Atkinson ° and others. Important and significant as these purely chemical observations are, they have helped little in explaining the nature of the processes going on in immune sera. The first actual light was thrown upon the mysterious phenomena of immunity by the investigations of Nuttall,6 v. Fodor, J3uchner, and others, who not only demonstrated the power of normal blood serum to destroy bacteria, but also showed that this property of blood serum became diminished with age and was destroyed completely by heating to 56° C. The thermolabile substance of the blood serum possessing this power was called by Buchner,7 alexin. Soon after this work, Behring, in collaboration with Kitasato 8 and Wernicke,9 in 1890 and 1892, made further important advances in the elucidation of the immunizing processes by showing that the blood sera of animals actively immunized against the toxins of diphtheria and tet- anus would protect normal animals against the poisons of these diseases. He believed, at the time of discovery, that such sera contained substances which had the power of destroying the specific toxins which had been 1 Nuttall, Zeit. f. Hyg., i, 1886. 2 v. Fodor, Deut. med. Woch., 1886. 3 Beljaeff, Cent. f. Bakt., xxxiii. 4 Joachim, Pfliigers Archiv, xciii. 5 Hiss and Atkinson, Jour. Exper. Med., v, 1900. 6 Nuttall, Zeit. f. Hyg., 1886. 7 Buchner, Cent. f. Bakt., i, 1889. 8 Behring und Kitasato, Deut. med. Woch., 1890, No. 49. 9 Behring und Wernicke, Zeit. f. Hyg., 1892. DEFENSIVE FACTORS OF THE ANIMAL ORGANISM 199 used in the immunization. He called these bodies antitoxins. While Behring's first conception of actual toxin destruction soon proved to be erroneous, his discovery of the presence in immune sera of bodies specifically antagonistic to toxins was soon confirmed and extended, and stands to-day as an established fact. Ehrlich,1 soon after Behring's announcement, showed that specific antitoxins could also be produced against the poisons of some of the higher plants (antiricin, antikrotin, antirobin) , and Calmette 2 produced similar antitoxins against snake poison (antivenin) . Stimulated by these researches, other observers have, since then, added extensively to the list of poisons against which antitoxins can be produced. Kempner 3 has produced antitoxin against the poison of Bacillus botulinus, and Wassermann,4 against that of Bacillus pyocyaneus. Antitoxin has been produced by Calmette 5 against the poison of the scorpion, and by Sachs 6 against that of the spider. Thus a large number of poisons of animal, plant, or bacterial origin have been found capable of causing the pro- duction of specific antibodies in the sera of animals into which they are injected. The formation of antitoxins directed against soluble poisons, how- ever, did not explain the immunity acquired by animals against bacteria like Bacillus anthracis, the cholera vibrio, and others which, unlike diph- theria and tetanus, produced little or no soluble toxin. It was evident that the antitoxic property of immune blood serum was by no means the sole expression of its protective powers. Much light was shed upon this phase of the subject by the discoveries of Pfeiffer in 1894, who worked along the lines suggested by the investigations of Nuttall and Buchner. Pfeiffer7 showed that when cholera spirilla were injected into the peritoneal cavity of cholera-immune guinea-pigs, the microorganisms rapidly swelled up, became granular, and often underwent complete solution. The same phenomenon could be observed when the bacteria were injected into a normal animal together with a sufficient quantity of cholera-immune 8 serum. 1 Ehrlich, Deut. med. Woch., 1891. ' 2 Calmette, Compt. rend, de la soc. de biol., 1894. 3 Kempner, Zeit. f. Hyg., 1897. 4 Wassermann, Zeit. f. Hyg., xxii. 5 Calmette, Ann. de Pinst. Pasteur, 1898. 6 Sachs, Hofm. Beit., 1902. 7 Pfeiffer, Zeit. f. Hyg., xviii, 1894. 8 Pfeiffer und Isaeff, ibid. 200 INFECTION AND IMMUNITY This process he observed microscopically by abstracting, from time to time, a small quantity of the peritoneal exudate and studying it in hanging-drop preparations. The reaction was specific in that the de- structive process took place to any marked extent only in the case of the bacteria employed in the immunization. Metchnikoff,1 Bordet, and others not only confirmed Pfeiffer's obser- vation, but were able to show that the lytic process would take place in vitro, as well as in the animal body. The existence of a specific destructive process in immune serum was thus established for the vibrio of cholera and soon extended to other microorganisms. The constitu- ents of the blood serum which gave rise to this destructive phenomenon were spoken of as bacteriolysins. Following closely upon the heels of Pfeiffer's observation came the discovery of another specific property of immune serum by Gruber and Durham.2 These workers noticed that certain bacteria, when brought into contact with the serum of an animal immunized against them, were clumped together, deprived of motility, and firmly agglutinated. They spoke of the phenomenon as agglutination and of the substances in the serum giving rise to it as agglutinins. The list of antibodies was again enlarged by Kraus,3 who in 1897 showed that precipitates were formed when filtrates of cultures of cholera, typhoid, and plague bacilli were mixed with their specific immune sera. He called the substances which bestowed this property upon the sera precipitins. The treatment of the animal body, therefore, with bacteria or their products gives rise to a variety of reactions which result in the presence of the " antibodies " described above. Extensive investigation has shown, however, that the power of stimulating antibody production is a phe- nomenon not limited to bacteria and their products alone. Antitoxins, we have already seen, may be produced with a variety of poisons of plant and animal origin. Lysins, agglutinins, and precipitins, likewise, may be produced by the use of a large number of different substances. Chief among these, because of the great aid they have given to the theo- retical investigation of the phenomena of immunity, are the red blood cells. Bordet 4 and, independently of him, Belfanti and Carbone 5 showed, 1 Metchnikoff, Ann. de l'inst. Pasteur, 1895. ? Gruber und Durham, Munch, med. Woch., 1896. 3 Kraus, R., Wien. klin. Woch., 32, 1897. 4 Bordet, Ann. de l'inst. Pasteur, 1898. 5 Belfanti et Carbone, Giornale della R. Acad, di Torino, July, 1898. DEFENSIVE FACTORS OF THE ANIMAL ORGANISM 201 in 1898 that the serum of animals repeatedly injected with the defibri- nated blood of another species exhibited the specific power of dissolving the red blood corpuscles of this species. This was the first demonstration of "hemolysis" — a phenomenon which, because of the ease with which it can be observed in vitro, has much facilitated investigation. The knowledge that specific " cytotoxins " or cell-destroying anti- bodies could be produced by injection of red blood cells naturally sug- gested the possibility of analogous reactions for other tissue cells. It was not long, therefore, before Metchnikoff 1 and, independently of him, Landsteiner 2 succeeded, by repeated injections of spermatozoa, in producing a serum which would seriously injure these specialized cells. Von Dungern 3 obtained similar results with the ciliated epithelium of the trachea. Since then a host of cytotoxins have been produced with the cells of various organs and tissues. Thus, Neisser and Wechsberg 4 produced leucotoxin (leucocytes) ; Delezenne,5 neurotoxin and hepa- totoxin; Surmont,6 pancreas cytotoxin; and Bogart and Bernard,7 su- prarenal cytotoxin. One of the most interesting of the cytotoxins, moreover, is nephro- toxin — produced by the treatment of animals with injections of emul- sions of kidney tissue. In all cases it was supposed by those first working with these bodies, that the injection of the sera of animals previously treated with any particular tissue substance would produce specific injury upon the or- gans homologous to the ones used in immunization. It need hardly be pointed out how very important such phenomena would be in throwing light upon the degenerative pathological lesions occurring in disease. As a matter of fact, however, sera so produced have been shown to be specific for certain organs in a limited sense only. The question of specific cytotoxins has been of especial importance in the case of nephritis, where Ascoli and Figari 8 and others have suggested an autonephrotoxin as the basis of the pathology of this disease. In the hands of Pearce and others, however, the strict specificity of 1 Metchnikoff, Ann. de l'inst. Pasteur, 1898. 2 Landsteiner, Cent. f. Bakt., i, 25, 1899. 3 v. Dungern, Munch, med. Woch., 1899. 4 Neisser und Wechsberg, Zeit. f. Hyg., xxxvi, 1901. 5 Delezenne, Ann. de l'inst. Past. 1900; Compt. rend, de l'acad. des sci. 1900. 6 Surmont, Compt. rend, de la soc. de biol., 1901. 7 Bogart et Bernard, ibid., 1891. 8 Ascoli and Figari, Berl. klin. Woch., 1902. 202 INFECTION AND IMMUNITY nephrotoxin could not be upheld and the subject is still in the ex- perimental stage. Recent experiments by Pearce i suggest that at least a part of the local injury to organs exerted by such " cytotoxic" sera may not be due to a specific action upon the organ cells so much as upon the hemagglutinating action of the sera causing embolism and necrosis. It is a fact also that most cytotoxic sera are usually hemolytic as well. It is not easy to decide, therefore, how much of the action upon the organs is due to their true cytotoxic properties and how much is attributable to the concomitant action upon blood cells. The extrav- agant hopes at first based upon cytotoxin investigation, especially in regard to the problem of malignant tumors, have been disappointed, and much is still obscure in regard to the cytotoxins which calls for further research. The many points of similarity existing between bacterial toxins and digestive ferments, by animal inoculation, suggested to several observ- ers the possibility of producing antibodies against the latter. As a result, a number of antiferments have been obtained, chief among which are antilab (Morgenroth 2) , antipepsin (Sachs 3) , antisteapsin (Schiitze 4) , and antilactase (Schiitze). The stimulation of antibody formation in the sera of animals is a consequence, therefore, of the injection of a large variety of substances — ■ some of them poisonous, some of them entirely innocuous. The sub- stances possessing this power have been conveniently named antigens or antibody-producers by German writers. The term antigen — though ety- mologically wrong, nevertheless is convenient and has crept into general usage. It signifies simply a substance which can stimulate the pro- duction or formation of an antibody. Such substances, so far as is known, belong to the group of proteids and are derivatives of animal or plant tissues. Being proteids, all antigens are colloids. Recently, how- ever, some crystalloidal substances have been described as possessing antigenic properties. 1 Pearce, Jour. Exper. Med., viii, 1906. 2 Morgenroth, Cent. f. Bakt., 1899. 3 Sachs, Fort. d. Med., 1902. * Schiitze, Deut. med. Woch., 1904; Zeit. f. Hyg., 1905. CHAPTER XIII TOXINS AND ANTITOXINS The Toxin-Antitoxin Reaction. — Apart from the therapeutic possi- bilities disclosed by the discovery of antitoxins, new light of inestimable value was thrown by these observations upon the biological processes involved in immunization. The most vital problem, of course, which immediately thrust itself upon all workers in this field was the question as to the nature of the reaction in which toxin was rendered innocuous by antitoxin. The simplest conception of this process would be an actual destruction of the toxin by its specific antitoxin, and it is not unnatural, therefore, that this was the view which, for a short time, found favor with some observers. Roux, and more particularly Buchner,1 however, under the sway of cellular pathology, advanced the opinion that the antitoxins in some way influenced the tissue cells, rendering them more resistant against the toxins. Antitoxin, according to this theory, did not act directly upon toxin, but affected it indirectly through the mediation of tissue cells. Ehrlich,2 on the other hand, conceived that the reac- tion of toxin and antitoxin was a direct union, analogous to the chem- ical neutralization of an acid by a base — an opinion in which Behring soon joined him. The conception of toxin destruction received unanswerable refuta- tion by the experiments of Calmette.3 This observer, working with snake poison, found that the poison itself (unlike most other toxins) possessed the property of resisting heat even to 100° C, while its specific anti- toxin, like other antitoxins, was delicately thermolabile. He noted, furthermore, that non-toxic mixtures of the two substances, when sub- jected to heat, regained their toxic properties. The natural inference from these observations could only be that the toxin in the original mix- ture had not been destroyed, but had been merely inactivated by the 1 Buchner, " Schutzimpfung," etc., in Penzoldt u. Stinzing, "Handbuch d. spez. Therap. d. Infektkrank.," 1894. 2 Ehrlich, Deut. med. Woch., 1891. *Calmette, Ann. de l'inst. Past., 1895. 203 204 INFECTION AND IMMUNITY presence of the antitoxin, and again set free after destruction of the antitoxin by heat. A similar observation, made soon after by Wasser-' mann ' in the case of pyocyaneus toxin and antitoxin, fully supported the results of Calmette. An ingenious proof of the direct action of antitoxin upon toxin was obtained by Martin and Cherry.2 It was found by them that veiy dense filters, the pores of which had been filled with gelatin, permitted toxin to pass through under high pressure, while the presumably larger antitoxin molecule was held back. Through such filters the}' forced toxin-antitoxin mixtures, under a pressure of fifty atmospheres, at vary- ing intervals after mixing. They found that, if filtered immediately, all the toxin in the mixtures came through, but that, as the interval elapsing between mixing and filtration was prolonged, less and less toxin appeared in the filtrate, until, finally, two hours after mixing, no toxin whatever passed through the filter. Besides demonstrating the direct action of antitoxin upon toxin, this work of Martin and Cherry showed that the element of time entered into the toxin-antitoxin reaction, just as it enters into reactions of known chemical nature. The absolute non- participation of the living tissue cells in these, reactions was demonstrated by Ehrlich himself. Kobert and Stillmarck 3 had shown that ricin pos- sessed the power of causing the red blood cells of defibrinated blood to agglutinate in solid clumps, a reaction which could easily be observed in vitro. Ehrlich,4 who had obtained antiricin in 1891 by injecting rabbits with increasing doses of ricin, found that this antibody pos- sessed the power of preventing the hemagglutinating action of ricin in the test tube. By a series of quantitatively graded mixtures of ricin and antiricin, with red blood cells as the indicator for the reaction, he succeeded in proving not only that the toxin-antitoxin neutralization was in no way dependent upon the living animal body, but that definite quantitative relations existed between the two substances entirely analogous to those which, according to the law of multiple proportions, govern reactions between different substances of known chemical nature. Similar quantitative results were subsequently obtained by Stephens and Myers 5 for cobra poison and its antitoxin, by Kossel 6 1 Wassermann, Zeit. f. Hyg., xxii, 1896. 2 Martin and Cherry, Proc. Royal Soc, London, briii, 1898. 3 Kobert und Stillmarck, Arb. d. phar. Inst. Dorpat; 1889. * Ehrlich, Fort. d. Med., 1897. 5 Stephens and Myers, Jour, of Path, and Bact., 1898. • Kossel, Berl. klin. Woch., 1898. TOXINS AND ANTITOXINS 205 for the toxic eel blood serum, and by Ehrlich * for the hemolytic tetanus poison known as tetanolysin. The introduction of the test-tube experiment into the investigation of these reactions permitted of much more exact observations, and by this means, as well as by careful, quantitatively graded, animal experi- ments, the further facts were ascertained that toxin and antitoxin com- bined more speedily in concentrated than in dilute solutions, and that warmth hastened, while cold retarded, the reaction — observations 2 which in every way seem to bear out Ehrlich/s conception of the chemi- cal nature of the process. Ehrlich's Analysis of Diphtheria Toxin. — Shortly after the discovery and therapeutic application of diphtheria antitoxin, it became apparent that no two sera, though similarly produced, could have exactly the same protective value. It was necessary, therefore, to establish some measure or standard by which the approximate strength of a given anti- toxin could be estimated. Von Behring 3 attempted to do this for both tetanus and diphtheria antitoxins by determining the quantity of immune sera which, in each case, was needed to protect a guinea-pig of known weight against a definite dose of a standard poison. He ascer- tained the quantity of standard toxin-bouillon which would suffice to kill a guinea-pig of 250 grams, and called this quantity the "toxin unit." This unit was later more exactly limited by Ehrlich, who, considering the element of time, stated it as the quantity sufficient to kill a guinea- pig of the given weight in from four to five days. Appropriating the terminology of chemical titration, v. Behring spoke of a toxin-bouillon which contained one hundred such toxin units in a cubic centimeter, as a "normal toxin solution" ("DTN1 M250"), and designated as " normal antitoxin " a serum capable of neutraliz- ing, cubic centimeter for cubic centimeter, the normal poison.4 A cubic centimeter of such an antitoxic serum was sufficient, therefore, to neu- tralize one hundred toxin units, and was spoken of as an "antitoxin unit." In the experiments of v. Behring, toxin and antitoxin had been separately injected. Ehrlich 5 improved upon this method by mixing toxin and antitoxin before injection, thereby obviating errors arising i Ehrlich, Berl. klin. Woch., 1898. 2 Knorr, Fort. d. Med., 1897. 3 v. Behring, Deut. med. Woch., 1893. 4 DTN » M25o signifies: D, Diphtheria; TN1, Normal Toxin solution; M250, Meer- schweinchen or guinea-pig weighing 250 grams. 5 Ehrlich, Kossel und Wassermann, Deut. med. Woch., 1894. 206 INFECTION AND IMMUNITY from differences which may have existed in the depth of injection or rapidity of absorption. In order, however, that any such method of standardization of an- titoxin may be practically applicable, it is necessary to produce either a stable toxin or an unchangeable antitoxin. This Ehrlich achieved for antitoxin by drying antitoxic serum in vacuo and preserving it in the dark, at a low temperature and in the presence of anhydrous phosphoric acid. By the use of such a stable antitoxin, various toxins may be measured and other antitoxic sera estimated against these. Given thus a constant antitoxin, the standardization of toxins would be a comparatively simple matter were the poison obtainable in a per- fectly pure state. Unfortunately for the ease of measurement, how- ever, this is not the case. The problem is rendered difficult by a number of complicating factors, many of which have been brought to light by Ehrlich 1 in his laborious researches into the quantitative relationship between the two reacting bodies. As previously stated, it had been noted by Ehrlich and others that toxin solutions would deteriorate with time; that is, a toxin-bouillon ^ToxopKore ^roixp Immuninin Substance Body Cell Fig. 55. — Toxin and Body Cell. which was found soon after production to contain, say, eighty toxin units in each cubic centimeter, would, after four or five months, be found to contain but forty units in the same gross quantity. It had lost, there- fore, in this case, just one-half of its toxic power. In spite of this loss, however, Ehrlich found that such bouillon had retained its full original power of neutralizing antitoxin. If the reaction was purely one of chemical neutralization, there seemed to,be but one explanation of this. The toxin molecule must contain two separate atom groups. One of these must possess the power of binding antitoxin and be stable; this Ehrlich, Klin. Jahrbuch, vi, 1897; Deut. med. Woch., 1898. TOXINS AND ANTITOXINS 207 he designates as the "haptophore" or " anchoring" group. The other, the one by which the ioxin molecule exerts its deleterious action, must be more easily changed or destroyed; this he calls the "toxophore" or " poison7' group. In the altered toxin-bouillon in which a part of the poisonous action has been lost while the antitoxin-neutralizing power is intact, the toxophore group of some of the toxin must have been changed or destroyed. Such altered toxin he speaks of as "toxoid." In support of this hypothesis and for the purpose of perfecting the methods of standardization, Ehrlich was led to determine, for a large variety of specimens of diphtheria toxin, the precise quantity, in cubic centimeters, which was necessary to neutralize exactly one unit of his standard antitoxin. This he accomplished by making a series of toxin-antitoxin mixtures, in each of which the quantity of antitoxin was exactly one unit, while the amount of toxin was gradually increased. These mixtures were injected into guinea-pigs of 250 grams weight. It is self-evident that in such an experiment the mixtures containing the smaller quantities of toxin would have no effect upon the guinea- pigs. Soon, however, a mixture would be reached in which toxin would be sufficiently in excess of antitoxin to produce the symptoms of slight poisoning, as evidenced in local edema, rise of temperature, etc. The largest quantity of toxin which could be added without producing such symptoms was then regarded as exactly neutralizing one antitoxin unit. This quantity of toxin Ehrlich speaks of as "-Limes zero" (Limes = threshold) or, briefly, "L0." For instance: One antitoxin unit -f- 0.6 c.c. toxin No symptoms of poisoning. 0.8 c.c 0.9 c.c " " " " it ti a i _ „ tt a a a " 1.1 c.c , Local edema. Paralysis in 30 days. " 1.2 c.c Death in 10 days. In this example, L0, therefore, equals 1 c.c. It is obvious, however, that because of the great difficulty in esti- mating the very slightest evidences of toxic action in guinea-pigs, a more exact method of standardizing the poisons against antitoxin would be to determine how much toxin would be required to neu- tralize one antitoxin unit and still be sufficiently in excess to cause the death of a guinea-pig of 250 grams in four to five days . This would then correspond to the action of one toxin unit, unmixed with antitoxin. A priori it would seem that this value (expressed by Ehrlich as " Limes 20S INFECTION AND IMMUNITY death " or " L+) must simply be L0 plus one toxin unit. This, however, was found not to be the case. Thus, in the example given, in which T (the toxin unit — the quantity of the bouillon killing a guinea-pig of 250 grains in four to five days) was equal to O.Olc.c, L0 (the quantity of toxin completely neutralizing one antitoxin unit) was found to be 1 c.c. or 100 T. In this same poison, however, L+ (the quantity of toxin neces- sary both to neutralize one antitoxin unit and yet to be sufficiently in excess of neutralization to kill a guinea-pig of 250 grams in four or five days) was not found to be merely L0 + IT; but on actual experi- ment proved to be L0 + 101 T. Expressed graphically, the conditions may be stated as follows : T= .01 c.c. of the toxin bouillon. L + (neutral, of 1 antitox. unit yet killing 1 pig) = 2.01 c.c. or 201 T. LQ (complete neutral, of 1 antitox. unit) = 1. c.c. or 100 T. Difference - 1.01 c.c. or 101 T. Ehrlich, at first, endeavored to explain this surprising phenomenon on the basis of toxoids. He argued that the toxoids formed by de- terioration of toxin might be conceived as possessing three different degrees of affinity for antitoxin. If their affinity for antitoxin were equal to, or more marked than, that of the toxin itself, they could have no influence upon the dose L+ . If, however, their affinity for antitoxin were weaker than that of toxin, each fresh toxin unit added to the dose L0 would, first uniting with antitoxin, replace a corresponding quan- tity of these nontoxic substances of weaker affinity, and L+ would be reached only after all of these " epitoxoids," as Ehrlich called them, had been replaced, and toxin became free in the mixture. Thus, in analyzing our example, we have: 100 tox.-antitox. + 100 epitox.-antitox. = L0 ; add 1 T, and we have 101 tox.-antitox. + 99 epitoxoid-antitoxin + 1 epitoxoid free; add 101 T and we have 200 toxin-antitoxin + 100 epitoxoid free +\ T free = L + . Two facts, however, led Ehrlich to abandon the opinion that epi- toxoid was merely a variety of toxoid. He found, in the first place, that the stated relations between L0 and L+ were true for perfectly, fresh toxin-bouillon in which little or no deterioration had taken place. He observed, furthermore, that in old, altered toxin bouillon, while T was very much affected, the quantity needed to kill a pig con- stantly increasing, and the number of actual fatal doses in L0 con- TOXINS AND ANTITOXINS 209 stantly decreasing (by reason of toxoid formation), L+ remained practically unchanged. Simply stated, this means that the epitoxoids or substances which have weaker affinity for antitoxin than toxin itself are already present in fresh bouillon and are not increased with time. For this reason, Ehrlich has separated these substances from toxoids. He calls them " tox- on" and believes them to be, like toxin, primary secretory products of the diphtheria bacilli. The toxoids themselves, Ehrlich believes, are of two kinds, those with a stronger affinity for antitoxin than toxin it- self (protoxoicls) , and those whose affinity for antitoxin is equal to that of toxin. These latter he calls "syntoxoids." The toxon (epitoxoid originally), as Ehrlich believes, has a hapto- phore or "binding" group similar to that of toxin, but a different toxophore or "poisoning" group. Qualitatively it has been shown to differ from toxin in that, lacking the power to produce acute symp- toms, it causes gradual emaciation and paresis in animals. That this difference in the poisonous action of toxin and toxon is not merely a quantitative difference, referable to small quantities of toxin, was proved by Dreyer and Madsen,1 who showed that if they made a toxin-antitoxin mixture in which after injection the only evidence of incomplete neutralization lay in the emaciation and final paralysis of the test animals, the quantity of such a mixture could be increased five- and tenfold, without producing the true toxin symptoms in ani- mals. These authors, too, claim to have been able to immunize against toxin with such mixtures, thereby proving the identity of the haptophore groups of the two substances. The importance of this observation will become more evident in connection with the section on the "side- chain theory." Method of Partial Absorption of Toxin. — Ehrlich 2 has gathered more exact data in support of his views from what he terms the " Method of Partial Absorption " of toxin by antitoxin. In order to understand this method clearly, it is necessary to re- member that Ehrlich 3 believes the union of toxin with antitoxin to take place according to the chemical laws of valency. Just as in H20 oxygen has an atomic valency of 2 for hydrogen, so, in the fully neutralized toxin-antitoxin compound, he believes antitoxin to have a 1 Dreyer iind Madsen, Zeit. f. Hyg., xxxvii, 1901. 2 Ehrlich, "Gesammelte Arbeiten zur Immunitatsforsch.," Berlin, 1904. ^ Ehrlich, Deut. med. Woch., 1898. 14 210 INFECTION AND IMMUNITY valency of 200 for toxin. It would require, according to this, 200 T or toxin molecules to satisfy the affinities of one antitoxin molecule.1 This belief is based upon the following consideration : In determining the L0 dose, or fully neutralized toxin-antitoxin union, Ehrlich, as well as Madsen, found that the number of T units contained in such a dose was almost regularly a multiple of one hundred, recurring again and again as 25, 33, 50, 75, etc. This pointed to more or less regularity in the deterioration of toxin into toxoid, and to a more or less regular relation of toxin to toxon. Now, as we have seen before, if we could procure a perfectly pure toxin, the L0 dose plus one toxin unit would give us the L+ dose; that is, one toxin unit in excess of full neutraliza- tion would suffice to kill a guinea-pig of 250 grams in four to five days. Since a perfectly pure toxin, however, has not been obtainable up to the present time, it is clear that the number of pure toxin bonds contained in L+ must be less than the actual number of neutralizing units in the combination, a part of the antitoxin being bound by toxon and toxoid. The actual values obtained for the number of T units in L+ has never exceeded 200, and has usually been more than 100, the highest value ascertained by Madsen being 160. Given, therefore, a combining value which, being a multiple of one hundred, is often more than one hundred, but in an obviously impure state has never reached 200, it is most likely that 200 represents the actual value sought for. Assuming, therefore, upon the foregoing considerations, that the valency of antitoxin for toxin is 200, Ehrlich carries out his experi- ments in the following way: Given a toxin, the unit (T) of which is 0.024 c.c, he first deter- mines the L+ dose which, tested against the standard antitoxin unit, in this case is 2.05 c.c. But 2.05 c.c. = 85 T. (or. 2.05 4- .024) units. By mixing the L+ dose of toxin and antitoxin in such a way that the quantity of antitoxin is gradually increased, while the toxin remains always L+, and determining upon animals the amount of free toxin contained in each mixture, the following table may be constructed:2 0 antitox. unit representing 0 valencies + L + = 85 free T units. .1 " " " 20 " + L + = 85 " " " .25 " " " 50 " + L + - 60 " " " .8 " " " 160 " +L+ = 10 " " " .9 " " " 180 " +L+ = 3.5 " " " 1 Ehrlich, " Schlussbetrachtungen," Nothnagel's System. 2 Example taken from Ehrlich, Deut. med. Woch., 1898. TOXINS AND ANTITOXINS 211 It is plain that the substances with the strongest affinity for antitoxin must be bound first by the antitoxin. This does not diminish the toxic value of the mixture; and these are the protoxoids. Next are bound syntoxoids and toxins, and, finally, the toxons. It is plain that, by this method, the constitution of any given toxin may be ascertained, and Ehrlich has constructed, on the basis of these observations, what he terms his toxin spectrum. Minor differences of toxicity and affinity for the antibody have caused him, by the partial saturation method de- scribed, still further to divide toxin into proto-, deutero-, and trito-toxin. His spectra graphically describe the constitution of any given toxic bouillon and trace its deterioration as follows: Fty.t F/vtotoxoids Toxons \ 1 T 1 1 1"|"|' 'f M'I"i"i"i"i"|i | 1— | 1 1 0 10 20 JO 90 JO SO 70.60 90 100 ffO f20 130 190 150 160 170 ISO *M 200 Fig.2. Prototoxins Deutero- toxins Tritotoxins Deutero- FC9"3' Prototoxoids cc toxoids cc Tritotoxoids & Prototoxins ft Deutero* Tritotoxins ft toxins/3 Ehrlich's opinion as to the constitution of toxin is by no means fully accepted. Arrhenius,1 the great physical chemist, and Madsen, lArrheniusundMadsen, Zeit. f.physik.Chem., 1902; Festschrift, Kopenhagen, 1902. 212 INFECTION AND IMMUNITY a bacteriologist who was once a pupil of Ehrlich, have recently opposed Ehrlich's theory on grounds of physical chemistry. Modern theories of solution maintain that substances in solution are broken up into their atoms or atom-groups, known as ions. Thus, NaCl in solution would be " dissociated " into its Na ion and its CI ion, the completeness of the dissociation depending upon the concentra- tion of the solution. A solution of NaCl, therefore, contains, according to this view, three substances, NaCl undissociated and free ions of Na and CI, the relative quantities of the three present in any given solution being calculable, and depend upon a law known as the law of mass- action of Guldberg and Waage. These free ions are the elements, there- fore, which are active in the formation of further chemical combination. When a strong acid, in solution, acts upon a base, say HC1 upon am- monia (NH3), strong acid having the property of quite complete dis- sociation in relatively concentrated solutions, little or no ammonia would remain unbound. A weak acid, like boric acid, however, not being •as completely dissociated, would leave some ammonia uncombined even after more than quantitatively sufficient boric acid had been added. Arrhenius and Madsen, on the basis of careful researches into the re- action between tetanolysin and its antibody, believe that toxin and anti- toxin possess weak chemical avidity for each other, their interaction being comparable to that taking place between a weak acid and a base. Toxin-antitoxin solutions, therefore, would contain the neutral com- pound, but at the same time uncombined toxin and antitoxin. The qualities which Ehrlich ascribes to toxon, they believe, are due to the unbound toxin present in such mixtures. In careful studies in which they inhibited the hemolytic action of ammonia by gradual addition of boric acid, they were able to show complete parallelism between the conditions governing this neutralization and those con- cerned in their tetanus experiments. Their explanation has the advantage of extreme simplicity oyer that of Ehrlich, but since the differences of opinion are now the subject of active experimental controversy, a critical discussion must rest until further facts are revealed. The Side-Chain Theory. — We have seen that the extensive researches of Ehrlich into the nature of the toxin-antitoxin reaction led him to believe that the two bodies underwent chemical union, forming a neu- tral compound. The strictly specific character of such reactions, further- more, diphtheria antitoxin binding only diphtheria toxin, tetanus antitoxin only tetanus toxin, etc., led him to assume that the chemical TOXINS AND ANTITOXINS 213 affinity between each antibody and its respective antigen depended upon definite atom groups contained in each. Ehrlich * had, in 1885, published a treatise in which he discussed the manner of cell-nutrition and advanced the opinion that in order to nourish a cell, the nutritive substance must enter directly into chemical combination with some elements of the cell protoplasm. The great number and variety of chemical substances which act as nutriment led him to believe that the highly complex protoplasmic molecules of cells were made up of a central atom-group (Leistungs-Kern) upon which depended the specialized activities of the cell, and a multi- plicity of side chains (a term borrowed from the chemistry of the benzol group), by means of which the cell entered into chemical relation with food and other substances brought to it by the circulation. If we illustrate graphically by the chemical conception from which the term side chain was borrowed, in salicylic acid, the formula given, the OH I C /\ H— C C— COOH I I H— C C— H V c I H benzol ring represents the " Leistungs-Kern/ ' or radicle, while GOOH and OH are side chains by means of which a variety of other substances may be brought into relation with the " radicle, " for instance, as in methyl salicylate. OH I C /\ C02CH3 Just as nutritious substances are thus brought into workable re- lation with the cell by means of the atom-groups corresponding to side chains, so Ehrlich believes toxins exert their deleterious action only because the cells possess side chains by means of which the toxin can be chemically bound. These side chains, Ehrlich in his later work calls "receptors." The receptors or side chains present in the cells and 1 Ehrlich, " Das Sauerstoffbediirfniss des Organismus," Berlin, 1885. 214 INFECTION AND IMMUNITY Toxin -AnHioxin Fig. 56. — Toxin and Antitoxin. possessing by chance specific affinity for a given toxin, are, by their union with toxin, rendered useless for their normal physiological func- tion. By the normal reparative mechanism of the body these recep- tors are probably cast off and regenerated. Regenerative processes of the body, however, do not, as a rule, stop at simple replacement of lost elements, but, according to the Irypothesis of Weigert,1 usually tend to overcompensation. The receptors eliminated by toxin absorption are not, therefore, simply reproduced in the same quantity in which they are lost, but are reproduced in excess of the simple physiological needs of the cell. Continuous and increasing dosage with the poison, consequently, soon leads to such excessive production of the particular re- ceptive atom-groups that the cells involved in the process become overstocked and cast them off to circulate freely in the blood. These freely circulating re- ceptors— atom-groups with specific affinity for the toxins used in their production — represent the antitoxins. These, by uniting with the poison before it can reach the sensitive cells, prevent its deleterious action. (Fig. 56.) The theory of Ehrlich, in brief, then, depends upon the assumptions that toxin and antitoxin enter into chemical union, that each toxin possesses a specific atom-group by means of which it is bound to a pre- existing side chain of the affected cell, and that these side chains, in ac- cordance with Weigert's law, under the influence of repeate-d toxin stimu- lation, are eventually overproduced and cast off by the cell into the circulation. It stands to reason that this theoretical conception would be vastly strengthened were it possible to show that such receptors or toxin- binding atom-groups actually pre-existed in the animal body, and such support was indeed given by the experiments of Wassermann and Taka- ki.2 These observers succeeded in showing that tetanus toxin could be rendered innocuous if, before injection into animals, it was thoroughly mixed with a sufficient quantity of the fresh brain substance of guinea- pigs. Similar observations were independently made by Asakawa,3 and 1 Weigert, Verhandl. d. Ges. Deutsch. Naturf. u. Aerzte, Frankfurt, 1896. 2 Wassermann imd Takaki, Berl. klin. Woch., 1898. s Asakawa, Cent. f. Bakt., 1898. TOXINS AND ANTITOXINS 215 variously confirmed. Kempner and Schepilewsky l showed a similar relation to exist between brain tissue and botulismus toxin, and Myers 2 brought proof of analogous conditions in the case of suprarenal tissue and cobra poison. In the discussion of Ehrlich's toxin analysis, we have seen that he accounted for variations in the quantitative relations by the existence of toxoids and toxons. He explained the striking fact that toxoids had lost their poisonous nature and yet retained full powers to neutralize antitoxin by the assumption that toxin was made up of two separate atom-groups; one, the haptophore group which possessed the specific affinity for the antitoxin or cell receptor; the other, the toxophore group by means of which the actually harmful effects were produced. The haptophore groups of all three of these substances, toxin, toxoid, and toxon, by virtue of their antitoxin-binding power, he assumed to be alike; in toxoid, the toxophore group has been destroyed or altered; in toxon, the toxophore group is qualitatively different from that of toxin. The haptophore group, however, being alone concerned in neu- tralizing receptors, all three of these substances should, if Ehrlich's theory is to be tenable, produce antitoxin. Dreyer and Madsen,3 ac- cordingly, actually succeeded in producing diphtheria antitoxin by im- munization with toxon. Attempts to produce antitoxin with toxoids have succeeded in the hands of Ehrlich and others. Such experiments have not, however, been always successful, a notable failure being that of Bruck.4 On the basis of such negative results the theory was advanced by Wassermann that overproduction of receptors was stimulated by the irritation (Zellreiz) produced by the toxophore group — a stimula- tion not present in the case of toxoids. 1 Kempner und Schepilewsky, Zeit. f. Hyg., 1898. 2 Myers, Cent. f. Bakt., i, 1899. 3 Dreyer und Madsen, Zeit. f. Hyg., 1901. 4 Bruck, Zeit. f. Hyg., 1904. CHAPTER XIV PRODUCTION AND TESTING OF ANTITOXINS DIPHTHERIA ANTITOXIN In spite of the great advances in our theoretical knowledge of anti- bodies, gained during the last three decades, extensive therapeutic application has been made of the antitoxins only. Pre-eminent among these from a practical point of view are the antitoxins against diphtheria and tetanus toxin. For diphtheria, careful statistical studies have demonstrated, beyond doubt, the therapeutic value of the serum treatment. Biggs and Guerard, in a general statistical review, ar- rived at the conclusion that the death rate of diphtheria had been re- duced fifty per cent by the use of antitoxin. Approximately the same estimate is made by Dieudonne1 who studied almost 10,000 treated cases. Production of Diphtheria Antitoxin. — The methods for producing diphtheria antitoxin vary only in minor technical details. The first requisite for successful antitoxin production is the possession of a strong toxin. The various means of obtaining this are outlined in the section on diphtheria toxin. The toxin used should be of such potency that less than 0.01. cc. will kill a guinea-pig of 250 grams weight in four to five days.2 For experimental purposes, goats or sheep may be used for immuniza- tion; for antitoxin production on a large scale, horses have been found to be the most useful animals. The horses should be young, four to six years old, vigorous, and healthy. It is advisable that they be sub- jected to the mallein test to exclude possible infection with glanders. The toxin injections are made subcutaneously. Because of the dif- ferences in susceptibility noted in various horses, it is advisable that the first doses of toxin should be either very small or weakened by chemicals or heat, or combined with antitoxin. In the Pasteur Institute in Paris, the small initial dose of toxin (0.5 cc.) is mixed before injection with an equal quantity of Lugol's solution (iodin-potassium iodid solution) . 1 Dieudonne, Arb. a. d. kais. Gesundheitsamt, 1895 and 1897. * Park, " Pathog. Bacteria and Protozoa," N. Y., 1908. 216 PRODUCTION AND TESTING OF ANTITOXINS 217 Park l advises an initial dose of 5,000 toxin units (about 20 c.c. of a strong toxin) combined with 100 units of antitoxin. The same amount is given with the second and third doses of toxin. The intervals between injections are from five days to a week, depending upon the time necessary for complete subsidence of the reaction (temperature). The doses of toxin are gradually increased until, at the end of two or three months, more than ten times the original dose is given (50,000 units) . Horses vary greatly in the strength of antitoxin which they will pro- duce. At the end of three or four months in favorable animals one cubic centimeter of serum may contain 250 to 800 antitoxin units. Fur- ther immunization will often increase the antitoxin output to 1,000 and more units to the cubic centimeter of serum. Park states that none of the horses used by him has ever yielded 2,000 units to the cubic centi- meter. The same writer advises a three months' period of rest from immunization at the end of every nine months. Given such resting periods, some horses have continued to furnish high-grade antitoxin for from two to four years. In order to obtain serum from horses, a sharp cannula is introduced into the jugular vein. After leading the horse into a specially con- structed stall, its head is slightly deflected and pressure is made upon the jugular vein below the point into which the needle is to be plunged. Compression can also be made by surrounding the neck of the horse close to the shoulders with a leather strap over a pad laid directly upon the vein. The vein becomes visible along the lower margin of the neck in a line running from the angle of the jaw to the edge of the scapula. The skin, of course, is previously shaved and sterilized. The cannula is then plunged into the vein, either with or without previous incision through the skin, and, through a sterile rubber tube, the blood is allowed to flow into high glass cylinders or slanted Erlenmeyer flasks. In this way, large quantities of blood may be obtained and, according to Kretz,2 as much as six liters may be taken at a time at intervals of a month, without injuring the animal. Ligature of the vein after bleeding is unnecessary. The cylinders and flasks are allowed to remain standing for two or three days in a cool place, preferably at or below 10° C. At the end of this time, the serum may be pipetted or siphoned away from the 1 Park, loc. cit., p. 212. 2 Kretz, in" Handb. der Techn. u. Meth. d. Immun./'Kraus and Levaditi, vol. ii, 1908. 2 IS INFECTION AND IMMUNITY clot and stored in the refrigerator. In order to diminish the chances of contamination, five-tenths per cent of carbolic acid or four-tenths per cent of tri-cresol may be added. Antitoxin is fairly stable and if kept in a cool, dark place, may re- main active, with but slight deterioration, for as long as a year. Kept in a dry state, in vacuo, over anhydrous phosphoric acid, by the method of Ehrlich, it retains its strength indefinitely. Standardization. — Since antitoxin units are measured in terms of toxin, it is obvious that uniformity of measurement necessitates the possession by the various laboratories of a uniform toxin. Antitoxin being more stable than toxin, uniformity of toxin is obtained by means of a standard antitoxin distributed from a central laboratory. This was first done by Ehrlich in Germany, and is now done for the United States by the Public Health and Marine Hospital Service laboratories. Bottles of the distributed antitoxin are marked with the number of units con- tained in each cubic centimeter. Dilutions of this antitoxin are mixed with varying quantities of the toxin to be tested, the mixtures are al- lowed to stand for fifteen minutes to permit union of the two elements, and injections into guinea-pigs of 250 grants weight are made. In this way, the L+ dose of the toxin is determined. (The L+ dose, as we have seen in a previous section [p. 208], is the quantity of poison not only suf- ficient to neutralize one antitoxin unit,1 but to contain an excess beyond this sufficient to kill a guinea-pig of 250 grams in four to five days. L+ is chosen rather than L0, the simple neutralizing dose, because of the difference between toxins in their contents of toxoid and toxon.2) The L+ dose of the toxin having thus been determined, this quantity is mixed with varying dilutions of the unknown antitoxin.3 Thus, given an antitoxin in which 300 to 400 units to the cubic centimeter are suspected, dilutions of 1 : 200, 1 : 250, 1 :300, etc., are made. One cubic centimeter of each of these is mixed with the L+ dose of the toxin, and the mixtures are injected into guinea-pigs of about 250 grams. If the guinea-pig receiving L+ plus the 1 : 250 dilution lives and the one re- ceiving L+ plus the 1 : 300 dilution dies in the given time, we know that the unit sought must lie between these two values, and further similar experiments will easily limit it more exactly. The possibility of error in 1 A unit of diphtheria antitoxin is a quantity of antitoxin sufficient to protect a guinea-pig of 250 grams against 100 times the fatal dose of diphtheria toxin. 2 Madsen, in Kraus u. Levaditi, " Handbuch," etc., 1907. 3 Donitz, " Die Werthbem. der Heilsera," in Kolle u. Wassermann, " Hand- buch," etc. PRODUCTION AND TESTING OF ANTITOXINS 219 measurement is much diminished by the use of larger quantities of dilu- tions higher than those given. Four c.c. is the volume usually injected. Since 1902, the production and sale of diphtheria antitoxin has been regulated by law in the United States. From time to time, antitoxin is bought in the open market and examined at the hygienic laboratories of the United States Public Health and Marine Hospital Service. Anti- toxic serum which contains less than two hundred units to each cubic centimeter is not permitted upon the market. In a previous section we have seen that Hiss and Atkinson 1 and others have shown an increase in the globulin contents of blood serum of immunized animals. It has been shown, furthermore, that the pre- cipitation of such serum with ammonium sulphate carried down in the globulin precipitate all the antitoxic substances contained in the serum. Upon a basis of globulin precipitation, Gibson 2 has recently perfected a method of concentrating and purifying diphtheria antitoxin for thera- peutic use. This procedure, as carried out at the New York Depart- ment of Health, is, in principle, as follows: The serum, as taken from the horse, is heated to 56° C. for twelve hours. This converts about half of the pseudoglobulin into euglobulin, the antitoxin remaining in the pseudoglobulin fraction.3 It is then 4 precipitated with an equal volume of a saturated ammonium sulphate solution. After two hours, the precipitate is caught in a filter and redissolved in a quantity of water corresponding to the original quantity of serum. After filtration, this solution is again precipitated with saturated ammonium sulphate solution and the precipitate again fil- tered off. The precipitate is then treated with a saturated solution of sodium chloride of double the volume of the original serum. This is allowed to stand for about twelve hours. At the end of this time the antitoxin-containing globulin is in solution and is pipetted away from the precipitate and filtered. This salt-solution extract is then pre- cipitated with twenty-five hundredths per cent acetic acid. The re- sulting precipitate of globulin is thoroughly dried by pressure between filter papers and placed in a parchment dialyzer. Dialysis with run- ning water is continued for seven to eight days, after neutralization with sodium carbonate, in order to remove the sodium chloride. At the end of this time, the globulin solution remaining in the dialyzer is fil- iHiss and Atkinson, Jour. Exper. Med., v, 1900. 2 Gibson, Jour, of Biol. Chem., i, 1906. 3 Dr. Banzhaf, personal communication. i Gibson and Collins, Jour, of Biol. Chem., iii, 1907. 220 INFECTION AND IMMUNITY ■ tered through a Berkefeld candle for the purpose of sterilization, after the addition of 0.8 per cent sodium chloric!. According to Gibson, this method produces a yield of antitoxin which equals about four-fifths of the original quantity but is concentrated five- to seven-fold. The method has more recently been modified as follows: After heating to 56° C, as above, and cooling, ammonium sulphate is added to the serum to thirty per cent saturation. This brings down all the euglobulins. This is then filtered and the filtrate, which contains the pseudoglobulins with the antitoxin, is again precipitated with ammonium sulphate in a concentration of fifty-four per cent of satura- tion. The precipitate is then separated on a paper, pressed to dryness, and directly diary zed.1 Park and Thome 2 have found that the use of such concentrated antitoxin is, therapeutically, equally efficient as the unconcentrated, and possesses the advantage of less frequently giving rise to the sec- ondary reactions in skin and mucous membranes occasionally noticed after the use of ordinary antitoxin, and referable, probably, to some other constituent of the horse serum. Diphtheria antitoxin is therapeutically used in doses ranging from 3,000 to 20,000 units. For prophylactic immunization of healthy individuals, about 500 units should be used. TETANUS ANTITOXIN Production of Tetanus Antitoxin. — The production of tetanus anti- toxin is, in every way, analogous to that of diphtheria antitoxin. It is necessary in the first place to produce a powerful tetanus toxin. The methods of procuring this will be discussed in the section upon tet- anus toxin, page 458. Suffice it to say here that the most satisfactory method of obtaining toxins consists in cultivating the bacilli upon veal broth containing five-tenths per cent to two per cent sodium chloric! and one per cent pepton. It has been advised, also, that the broth should be neutralized by means of magnesium carbonate rather than with sodium hydrate. The bacilli are cultivated for eight to ten days at incu- bator temperature and the broth filtered rapidly through Berkefeld filters. The toxin may be preserved in the liquid form with the ad- dition of five-tenths per cent carbolic acid, or may be preserved in the dry state after precipitation with ammonium sulphate. 1 Dr. Banzhaf, personal communication. 2 Park and Throne, Amer. Jour. Med. Sci., Nov., 1906. PRODUCTION AND TESTING OF ANTITOXINS 221 It is necessary to determine the strength of the poison. This is done according to Behring * by determining the smallest amount of toxin which will kill a white mouse of twenty grams weight within four days. This is most easily done by making dilutions of the toxin ranging from 1 : 100 to 1 : 1,000, and then injecting quantities of 0.1 c.c. of each of these dilutions subcutaneously into white mice. In this way, the mini- mal lethal dose is ascertained. For the actual production of antitoxin, horses have been generally found to be the most favorable animals. The horses should be healthy and from five to seven years old. The first injection of toxin admin- istered to these animals should be attenuated in some way. Vari- ous methods for accomplishing this have been in use. In America, the first injection of about ten to twenty thousand minimal lethal doses 2 (for mice of twenty grams weight) is usually made subcutaneously to- gether writh sufficient antitoxin to neutralize this quantity. In Germany, v. Behring uses, for his first injection, a much larger dose of toxin to which about 0.25 per cent of terchlorid of iodin has been added. Immediately after an injection, the animals will usually show a reac- tion expressed by a rise of temperature, refusal of food, and some- times muscular twitching. A second injection should never be given until all such symptoms have completely subsided. This being the case, after five to eight days double the original dose is given together wTith a neutralizing amount of antitoxin or with the addition of terchlorid of iodin. Again after five to eight days, a larger dose is given and there- after, at similar intervals, the quantity of toxin is rapidly increased. In America the neutralizing antitoxin is omitted after the third or fourth injection; in v. Behring's laboratory the quantity of terchlorid of iodin is gradually diminished. The increase of dosage is often controlled by the determination of the antitoxin contents of the animal's blood serum. The immunization is increased until enormous doses (500 c.c.) of a toxin in which the minimal lethal dose for mice is represented by 0.0001 c.c, or less, is borne by the horse without apparent harm. The antitoxic serum is then obtained by bleeding from the jugular vein, as in the case of diphtheria antitoxin. It may be preserved in the liquid state by the addition of five-tenths per cent of carbolic acid or four-tenths per cent of tricresol. 1 v. Behring, Zeit. f. Hyg., xii, 1892 ; Deut. med. Woch., 1900. 2 According to Park the "horses receive 5 c.c. as the initial dose of a toxin of which 1 c.c. kills 250,000 grams of guinea-pig, and along with this a sufficient amount of antitoxin to neutralize it." 222 INFECTION AND IMMUNITY Standardization. — The universal prophylactic use of tetanus antitoxin has, as in the case of diphtheria antitoxin, necessitated its standardiza- tion. A variety of methods are in use in different parts of the world. In the following description the American method only will be consid- ered as laid down under the law of July, 1908, and based upon the work of Rosenau and Anderson * at the United States Hygienic Laboratories at Washington. In conjunction with a committee of the Society of American Bac- teriologists, these authors have defined the unit of tetanus antitoxin as follows : The unit shall be ten times the least amount of serum necessary to save the life of a 350 gram guinea-pig for ninety-six hours against the official test dose of standard toxin. The test dose consists of 100 minimal lethal doses of a precipitated toxin preserved under special conditions at the hygienic laboratory of the Public Health and Marine Hospital Service. (The minimal lethal dose is in this case, unlike Behring's minimal lethal dose, measured not against 20 gram mice, but against 350 gram guinea-pigs.) In the actual standardization of tetanus antitoxin, as in that of diph- theria antitoxin, the L+ dose of toxin is employed. The L+ dose is, however, in this case, defined as the smallest quantity of tetanus toxin that will neutralize one-tenth of an immunity unit, plus a quantity of toxin sufficient to kill a 350 gram guinea-pig in just four days. At the Hygienic Laboratory at Washington, a standard toxin and antitoxin are preserved under special conditions, and standard toxin and anti- toxin, arbitrary in their first establishment, are kept constant by being measured against each other from time to time. In measuring the anti- toxic serum thus preserved, at the Hygienic Laboratory, a mixture of one-tenth of a unit of antitoxin and 100 minimal lethal doses of the standard toxin must contain just enough free poison to kill the guinea- pig in four days. This L+ dose of the standard toxin is given out to those interested commercially or otherwise in the production of antitoxin. In measuring an unknown antitoxic serum against this L+ dose of toxin, a large number of mixtures are made, each containing the L+ dose of the toxin and varying quantities of the antitoxin. Dilu- tions must always be made with 0.85 per cent salt solution and the total quantity injected into the animals should always be brought up to 1 Rosenau and Anderson, Pub. Health and Mar. Hosp. Serv. U. S., Hyg. Lab. Bull. 43, 1908. PRODUCTION AND TESTING OF ANTITOXINS 223 4 c.c. with salt solution in order to equalize the conditions of concen- tration and pressure. The mixtures are then kept for one hour at room temperature in diffused light. After this they are subcutaneously injected into a series of guinea-pigs weighing from 300 to 400 grams. The following example of a test is taken from the article by Rosenau and Anderson quoted above. No. of Guinea- Weight of Guinea-pig (Grams.) Subcutaneous Injection of a Mixturp: of Time of Death. pig- Toxin (Test Dose). Antitoxin. 1 2 3 4 5 360 350 350 360 350 Gram. 0.0006 .0006 .0006 .0006 .0006 c.c. 0.001 .0015 .002 .0025 .003 2 days 4 hours 4 days 1 hour Symptoms Slight symptoms No symptoms In this series the guinea-pig, receiving 0.0015 c.c. of the antitoxin, died in approximately four days ; 0.0015 c.c. therefore represents one-tenth of an immunity unit. In therapeutically employing antitoxin for prophylactic purposes, about 1,500 units should be employed. CHAPTER XV LYSINS, AGGLUTININS, PRECIPITINS, AND OTHER ANTIBODIES LYSINS In the immediately preceding sections, we have dealt solely with immunity as it occurs where soluble toxins play an important part and in which antitoxins are developed in the immunized subject. There are many species of pathogenic bacteria, however, which stimulate the production of little or no antitoxic substance when introduced into animals, and the resistance of the immunized animal can not, therefore, be explained by the presence of antitoxin in the blood. v. Foclor,1 Nuttall,2 Buchner,3 and others had in 1886 and the years following carried on investigations which showed that normal blood serum possessed the power of killing certain of the pathogenic bacteria. Nuttall, working under the direction of Fliigge, made the important dis- covery that this bactericidal power became gradually diminished with time, and could be experimentally destroyed by exposure of the serum to a temperature of 56° C. for one-half hour. Buchner, who confirmed and extended the observations of Nuttall, called this thermolabile sub- stance upon which the bactericidal character of the serum seemed to depend " alexin." Our knowledge of the bactericidal action of serum was, soon there- after, extensively increased by the discovery, by Pfeiffer and Ioaeff,4 that cholera spirilla injected into the peritoneal cavity of a cholera- immune guinea-pig were promptly killed and almost completely dis- solved. The same phenomenon could be observed when the spirilla, mixed with fresh immune serum, were injected into the peritoneum of a normal guinea-pig. The processes observed by Pfeiffer as taking place intraperitoneally were soon shown by Metchnikoff,5 Bordet,6 and others to take place, though to a lesser extent, in vitro. Bordet, furthermore, observed that 1 v. Fodor, Deut. med. Woch., 1886. 2 Nuttall, Zeit. f. Hyg., 1886. 3 Buchner, Cent, f . Bakt. , 1889. 4 Pfeiffer und Isaeff, Zeit. f . Hyg., 1894. 5 Metchnikoff, Ann. de l'inst. Pasteur, 1895. 6 Bordet, ibid., 1895. 224 LYSINS, AGGLUTININS, PRECIPITINS, ETC. 225 the bacteriolytic digestive power of such immune serum, when destroyed by heating, or after being attenuated by time, could be restored by the addition to it of small quantities of normal blood serum. It could, in other words, be " reactivated " by normal serum. From this obser- vation Bordet drew the conclusion that the bactericidal or bacteriolytic action of the serum depended upon two distinct substances. The one present in normal serum and thermolabile, he conceived to be identical with Buchner's alexin. The other, more stable, produced or at least increased in the serum by the process of immunization, he called the " sensitizing substance.'' This substance, he believed, acting upon the bacterial cells, rendered them vulnerable to the action of the alexin. Without the previous preparatory action of the "sensitizing substance" the alexin was unable to act. Without the co-operation of alexin, the " sensitizing substance " produced no visible effects. Bordet's interpretation of the phenomenon of lysis differs essentially from that of Ehrlich, in that both active serum components are con- ceived by him, though independent, to act directly upon the bacterial cell. A few years later, Bordet was able to show that exactly analogous conditions governed the phenomenon known as " hemolysis" or dis- integration of red blood cells. It had been known for many years that in the transfusion of blood from an animal of one species into an animal of another species, in- jury was done to the red corpuscles which were introduced. Observed in the test tube, the red cells in the heterologous serum were seen to give up their hemoglobin in the fluid, the mixture taking on the red transparency characteristic of what is known as "laked" blood. Buch- ner,1 in his alexin studies, had shown that the blood-cell destroying action of the normal serum was subject to the same laws as the bac- tericidal power of similar serum, in that it was destroyed by heating, and he assumed that both the bacteriolytic and the hemolytic action of normal serum were due to the same "alexin." Metchnikoff,2 more- over, had pointed out the striking analogy between the two phenomena as early as 1889. Bordet 3 now observed that the blood serum of guinea-pigs previously treated with the defibrinated blood of rabbits developed marked powers of dissolving rabbits' corpuscles, and that this hemolytic action could 1 Buchner, Arch. f. Hyg., xvii, 1893; Waremberg, Arch. d. med. exper., 1891. 2 Metchnikoff, Ann. de l'inst. Pasteur, 1889. 3 Bordet, Ann. de l'inst. Pasteur, t. xii, 1898. 15 226 INFECTION AND IMMUNITY be destroyed by heating to 56° C, but " reactivated " by the addition of fresh normal serum. He had thus produced an immune hemolysin, just as Pfeiffer had produced immune bacteriolysin, and had demon- strated the complete parallelism which existed between the two phe- nomena. A practical test-tube method was thus given for the investigation of the lysins, just as a practical test-tube method for antitoxin researches had been developed by Ehrlich in his ricin-antiricin experiments. The path of investigation thus pointed out by Bordet was soon ex- plored in greater detail by Ehrlich and Morgenroth.1 The reasoning which Ehrlich had applied in explaining the production of antitoxins was thought, by these observers, to be equally applicable to the phe- nomena of bacteriolysis and hemolysis. Since the thermolabile substance or alexin, renamed by Ehrlich i " complement," was already present in normal serum and had been shown to be little, if at all, increased during the process of immunization, this substance could have but little relation to the changes taking place in the animal body as immunity was acquired. The more stable serum- component, however, the "substance sensibilisatrice " of Bordet, or, as Ehrlich now called it, the "immune body," was the one which seemed specifically called forth by the process of active immunization. Ehrlich argued, therefore, that when bacteria or blood cells were injected into the animal, certain atom-groups or chemical components of the injected substances were united to other atom-groups or "side chains" of the protoplasm of the tissue cells. These " side chains " or receptors, then reproduced in excess and finally thrown free into the circulation, con- stituted the "immune body." The immune body, therefore, he con- cluded, must possess atom complexes which endow it with specific chemical affinity for the bacteria or red blood cells used in its produc- tion. This contention was supported by Ehrlich and Morgenroth by an ingenious series of experiments. Having in their possession, at that time, the blood serum of a goat immunized against the red blood cells of a sheep, they inactivated it (destroyed the complement or alexin) by heating to 56° C. The serum then contained only the "substance sensibilisatrice" or immune body. To this inactivated serum they added sheep's red corpuscles, without obtaining hemolysis. Having left the inactive serum and the sheep's corpuscles in contact with each other for some time, they separated 1 Ehrlich und Morgenroth, Berl. klin. Woch., 1, 1899. LYSINS, AGGLUTININS, PRECIPITINS, ETC. 227 them by centrifugalization. To the supernatant fluid, they now added sheep-blood corpuscles and normal goat serum (complement) and found that no hemolysis took place. The immune body had apparently gone out of the serum. The red cells which had been in contact with the serum and separated by the centrifuge were then washed in salt solution and to them complement was added in the form of fresh normal serum. Immunising Substance Complement Body Cell Fig. 57. — Ehrlich's Conception of Cell-Receptors, Giving Rise to Lytic Immune Bodies (Haptines of the Third Order). Hemolysis occurred. It was plain, therefore, that the immune body of the inactivated serum had gone out of solution and had become at- tached to the red blood cells, or, as Ehrlich expressed it, the immune body by means of its " haptophore " atom-group had become united to the corpuscles. In contrast to this, if normal goat serum (containing Complement Immune body Cell used for immun'^in^ Fig. 58. — Complement, Amboceptor or Immune Body, and Antigen on Immunizing Substance. complement only) was added to sheep corpuscles and separated again by centrifugalization, the supernatant fluid was found to be still capable of reactivating inactivated serum (immune body) . This he interpreted as proving that the complement was not bound to the corpuscles directly. If the three factors concerned — corpuscles, immune body, and complement — were mixed and the mixture kept at 0° C, no hemolysis 228 INFECTION AND IMMUNITY occurred; yet, centrifugalized at this temperature, immune body was found to have become bound to the corpuscles, the complement re- maining free in the supernatant fluid. If the same mixture, however, was exposed to 37° C, hemolysis promptly occurred. From these facts, Ehrlich concluded that complement did not direct- ly combine with the corpuscles, but did so through the intervention of the immune body. This immune body, he reasoned, possessed two distinct atom-groups or haptophores; one, the cytophile haptophore group, possessing strong chemical affinity for the red blood cell; the other, or complementophile haptophore group, with weaker avidity for the complement. Because of this double combining power, Ehrlich speaks of the immune body as " amboceptor." His views as to the nature and action of immune body and complement are graphically represented in Figs. 57 and 58 (p. 227). From what has been said before, it will be seen that the fundamental difference between the conceptions of the mechanism of the lytic proc- esses as held by Bordet and by Ehrlich lies in the ability of the alexin or complement to act directly upon the antigen, as claimed by Bor- det, or, as Ehrlich holds, only through the intermediation of the im- mune body. Bordet's views,1 by no means disproved and still held by many bacteriologists, may be summed up in his own words as follows: "Neither immune body nor antigen (bacterium, blood cell, etc.) alone has any manifest affinity for alexin (complement) ; but, united, they form a complex which can absorb alexin. " The absorption of comple- ment is thus conceived as a property of the immune body or amboceptor (or, in Bordet's language, sensitizer) plus its specific antigen — acting as a complex and not through a complementophile group of the immune body. AGGLUTININS Although Metchnikoff 2 and Charrin and Roger 3 had noticed pecul- iarities in the growth of bacteria when cultivated in immune sera, which were unquestionably due to agglutination, the first recognition of the agglutination reaction as a separate function of immune sera was the achievement of Gruber and Durham. While investigating the Pfeiffer reaction with B. coli and the cholera vibrio, Gruber and Durham 4 1 Bordet, A Resume of Immunity in " Studies in Immunity." Transl. by Gay, Wiley & Son, 1909. 2 Metchnikoff, " Etudes sur Fimmunite," IV Memoir, 1891. 3 Charrin et Roger, Compt. rend, de la soc. de biol., 1889. 4 Gruber und Durham, Munch, med. Woch., 1896. LYSINS, AGGLUTININS, PRECIPITINS, ETC. 229 noticed that if the respective immune sera were added to bouillon cul- tures of these two species, the cultures would lose their turbidity and flake-like clumps would sink to the bottom of the tube, the supernatant fluid becoming clear. Gruber, at the same time, called attention to the fact that immune sera would affect in this way not only the microor- ganism used in their production, but, to a less energetic extent, other closely related bacteria as well. Widal, very soon after Gruber and Durham's announcement, ap- plied the agglutination reaction to the practical diagnosis of typhoid fever, finding that the serum of patients afflicted with this disease showed agglutinating power over the typhoid bacillus at early stages in the course of the fever. The reaction, thus practically applied to clinical diagnosis, was soon shown to be of great importance in its Fig. 59. — Microscopic Agglutination Reaction. bearing on bacteriological species differentiation. Since animals im- munized against a definite species of bacteria acquire in their sera specific agglutinating powers for these bacteria and at best only slight agglutinating powers for other species, immune sera can be used ex- tensively in differentiating between bacterial varieties. Agglutination may be observed microscopically or macroscopically. Bacteria brought into contact with agglutinating serum in the hanging drop rapidly lose their motility, if motile, as in the case of typhoid bacilli, and gather together in small clumps or masses. The microscopic picture is striking and easily recognized and the reaction takes place with varying speed and completeness, according to the strength of the agglutinating serum. As the reaction approaches completeness, the clumps grow larger, 230 INFECTION AND IMMUNITY individual microorganisms become more and more scarce, finally leaving the medium between clumps entirely clear. While the clumping of a motile organism suggests that motility has something to do with the coming together in clumps, it nevertheless has no relation whatever to agglutination, motile and non-motile organisms alike being subject to the reaction. Microscopically observed, in small test tubes or capillary tubes, ag- glutination evidences itself by the formation of flake-like masses which Fig. 60. — Macroscopic Agglutination. Dilutions from 1 in 10 to 1 in 1,000. The first tube contains a 1 : 20 control with the bacteria and normal serum. Agglutination complete in the tubes marked 10, 20, 50, 100. settle into irregular heaps at the bottom, leaving the supernatant fluid clear, in distinct contrast to the even flat sediment and the clouded supernatant fluid of the control. Macroscopically, too, agglutination is evidenced when bacteria are grown in broth to which immune serum has been added. Instead of evenly clouding the broth, the micro- organisms develop in clumps or chains. Another phenomenon probably produced by agglutinins is the so- LYSINS, AGGLUTININS, PRECIPITINS, ETC. 231 called " thread-reaction " of Pfaundler.1 This consists in the forma- tion of long convoluted threads of bacterial growth in the hanging drop of dilute immune serum after twenty-four hours. Very strict speci- ficity is attributed to this reaction by Pfaundler. Agglutinins act upon dead as well as upon living bacteria. For the microscopic tests bacterial emulsions killed by formalin were intro- duced by Neisser. Ficker 2 has recently succeeded in preparing an emulsion of typhoid bacilli, which is permanent and may be kept indefinitely, and may be employed for macroscopic agglutinations.3 Attention has been called by various workers to a source of error in all these methods, known as pseudo-clumping.4 The causes for such clumping not due to agglutinins seem to lie in the presence of blood cells in the serum or excessive alkalinity of the culture medium.5 While the microscopic methods are more suitable for clinical-diag- nostic purposes, because of the smaller amounts of blood required, the macroscopic tests are far preferable for the purposes of bacterial differen- tiation and research. Greater exactitude of dilution is possible when dealing with larger quantities; microscopic unevenness in the bacterial emulsion does not become a source of error; and positive and negative reactions are more sharply defined. Nature of Agglutinins. — Gruber and Durham,6 the discoverers of ag- glutinins, at first advanced the opinion that the agglutinins were identical with the immune body concerned in the Pfeiffer reaction, which by in- juring the bacteria rendered them susceptible to the alexins. Pfeiffer 7 and Kolle 8 soon showed, however, that by the addition of cholera vibrio to immune serum, the agglutinins could be completely absorbed, or used up, while bacteriolytic substances still remained. The same authors demonstrated that immune serum, preserved for several months, would lose its agglutinins without a corresponding loss of bacteriolytic power. It has been variously shown since then, by these and other authors, that the agglutinins and the bactericidal substances are in no way parallel 1 Pfaundler, Cent. f. Bakt., xxiii, 1898. 2 Ficker, Berl. klin. Woch., 1903. 3 The exact method of production of "Ficker's Diagnosticum" is a proprietary secret. 4 Savage, Jour, of Path, and Bact., 1901. 5 Biggs and Park, Amer. Jour, of Med. Sci., 1897; Block, Brit. Med. Jour., 1897. 6 Loc. cit. ? Pfeiffer, Deut. med. Woch., 1896. 8 Pfeiffer und Kolle, Cent. f. Bakt., xx, 1896. 232 INFECTION AND IMMUNITY in their development, and that strongly agglutinating sera may be ex- tremely weak in bactericidal substances and vice versa, the relative quan- tity of either of these substances depending to a certain extent upon the method of immunization. Whether or not agglutinins possess any direct protective function can not at present be stated with certainty. Metchnikoff * assigns to them a purely secondary role. As a matter of fact, agglutinated bacteria 2 are not killed by the act of agglutination and are often as virulent as non-agglutinated cultures. The agglutinins, furthermore, unlike the bactericidal substances in sera, remain active after exposure to temperatures of over 55° C, some of them withstanding even 65° to 70°, and can not be reactivated by the subsequent addition of normal serum. These facts definitely preclude the participation in the reaction of the alexin or complement and have an important bearing upon Ehrlich's views of their structure 3 (see page 238). As a result of these and a multitude of other studies, the agglu- tinins have come to be regarded as separate antibodies, closely related to the precipitins. The agglutinins may be chemically precipitated out of serum together with the globulins. They do not dialyze. Bordet 4 made the observa- tion that agglutinins do not act in the absence of NaCl. Whether the presence of the salt aids the reaction in a chemical or purely physical way, as Bordet supposed, is uncertain. Production of Agglutinins. — Just as normal sera contain small quan- tities of bactericidal substances, so do they contain agglutinins in small amount. In a general way these "normal agglutinins" have the same nature as the immune agglutinins, and it is probable that their presence is traceable to the various microorganisms parasitic upon the human >and animal body. As a matter of fact, the blood serum of new-born guinea-pigs hardly ever contains agglutinin for B. coli, while that of adults acts upon these bacilli in dilutions of 1 : 20.5 Similarly, infants show lower normal ag- glutinating values than adults.6 ' Metchnikoff, " L'immunite," etc., 1901, p. 214. 2 Mesnil, Ann. de l'inst. Pasteur, 1898. 3 Pane, Cent. f. Bakt., 1897; Trumpp, Arch. f. Hyg., 1898; Fbrster, Zeit. f. Hyg., xxiv. 4 Bordet, Ann. de Tinst. Pasteur, 1899. 6 Kraus und Low, Gesell. d. Aerzte, Wien, 1899. « Pfaundler, Jahrb. f. Kinderheilk., Bd. 50. LYSINS, AGGLUTININS, PRECIPITINS, ETC. 233 Agglutinins may be produced in the sera of animals by the intro- duction of microorganisms subcutaneously, intravenously, or intraperi- toneally. The intravenous method seems to give the most abundant and speedy results.1 The formation of agglutinins is a reaction to the body- substances of the bacteria themselves, rather than to their toxic prod- ucts. Thus agglutinins are produced in response to the introduction of dead bacteria and soluble extracts of cultures. Pathogenicity 2 does not influence agglutinin formation to any great extent, non-pathogenic as well as pathogenic giving rise to these substances in serum. As a rule, however, agglutinins are more easily produced against avirulent than against fully virulent strains of bacteria of the same species. While agglutinins can be produced with almost all the known bac- teria, there are great differences between various species in the quantity and speed of production, and Nicolle and Thenel 3 have classified bac- teria in three groups according to their power of stimulating the pro- duction of agglutinins in immunized animals. As a rule, the agglutinins appear in the blood of animals three to six days after the introduction of bacteria. From the third to the sixth day they rapidly increase to a maximum at the seventh to thirteenth day. They then fall off rapidly until they reach a level at which they remain for a long period without very considerable change. Curves to illustrate these phases have been constructed by Jorgensen and Madsen.4 The Reaction between Agglutinin and Agglutinin-Stimulating Sub- stances (Agglutinogen) . — The fact that agglutinin can be removed from, or absorbed out of, serum by the specific bacilli which have led to its formation indicates that there is in the act of agglutination a com- bination between the agglutinin and the agglutinin-stimulating sub- stance (agglutinogen) . It is likely that this combination is of a chemical nature, since, as we have mentioned, agglutinins result from the in- jection of bacterial extracts as well as from the introduction of living bacteria. The probability that the process follows chemical laws of combination is furthermore strengthened by the work of Joos 5 and others, who have demonstrated that definite quantitative relations ex- ist between the agglutinin-stimulating substances and the agglutinins. Every agglutination reaction, therefore, will vary in its degree of com- 1 Hoffmann, Hyg. Rundschau, 1903. 2 Nicolle, Ann. de l'inst. Pasteur, 1898. 3 Nicolle et Thenel, Ann. de l'inst. Pasteur, 1902. 4 Jorgensen and Madsen, Festschrift, Kopenhagen, 1902. 5 Joos, Zeits. f. Hyg., xxxvi, 1901. 234 INFECTION AND IMMUNITY pleteness with the quantities of agglutinin and agglutinogen, a fact which makes it necessary, especially for clinical tests, to preserve a certain uniformity in the quantity and density of the bacterial culture or emulsion employed. Specificity. — From the very beginning, Gruber and Durham * had claimed specificity for the agglutination reaction, and in this sense it was clinically utilized by Widal for the diagnosis of typhoid fever. It was noticed, however, even by these earliest workers, that the serum of an animal immunized against one microorganism would often agglutinate, to a less potent degree, other closely related species. Thus, the serum of a typhoid-immune animal may agglutinate the typhoid bacillus in dilutions of 1 : 1,000, and the colon bacillus in dilutions as high as 1 : 200; while the agglutinating power of normal serum for the colon bacillus rarely exceeds 1 : 20. The specificity of the reaction for practical pur- poses, thus, is not destroyed if proper dilution is carried out, the degree of agglutinin formation being always far higher for the specific organism used in immunization than it is for allied organisms. The specific immune-agglutinin in such experiments is spoken of as the chief ag- glutinin (haupt agglutinin) , and the agglutinins formed parallel with it, as the partial agglutinin (metagglutinin) , terms introduced by Wasser- mann. Hiss has spoken of these as major and minor agglutinins. The relative quantities of the specific chief agglutinin and partial agglutinins present in any immune serum depend upon the individual cultures used for immunization, and the phenomenon is probably dependent upon the fact that certain elements in the complicated bacterial cell-body may be common to several species and find common receptors in the animal body. Whenever an immune serum agglutinates a number of members of the group related to the specific organism used for its produc- tion, the reaction is spoken of "group agglutination. " The partial agglutinins (metagglutinins) have been extensively studied by Castellani 2 and others, by a method spoken of as the " ab- sorption method." This consists in the separate addition of bacterial emulsions (agglutinogens) of the various species concerned in a group agglutination, to the agglutinating serum. In this way, specific and par- tial agglutinins can be separately removed from the immune serum by absorption — each by its corresponding agglutinogen. In such experi- ments all agglutinins will be removed by the organisms used for im- 1 Gruber und Durham, loc. cit. 2 Castellani, Zeits. f. Hyg., xl, 1902. LYSINS, AGGLUTININS, PRECIPITINS, ETC. 235 munization, a partial removal only resulting from the addition of allied strains. This method has thrown much light upon the intimate relations existing between members of various bacterial species, and has been particularly valuable in the study of the typhoid-colon-dysentery group. It is important to mention, however, that " groups" as de- termined by agglutination tests, do not always correspond to classi- fications depending upon morphological and cultural characteris- tics. An interesting phenomenon of great practical importance, which has been noticed by a number of observers, and which may often be encountered in routine agglutination tests, is the frequent failure of a strongly agglutinating serum to produce agglutination if used in concen- tration, while in dilutions it produces a characteristic reaction. This has been explained theoretically by what is known as the " proagglutinoid zone." It is assumed that agglutinins may deteriorate as do toxins and be converted into substances which are capable of combining with agglu- tinogen without causing agglutination. Such substances, as we will see in discussing Ehrlich's views on the structure of agglutinins, may have a stronger affinity for agglutinogen than the agglutinins them- selves, and are, therefore, termed "proagglutinoids." In strongly agglutinating sera these proagglutinoids may be present in considerable quantities and prevent the combination of agglutinin with agglutinogen. In dilution, this proagglutinoid action would naturally become weaker and of no actual significance in obscuring the reaction. Agglutination, like other immune phenomena, is a manifestation of broad biological laws and not limited to bacteria. Thus, as hemolysins are produced by the injection of red blood cells, so hemagglutinins, or substances which clump together red blood cells, are formed by the same process. PRECIPITINS (Coagulins) In the year 1897, R. Kraus,1 of Vienna, demonstrated that the sera of animals immunized against B. pestis, B. typhosus, and Vibrio choleras, when mixed with the clear filtrate of bouillon cultures of the respective organisms, gave rise to macroscopically visible precipitates. The precipitates occurred only when filtrate and immune serum were homologous, that is to say, the animal from which the serum was ob- 1 Kraus, Wien. klin. Woch., 1897. 236 INFECTION AND IMMUNITY tained was immunized by the same species of microorganism as that used in the test ; and for this reason Kraus spoke of them as " specific precipitates." It was evident, therefore, that during the process of active immunization with these organisms, a specific antibody had been pro- duced in the serum of the treated animal, which, because of its precipitat- ing quality, was named "precipitin." This peculiar reaction was soon found to hold good, not only for the bacteria used by Kraus, but also for other bacteria, few failing to stimulate the production of specific precipi- tins in the sera of immunized animals. The phenomenon of precipitation, however, is not limited to bacterial immunization, but has been found, like the phenomena of agglutination and lysis, to depend upon biolog- ical laws of broad application. Thus, Bordet 1 found that the blood serum of rabbits treated with the serum of the chicken gave a specific precipitate when mixed with chicken serum. Tchistovitch 2 demon- strated a similar reaction with the sera of rabbits treated with horse and eel sera. By the injection of milk, Wassermann,3 Schiitze,4 and others produced an antibody which precipitated the casein of the particular variety of milk employed for immunization. The reaction was thus found applicable to many substances of albuminous nature. These substances, because of their precipitin-stimulating quality, are spoken of as "precipitinogens." Nature of Precipitins. — The precipitins, like the agglutinins, may be inactivated by heating to from 60° to 70° C, and can not be reactivated by the addition of normal serum or by any other known method. Such inactivated precipitin, however, while unable to produce precipi- tates, has not lost its power of binding the precipitinogen. This is shown by the fact that the inactivated precipitin, when mixed with pre- cipitinogen, will prevent subsequently added fresh precipitin from caus- ing a reaction. From these facts the conclusion has been drawn that precipitin, like toxin, is built up of two atom groups,5 a stable hap- tophore and a labile precipitophore group. By the destruction of the latter, an inactive, yet neutralizing substance is produced which is spoken of as "precipitoid." The precipitoids, like protoxoids, have a higher affinity for precipitinogen than the unchanged precipitin, and thus are able to prevent the action of these. 1 Bordet, Ann. de l'inst. Pasteur, 1899. 2 Tchistovitch, Ann. de l'inst. Pasteur, 1899. 3 Wassermann, Deut. med. Woch., 29, 1900. *Schutze, Zeit. f. Hyg., 1901. 5 Kraus und v. Pirquet, Cent, f, Bakt., Orig. Bd., xxxii. LYSINS, AGGLUTININS, PRECIPITINS, ETC. 237 Specificity. — The specificity of precipitins is a question of the greatest importance, since, as we shall see, these bodies have been used extensively for the differentiation of animal proteids. In regard to bacterial precipitins it may be said that, just as in agglutination, there is in precipitation a certain degree of "group reaction/' The pre- cipitin obtained with a colon bacillus, for instance, will cause precipita- tion with culture-filtrates of closely allied organisms, though in a less marked degree. According to Kraus, such confusion may be easily overcome by the proper use of dilution and quantitative adjustment, similar to that used in agglutination tests. Similar results were ob- tained by Norris,1 who found that the precipitates given by immune sera with the filtrates of the homologous bacteria were invariably heavier than those given with allied strains and that the latter could be eliminated entirely by sufficient dilution. Specificity becomes of still greater importance in the forensic use of the precipitin reaction introduced by Uhlenhuth,2 Wassermann and Schiitze,3 and Stern.4 These authors found that the precipitin reaction furnished a means of distinguishing the blood of one species from that of another. Thus, blood spots, dissolved out in normal salt solution, could be recognized by this reaction as originating from man or from an animal, even after months of drying and in dilutions as high as 1 : 50,- 000. Since the value of this test depends entirely upon the strict specificity of the reaction, this question has been studied with especial care, notably by Nuttall.5 All who have investigated the subject find the only important source of confusion in the blood of the anthropoid apes. The specificity of the reaction, too, has been found to depend very closely upon the amount of precipitin in the serum employed. If a highly immune serum is insufficiently diluted, the reaction loses much of its specific value.6 This source of error, however, is easily elimi- nated in practical application by careful control and titration of the sera used for the tests. Unlike agglutinins, precipitins have, so far, not been demonstrated in normal sera.7 1 Norris. Jour. Inf. Dis., i, 3, 1904. 2 Uhlenhuth, Deut. med. Woch., xlvi, 1900; vi and xvii, 1901. 3 Wassermann und Schiitze, Berl. klin. Woch., vi, 1901. * Stern, Deut. med. Woch., 1901. 5 Nuttall, Brit. Med. Jour., i, 1901; ii, 1902. e Kister und Wolff, Zeit. f. Medizinal-Beamte, 1902. 7 Kraus, loc. cit., and Norris, loc. cit. 238 INFECTION AND IMMUNITY Theoretical Considerations Concerning Agglutinins and Precipitins. — We have seen that Ehrlich evolved his theories of antibody forma- tion from his early views upon the absorption of nutritive substances by the body cells, and we have followed, in more or less detail, the steps of his reasoning as he developed his hypothesis in its application to the antitoxic and the lytic substances. There still remained the agglutinins and precipitins, bodies which because of their individual characteris- tics can be classed neither with the group of antitoxic, nor with that of the lytic substances. These two antibodies, while by no means identical, possess the common characteristics of being more thermostable than the bacteriolytic substances, and of being insusceptible to reactivation by normal serum. It is plain, therefore, that both agglutinating and precipitating reactions take place without the co-operation of comple- ment. The substances which give rise to precipitins and agglutinins, A43l.v1.tvn. in Cell vised for immunising Fig. 61. — Ehrlich's Conception of the Structure of Agglutinins and Precipitins. moreover, are not of the relatively simple soluble character of the toxins, but are intrinsic portions of complex albuminous molecules, comparable to and often identical with the true nutritive substances. For these reasons Ehrlich believes that the cell-receptors for the various substances which give rise to agglutinins and precipitins are neither of the simple structure of the toxin receptor, nor of the double-haptophore nature of the bacteriolytic receptors, but contain a single haptophore group for the anchorage of the ingested material and at the same time a constantly attached zymophore group or ferment by means of which the an- chored substance is transformed preparatory to its absorption by the cell protoplasm. For the sake of clearness, this form of receptor may be compared to a bacteriolytic or hemolytic amboceptor with a per- manently attached and inseparable complement. Three forms of receptors, then, are proposed by Ehrlich in explana- tion of all known varieties of antibodies. The first, the simplest side 239 240 INFECTION AND IMMUNITY chains of the body cells, he calls " receptors or haptines of the first order." These, overproduced and cast off, constitute the antitoxin and antifer- ments. Next " haptines of the second order " are the receptors planned both for the anchorage and further digestion of antigens. These, free in the circulation, are the precipitins and agglutinins. "Haptines or receptors of the third order" are merely able to anchor a suitable sub- stance, but exert no further action upon it until re-enforced by the com- plement normally present in the serum. These, free in the circulation, with a chemical group having avidity for the antigen, and another complementophile group, are the amboceptors or immune bodies of bacteriolytic, cytolytic, and hemolytic sera. (See Fig. 62.) It is plain that all these receptors while still parts of their respec- tive cells, serve by their chemical affinity to attract and hold the foreign substances injected; freely circulating, on the other hand, they serve in preventing these substances from reaching the cells. As Behring has aptly expressed it, the very elements which situated in the animal cells render the body susceptible to toxic substances serve to protect when circulating freely in the blood. Bordet,1 at present the strongest antagonist of Ehrlich/s point of view, claims that the conception of Ehrlich rests upon the basis of a number of undemonstrated hypotheses. He asserts, and with justice, that it has never been shown beyond question that the antibodies, free in the serum, are identical with the receptors of the body cells upon which the antigen originally acts. In regard to agglutinins, Ehrlich, as we have seen, believes that it is the agglutinin itself which, first uniting with its antigen by its hap- tophore group, then causes clumping by its zymophore group. Now, as a matter of fact, Bordet2 has shown that it is not the agglutinin itself which agglutinates, but that agglutinin with its antigen forms a com- plex which is then aggiutinable by the salt present in the solution. This conclusion seems borne out by the later work of Gengou,3 Landsteiner and Jagic,4 and others, who have shown that bacteria which have ab- sorbed other substances, such as uranium compounds, colloidal silicic acid, etc., are subsequently aggiutinable by salts. In consequence, from these and other observations, Bordet concludes that it is neither necessary nor accurate for the explanation of these phenomena, to 1 Bordet, Resume of Immunity in Bordet's "Studies in Immunity," transl. by Gay, Wiley & Sons, 1909. 2 Bordet, Ann. de l'inst. Pasteur, 1899. 3 Gengou, Annal. Past., 1904. 4 Landsteiner und Jagic, Wien. klin. Woch., iii, 1904. LYSINS, AGGLUTININS, PRECIPITINS, ETC. 241 assume the conditions conceived by Ehrlich, but that the phenomenon of agglutination consists primarily of the union of the antibody with its antigen in a colloidal solution, and that the actual subsequent agglu- tination is a purely secondary phenomenon which depends possibly upon a change in the physical properties of the emulsion — upon, as he expresses it, its colloidal stability. A similar condition he assumes for precipitins. FURTHER FACTS AND THEORIES CONCERNING ANTIBODIES Multiplicity of Amboceptors. — Fresh normal serum, as Nuttall * was first to show, possesses moderate bactericidal powers which are ' lost when the serum is subjected to heat. Since such inactivated normal serum can be reactivated by the addition of fresh peritoneal exudates, as the experiments of Moxter 2 have demonstrated, it is plain that the bactericidal power of normal serum must depend, like that of immune serum, upon amboceptor and complement. But normal serum often exerts lytic powers upon several species of bacteria, or, in the case of hemolytic tests, upon the red blood cells of several species of animals. It is supposed that this multiplicity of action is due to the presence in the normal serum of a variety of different amboceptors or immune bodies. The method for proving this was devised by Ehrlich and Morgenroth.3 They worked with normal goat's serum, which has the power of hemolyzing the red blood cells of guinea-pigs as well as those of rabbits. Goat serum, inactivated by heat, was mixed with rabbits' corpuscles. After the mixture had been allowed to stand for a short time, the corpuscles were removed by centrifugalization. The serum was then reactivated and found still to possess its hemolytic power for guinea-pigs' blood, but to have lost this power for rabbits' blood. By a similar technique, Pfeiffer and Friedberger 4 were able to demonstrate the multiplicity of bactericidal immune bodies in normal era. The immunity acquired by an animal as the result of treatment with any of the various antigens is specific. An animal immunized against the cholera vibrio, for instance, possesses marked bactericidal powers for the cholera vibrio only. 1 Nuttall, loc. cit. 2 Moxter, Cent. f. Bakt., xxvi, 1896. 3 Ehrlich und Morgenroth, Berl. klin. Woch., 1901. * Pfeiffer und Friedberger, Deut. med. Woch., 1901. 16 242 INFECTION AND IMMUNITY According to Ehrlich's views, the amboceptor or immune body alone enters into direct relation with the substance used for immunization, and it would seem natural therefore that the specificity of immune sera depends entirely upon the increase of amboceptor or immune body. Von Dungern,1 indeed, was able to show that the specific amboceptor was increased as immunity was acquired, without there being a cor- responding enhancement of the complement. The chief difference be- tween a normal and an immune serum in this respect, therefore, con- sists in an enormous increase, in the latter, of the specific amboceptor.. Multiplicity of the Complement. — A number of very complicated ex- periments have been carried out by Ehrlich, Morgenroth,2 Sachs,3 and others, which seem to show that the same serum may contain a variety of complements. Similar conclusions have been drawn by Wechsberg 4 and by Wassermann,5 who demonstrated separate complements for bac- tericidal and hemolytic amboceptors in the same serum. Bordet 6 and his school, on the other hand, deny the multiplicity of the complement, and, basing their views upon numerous experimental data, contend that any given serum contains but one alexin or complement. Buchner and Gruber share the views of Bordet, and, in the light of recent work, especially with complement fixation (see below), it seems more likely that one and only one alexin exists in any given serum. Anticomplements and Antiamboceptors. — Hemolytic sera, having the power of destroying red blood cells, must necessarily prove in the presence of sufficient complement to be powerful poisons when intro- duced into animals whose corpuscles they are able to injure. By care- ful and gradual dosage with such hemolytic sera, Ehrlich and Morgen- roth,7 as well as Bordet,8 have been able to produce immunity against the hemolytic action. Thus antihemolytic sera have been produced, the action of which may depend either upon the presence of anticomple- ment or of antiamboceptor. The presence of anticomplement in such sera, it is believed, has been demonstrated by mixing inactivated hemolytic serum with its respective red blood cells, then adding the 1 v. Dungern, Munch, med. Woch., xx, 1900. 2 Ehrlich und Morgenroth, Berl. klin. Woch., 1900. 3 Ehrlich und Sachs, Berl. klin. Woch., 1902. 4 Wechsberg, Zeit. f. Hyg., 1902. 6 Wassermann, Zeit. f. Hyg., 1901. 6 Bordet, Ann. de Tinst. Pasteur, 1900 and 1901. 7 Ehrlich und Morgenroth, Berl. klin. Woch., xxxi, 1900. 8 Bordet, Ann. de Tinst. Pasteur, t. 14, 1900. LYSINS, AGGLUTININS, PRECIPITINS, ETC. 243 antiserum and later complement. After centrifugalization and sepa- ration of the corpuscles, these may be dissolved by the addition of fresh complement. This proves conclusively that there was no obstacle in the original mixture to the absorption of the immune body by the red blood cells, and that the antihemolytic properties of the serum must be attributed to an anticomplement. This was the method of experimentation employed by Ehrlich and Morgenroth.1 Antiambocep- tors have been produced by the same authors as well as by Bordet 2 and Miiller,3 against hemolytic amboceptors. Complementoids. — Ehrlich and Morgenroth and Miiller have suc- ceeded in producing anticomplements by the treatment of animals with normal heated serum. They explain this by assuming that the heating has not entirely destroyed the complement in the normal serum, but that this, analogous to toxin, possesses two groups, a haptophore and a zymo- phore group. Heating destroys the zymophore without affecting the haptophore group. The resulting body, which corresponds to toxoid, they call "complementoid." Further evidence for the existence of such complementoids has been claimed by Ehrlich and Sachs 4 in working with dog serum. Unheated dog serum hemolyzes guinea-pig corpuscles. Heated to 52° C. for thirty minutes, however, it no longer hemolyzes these corpuscles ow- ing to complement destruction. Such heated dog serum can be reacti- vated by fresh guinea-pig serum (fresh complement) . If, however, the corpuscles are left in contact with the heated dog blood for two hours, reactivation by the guinea-pig serum no longer occurs — that is, the ad- dition of guinea-pig serum no longer causes hemolysis. They conclude from this that the hemolytic amboceptor of the dog serum has been attached by its complementophile group to complementoids produced in the heating — leaving no point of attachment for the complement added later. These experiments have failed of confirmation by Gay 5 — who with Bordet denies the existence of complementoids. Muir, on the other hand, claims to have demonstrated the existence of complementoids by experiments too complicated to be detailed in this place. The question of complementoids must be left undecided until further work has been done. 1 Ehrlich und Morgenroth, loc. cit. 2 Bordet, loc. cit. 3 P. Th. Miiller, Cent. f. Bakt., 1901. 4 Ehrlich and Sachs, :t Ehrlich Collected Studies on Immunity/' trans, by Boldnau. 5 Gay, Cent. f. Bakt., I, xxxix, 1905. 244 INFECTION AND IMMUNITY Filtration of Immune Body and Complement. — Muir and Browning have recently shown that, on the filtration of serum, amboceptor or immune body will pass through the filter, whereas alexin or comple- ment is held back. The amboceptor filters equally well, whether or not mixed with the complement. The Fixation of Complement by Precipitates. — It has been found by Gengou l and confirmed by Gay 2 and others, that when the serum of an animal immunized with the serum of another species or with a foreign albumin is mixed with a solution of the substance used in the immu- nization, the precipitate formed will remove complement from the mix- ture. In other words, precipitates formed by the reaction of precipitin with its antigen will fix complement. This phenomenon is of great im- portance in complement-fixation tests such as those of Wassermann or Noguchi (see below) ; for because of insufficient washing, the blood cells used in producing the hemolytic amboceptor, may, from the presence of serum, give rise to a precipitin as well as a hemolysin. In the test done subsequently, a precipitin reacion may take place and by thus removing complement may give a false result. The ab- sorption of complement by such precipitates takes place when the two reacting factors, the precipitin and its antigen, are in extremely high dilution — in fact, when a visible precipitate can not be observed. Quantitative Relationship Between Amboceptor and Complement. — Morgenroth and Sachs,3 in studying the quantitative relationship ex- isting between hemolytic amboceptor and its complement, have suc- ceeded in showing that within certain limits an inverse relationship exists between these two bodies. If for a given quantity of red blood cells a certain quantity of amboceptor and complement suffices to produce complete hemolysis, reduction of either the complement or the ambo- ceptor necessitates an increase of the other factor. As amboceptor is increased, in other words, complement may be reduced and vice versa. This result is of great importance in arguing against the original con- ception of Ehrlich in supposing these substances to act together mul- tiple for multiple as do compounds in chemical reactions. Deviation of the Complement (Complement-Ablenkung) . — It wTas no- ticed by Neisser and Wechsberg 4 that in mixing together bacteria, 1 Gengou, Ann. Past., 1902. 2 Gay, Cent. f. Bakt., I, xxix, 1905. 3 Morgenroth und Sachs, " Gesammel. Arb. fur Immunitatsforschung. " Berlin, Hirschwald, 1904. 4 Neisser und Wechsberg, Munch, med. Woch., xviii, 1901. LYSINS, AGGLUTININS, PRECIPITINS, ETC. 245 inactivated bactericidal immune serum (immune body), and comple- ment in the test tube, a great excess of immune body hindered rather than helped bactericidal action. As the amount of immune body in the mixture was carried beyond the experimental optimum, bac- tericidal action became less and less pronounced, and was finally completely suspended. They explain this by assuming that free im- mune body, uncombined with complement, has a greater affinity for the bacterial receptor than the immune body combined with complement. The complement is consequently diverted and prevented from activating the amboceptor attached to the bacterial cell. Graphically, the con- ditions may be illustrated as follows: AJ IaI vv IAI IaI (a VI /V» |N/1 4aJ IaI IaI Fig. 63. — Neisser and Wechsberg's Conception of Complement Deviation. The above theory of Neisser and Wechsberg is here stated simply because of the wide discussion it has aroused. In the light of our present knowledge concerning the relations between antigen, amboceptor, and complement, their conception is obviously erroneous. 246 INFECTION AND IMMUNITY Fixation of the Complement. — Bordet and Gengou 1 in 1901, devised an ingenious method of experimentation by which even very small quantities of any given immune body (amboceptor) can be demon- strated in serum. The term "fixation of complement," by which their method of investigation is now generally known, explains itself, as the steps of experimentation are followed. They prepared the following mixtures: (a) (b) Bacteriolytic amboceptor Normal serum, heated (Plague immune serum, heated) + + Plague emulsion Plague emulsion + + Complement Complement (Fresh normal serum) (Fresh normal serum) To both of these after five hours was added Hemolytic amboceptor (Heated hemolytic serum) + Red blood cells Results : (a) showed no hemolysis. (b) showed hemolysis + . The conclusion to be drawn from this was that in (a) the presence of immune body had led to absorption of all the complement. In (b) , there being no bacteriolytic immune body to sensitize the bacteria and enable them to absorb complement, the latter substance was left free to activate the subsequently added hemolytic ambocep- tors. The Bordet-Gengou phenomenon has been extensively used by Wassermann and Bruck,1 Neisser and Sachs,2 and others to demon- strate the presence of immune bodies in various sera. (See p. 262.) It should be noted that this method, if valid, must presuppose the identity of the hemolytic and bactericidal complement in the activating serum. Complement fixation will be more extensively discussed in the section dealing with the Wassermann reaction. 1 Bordet et Gengou, Ann. de l'inst. Pasteur, 1901. 1 Wassermann und Bruck, Med. Klin., 1905. 2 Neisser und Sachs, Berl. klin. Woch., xliv, 1905, and i, 1906. LYSINS, AGGLUTININS, PRECIPITINS, ETC. 247 The Specificity of Hemolysins. — In the sections preceding we have seen that the blood cells of one animal, injected into an animal of another species, give rise to a hemolytic substance in the blood serum of the second animal, which is strictly specific for the variety of cells injected. Such hemolysins, when produced in one animal 1, Wr Complement" Syphilitic preserdf Together at immune or* ? or 1 37. SO C. antibody not > for one "hour 3. pfl Antigen vsl _ _Haemolyfic Amboceptor i Red blood cell . If (2) present , + kaemolys is. If (2) not present.no Haemolysis. Fig. 64. — Schematic Representation of Complement Fixation in the Bordet-Gengou Reaction. against blood cells of another species, are spoken of as heterolysins. In studying the nature of hemolysis, Ehrlich and Morgenroth 3 now discovered that hemolysins could also be produced if an animal was injected with red blood cells of a member of its own species. Such hemolytic substances they called isolysins. In their experiments they injected goats with the washed red blood corpuscles of other goats and found that the serum of the recipient developed the power of 3 Ehrlich und Morgenroth, Berliner klin. Woch., xxi, 1900. 248 INFECTION AND IMMUNITY causing hemolysis of the red blood cells of the particular goat whose blood had been used for injection. It did not, however, possess the power of producing hemolysis in the blood of all goats, nor did it pro- duce hemolysis with the red corpuscles of its own blood. It is thus shown that the specificity of the hemolysins extends even within the limits of species, and is, to a certain extent at least, an individual property. The production of autolysins, that is, of substances in the blood serum which will produce hemolysis of the individual's own corpuscles, has, so far, been unsuccessful. Ehrlich and Morgenroth, in the course of these experiments, further- more succeeded in showing that the injection of isolysins into animals produced antiisolysins, and that these again were strictly specific. The almost universal failure of autolysin production has found no satisfactory explanation. It is supposed by Ehrlich and Morgenroth that autolysins may be formed, but are probably speedily neutralized by the production of antiautolysins. The clinical significance of the presence of isolysins and possibly of autolysins in human beings, is too evident to require much discussion. A practical and extremely interesting result which these investigations have yielded is that of Donath and Landsteiner,1 who discovered an autolysin in the blood serum of patients suffering from paroxysmal hemoglobinuria. In these cases the sensitizing substance or ambo- ceptor appeared to be absorbed by the red blood cells only at low tem- peratures— probably in the capillaries during exposure to the cold, and hemolysis subsequently resulted in the blood stream by the action of complement. These observations have been confirmed by other writers, but the phenomenon is surely not present in all cases of paroxys- mal hemoglobinuria. The writers have had occasion to examine care- fully several clinically typical cases with negative results. 1 Donath und Landsteiner, Mtinch. med. Woch., xxxvi, 1904. CHAPTER XVI THE TECHNIQUE OF SERUM REACTIONS Obtaining Serum from Animals and Man. — To obtain blood serum from man, the blood may be taken from the finger or the ear; either into a sterile centrifuge tube or into a Wright capsule. (See section on Opsonins, page 284.) When taken into a centrifuge tube, the blood is allowed to clot and the serum separated from the coagulum by a few revolutions of the centrifuge. When larger quantities of blood are desired, it may be taken with a syringe from the median basilic vein and either slanted in sterile test tubes in the ice chest or put into centrifuge tubes and centrifugalized. In bleeding small laboratory animals, a number of methods may be employed, depending upon whether a large or small amount of serum is required. The animals most frequently used for laboratory purposes are rabbits. To obtain small quantities of serum from rabbits, the animals may be bled from the marginal vein of the ear. In doing this, a satis- factory yield of blood may be obtained by following a simple method devised by Wadsworth. The animal is strapped upon a tray and under- neath it is placed a rubber bag filled with warm water. This keeps the body temperature of the rabbit somewhat higher than normal, causes dilatation of the vessels, and thus facilitates the flow of blood. The tray is then placed upon a simply constructed easel so that the animal's head hangs downward. The skin over the ear vein is shaved and sterilized, and an incision is made into the vein in its long axis with a sharp knife. The blood is caught in test tubes or centrifuge tubes. When larger quantities of blood are desired, the animal is strapped down, anesthetized, and the neck shaved and sterilized. The carotid artery is then isolated by dissection. In rabbits, the carotid artery may be found lying just lateral to the trachea and deeply placed, and must be carefully separated from the pneumogastric nerve by blunt dissection. The distal end of the artery is then tied off and the proximal end tem- porarily closed with a small clamp. (This clamp should be rather weak and not exert sufficient pressure to injury the artery and cause throm- 249 250 INFECTION AND IMMUNITY bosis.) The artery is then raised out of the wound on a knife or forceps handle and, with sharp-pointed scissors, a small incision is made into but not through the vessel. A small glass cannula is now introduced into the proximal end of the artery through the incision and tied into place by a thread. To this cannula a small rubber tube fitted with a pinch- cock should have been attached, the whole being sterilized. The clamp on the artery is then released and the blood allowed to flow into sterile test tubes which are slanted and placed in the cold for separation of the serum. A larger yield of serum will be obtained if, after coagulation, the clot is separated from the glass with a sterile platinum wire. In obtaining blood from larger animals, horses, sheep, etc., a cannula may be introduced into the jugular or internal saphenous veins. The skin is shaved and sterilized and a rubber tourniquet placed about the neck or thigh, as the case may be, in order to cause the vein to stand out. A small incision may be made through the skin over the vein, but is not necessary. A cannula, with rubber tubing attached, is then plunged into the vein and the blood caught in sterile high cylindrical jars, allowed to clot, and placed in the refrigerator. The serum is taken off after twenty-four to forty-eight hours with sterile pipettes. Agglutination Tests. — For the determination of the agglutinating power of serum it is necessary to make suitable dilutions of the serum, and to prepare an even emulsion of the microorganisms to be tested. The test may be made microscopically or macroscopically. The micro- scopic test is the one in general use in the diagnosis of typhoid fever, and is occasionally applied to some other diseases. In its application to typhoid fever it is usually spoken of as the Gruber-Widal reaction. Twelve- to eighteen-hour broth cultures of the typhoid bacillus, grown at incubator temperature, may be used. It is preferable, how- ever, to use an emulsion of a twelve to twenty-four hour old agar culture in physiological salt solution (0.85 per cent) . The salt-solution emulsion is made by adding about 10 c.c. of normal salt solution to the fresh agar slant culture, carefully detaching the culture from the surface of the agar with a flexible platinum wire, and pipetting off the emulsion thus made. With some microorganisms it is sufficient simply to allow the larger clumps to settle and to pipette off the supernatant turbid emulsion. With other microorganisms, the tendency to form clumps makes it necessary to resort to further methods of securing an even distribution of the bacteria. This may be done either by sucking the emulsion in and out through a narrow pipette held perpendicularly against the bottom of a watch glass, as in Wright's technique for the opsonic test (see section THE TECHNIQUE OF SERUM REACTIONS 251 on Opsonins, p. 285), or by carefully rubbing the clumps against the watch glass with a stiff platinum wire. In the case of the tubercle ba- cillus not even this suffices, but it becomes necessary to grind the moist bacillary masses in a mortar before emulsifying. With the tubercle bacil- lus, too, it is preferable to use salt solution at 1.5 per cent concentration. In preparing cultures of streptococcus and pneumococcus for agglutination tests, it has been found convenient by Hiss to grow microorganisms for about four days in flasks of a one per cent glucose, two per cent pepton meat-infusion broth, to which has been added one per cent of calcium carbonate. (See page 126.) The insoluble calcium carbonate sinks to the bottom, but by neutralizing the inhibiting acid formed in the broth by the microorganisms, permits the development of a mass culture. The flasks should be shaken thoroughly at least once a day. The broth may be pipetted off and the clumps may be removed by a few revolutions of a centrifuge. Without this technique it is sometimes difficult to get sufficient growths of these bacteria for any quantity of emulsion unless large surfaces of agar are employed in special receptacles or by making many slant cultures. The serum dilutions are obtained by first making a one to ten dilution of serum with normal salt solution. The serum used for this purpose may be cleared of red blood corpuscles by centrifugalization. From the one to ten dilution any number of higher dilutions may be made, simply by mixing given parts of the one to ten dilution with normal salt solution; thus one part of a one to ten dilution plus an equal quantity of salt solution gives a dilution of one to twenty. One part of one to ten dilution plus two parts of normal salt solution gives one to thirty, and one part of one to twenty dilution plus one part normal salt solution gives one to forty, etc. It must not be forgotten that, when equal parts of the serum and bacillary emulsion have been mixed, each one of these dilutions is doubled. In making the microscopic agglutination test, minute equal quanti- ties of serum dilution and bacterial emulsion are mixed upon the sur- face of a cover-slip. The mixture may be made either by measuring out a drop of each substance with a standard platinum loop, depositing them close together on the cover-slip, and mixing; or, more exactly, equal quan- tities may be sucked up, each to a given mark, in a capillary pipette, mixed by suction in and out, and then deposited upon the cover-slip. The cover-slip is inverted over a hollow glass slide, the rim of which has been greased with vaseline. The drop is then observed, preferably through a (Leitz) No. 7 lens, ocular No. 3. 252 INFECTION AND IMMUNITY The macroscopic agglutination test, always preferable for exact laboratory research, is made in narrow test tubes especially designed for the purpose, measuring about 0.5 cm. in diameter and about 5 cm. in length (Fig. 60, p. 230). In these test tubes equal quantities, usually 1 c.c. each, of serum dilution and emulsion are mixed. A series of tubes is prepared, in each subsequent one of which the dilution is higher. These mixtures may be placed in the incubator for a few hours and then kept at room tem- perature. It has been observed by Hiss that after removal from the in- cubator agglutination is in some instances hastened by transference to the ice chest. When agglutination takes place in these tubes, clumps of bacteria may be seen to form, which settle to the bottom of the tube, very much like snow-flakes. The surface of the sediment is heaped up and irregular. The supernatant fluid becomes entirely clear. When the reaction does not occur the sediment is an even, granular one with a flat surface, and the emulsion remains turbid. Instead of using test tubes as described above, Wright has sug- gested the use of throttle pipettes of comparatively large diameter into each of which at least three or four different dilutions can be sucked with a nipple, a small air bubble being left between the mixtures. By sealing the distal end of these pipettes in a flame the various dilutions are kept at a distance from each other, and the pipettes may be set on end in a tumbler and observed just as are the test tubes (Fig. 68, p. 285). Precipitin Tests. — In an earlier section on precipitins we have seen that precipitates are formed when clear filtrates of bacterial extracts or of broth cultures are mixed with their specific immune sera. Such precipitin reactions are not limited to the realm of bacteria, but have a broad biological significance, in that specific precipitating sera may be produced with proteids of varied source. For actually carrying out a precipitin test, the following reagents are required : 1. A specific precipitating antiserum (antibacterial or antiproteid) ; 2. A bacterial filtrate or proteid solution. The Production of Precipitating Antisera.1 — Antibacterial precipitins may be produced in animals by any one of a variety of methods. Animals, preferably rabbits, are injected either with broth cultures or with salt solution emulsions of agar cultures of the bacteria, in gradually increasing quantities. Five or six injections are given in- 1 R. Kraus, Wien. klin. Woch., 1897; Norris, Jour. Inf. Dis., 1 and 3, 1904. THE TECHNIQUE OF SERUM REACTIONS 253 traperitoneally or intravenously, at intervals of from five to six days, the dosage and mode of administration being adapted in each case to the pathogenic properties of the microorganisms in question. It has been asserted by Myers 1 that when pepton-broth cultures are used for immunization a specific precipitin for pepton may be formed which by giving a precipitate with a culture filtrate containing pepton may lead to error. This observation could not be confirmed by Norris.2 The immunized animals should be bled about seven to twelve days after the last injection of bacteria. Specific precipitating antisera against proteid solutions are prepared by methods analogous to those employed for the production of anti- bacterial sera. A variety of methods have been described. The sera or proteid solutions used should be sterile. This may be accomplished by filtration through small porcelain filters. The injections into animals may be made subcutaneously, intraperitoneally, or intravenously. The subcutaneous route has no advantages unless the substances to be used are contaminated. Nuttall advises the use of rabbits. The animals should be weighed from time to time, and if considerable loss of weight ensues during im- munization, the intervals between injections should be increased. Dosage should be carefully graded, beginning, in the case of an animal serum, for instance, with 2 c.c. and increasing gradually through 3, 5, and 8 c.c. to possibly 15 c.c. at the last injection. A single injection of a large quantity has occasionally yielded a precipitating serum of con- siderable strength,3 but this method is not usually successful. Injec- tions are made at intervals of from five to seven days. Seven to twelve days after the last injection the animals may be bled, and a preliminary test made to ascertain the precipitating value of the serum. If this is insufficient for the desired purposes, more injections may be made before the animal is finally bled. Bleeding should be done seven to twelve days after the last injection. Such sera may be preserved by sealing in glass bulbs and keeping in the dark and at a low temperature. If a preservative is to be added, Nuttall recommends chloroform, but dis- approves of the phenols, because of occasional turbidity produced by these. The precipitating antisera used for the tests should be absolutely 1 Myers, Lancet, ii, 1900. 2 Norris, loc. cit. 3 Michaelis, Deut. med. Woch., 1902. 254 INFECTION AND IMMUNITY clear. If turbidity is present, the sera should be filtered through small Berkefeld or porcelain candles. Preparation of Bacterial Filtrates and Proteid Solutions for Precipitin Tests. — Bacteria may be grown in broth made of Liebig's beef extract five-tenths per cent, pepton one per cent, NaCl five- tenths per cent, and having an initial reaction of neutrality or five-tenths per cent acidity to phenolphthalein. The cultures are incubated for times varying from a week to several months, and are then filtered through porcelain or Berkefeld candles until perfectly clear. Bacterial extracts may also be made by emulsifying agar cultures in salt solution, placing at 37.5° C. in the incubator for a week or longer, and filtering. More rapid extraction of bacteria may be accomplished by repeated, rapid freezing and thawing of salt-solution emulsions. This is easily and simply done by placing the test tubes in battery jars filled with brine and cracked ice. Proteid solutions to be tested should be made in salt solution. When dealing with blood stains, as is frequently the case in doing the test for forensic purposes, the stains should be dissolved out in salt solution, an approximate dilution of one in five hundred being aimed at. This solution if turbid should be filtered through a small porcelain filter. Before use it should be perfectly clear and colorless, should show a faint cloud on boiling with dilute acetic acid, and, according to Muller, should show distinct frothing when shaken. When the reaction is to be done with the purpose of determining the nature of meat (detection of horse-meat substitution for beef, etc.), about 20 to 40 grams of the suspected meat are macerated by bein^ placed in a flask, and covered with 100 c.c. of physiological salt solution. This mixture is allowed to infuse at room temperature for three to four hours, and is then placed in the refrigerator for twelve hours or more. At the end of this time 2 c.c. may be poured into a test tube and shaken. If frothing 1 appears easily and profusely, the extract is ready for use. It is then filtered clear, either through paper, or, if this is unsuccessful, through infusorial earth in a Buchner or Nutsche filter. Berkefeld filters may also be used, but their use is less simple. The clear solution is then further diluted until the addition of concentrated HN03 produces only a slight even turbidity. Before use, furthermore, the reaction of the meat extract should be tested, and if necessary adjusted to neutrality or slight acidity or alkalinity. 1 P. Th. Midler, " Technik d. serodiagnos. Methoden." THE TECHNIQUE OF SERUM REACTIONS 255 In the actual test with bacterial filtrate, the procedure is as follows: In a series of narrow test tubes, the following mixtures are made: Tube 1. Antibacterial serum .5 c.c. + bacterial filtrate 1. c.c. 4 " 2. Normal serum .5 c.c. + bacterial filtrate 1. c.c. " 3. Antibacterial serum .5 c.c. + salt solution 1. c.c. " 4. Salt solution .5 c.c. + bacterial filtrate 1. c.c. Place the tubes in the incubator at 37.5° C. In a positive test, tube 1 only should show a haziness which develops into a distinct cloudiness or even a flocculent precipitate within one hour. Tubes 2, 3, and 4 should remain clear. For the testing of an unknown proteid with the serum of an animal immunized with the proteid sought for, the technique of the test is as follows : 1. 0.1 c.c. immune serum + 2 c.c. unknown proteid solution. 2. 0.1 c.c. immune serum + 2 c.c. known proteid solution of variety suspected (similarly diluted). 3. 0.1 c.c. immune serum + 2 c.c. proteid solution of different nature (similarly diluted). 4. 0.1 c.c. immune serum + 2 c.c. salt solution. 5. 2 c.c. unknown proteid solution. The test is positive when a precipitate appears in tube 1 and in tube 2, but not in any of the others. The precipitate should appear definitely within fifteen to twenty minutes. Bactericidal and Bacteriolytic Tests. — The bactericidal and bac- teriolytic powers of serum may be tested either in the animal body or in the test tube. The most common bacteriolytic test, in vivo, is that which is known as Pfeiffer's test. This test depends upon the fact considered in a previous section, that bacteria, when injected into the peritoneal cavity of a guinea-pig, together with a homologous immune serum, undergo dissolution. As practiced in bacteriological work, the test finds a double appli- cation. It may be carried out either for the determination of the specific bacteriolytic power of a given serum against a known micro- organism, or for the identification of a particular microorganism by means of its susceptibility to lysis in a known immune serum. 1. Determination of the bacteriolytic power of serum against a known microorganism in vivo : 1 1 P. Th. MiLller, " Technik d. serodiagnos. Methoden," Jena, 1909. 256 INFECTION AND IMMUNITY A number of dilutions of the serum are made with sterile neutral bouillon or salt solution, ranging from 1 in 20 to 1 in 500, or higher. It is convenient to make a first solution of 1 in 20. One c.c. of this mixed with 4 c.c. of broth will give 1 in 100. One c.c. of the 1 in 100 dilution with 1 c.c. of broth, 2 c.c. of broth and 4 c.c. of broth will give 1 in 200, 1 in 300, and 1 in 500, respectively. Into one cubic centimeter of each of these dilutions there is placed one platinum loopful of a twenty-four- hour agar culture of the microorganism against which the serum is to be tested. Into another test tube is placed 4 c.c. of broth, without serum, and with one loopful of the microorganisms. The mixtures are thoroughly emulsified in each case by rubbing the bacteria against the sides of the tube with the platinum loop. Intraperitoneal injections into guinea-pigs are then made of 1 c.c. of each of the serum-dilution-bacterial-emulsions. A control guinea-pig (better two or three) receives lc. c. of the broth emulsion — one-fourth as many bacteria, therefore, as the animals receiving the serum dilutions. Before making the injections, areas on the lateral abdominal walls of the guinea-pigs are shaved, and small incisions made through the Fig. 65. — Capillary Pipette for Removal of Exudate in doing the Pfeiffer Test. skin, down to the muscular layers. The needle of the syringe is then introduced perpendicular to the skin until it has penetrated the peri- toneum, and then carefully slanted to avoid puncturing the gut. The animals need not be strapped down during this procedure and after- ward may be allowed to run about. After one-half hour, and again after one hour has elapsed, a drop of peritoneal exudate is removed from each guinea-pig and examined in the hanging drop for granulation and swelling of the bacteria. The method of obtaining the peritoneal exudate is as follows: Small glass tubing is drawn out into capillary pipettes, the ends of the capillaries being again drawn to fine points in a small yellow flame. A number of such pipettes should be prepared before the test is begun. The guinea- pig is then held down upon a table, either by an assistant or by the left hand of the operator, and the point of the pipette pushed through the cut in the abdominal wall into the peritoneum by a sharp, quick thrust- THE TECHNIQUE OF SERUM REACTIONS 257 ing motion. A column of peritoneal fluid will run into the glass tubing by capillary attraction; this can then be blown out upon a cover-slip for hanging-drop examination or may be blown upon a slide, smeared, and examined after staining. The reaction is regarded as positive if within thirty minutes to an hour the peritoneal exudates of the animals receiving immune sera contain only swollen or disintegrated microor- ganisms, while in that of the control animals only well-preserved and undegenerated bacteria are found. In dealing with typhoid bacilli and cholera spirilla, in connection with which the test is most often used, active motility in the controls is of much help. Should there be extensive degeneration of the bacteria in the exudate of the control animals the test is of no value. 2. Identification of a microorganism by observing its a susceptibility to lysis in a known immune serum in vivo: The technique for this test is practically the same as that of the preceding except that in this case we require a potent known immune serum and normal serum for control. It is necessary, furthermore, that by previous tests we should know the degree of dilution in which the immune serum will cause complete bacteriolysis of the microorganism used in its production. Thus, if we are. employing a typhoid immune serum and are about to test by this method an unknown Gram-negative bacillus, we must know the titer of the serum for the typhoid bacillus itself. Mixtures are then made of dilutions of this serum and definite quantities of the microorganism to be tested. It is best, always, to employ from ten to one hundred times the amount of immune serum which suffices to produce lysis with its homologous microorganism. Thus, if the serum has been found to be active in dilutions of 1 : 1,000, it is employed in the test in dilutions of 1 : 1,000, 1 : 100, and 1 : 10. These dilutions are then injected into guinea-pigs in quantities of 1 c.c. together with the bacteria to be tested, and control guinea-pigs are injected with undiluted normal serum mixed with the bacteria and with salt solution and the bacteria. The exudates are then observed in the same way as in the preceding experiment. Bactericidal Reactions in the Test Tube. — Bactericidal reactions in the test tubes may be made by mixing in small sterile test tubes, definite quantities of the bacteria with inactivated serum and com- plement, the latter in the form of unheated normal serum. The mixtures, diluted with equal volumes of neutral broth or salt solution, are set away for a definite time three to four hours in an incubator at 17 258 INFECTION AND IMMUNITY 37.5° C, and equal quantities from all the tubes are then inoculated into melted agar at 40° C, and plates are poured. Control plates must be made in each case with mixtures of similar quantities of bacteria in salt solution, and similar quantities of bacteria in normal serum. By colony counting after the plates have developed, it is then possible to estimate the degree of bacterial destruction in any of the given dilutions. In actually carrying out the test, dilutions of the inactivated serum are first made, ranging from 1 : 10 to 1 : 1,000 and over. An emulsion of bacteria from a twenty-four-hour agar slant is then made in salt solu- tion, or a twenty-four-hour broth culture properly diluted may be used. Complement is obtained by taking fresh normal rabbit serum and dilut- ing it with salt solution 1 : 10 or 1 : 15. Into a series of test tubes, then, 1 c.c. of each of the serum dilutions is placed, and to each tube is added 0.5 c.c. of the diluted fresh normal rabbit serum (complement) . To these mixtures the bacteria are then added. In adding the bacterial emulsion to these tubes, the writers have found it more accurate to discard the use of the platinum loop and to measure' the bacterial emulsion in a marked capillary pipette such as that used in the opsonin test. (See page 285, Fig. 68.) The controls are set up in a similar way, all of them con- taining a similar quantity of bacterial emulsion, one control containing 1.5 c.c. of salt solution, another control containing 1 c.c. of salt solution + 0.5 c.c. of the diluted complement, and the third control containing in- activated normal serum 1 c.c. +0.5 c.c. of diluted complement. Defi- nite quantities of these mixtures, taken with a standard loop, or preferably with a capillary pipette, are platecl in agar immediately after mixing. BACTERICIDAL TEST IN VITRO (To Determine the Bactericidal Power of a Typhoid Immune Serum against Typhoid Bacilli). Plates Poured After 3 Hrs. at 37° C. c.c. Immune Typh. Ser. 1:200 1:400 1:800 +0.5 1:1600 +0.5 1:3200 +0.5 1:6400 +0.5 1:12800 + 0.5 1:25600 + 0.5 la ( 1.5 c.c. NaCl + 0.5 Typh. Emulsion ■(1.5 " " +0.5 " lib 1. " " +0.5 " + 0.5 c.c. Typh. Emulsion + 0.5 c.c. Rab. Ser. 1:15 I + 0.5 " " " +0.5 " " " " f + 0.5 + 0.5 + 0.5 + 0.5 + 0.5 + 0.5 + 0.5 0 Colonies. 100-1,000 Colonies More than 10,000 Colonies Controls + 0.5 c.c. Rab. Ser. 1:15 Platecl immediately ) More than after 3 hrs. } 10,000 " " " ) Colonies THE TECHNIQUE OF SERUM REACTIONS 259 After incubation for two or three hours similar quantities are again measured into tubes of melted agar with the capillary pipette. With a little practice, great accuracy in these measurements can be acquired. The inoculated agar tubes are very thoroughly mixed, and plates are poured. At the end of twenty-four hours' incubation, an enumeration of the colonies in the various plates is made and the results are compared. The in vitro bactericidal tests have been employed, practically, chiefly in the diagnosis of typhoid fever by Stern and Korte.1 While the serum of normal individuals shows practically no bactericidal power for typhoid bacilli, the sera of typhoid patients may be actively bacteri- cidal in dilutions as high as 1 : 50,000. Hemolytic Tests. — Determination of the hemolytic action of blood serum, bacterial filtrates, and of a variety of other substances, such as tissue extracts and animal and plant poisons, is frequently made in bacteriological laboratories. Familiarity with the methods of carrying out such tests is especially essential since hemolytic tests are also em- ployed in determining other serum reactions, such as the " complement- fixation tests " discussed in another section. For these tests it is necessary to prepare washed red corpuscles of the species of animal against which the hemolysins are to be tested, and to obtain these, blood may be taken in one of the following ways : A. If small quantities of blood corpuscles are desired, the blood may be received into a sterile test tube into which a copper or other wire bent into a loop at the lower end has been introduced. This is used to prevent clotting and to remove the fibrin. Immediately after receiving the blood into this tube, the wire is twirled between the fingers so that the blood is beaten by the wire as by an egg-beater. At the end of five minutes of continuous agitation, the fibrin adhering in a mass to the wire may be lifted out. The corpuscles are then washed and centrifugalized in several changes of salt solution to remove all traces of serum, and are finally emulsified in salt solution. B. The blood may be taken into a centrifuge tube and immediately centrifugalized before clotting has taken place. The plasma is then poured off and the corpuscles are washed with salt solution, as before, to remove the serum. C. The blood may be taken directly into a solution containing five-tenths per cent sodium chlorid and one per cent sodium citrate. 1 Stern und Korte, Berl. klin. Woch., 1904. 260 INFECTION AND IMMUNITY The corpuscles are concentrated by centrifugalization, the citrate solu- tion is decanted, and corpuscles are washed with salt solution, as before, to remove the serum. D. When large quantities of blood are desired, either from man or from an animal, the blood may be received directly into a flask into which a dozen or more glass beads or short pieces of glass tubing have been placed. The flask is shaken for five or ten minutes, immediately after the blood has been taken and, in this way, defibrination is accom- plished. Since, for comparative tests, it is necessary to establish some stand- ard concentration of red blood cells, it is customary in these tests to employ a five per cent emulsion of corpuscles in salt solution. .To obtain this, the thoroughly washed corpuscles from one volume of the original blood are mixed with nineteen parts of 0.85 per cent salt solu- tion.1 Such an emulsion, if kept sterile and in the refrigerator, will serve for hemolytic tests for from one to three days. An emulsion should not be used if the supernatant salt , solution shows any transpa- rent redness, as this indicates hemolysis. If the substance in which hemolysins are to be determined is serum, this may be used either as such or it may be inactivated by exposure to 56° C. in a water bath, and to each test, complement may be added in the form of fresh guinea-pig or rabbit's serum. No absolute rule for the quantity of complement to be used in these tests can be given. As a starting-point, however, when 1 c.c. of a 5 per cent emulsion of red corpuscles is used, it is best to use about 0.1 to 0.2 c.c. of fresh guinea- pig serum as complement. In the actual test, mixtures are made of the corpuscle emulsion, the inactivated immune serum, and complement in small test tubes and the volumes of the various tubes made equal by the addition of definite quantities of salt solution. The contents of the tubes are thoroughly mixed and the tubes put in the incubator or in a water bath at 37.5° C. If complete hemolysis occurs, the fluid in the tube will as- sume a deep Burgundy red. If no hemolysis occurs, the fluid will remain uncolored and the corpuscles will settle out. Incomplete hemo- lysis will be evidenced by a lighter tinge of red in the tube and by the settling out of a varying quantity of blood corpuscles. 1 The method here given was formerly much employed. It is now the general practice, however, to use one volume of the actual sediment to nineteen volumes of salt solution. THE TECHNIQUE OF SERUM REACTIONS 261 In all hemolytic tests the time element is important. No hemo- lysis should be adjudged as incomplete unless at least one hour has elapsed. It is often necessary to carry out hemolytic tests on the blood corpuscles of one human being with the serum of another in order to determine the advisability of performing transfusion. In this case, the serum of the recipient is mixed with a corpuscle emulsion of the cells of the donor, and vice versa. Since it is often difficult to obtain much blood for these tests, the writers have found it convenient to make the test in throttle pipettes, instead of in test tubes. By this technique, ten or fifteen drops of blood and a very small amount of serum will suffice. It should be stated, however, that whenever sufficient quantities of serum can be obtained this technique should not be employed. The Determination of Antibodies in Sera by Complement Fixation. — The principle of complement fixation, discovered by Bordet and Gengou1 in 1901, has been utilized both in bacteriological investigations, and in practical diagnosis for the determination in serum of the presence of specific antibodies. Although spoken of in another section of this book, it may be well to review, briefly, the principles of the Bordet-Gengou phenomenon. The reaction depends upon the fact that when an antigen, i.e., a substance capable of stimulating the formation of antibodies in animals or man, is mixed with its inactivated antiserum, in the presence of complement, the complement is firmly fixed by the combined immune body and antigen in such a way that it can no longer be found free in the mixture. If such a mixture is allowed to stand at a suitable tem- perature for an hour or more, and to it is then added an emulsion of red blood cells together with a suitable quantity of inactivated hemolytic serum, no hemolysis will take place, since there is no free complement available to complete the hemolytic system. If, on the other hand, the original mixture contains no antibody for the antigen used, the comple- ment present is not fixed and is available for the activation of the hemolytic serum later added. The reaction thus depends, in principle, intimately upon the fact that neither antigen 2 alone, nor amboceptor (antibody) alone, can fix complement, but that this fixation is carried out only by the combina- tion of antigen plus amboceptor. Any specific amboceptor can be deter- mined by this method, provided the homologous or stimulating antigen 1 Bordet and Gengou, Ann. de l'inst. Pasteur, xv, 1901. 2 Bordet and Gay, Ann. de l'inst. Pasteur, xx, 1906. 262 INFECTION AND IMMUNITY is used; and vice versa, by the use of a known antibody a suspected antigen may be determined. When testing immune sera for amboceptors given rise to in man or animals by microorganisms which can be cultivated, either the whole bacteria or extracts of the bacteria may be used as an antigen. For the diagnosis of syphilis by this method, in the so-called " Wasser- mann reaction," the antigen employed was originally obtained by the extraction of syphilitic organs, in which free syphilitic antigens, i.e., uncombined products of Spirochete pallida, were assumed to be present. As this reaction has recently become prominent and has proven of no inconsiderable diagnostic value, the technique given below for immune- body determination by complement fixation will be that utilized in the Wassermann test for syphilis. The reader will, however, bear in mind that the test may be applied to other diseases simply by the substitution of the suitable, specific antigen. Thus, when cultivatable bacteria are used as antigens, Bordet and Gengou make use of a thick salt-solution emulsion of a twenty-four-hour agar-slant culture of the microorganisms. In the case of the tubercle bacilli, these authors emulsify 80 milligrams of the bacilli in 1 c.c. of the salt solution. Wassermann and Bruck,1 on the other hand, prepare their bacterial antigen in the following way: The growths of about ten agar slant cultures are emulsified in 10 c.c. of sterile, distilled water. This emulsion is shaken for twenty-four hours in a shaking apparatus. At the end of this time 0.5 per cent of carbolic acid is added and the fluid cleared by centrifugalization. These antigens become slightly weaker during the first ten or fourteen days, but after that remain fairly constant. For the determination of tuber- culosis antibody, these authors make use of either old tuberculin or the new tuberculins UTR" or "Bazillen Emulsion." The Wassermann Test for the Diagnosis of Syphilis.2 — The sub- stances for the test are the following: I. The Antigen. — In their original experiments, Wassermann and his collaborators made use of salt-solution extracts of the organs (chiefly of the spleen) of a syphilitic fetus. The tissue substance was cut in- to small pieces and to one part by weight of this substance, four parts of normal salt solution and 0.5 per cent of carbolic acid were added. This 1 Wassermann und Bruck, Med. Klinik, 55, 1905, and Deut. med. Woch., xii, 1906. 2 Wassermann, Neisser und Bruck, Deut. med. Woch., xix, 1906; Wassermann, Neisser, Bruck und Schucht. Zeit. f. Hyg., lv, 1906. THE TECHNIQUE OF SERUM REACTIONS 263 mixture was shaken in a shaking apparatus for twenty-four hours, and after this the coarser particles were removed by centrifugalization. The reddish supernatant fluid was used as the antigen and could be preserved for a long time in dark bottles in the ice chest. Michaelis * obtained the antigen in the following way : The liver of a syphilitic fetus was preserved in a frozen state and from time to time small quantities of extract were prepared for the purpose of obtaining antigen. This was obtained by thoroughly grinding up a small piece of the liver in a mortar and adding five parts of salt solution and about 0.5 per cent of carbolic acid. This mixture was shaken in a shaking apparatus for several hours and was then allowed to stand at a tem- perature slightly above 0° C. for several days. Finally it was cleared by filtration or centrifugalization. Alcoholic extracts of syphilitic organs have been used by a number of authors. Porges and Meier 2 extract the chopped-up syphilitic liver for twenty-four hours with five times the volume of absolute alcohol. This is then filtered through paper and the alcohol evaporated in vacuo at a temperature not above 40° C. The greenish sticky residue should have an alkaline or neutral reaction. About 1 gram of this material is then emulsified in 100 c.c. of salt solution to which 0.5 per cent of carbolic acid has been added. The fine emulsion which results is filtered through thin paper and the filtrate used as the antigen. Porges and Meier, as well as a number of others, have discovered that in actual practice it is not necessary to make use of syphilitic organs in order to obtain an antigen which will combine with syphilitic immune body. This fact, of course, has thrown much suspicion upon the specificity of the phenomenon. In practice, however, it appears as a purely empirical fact that many of the non-specific antigens, neverthe- less, give reasonably reliable results. The authors mentioned above have found that a 1 per cent emulsion of commercial lecithin (Kahlbaum) in carbolized salt solution furnishes a suitable antigen. This has not been universally confirmed. The same authors have obtained good results by extracting a normal fetal liver by alcohol in the same way as they extracted the syphilitic organ. Landsteiner, Muller, and Poetzl 3 have successfully employed an alcoholic extract of the heart substance of a guinea-pig. 1 Michaelis, Berl. klin. Woch., 1907. 2 Porges und Meier, Berl. klin. Woch., xv, 1908. 3 Landsteiner, Muller und Poetzl, Wien. klin. Woch., 50, 1907. 264 INFECTION AND IMMUNITY Similar alcoholic extracts of normal human spleen or of normal rabbit's liver may be employed. Although often claimed that the anti- gen in such extracts is furnished by the lipoids, as a matter of fact it is at the present day unknown to which ingredient the immune-body binding power is to be attributed. The antigen used in several hundred reactions by the writers with satisfactory result is one prepared according to the method of Noguchi,1 as follows: Fresh normal liver or spleen is covered and thoroughly macerated with five times its volume of absolute alcohol. This is allowed to extract in the incubator for six to eight days, being thoroughly stirred up at least once a day. It is then pressed through cheese-cloth and filtered through paper. This alcoholic extract is evaporated to dryness at room temperature with the aid of a wind fan. The sticky, brownish residue resulting is taken up in a small quantity of ether and the solution poured into four times its volume of C. P. acetone. A heavy flocculent precipitate forms which settles to the bottom as a sticky brown mass. This is retained as antigen and may be preserved under acetone. The acetone-soluble fraction is thrown away. For use, about 0.2 gram of the sticky paste is dissolved in about 5 c.c. of ether and 100 c.c. of salt solution added. This is shaken until the ether has evaporated. The resultant antigen, ready for use, is a slightly opalescent greenish fluid from which nothing settles out on standing. Before an antigen can be used for the actual test, it is necessary to determine the quantity which will furnish a valid result. The substances which are used as antigens often have the power, if used in too large quantity, of themselves binding complement. It is necessary, there- fore, to determine the largest quantity of each given antigen which may be used without exerting an anti-complementary action, i.e., which will not inhibit in the presence of normal serum but which will at the same time inhibit hemolysis when syphilitic serum is used. This is done by mixing graded quantities of the antigen with a constant quantity of complement (0.1 c.c. of fresh guinea-pig serum), in duplicate sets, adding to each tube of one set 0.2 c.c. of a normal serum, and to the other 0.2 c.c. of a known syphilitic serum. These substances are allowed to remain together for one hour and then red blood corpuscles and inac- tivated hemolytic serum are added. The quantity which has given complete inhibition with the syphilitic serum, but absolutely no inhibi- 1 Noguchi, Personal communication. THE TECHNIQUE OF SERUM REACTIONS 265 tion with normal serum, is the one to be employed in subsequent re- actions. Before actual use, it is convenient to make a dilution of antigen in salt solution in such a way that 1 c.c. shall contain the amount re- quired. Thus if 0.05 c.c. is wanted, mix 0.5 c.c. with 9.5 c.c. salt solu- tion. Then 1 c.c. of this can be added to each tube in the test. II. The Hemolytic Serum. — The hemolytic amboceptor, for the reaction, is obtained by injecting into rabbits the washed red blood corpuscles of a sheep. A 5 per cent emulsion of the corpuscles is made and of this 5 c.c, 10 c.c, 15 c.c, etc., are injected at intervals of five or six days. Three or four graded injections of this kind are usually sufficient to furnish a serum of adequate hemolytic power. The injec- tions may be made intraperitoneally or intravenously. About nine or ten days after the last injection of corpuscles, the rabbit is bled from the carotid artery and the serum obtained by pipetting it from the clot. It is best to have a hemolytic serum of high potency in order that the quantities used for the reaction may be as small as possible. This is desirable because of the fact that the serum may contain small amounts of precipitins for sheep's serum, due to insufficient washing of the cor- puscles employed in the immunization. If such precipitins should be present in any quantity in the serum used for the reaction, precipitates might be formed, and these, as we know, have a tendency to carry down complement from a mixture. While the quantitative relations of the complement and antigen in the Wassermann reaction are important, they are vastly more so in the case of the hemolytic amboceptor. For the actual reaction most observers make use of two hemolytic units. A hemolytic unit is the quantity of inactivated immune serum which, in the presence of com- plement, suffices to cause complete hemolysis in 1 c.c of a 5 per cent emulsion of washed blood corpuscles. Noguchi * has pointed out very clearly the dangers of not delicately adjusting the quantity of ambo- ceptor used in the reaction. In a recent communication upon the subject, he has called attention to the experiments of Morgenroth and Sachs 2 who have shown that the relationship between complement and amboceptor necessary for hemolytic reactions is one of inverse propor- tions. To state it more clearly, in their own words, "in the presence of larger quantities of amboceptor, smaller quantities of complement suf- fice," and vice versa. Noguchi, in his work, has found that, while, in the 1 Noguchi, Proc. Soc. for Exper. Biol, and Med., VI., 3, 1909. 2 Morgenroth und Sachs, in Ehrlich's " Gesammelte Arbeiten," etc., Berlin, 1904. 266 INFECTION AND IMMUNITY presence of one unit of amboceptor, 0.1 c.c. of guinea-pig's complement is required to produce hemolysis, by using four, eight, and twenty units of amboceptor, complete hemolysis is obtainable with one-third, one- fifth, and one-tenth of the 0.1 c.c. of complement, respectively. For this reason an excess of amboceptor might result in complete hemolysis in a test, if a small fraction of the complement were left unfixed by the syphilitic antibody. Another result of an excess of amboceptor would consist in a partial dissociation of the complement from its com- bination with the antigen-antibody compound. As Noguchi puts it, "a quantity of syphilitic antibody just sufficient to fix 0.1 c.c. of the complement against two units of the amboceptor is no longer efficient in holding back the complement from partial liberation against the influence exerted by more than four units of the amboceptor." From these considerations it follows that the serum from rabbits immunized against sheep corpuscles must, in each case, be titrated in order to determine the hemolytic unit. For this purpose a number of mixtures are made in test tubes, containing each 0.1 c.c. of complement (fresh guinea-pig serum), 1 c.c. of a 5 per cent emulsion of sheep's cor- puscles, and diminishing quantities of the inactivated hemolytic serum, thus : .1 c.c. of ^| complement fresh y + -< guinea-pig serum. 1. c.c. of 5 per cent emul- sion sheep's corpus- cles. y + 1 Inac- tivated hemo- lytic L serum. .01 c.c. = complete hemolysis. .009 c.c. = complete hemolysis. .005 c.c. = complete hemolysis. .003 c.c. = complete hemolysis. .001 c.c. = complete hemolysis. .0009 c.c. = partial hemolysis. .0005 c.c. = no hemolysis. .0003 c.c. = no hemolysis.1 In the given case, 0.001 c.c. of the serum represents one unit, and 0.002 c.c, two units, is the quantity to be used for each test. III. The Complement. — The complement for the Wassermann re- action is used in the form of fresh guinea-pig serum. This may be obtained in one of the following ways: A guinea-pig may be killed by an incision in the throat and the blood allowed to flow into a large Petri dish. This is set away in the ice chest until clear beads of serum have formed upon the surface, and these are then carefully removed with a pipette. The writers have found it convenient, however, to anesthetize the guinea-pigs, then, by a longitudinal incision into the 1 In each tube the volume of the mixture should be made up to 5 c.c. with 0.85 per cent salt solution. THE TECHNIQUE OF SERUM REACTIONS 267 neck to lay bare the carotid artery and, severing this, to allow the blood to flow into a sterile centrifuge tube. When clotting has occurred, the clot is loosened from the glass with a platinum needle and the serum separated by centrifugalization. Such serum should be used for no longer than three days after being taken and should be kept, except when in actual use, at a low temperature. The complement in guinea-pig serum is sufficiently constant in quantity for practical purposes. IV. The Sheep Corpuscles. — The sheep corpuscles for the actual reaction are obtained by receiving the blood of a sheep in a small flask containing a sterile solution of a 0.5 per cent sodium citrate and 0.85 per cent sodium chloride, or into one containing glass beads or short pieces of glass tubing. In the former case, the citrate solution prevents clotting and the corpuscles may be washed free from the citrate solution and emulsified in salt solution before use in the test. In the latter case, it is necessary to shake the blood in the flask immediately after taking, and to continue the shaking motion for about ten minutes. At the end of this time, the blood will be defibrinated and the corpuscles are washed free from serum by centrifugalization in salt solution. A 5 per cent emulsion of the corpuscles in salt solution is employed for the test, made by measuring the bulk of centrifugalized corpuscles and adding nineteen parts of sterile salt solution. Thorough washing of the cor- puscles is essential both in order to preclude the occurrence of pre- cipitates and to remove any traces of complement present in the serum. V. The Serum to be Tested for Syphilitic Antibody. — The serum of the patient upon whom the test is to be made is best obtained in the same way that blood is obtained for blood cultures. After surgical precautions as to sterilization, a needle is plunged into the median basilic vein and 3 or 4 c.c. of blood are removed. Whenever circum- stances do not permit such procedure, blood may be obtained from the finger or the ear, always in sufficient quantity to furnish at least 1 c.c. of clear serum. Before use for the test, the patient's serum must be inactivated by heating in a water bath to 56° C. for twenty minutes to half an hour. As, according to some observers, 56° C. destroys the syphilitic antibody in part, Noguchi advises inactivation at 54° C. The Test. — The actual test for antibody in a suspected serum is carried out in the following way: In a test-tube of suitable size, 0.1 c.c. of complement, 0.2 c.c. of the inactivated suspected serum, and the antigen, in quantity determined by titration, are mixed, and th^ total 268 INFECTION AND IMMUNITY volume brought up to 3 c.c. with normal salt solution. This mixture is thoroughly shaken, and placed for one hour in a water bath or in the incubator at 37.5° C. At the end of this time, there is added 1 c.c. of a 5 per cent emulsion of sheep's corpuscles, and two units of hemolytic amboceptor, determined by a titration of the inactivated hemolytic rabbit serum, as described above. This mixture is again placed at 37.5° C. for one to two hours. If the antibody is present in the suspected serum, no hemolysis takes place. If absent, haemolysis is complete. No test is of use unless suitable controls are made. The controls set up should be as follows: Control 1. For each serum tested the mixture described above, omitting antigen. Controls 2 and 3. The mixture made as in the test but with known syphilitic serum (2) with and (3) without antigen. Controls 4 and 5. The mixture made as in the test, but with normal serum (4) with and (5) without antigen. Control 6. Antigen and complement alone, left together for an hour before the addition of blood cells and amboceptor in order to preclude the possibility of the antigen itself fixing complement. When working with a well-controlled antigen this control may be omitted. Controls 7 and 8. The hemolytic system, complement, blood cells and amboceptor, set up in order to show that the system is in working order (7) with and (8) without antigen. It is convenient to set the tubes in two rows in a rack, the front row containing antigen, the back row containing the same mixture without antigen. In a positive test, the test itself, and Control 2, alone, should show inhibited hemolysis. The other tubes should show complete solution of the hemoglobin. (See scheme, p. 259.) Modifications of the Wassermann Test. — Bauer's Modification. — Bauer1 utilizes the fact that normal human serum contains a certain amount of hemolytic amboceptor for sheep's corpuscles. In consequence he omits in his reaction the use of specifically immunized hemolytic rabbit serum. In carrying out the test he uses but four tubes: 1. Contains 0.1 c.c. of complement, the titrated amount of antigen, and 0.2 c.c. of the inactivated serum to be tested. 2. Contains the same mixture without antigen. 3. Is like one, except that normal serum is substituted for that of the patient. 1 Bauer, Dent. med. Woch., xii, 1908, and Berl. klin. Woch., xvii, 1908. THE TECHNIQUE OF SERUM REACTIONS 269 4. Is like three, except that antigen is omitted. If this modification is used at all (and its value is by no means established), a fifth control should be added in which known syphilitic serum is used. These tubes are exposed to 37.5° C. for one hour, at the end of which time 1 c.c. of a 5 per cent emulsion of sheep's corpuscles are added. If the test is positive, tube one should be without hemolysis, as well as the fifth control with known syphilitic serum. Tubes two, three, SCHEME FOR WASSERMANN TEST. ADAPTED TO ORIGINAL WASSERMANN SYSTEM AFTER SCHEME OF NOGUCHI. Test with Unknown Serum. Test with Known Positive Syphilic Serum. Test with Known Negative Normal Serum. Test without Serum to Control Efficiency of Hemolytic System. Serum .2 c.c. d d Serum .2 c.c. Serum .2 c.c. 4- CO + + as Q Complement a 2 Q Complement Q Complement 0 Complement 5 3 .1 c.c. 'o .1 c.c. .1 c.c. .1 c.c. < 2 + > + + + S Salt sol. 3. c.c. o Salt sol. 3. c.c. Salt sol. 3. c.c. Salt sol. 3. c.c. 2. H 4, Serum .2 c.c. 6. 8. Serum .2 c.c. Serum .2 c.c. + d + + o

■ quantity determined by titration. 2. Antiserum )^ J J 3. Diminishing quantities of the substance in which the antigen is suspected, ranging from 0.1 c.c. downward to 0.0001 c.c. Salt solution is added as a diluent up to 3 c.c. and the tubes are placed in the incubator or water-bath at 37.5° to 40° C. At the end of this time red blood cells and amboceptor are added as before. The tubes are controlled by a series containing all the above ingredi- ents except the antiserum. CHAPTER XVII PHAGOCYTOSIS The studies on immunity which we have outlined in the preceding sections have dealt entirely with the phenomena occurring in the re- action between bacteria or bacterial products and the body fluids. These studies, we have seen, have formed the basis of a theoretical conception of immunity formulated chiefly by the German school of bacteriologists under the leadership of Ehrlich, Pfeiffer, Kruse, and others. Parallel with these developments, however, investigations on immunity have been carried on which have brought to light many important facts con- cerning the participation of the cellular elements of the body in its resistance to infectious germs. The inspiration for this work and the greater part of the theoretical considerations which have been based upon it have emanated from Metchnikoff1 and his numerous pupils at the Pasteur Institute in Paris. The phenomenon which these observers have studied in great detail and upon the occurrence of which they have based their conceptions of immunity, is known as phagocytosis. It is well known that among the lowest unicellular animals the nutritive process consists in the ingestion of minute particles of organic matter by the cell. The rhizopods, which may be found and studied in water from stagnant pools or infusions, when observed under the microscope, may be seen to send out short protoplasmic processes, the pseudopodia, by means of which they gradually flow about any foreign particle with which they come in contact. If the ingested particle is of an inorganic nature and indigestible, it will be again extruded after a varying period. If, however, the ingested substance is of a nature which can be utilized in the nutrition of the protozoon, it is rapidly surrounded by a small vacuole within which it is gradually dissolved and becomes a part of the cellular protoplasm. This digestion within the unicellular organism is probably due to a proteolytic enzyme2 which acts in the Metchnikoff, " LTmmunite dans les maladies infectueuses. " 2 Mouton, Ann. de l'inst. Pasteur, xvi, 1902. 275 276 INFECTION AND IMMUNITY presence of a weakly alkaline reaction. This has been shown by the actual extraction, from amebae, of a trypsin-like ferment. As we proceed higher in the scale of the animal kingdom, we find that this power of intracellular digestion, while not uniformly an attribute of all the body cells, is still well developed and a necessary physiological function of certain cells which have retained primitive characters. In animals like the ccelenterata, in which there are two cell layers, an entoderm and an ectoderm, the ectodermal cells have lost the power of intracellular digestion, while the entodermal cells are still able to ingest and digest suitable foreign particles. It is only as we proceed to animals of a much higher organization that the function of cell ingestion of crude food is entirely removed from the process of general nutrition. Never- theless, in these animals also, the actual cell ingestion of foreign particles occurs, but it is now limited entirely to a definite group of cells. In the higher animals and in man, this function of phagocytosis is limited to the white blood cells of the circulation, or leucocytes, to certain large endothelial cells lining the serous cavities and blood-vessels, and to cells of a rather obscure origin which contribute to the formation of giant cells within the tissues. A convenient division of these phagocytic cells is that into "wandering cells" and "fixed cells." The wandering cells are the polymorphonuclear leucocytes, called " microphages " by Metchnikoff, and certain large mononuclear elements or "macrophages/' Fixed cells, also called macrophages by Metchnikoff and possessing the power of ameboid motion, include the cells lining the serous cavities, and the blood and lymph spaces. The small lymphocytes, so far as we know, have no phagocytic functions. In studying the cellular activities which come into play whenever foreign material of any description gains entrance into the animal body, a definite reaction on the part of the phagocytic cells may be observed. When we inject into the peritoneal cavity of a guinea-pig a small quan- tity of nutrient broth, and examine the exudate within the cavity from time to time, we can observe at first a diminution from the normal of the cells present in the peritoneal fluid. This may be due either to an injury of the leucocytes by the injected substance, or to an actual repel- lent influence which the injected foreign material exerts upon the wan- dering cells.1 Very soon after this, however, the exudate becomes ex- tremely rich in leucocytes, chiefly of the polymorphonuclear variety, the maximum of the reaction being reached about eighteen to twenty-four 1 Pierrallini, Ann. de l'inst. Pasteur, 1897. PHAGOCYTOSIS 277 hours after the injection. After this, there is a gradual diminution in the leucocytic elements until the fluid in the peritoneal cavity again reaches its normal condition. It is plain, therefore, that the presence of the foreign material in the peritoneal cavity has, after a primary repellent action upon the phagocytes, attracted them in large numbers to the site of the foreign substance. Such repelling or attracting in- fluences upon the leucocytes are spoken of as negative or positive chemotaxis. The reasons for chemotaxis are not well understood. In the case of bacteria, which chiefly interest us in the present connection, chemotactic attraction or repulsion is intimately dependent upon the nature of the microorganism, and very probably has a definite relation- ship to its virulence. Whether or not the principles of chemotaxis may serve to explain the hypo- and hyper-leucocytoses, observed and diag- nostic ally utilized in clinical medicine, is by no means positive. It is likely, however, that the two phenomena are closely associated. Leva- diti 1 believed that he obtained some evidence that negative chemotaxis may take place within the blood-vessels when he noticed that the intra- venous injection of cholera spirilla into immunized guinea-pigs resulted in an immediate disappearance of leucocytes from the circulating blood, and their accumulation in the internal organs. On the other hand, this may possibly be more logically explained by a concentration of both bacteria and leucocytes in the capillary system of such an organ as the liver, as it is known that injected bacteria rapidly disappear from the general circulation, but may be demonstrated in the various organs for some time after injection. We have seen, therefore, that the invasion of the animal body by foreign material, living or dead, is followed by a prompt response on the part of the phagocytic cells. In the case of bacteria, when these are deposited in the subcutaneous areolar tissues, the inflammatory reaction which follows brings with it an emigration of microphages (polynuclear leucocytes) from the blood-vessels — and these are the so-called pus cells. When the injection of bacteria is intraperitoneal, after a primary diminution, there is an increase of leucocytes in the peritoneal cavity which soon results in the formation of a copious turbid exudate. If the pus of an abscess or the exudate from an infected peritoneum is ex- amined microscopically, it will be seen that many of the microphages have taken bacteria into their cytoplasm. That fully virulent living bacteria can be so taken up has been variously proven. The phago- Levaditi, Presse med., 1900. 278 INFECTION AND IMMUNITY cytosis is, therefore, not simply a removal of the dead bodies of bacteria previously killed by the body-fluids, but represents an actual attack upon living and fully virulent microorganisms. That the ingested bacteria are often alive after ingestion is proved by the fact that the injection of exu- date containing, so far as can be determined, only intracellular bacteria, has, in several instances, been found to give rise to infection. After the bacteria have remained for some time within the cytoplasm of the leucocyte, vacuoles may be seen to form about them, similar to those mentioned in discussing the digestive processes of amebse. If the preparations are, at this stage or later, stained with a one-per-cent solution of neutral red, it will be found that the bacteria, colorless under normal conditions, will be stained pink, an evidence of their beginning disintegration. At a later stage in the process of intracellular digestion, the bacteria will lose their form, and appear swollen, granular, and vacuolated, and finally will be no longer distinguishable. If, on the other hand, the ingestion of bacteria brings about the death of a leucocyte, the neutral red will not stain the bacteria, the digestive vacuoles will not form, and the leucocyte itself will disintegrate. It must not be forgotten, however, that not all microorganisms are equally susceptible to phagocytosis. Some may resist ingestion more energetically than others by agencies not fully understood. Others again, like the tubercle bacillus and the anthrax bacillus for instance, may, after ingestion, oppose great difficulties to intracellular digestion. To a certain extent, moreover, the variety of the bacterium deter- mines the variety of phagocyte attracted to the point of invasion. In the cases of most of the bacteria of acute diseases, the microphages or polymorphonuclear leucocytes are the ones upon which the brunt of the battle devolves. Other invaders, like the Bacillus tuberculosis, blastomyces, and others, find themselves opposed chiefly by the macro- phages. Cells of animal origin, such as the dead or injured cells of the animals' own body or the cells of other animals artificially introduced, are ingested by macrophages. This is true also of many parasites of animal nature. It is clear, thus, that the process of phagocytosis is a universal re- sponse on the part of the body to the invasion of foreign particles of dead material, of alien cells, and of living microorganisms. It remains to be shown upon what basis this process may be regarded as an essential feature in protecting the body against infection. The numerous researches of Metchnikoff have brought out the important fact that phagocytosis is regularly more active in cases in PHAGOCYTOSIS 279 which the infected animal or human being eventually recovers. In animals, furthermore, which show a high natural resistance against any given microorganism, phagocytosis is decidedly more energetic than it is in animals more susceptible to the same incitant. Thus, experiment- ing with anthrax infection in rats, Metchnikoff was able to show that, in these animals, a decidedly more rapid and extensive phagocytosis of anthrax bacilli takes place than in rabbits and guinea-pigs and other animals which are delicately susceptible to this infection. While different interpretations have been attached to this phenomenon, its actual occurrence may be accepted as a proven fact. In his later investigations, furthermore, MetchnikofT was able to show that a direct parallelism existed between the development of immunity in an artificially immunized animal and the phagocytic powers of its white cells. He showed that rabbits artificially immunized to anthrax, responded to anthrax infection by a far more active phagocy- tosis than did normal, fully susceptible animals of the same species. It is quite impossible, in the space allotted, to recount the many similar experiments by which the accuracy of these observations has been confirmed. While few bacteriologists at the present day harbor any doubt as to the truth of these contentions, the fundamental differences between the conclusions drawn from these various phenomena by the school of MetchnikofT and by that of the German workers may be clearly stated as follows: Metchnikoff believes that phagocytosis is the cardinal factor which determines immunity, while Pfeiffer and others maintain that the determining factors upon which recovery or lethal outcome depends, lie in the fluids of the body, the serous exudate and its contents of immune body and complement, while the phagocy- tosis occurring coincidently, is merely a means of removal of the bacteria after the outcome has already been decided. In the further developments of his theory, Metchnikoff has claimed that the immune body and complement — the presence of which in blood serum and exudates he by no means overlooks — are derivatives of the leucocytes. The immune body or " fixator," as Metchnikoff has named it, has been shown by . Wassermann and Takaki1 to be most plentiful in the spleen, lymph nodes, and bone marrow of animals — all of them organs in which large collections of leucocytic elements are found. MetchnikofT s opinions as to the leucocytic origin of the complement, or "cytase," 1 Wassermann und Takaki, Berl. klin. Woch., 1898. 280 INFECTION AND IMMUNITY have found support in the experiments of Levaditi,1 who was able to demonstrate the absence of complement in blood plasma, — i.e., where no destruction of leucocytes had taken place — and in those of Cantacuzene,2 who showed that cholera-immune guinea-pigs would succumb to intra- peritoneal injection of these bacteria when the diapedesis of leucocytes had been prevented by the administration of opium. The chapter of phagocytosis in its relation to bacterial immunity is by no means closed. The problems involved in it are intricate and will require much further study. The subsequent sections upon opsonins, aggressins, and upon leucocyte extract, incorporate the more recent studies which may be said to have followed logically in the footsteps of MetchnikofPs work. 1 Levaditi, Presse med., 1900. 2 Cantacuzene, Ann. de l'inst. Pasteur, 1897. \ CHAPTER XVIII OPSONINS, LEUCOCYTE EXTRACT, AND AOGRESSINS OPSONINS ALTHough the theories of immunity are, as we have stated, generally classified as the humoral and the cellular or phagocytic theories, the separation has never, even in the minds of the warmest partisans, been an absolute one. Thus, Buchner and his successors looked for the origin, first, of alexin, then of complement, in the leucocytes, and Metchnikoff attributed to immune serum the quality of stimulating the leucocytes (stimulins) to increased phagocytosis. The serum, accord- ing to Metchnikoff, acted, not directly upon the bacteria, in the nature of bactericidal or lytic substances, but rather upon the leucocytes, pre- paring or arming these for the fray. Denys and Leclef * were the first definitely to oppose this view. These authors, on the basis of ex periments done upon streptococcus immunity in rabbits, came to the conclusion that the serum aided phagocytosis rather by its action upon the bacteria than by its influence upon the leucocytes. Wright2 in 1903 and 1904 undertook a systematic study of the re- lation of the blood serum to phagocytosis, in a series of careful experi- ments. Using his own modifications of the technique of Leishman,3 he first determined the direct dependence of phagocytosis upon some substance contained in the blood serum. He further proved conclu- sively that this serum component acts upon the bacteria directly and not upon the leucocytes, is bound by the bacteria, and renders them subject to phagocytosis. The presence of these substances in sera, furthermore, which appear entirely free from bactericidal or lytic bodies, and the thermolabile character of the substances (60° for ten or fifteen minutes destroys them) seemed to exclude their identity with the immune bodies of other authors. 1 Denys et Leclef, La cellule, xi, 1895. 2 Wright and Douglas, Proc. Royal Soc. London, lxxii, 1904. 3 Leishman, Brit. Med. Jour., i, 1902. 281 282 INFECTION AND IMMUNITY Because of their action in preparing the bacteria for ingestion by the leucocytes, he named these bodies " opsonins " (o^wvlw, to prepare food). Neufeld and Rimpau1 soon after, and independently of Wright, described similar substances in the blood serum of streptococcus and pneumococcus immune animals, which they called bacteriotropins. Because of their greater thermostability it is not yet possible to identify these bacteriotropins absolutely with the opsonins. The importance of these opsonic substances in immunity was shown by Wright 2 in a series of experiments in which he determined that in persons ill with staphylococcus or tubercle-bacillus infections, the phago- cytic powers were relatively diminished toward these microorganisms, but could be specifically increased upon active immunization with dead bacteria or bacterial products. The results of Wright have been confirmed and elaborated by nu- merous workers. The diminished power of leucocytes to take up bacteria without the co-operation of serum was demonstrated, after Wright, by Hektoen and Ruediger,3 who worked with gradually increasing dilutions of serum. The contention of the Wright school, however, that leucocytes are en- tirely impotent for phagocytosis without the aid of serum, can not be regarded as proven, in face of the work of Lohlein 4 and others who have observed phagocytosis on the part of washed leucocytes. The specificity of opsonins and their multiplicity in a given serum were shown mainly by the work of Bullock and Atkin,5 Hektoen and Ruediger,6 and Bullock and Western.7 These authors showed that the opsonic substances in sera could be absorbed out of the sera, one by one, by treatment with various species of bacteria, a procedure analogous to the method of absorption used in the study of agglutinins. The increase of phagocytic power demonstrated by Wright in immune sera naturally led to the question whether this depended merely upon an increase of the normal opsonins or whether the newly formed immune opsonins were entirely different substances. The greater thermosta- bility of the opsonins in immune sera seemed, at first, to support the 1 Neufeld und Rimpau, Deut. med. Woch., xl, 1904. 2 Wright and Douglas, Proc. Roy. Soc, London, lxxiv, 1905 3 Hektoen and Ruediger, Jour. Inf. Dis., ii, 1905. 4 Lohlein, Ann. de l'inst. Pasteur, 1905 and 1906. •s Bullock and Atkin, Proc. Roy. Soc, London, lxxiv, 1905. 6 Hektoen and Ruediger, loc. cit. 7 Bullock and Western. Proc. Roy. Soc, loc. cit. OPSONINS 283 latter view. Dean/ however, showed that not all of the normal opsonins are thermolabile and that, by absorption experiments, bacteria treated with normal sera could be prevented from taking up opsonins from immune sera. These facts seem to point strongly toward the identity of normal and immune opsonic substances. Further study of the opsonins has led to numerous other questions regarding their structure, their relation to other immune bodies, etc., which are largely still in the stage of controversy, and for which the original monographs must be consulted. The controversial questions may be briefly reviewed as follows: As stated above, Wright believed originally that the bodies dis- covered by him in normal sera, the " normal opsonins," in other words, were distinct bodies that could not be identified with either the comple- ment or antibodies present in serum. Neufeld and Hiine,2 Levaditi and Inmann,3 and others, on the other hand, maintain that the opsonic action of normal serum, at least, is intimately related to the complement contents of such serum. They base this contention not only upon the thermolability of normal opsonins, but also upon the fact that opsonin may be removed from normal serum at the same time as complement by the method of com- plement fixation, detailed in another section (see pp. 245 and 261) .4 The contention of Wright that the thermostable opsonic substances of immune serum are distinct bodies, not identical with the ambocep- tors, is supported by the work of Hektoen,5 Neufeld and Topfer,6 and others. The problem, however, can by no means be regarded as finally settled, since other workers, notably Levaditi, are inclined to identify the immune opsonins with lytic amboceptors. As to the structure of the opsonic substances, moreover, dif- ferences of opinion still exist. Hektoen and Ruediger 7 who have investigated the question attribute to opsonins a complex constitution. They believe them to possess a thermostable haptophore group and a thermolabile " opsonophore " group and that heating beyond a definite temperature converts the opsonins into opsonoids by 1 Dean, Proc. Roy. Soc, London, lxxvi, 1905. 2 Neufeld and Hiine, Arb. a. d. kais. Gesundheitsamt, xxv. 3 Levaditi and Inmann, Compt. rend, de la soc. de biol., 62, 1907. 4 Levaditi, Presse medicale, 70, 1907. 5 Hektoen, Jour, of Inf. Dis., iii, 1906. 6 Neufeld und Topfer, Cent. f. Bakt., xxxviii, 1905. 7 Hektoen and Ruediger, Jour, of Inf. Dis., ii, 1905. 284 INFECTION AND IMMUNITY destruction or alteration of the " opsonophore " group. This view is not shared by all workers and has been disputed by Bullock and Atkin.1 The Technique of Wright. — The three factors necessary for the per- formance of an opsonic test are (1) the blood serum to be tested; (2) an even emulsion of bacteria, and (3) leucocytes. (1) Blood serum is obtained by bleeding from the ringer and receiving the blood into glass capsules (Fig. 66). These are sealed at both ends; the blood is allowed to clot ; and the separation of serum is hastened by a few revolutions of a centrifuge. (2) The bacterial emulsion is obtained by rubbing up a few loopfuls of a twenty-four-hour slant agar culture with a little physio- logical salt solution (0.85 per cent) in a watch glass. A very small amount of salt solution is used at first and more is gradually added, drop by drop, as the emulsion becomes more even. The final breaking up of the smaller clumps is best accomplished by -r. n/l „T , ^ cutting off very squarely the end of a Fig. 66. — Wright's Capsule for ... d . , . Collecting Blood. capillary pipette, placing it perpen- dicularly against the bottom of the watch glass, and sucking the emulsion in and out through the narrow chink thus formed. (Fig. 67.) Emulsions of tubercle bacilli are more difficult to make. The bacilli filtered off in the manufacture of old tuberculin are commonly used. These are washed in salt solution on the filter, and are then scraped off and sterilized. They are then, in a moist condition, placed in a mortar and thoroughly ground into a paste. While grinding, salt solution 1.5 cent) is gradually added until a thick emulsion appears. This emulsion may be diluted and larger clumps separated by centri- fugalization. (3) The leucocytes are obtained by bleeding from the ear or finger directly into a solution containing eighty-five hundredths per cent to one per cent of sodium chlorid and five-tenths to one and five-tenths per cent of sodium citrate. Ten or fifteen drops of blood to 5 or 6 c.c. of the solution will furnish sufficient leucocytes for a dozen tests. This mixture is then centrifugalized at moderate speed for five to six minutes. At the end of this time, the corpuscles at the bottom of the tube will be covered by a thin grayish pellicle, the buffy coat, consisting 1 Bullock and Atkin, Proc. Royal Soc, lxxiv, 1905. OPSONINS 285 chiefly of leucocytes. These are pipetted off with a capillary pipette (by careful superficial scratching movements over the surface of the buffy coat). There being, of course, no absolute scale for phagocytosis, whenever an opsonin determination is made upon an unknown serum, a parallel control test must be made upon a normal serum. This normal is best obtained by a " pool " or mixture of the sera of five or six supposedly normal individuals. The three ingredients — serum, bacterial emulsion, and leucocytes — having thus been prepared, the actual test is carried out as follows: Fig. 67. — Pipette for Opsonic Work. Capillary pipettes of about six or seven inches in length and of nearly even diameter throughout, are made. These are fitted with a nipple and a mark is made upon them with a grease-pencil about 2 to 3 cm. from the end (Fig. 68). Corpuscles, bacteria, and serum are then successively, in the order named, sucked into the pipette up to the mark, being separated from each other by small air-bubbles. Equal quantities of each having thus been secured, they are mixed thoroughly by repeatedly drawing them in and out of the pipette upon a slide. The mixture is then drawn into the pipette; the end is sealed; and incubation at 37.5° is carried on for an arbitrary time, usually fifteen to thirty minutes.1 The control with normal serum is treated in exactly Fig. 68. — Pipette with three Substances, Corpuscles, Bacteria, and Serum, as first taken up. the same way. After incubation the end of the pipette is broken off, the contents are again mixed, and smears are made upon glass slides in the ordinary manner of blood smearing. Staining may be done by Wright's modification of Leishman's stain, by Jenner's, or by any other of the usual blood stains. In these smears, then, the number of bacteria contained in each leucocyte is counted. The contents of about eighty 1 For the purpose of incubation, specially constructed water baths, marketed under the name of " opsonizers," may be used. 286 INFECTION AND IMMUNITY to one hundred cells are usually counted and an average is taken. This average number of bacteria in such leucocytes is spoken of as the "phagocytic index." The phagocytic index of the tested serum, divided by that of the "normal pool" (control) serum, gives the " opsonic index." Another method of estimating the opsonic content of a given blood serum has been contributed by Simon, Lamar, and Bispham.1 These authors employed dilutions both of the patient's serum and of normal serum ranging from one in ten to one in one hundred. With these dilutions, they carry out opsonic experiments with bacterial, emulsions and washed leucocytes in the same way as this is done in the Wright method, except that they recommend the employment of thinner bac- terial emulsions than are usually employed in the former method. In examining their slides, they do not estimate the number of bacteria found within the leucocytes, but rather the percentage of leucocytes which actually take part in the phagocytic process,2 i.e., which con- tain bacteria. By the same method of dilution, they determine what they have called "the opsonic coefficient of extinction," a phrase which is used to express the degree of dilution of the serum at which no further phagocy- tosis takes place. They claim for their methods the more delicate de- termination of variations in opsonic power. The method has not been sufficiently used to permit the expression of an opinion as to its value. The Vaccine Therapy of Wright. — In connection with his more theoretical work upon opsonins, Wright has laid much stress upon the value of active immunization in the treatment of infectious diseases. Beginning his work with staphylococcus and tubercle-bacillus infectiom, he has extended his methods, with the aid of many collaborators, to gonococcus, streptococcus, pneumococcus, and a number of other bacterial infections. In all these cases, when possible, he uses for therapeutic purposes a so-called "autogenous vaccine" which is mado with the bacteria isolated from the patient himself. In the case of tubercle-bacillus infections, he uses for treatment the new-tuberculin- bacillary-emulsion of Koch. The production of vaccine is, according to Wright, as follows: Production of Vaccines. — After isolation of the organisms from the patient, cultures are made with a view of obtaining considerable amounts 1 Simon, Lamar, and Bispham, Jour. Exp. Med., viii, 1906. 2 Simon and Lamar, Johns Hopkins Hosp. Bull., xvii, 1906. OPSONINS 287 of bacterial growth. In making vaccines with poorly growing organisms, large surfaces must be inoculated. Organisms are best grown for this purpose upon the surface of agar or glucose agar (the enrichment of the agar with sugar or acetic fluid, etc., depending upon the cultural require- ments of the organism in question), in square eight-ounce medicine bottles laid upon their sides. This furnishes a large area for inoculation. After sufficient growth has taken place upon the agar, two or three cubic centimeters of sterile normal salt solution are introduced into the bottles with a sterile pipette. With this the growth is gently- washed off the surface of the agar, more salt solution gradually being added as necessary. The emulsification may be facilitated by gently scraping the growth off the medium by means of a flexible platinum loop. This thick bacterial emulsion is then pipetted out of the bottles, during which process an equalization of the emulsion can be attained by repeated sucking in and out with the pipette. The emulsion is then placed in a sterile test tube which may then be drawn out at its open end into a capillary opening. It is a point of practical importance that, in pre- paring such capsules out of a test tube, a few inches of air space should be left above the surface of the emulsion, so that expansion during heating may not blow out the top of the glass tube. A dozen or so of sterile glass beads may be put into these tubes in order to aid in emul- sification. Shaking the beads in such a tube will help in breaking up small clumps of bacteria. The emulsion is then standardized ; that is, a numerical estimation of bacteria per cubic centimeter must be made. This standardization is best done before sterilization, because during the latter process a number of bacteria may be broken up, and, while unrecognizable morphologically, are, nevertheless, represented in the emulsion by their products. The standardization may be accomplished by highly diluting a definite volume of the emulsion, planting plates with definite quan- tities of the dilution, and counting colonies. Wright prefers, as more exact, an enumeration of the bacteria against red blood cells. This is done in the following way: A little of the emulsion is placed in a watch glass and from it, with a pipette as used in the estimation of the opsonic index, one volume is taken and is mixed with an equal volume of blood from the finger and two or three volumes of salt solution. The salt solution is added in order to dilute the red cells so that they can be conveniently counted and to prevent clotting. These substances are thoroughly mixed in a pipette and spread upon a slide as in making a blood smear, and as even 288 INFECTION AND IMMUNITY and uniform a smear as possible should be made. They are then stained either by Jenner's or Wright's blood stain. The preparations are examined with an oil-immersion lens. In order to limit a definite microscopic field, it is convenient to use an Ehrlich diaphragm, or else, in lieu of this, to mark a circle with a blue pencil upon the lens of the eye-piece. The red blood cells and bacteria, in a number of these fields, are counted and the ratio between them is estimated. Knowing the number of red blood cells to the cubic milli- meter in the particular blood employed, by previous blood count, and knowing that equal volumes of blood and of bacterial emulsion have been used in the mixture, it is easy from this ratio to ascertain the number of bacteria contained in a cubic millimeter of the original emulsion. Thus, for instance, if in an average of twenty fields bacteria are to red blood cells as two is to one, and the blood employed con- tains five million red blood cells to each cubic millimeter, then a cubic millimeter of our emulsion contained ten million bacteria, and a cubic centimeter one hundred times as many. The vaccine, thus produced and standardized, is sterilized by sus- pension in a water bath at 60° C. for one hour on each of five or six consecutive days. Its sterility is then controlled by culture. From this stock emulsion small quantities may be drawn off and diluted for therapeutic use. The initial dose given by Wright in staphylococcus infections, in which the method has been most frequently employed, varies from fifty to one hundred millions of bacteria. In working with the tubercle bacillus, the ordinary tuberculin dosage is adhered to. Wright, in his work, makes use of the opsonic index in order to estimate changes in the resistance of the patient against the given in- x fection. In other words, he bases his judgment as to whether the patient is improving or not, upon the opsonic power of the patient's serum. In following the opsonic index of a patient during systematic treatment with vaccine, Wright has found definite changes upon the basis of which he constructs a curve of opsonic power. Immediately after the injection of vaccine, he finds that there is a brief period during which the opsonic power of the patient is depressed below its original state. This he calls the negative phase. The length of time occupied by this negative phase depends both upon the condition of the patient and upon the size of the dose given. It is usually completed within twenty-four hours. After this, there is a gradual rise in the opsonic power, at first rapid, later more slow, until a maximum is reached after a vary- LEUCOCYTE EXTRACT 289 ing number of days. This period of rise represents the positive phase. The second inoculation with vaccine should, according to Wright, be made when the opsonic power is again beginning to sink after the highest point of the positive phase. The facts of Wright's investigations have been given in the preceding pages, purposely without critical considerations. The existence of opsonic or phagocytosis stimulating substances in blood serum may be accepted as fact. It is also of unquestionable value to the science of immunity that renewed vigor has been infused into the investiga- tion of active therapeutic immunization. The far-reaching claims of therapeutic benefit, which have been made by Wright and his school, however, have not yet received sufficient support by clinical observa- tion to be fully accepted, although intelligent application of this treat- ment in suitable cases has undoubtedly proved its therapeutic value. LEUCOCYTE EXTRACT In the foregoing sections upon Phagocytosis and Opsonins, we have discussed the protective action exerted by the living leucocytes against bacterial infection and the relation of these cells to the blood serum. We have seen, furthermore, that, while our knowledge of the blood serum, as developed at present, shows that phagocytes may be aided by this in the ingestion of bacteria, the subsequent digestion of the germs, and possibly the neutralization or destruction of their intracel- lular poisons, is, as far as we know, largely accomplished by the unaided -phagocytic cell. It is an obvious thought, therefore, that, in the struggle with bacterial invaders, the leucocytic defenders might be considerably re-enforced if they were furnished, as directly as possible, with a further supply of the very weapons which they were using in the fight with the microorganisms: With this thought as a point of departure, Hiss 1 conceived the plan of injecting into infected subjects the substances composing the chief cells or all the cells usually found in exudates, in the most diffusible form and as little changed by manipulation as possi- ble; and he also assumed that extracts would be more efficacious than living leucocytes themselves, since if diffusible they would be distrib- uted impartially to all parts of the body by the circulatory mechan- ism. They would then, as quickly as absorption would permit, relieve the fatigued leucocyte and also protect by any toxin-neutralizing or other power they might possess, the cells of highly specialized functions. 1 Hiss, Jour. Med. Res., N. S., xiv, 3, 1908. 19 290 INFECTION AND IMMUNITY The method of obtaining these substances as used both in animal experiments and in the treatment of human subjects is at present as follows: Rabbits, preferably of 1,500 grams weight or heavier, receive intra- pleural injections of aleuronat. This is prepared by making a three per cent solution of starch in meat-extract broth, without heating, and adding to this, after the starch has gone into thorough emulsion, five per cent of powdered aleuronat. This is thoroughly mixed, boiled for five minutes, and filled into sterile potato tubes, 20 c.c. into each tube. Final sterilization is done preferably in an autoclave. The rabbit in- jections are carried out by injecting 10 c.c. into each pleural cavity in the intercostal spaces at the level of the end of the sternum, in the anterior axillary line, great care being exerted to avoid puncturing of the lungs. The rabbits are left for twenty-four hours, at the end of which time a copious and very cellular exudate will have accumulated in the pleural cavities. This is removed, after killing the animals with chloroform, by opening the anterior chest wall under rigid precautions of sterility, and pipetting the exudate into sterile centrifuge tubes. Immediate centrifugalization before clotting can take place then per- mits the decanting of the supernatant exudate fluid. To the leuco- cytic sediment is then added about 2 c.c. of sterile distilled water, and the emulsion is thoroughly beaten up with a stiff bent platinum spatula. Smears are now made on slides, stained by Jenner's blood stain, and ex- amined for possible bacterial contamination. It is well also to take cultures. Sterile distilled water is then added to each tube, about twenty volumes to one volume of sediment, and the tubes are set away in the incubator for eight hours. At the end of this time the sterility is again controlled as above, and further extraction in the refrigerator continued until the extract is used. In experimenting upon animals, Hiss 1 observed that pneumococcus, staphylococcus, streptococcus, meningococcus, and typhoid, dysentery, and cholera infections in rabbits and guinea-pigs were profoundly modi- fied when injections of leucocyte extracts, prepared as above, were ad- ministered intraperitoneally or.subcutaneously during the course of the infection. In many cases animals were saved by these substances from infections which proved rapidly fatal in untreated control animals, even when the protective injections were made as late as twenty-four hours after intravenous infection. 1 Hiss, Jour. Med. Res., N. S., xiv, 3, 1908. AGGRESS1NS 291 In applying this method of treatment, by subcutaneous injections, to infections in man, Hiss and Zinsser observed distinctly beneficial results in cases of epidemic cerebrospinal meningitis, in lobar pneumonia, in staphylococcus infections, and in erysipelas.1 In experimenting with the leucocyte extracts in vitro the same au- thors were able to show that precipitates occurred when clear leucocyte extract and the clear extract of various bacteria were mixed.2 Further experiments, carried out both in animals and in the test tube, showed that while the leucocytic extracts possessed slight bactericidal powers for a variety of microorganisms, these attributes did not seem sufficient to explain the profound, modifying influences exerted upon bacterial infections by these extracts. Experiments have also shown that the leucocyte extracts possess some distinct power of neutralizing or destroying the poisonous products of typhoid and dysentery bacilli. Whether or not the final explanation of the action of these extracts will be found to lie in these endotoxin-neutralizing properties of the leuco- cytic substances, can not as yet be determined, and this problem must be left for further research to decide. That bactericidal substances can be extracted from leucocytes by various methods has been repeatedly shown by Schattenfroh, Petter- son, Korschun, and others.3 The researches of Petterson as well as, more recently, the work of Zinsser, have shown that these "endolysins," as Petterson has called them, have a structure quite different from that of the serum bacteriolysins in that they are not rendered inactive by temperatures under 80° C, but, when once destroyed by higher tem- peratures, can not be reactivated either by the addition of fresh serum or of unheated leucocyte extracts. The last-named authors, moreover, have shown that these endocellular bactericidal substances are not increased by immunization, the quantity present in each leucocyte being probably at all times simply sufficient for the digestion of the limited number of bacteria which can be taken up by the individual leucocyte. AGGRESSINS An extremely obscure chapter in our knowledge of the reaction of animals and man against infection is the one dealing with the questions 1 Hiss and Zinsser, Jour. Med. Res., N. S., xiv, 3, 1908; ibid., xv, 3, 1909. 2 Hiss and Zinsser, ibid., xiv, 3, 1908. 3 Schattenfroh, Arch. f. Hyg., 1897; Petterson, Cent. f. Bakt., I, xxxix, 1905, and ibid., xlvi, 1908; Korschun, Ann. de l'inst. Pasteur, xxii, 1908; Zinsser, Jour. Med. Res., xxii, 3, 1910. 292 INFECTION AND IMMUNITY of varying pathogenicity between different bacterial species and between different races of the same microorganism. We know that certain bac- teria may be injected into an animal or human being in considerable quantities, without producing anything more than the temporary local disturbance following the subcutaneous administration of any innocuous material. Other bacteria, on the other hand, such as the bacillus of an- thrax or the bacillus of chicken cholera, injected in the most minute dosage, may give rise to a rapidly fatal septicemia. Within the same species, furthermore, fluctuations in virulence may take place which may depend upon a variety of influences which have been discussed in another section and need not be recapitulated. Suffice it to say that variations in the susceptibility of inoculated subjects do not, in any way, furnish a sufficient explanation for these phenomena and we are forced to seek for the key to the problem in the activities of the bacteria themselves. In an effort to cast light upon this subject, Bail, following in the footsteps of his predecessors, Kruse,1 Deutsch and Feistmantel,2 has formulated his so-called " aggressm-theory." In its reasoning, this theory is indirectly an offspring of MetchnikofFs phagocytic theory' and is, in many of its phases, antagonistic to the purely humoral con- ception of immunity. Bail 3 was first led to the formulation of his theory by extensive re- searches which he had made in conjunction with Petterson 4 into an- thrax immunity. He had noted, as others before him had, that animals, highly susceptible to anthrax, often possessed marked bactericidal powers against this bacillus. When such animals, whose serum should surely be capable of bringing about the death of, at least, a few hundred anthrax bacilli, were injected with doses far less than this number they nevertheless succumbed rapidly and the bacilli multiplied enormously in their bodies. He argued from this that the injected microorganisms must possess some weapon whereby they were enabled to counteract the protective forces of the animal organism. In an anthrax-immune animal, as a matter of fact, no proliferation of bacteria took place and the injected germs were rapidly disposed of by the protective forces, foremost of which was phagocytosis. 1 Kruse, Ziegler's Beitrage, xii, 1893. - Deutsch und Feistmantel, " Die Impfstoffe in Sera," Leipzig, 1903. 3 Bail, Cent, f. Bakt. I, xxvii, 1900, and xxxiii, 1902. 4 Bail und Petterson, Cent. f. Bakt., I, xxxiv, 1903; xxxv, 1904; xxxvi. 1904. AGGRESSINS 293 The theory of Bail 1 as eventually formulated, after extended in- vestigations which need not be outlined, contains the following basic principles:2 Pathogenic bacteria differ fundamentally from non-pathogenic bacteria in their power to overcome the protective mechanism of the animal body, and to proliferate within it. They accomplish this by virtue of definite substances given off by them, probably in the nature of a secretion, which acts primarily by protecting them against phagocy- tosis. These substances (referred to by Kruse as " Lysins ") were named by Bail, " Aggressins." The production of aggressins by pathogenic germs is probably absent in test-tube cultures, or, at any rate, is greatly depressed under such conditions, but is called forth in the animal body by the onslaught of the germicidal or other influences encountered after inoculation. These aggressins can be found, according to Bail, in the exudates occurring about the site of inoculation in rapidly fatal infections. He obtained them, separate from the bacteria themselves, by the prolonged centrifugation and subsequent decanting of edema fluid, and pleural and peritoneal exudates. Two fundamental experimental observations are brought forward by Bail in support of the truth of his contentions. In the first place, he was able to show that fatal infection could be produced in animals by the injection of sublethal doses of bacteria, when, together with the germs, there was administered a small quantity of "aggressin." He inferred from this experiment that the injected aggressin had served in paralyzing the onslaught of phagocytic and other protective agencies, and had thus made it possible for the bacteria to gain a foothold and to proliferate. The second experimental support upon which Bail's theory is founded consists in the successful immunization of animals with aggres- sin. Animals were treated with aggressive exudates, from which all bac- teria had been removed by prolonged centrifugalization and which had been rendered entirely sterile by three hours' heating to 60° C. and by the addition of five-tenths per cent of phenol. Animals so treated were not only immune themselves, but contained a substance in their serum which permitted the passive immunization of other untreated animals. Bail explained this by assuming the production of antiaggressins in the 1 Bail, Arch. f. Hyg., Hi, 1905; liii, 1905; Wien. klin. Woch., xvii, 1905. 2 Bail und Weil, Wien. klin. Woch., ix, 1906; Cent. f. Bakt., I, xl, 1906; xlii, 1906. 294 INFECTION AND IMMUNITY treated subjects. His experiments and those of his pupils were con- ducted with a large variety of microorganisms, among which were the typhoid and dysentery bacilli, the bacilli of chicken cholera and of plague, the cholera spirillum, and various micrococci. According to whether a microorganism is capable of producing an aggressin and consequently of invading the animal body, he divides bacteria into "pure parasites/' "half parasites," and "saprophytes." The theory of Bail has been extensively attacked by a number of authors, chief among whom are Wassermann and Citron,1 Wolff,2 and Sauerbeck.3 The criticism which these investigators make of Bail's views is based upon laborious experimentation and has succeeded in placing the "aggressin" theory upon a very precarious footing. It is claimed by them, in the first place, that much of the "aggressive" character of Bail's exudates is due to their containing liberated bacterial poisons (endotoxins). This they have maintained both because the sterile "aggressin" exudates could be shown to possess a considerable degree of independent toxicity and because the aggressive action of such an exudate could be duplicated by aqueous extracts of bacteria. Citron,4 furthermore, was able to show, by the Bordet-Gengou method of com- plement fixation, that the exudates of Bail contained considerable quantities of free bacterial receptors, which, in taking up bacteriolytic immune body, would neutralize any lytic power on the part of the in- fected animal. By this antilytic action, he believes, Bail's first conten- tion, the virulence-enhancing action of the exudates, can* be explained. The nature of the immunity produced in animals by Bail's method of treatment is less easily explained and less exposed to adverse criticism. Bacteriolytic immunity alone probably can not account for the high degree of resistance imparted by a few injections of the aggressins. On the other hand, the establishment of an antiaggressive immunity has not been sufficiently supported to stand as a proven fact. Final judgment must be postponed until further investigation shall have brought a better understanding of the phenomenon. 1 Wassermann and Citron, Deut. med. Woch., xxviii, 1905. 2 Wolff, Cent. f. Bakt., I, xxxviii, 1906. 3 Sauerbeck, Zeit. f. Hyg., lvi, 1907. a Citron, Cent. f. Bakt., I, xl, 1905; xli, 1906; and Zeit. f. Hyg., Hi, 1905. CHAPTER XIX ANAPHYLAXIS OR HYPERSUSCEPTIBILITY PHENOMENA OF ANAPHYLAXIS The phenomena now grouped together under the heading of anaphy- laxis and hypersusceptibility have but recently become the subject of systematic experimentation. Nevertheless, manifestations now recog- nized as belonging to this category, had not escaped the attention of a number of the earlier workers in immunity. By anaphylaxis is meant the following train of phenomena: When a foreign proteid is introduced by subcutaneous, intraperitoneal, intra- venous, or subdural injection (or in some cases by feeding) into the animal body, after a time there will appear a specific hypersuscepti- bility of the animal for this proteid. After a definite interval, a second injection of the same substance, harmless in itself, will produce violent symptoms of illness and often rapid death in an animal so prepared. The phenomena are not limited to any given class of proteids, but are manifest in the case of animal, vegetable, and bacterial proteids, and within certain limits are specific. As early as 1893, Behring1 and his pupils 2 had noticed that animals, highly immunized against diphtheria toxin, with high antitoxin content of the blood, would occasionally show marked susceptibility to injections of small doses of the toxin. The phenomena observed by them was interpreted as an increased tissue susceptibility to the toxin, and Wassermann, reasoning on the basis of Ehrlich/s side-chain theory, formulated the conception that the increased susceptibility was due to toxin receptors, increased in number by immunization, but not yet separated from the cells that had produced them; the cells thereby becoming more vulnerable to the poison. In the same category belongs the observation of Kretz, who noticed that normal guinea-pigs did not show any reaction after injections of innocuous 1 Behring, Deut. med. Woch., 1893. 2Knorr, Dissert., Marburg, 1895; Behring und Kitashina, Berl. klin. Woch., 1901. 295 296 INFECTION AND IMMUNITY toxin-antitoxin mixtures, but that marked symptoms of illness often followed such injections when made into immunized guinea-pigs. Other phenomena which are now regarded, a posteriori, as probably depending upon the principles involved in anaphylaxis, are the tuberculin and mallein reactions, fully described in another place, and the adverse effects often following the injections of antitoxins in human beings, conditions spoken of under the heading of " serum sickness." The last- named condition has been made the subject of an exhaustive study by v. Pirquet and Schick.1 That the injection of diphtheria antitoxin in human beings is often followed, after an incubation time of from three to ten days, by ex- anthematous eruptions, urticaria, swelling of the lymph glands, and often albuminuria and mild pulmonary inflammations, has been noticed by many clinicians, who have made extensive therapeutic use of anti- toxin. It was recognized early that such symptoms were entirely inde- pendent of the antitoxic nature of the serum, but depended upon other constituents or properties peculiar to the antitoxic serum. Moreover, symptoms of this description were by no means regular in patients in- jected for the first time, but seemed to depend upon an individual pre- disposition, or idiosyncrasy, v. Pirquet and Schick, however, noticed that in those injected a second time, after intervals of weeks or months, the consequent evil effects were rapid in development, severe, and occurred with greater regularity. Many of the phases of such " serum sickness" are still obscure, since experimental conditions can not be controlled, and many modifying factors can not be excluded in observa- tions made upon human beings, and the grouping of the above conditions with the phenomena of anaphylaxis is still tentative. The fundamental observations from which our present knowledge of anaphylaxis takes its origin are those made in 1898 by Hericourt and Richet,2 who observed that repeated injections of eel serum into dogs gave rise to an increased susceptibility toward this substance instead of immunizing the dogs against it. Following up the lines of thought suggested by this phenomenon, Portier and Richet3 later made an in- teresting observation while working with actino-congestin — a toxic substance which they extracted from the tentacles of Actinia. This 1 v. Pirquet and Schick, " Die Serum Krankheit," monograph, Leipzig and Wien, 1905. 2 Hericourt and Richet, Compt. rend, de la soc. de biol., 53, 1898. s Portier and Richet, Compt. rend, de la soc. de biol., 1902; Richet, Ann. de l'inst. Pasteur, 1907 and 1908. ANAPHYLAXIS OR HYPERSUSCEPTIBILITY 297 substance in doses of 0.042 grams per kilogram produced vomiting, diarrhea, collapse, and death in dogs. If doses considerably smaller than this were given in quantities sufficient to, cause only temporary illness, and several days allowed to elapse, a second injection of a quantity less than one-quarter or one-fifth of the ordinary lethal dose would cause rapid and severe symptoms and often death. Similar observations were made soon after this by Richet with mytilo-conges- tin, a toxic substance isolated from mussels. In these experiments there remained little doubt as to the fact that the first injection had given rise to a well-marked increased susceptibility of the dogs for the poison used. It was Richet who first applied to this phenomenon the term " ana- phylaxis" (avd against, * .♦^ * Fig. 69. — Staphylococcus pyogenes aureus. (After Giinther.) grouped together, unseparated after cell cleavage. In this case, adjacent cocci are slightly flattened along their contiguous surfaces. Examined in smears from cultures or pus, the staphylococci may appear as single individuals, in pairs, or, most frequently, in irregular grape-like clusters. Occasionally, short chains of three or four may be seen. In very young cultures in fluid media, the diplococcus form may predominate. The staphylococci stain with all the usual basic aqueous anilin dyes, and, less intensely, with some of the acid dyes. Stained by the method of Gram, they retain the anilin-gentian- violet. Gram's method of staining is excellently adapted for demonstration of these cocci in tissue sections. STAPHYLOCOCCUS PYOGENES AUREUS 323 Although exhibiting marked Brownian movements in the hanging drop, staphylococci are non-motile and possess no flagella. They are non-sporogenous and form no capsules. Cultural Characters. — Staphylococci grow readily upon the usual laboratory media. The simpler media, made of meat extract, are quite as efficient for their cultivation as are the freshly made meat-infusion Fig. 70. — Staphylococcus Colonies, products. The optimum temperature for staphylococcus cultivation lies at or about 30° C, though growth readily takes place at tempera- tures as low as 15° C, and as high as 40° C. Slow but definite growth has been observed at a temperature as low as 10° C. While development is most characteristic and luxuriant under aerobic conditions, staphylococci are facultatively anaerobic on suitable media. They grow readily in an atmosphere of hydrogen. 324 PATHOGENIC MICROORGANISMS As to the reaction of media, staphylococcus develops most favorably upon those having a slightly alkaline titer. Moderately increased alkalinity or even moderate acidity of media does not inhibit growth. On gelatin plates, growth occurs readily at room temperature, form- ing within thirty-six to forty-eight hours, small, shining, pin-head shaped colonies, appearing, at first, grayish-white, and later assuming a yellowish hue, which intensifies into a light brown and often a bronze color as the colony grows older. The intensity of the color differs con- siderably in different races of staphylococci. Liquefaction of the gelatin occurs, and, shallow, saucer-shaped depressions are formed, about the colonies after forty-eight hours or more. These zones of nuidification grow larger as the colonies grow, finally becoming confluent. Micro- scopically, the colonies themselves, before liquefaction has destroyed their outline, are round, rather finely granular, with smooth edges. They are not flat, but rise from the surface of the medium as the seg- ment of a sphere. In gelatin stab cultures in tubes, nuidification leads to the formation of a funnel-shaped depression, with, finally, complete liquefaction of the medium and sedimentation of the bacteria. Lique- faction of gelatin by the staphylococcus is due to a ferment-like body elaborated by it, which is spoken of as "gelatinase." This substance can be obtained apart from the cocci by the filtration of cultures.1 It is an extremely thermolabile body. On agar plates the characteristics of the growth, barring liquefaction, are much like those on gelatin. Colonies do not show a tendency toward confluence, remaining discrete, and show a rather remarkable differ- ence in the size of the colonies occurring upon the same plate. L'pon slanted agar in tubes, rapid growth occurs, at first grayish- white, but soon covering the surface of the slant as a glistening, golden-brown layer. In broth, growth is rapid, leading to a general, even clouding of the medium, and giving rise, after forty-eight or more hours, to the formation of a thin surface pellicle. As growth increases, the bacteria sink to the bottom, forming a heavy, mucoid sediment. The odor of old cultures is often peculiarly acrid, not unlike weak butyric acid. In milk, staphylococcus causes coagulation usually within three or four days, with the formation of lactic and butyric acids. On potato, growth is abundant, rather dry and usually deeply pig- mented. 1 Loeb, Cent, f. Bakt., xxxii, 1902. STAPHYLOCOCCUS PYOGENES AUREUS 325 Upon coagulated animal sera, rapid growth takes place and eventually slight liquefaction of the medium is said to occur. In nitrate solutions, reduction of the nitrates to nitrites is caused. In Dunham's broth, indol is formed. In media containing the carbohydrates — dextrose, lactose, or sac- charose— acidification takes place with the formation chiefly of lac- tic, butyric, and formic acids. There is no gas formation, however. In proteid media free from sugars, the staphylococcus produces alkali. The reducing action of staphylococcus is shown by decolorization in cultures of litmus, methylene-blue, and rosanilin.1 Pigment Formation. — Differentiation between the various members of the staphylococcus group is based largely upon the formation of pigments. These pigments, so far as we know, seem to be species characteristics. Thus, Staphylococcus pyogenes aureus is recognized primarily by its production of a yellowish-brown pigment, varying in different strains from a pale brown hue to a deep golden yellow. Pro- longed cultivation upon artificial media may lead to a diminution in the depth of color produced.2 It appears only when cultivation is carried on under freely aerobic conditions, anaerobic cultivation resulting in unpigmented colonies. The coloring matter is insoluble in wTater but soluble in alcohol, chloroform, ether, and benzol.3 According to Schnei- der,4 the pigment belongs to the class of "lipochromes" or fatty pig- ments, and is probably composed of carbon, oxygen, and hydrogen, without nitrogen. Treatment with concentrated sulphuric acid changes it to a green or greenish-blue.5 Resistance. — Although not spore formers, staphylococci are more resistant to heat than many other purely vegetative forms. The thermal death point given for Staphylococcus pyogenes aureus by Sternberg 6 lies between 56° and 58° C, the time of exposure being ten minutes. The same author states that, when in a completely dried state, the coccus is still more resistant, a temperature of from 90° to 100° C. being re- quired for its destruction. Against low temperatures, staphylococci are extremely resistant, repeated freezing often failing to sterilize cultures. 1 Fr. Miiller, Cent. f. Bakt., xxvi, 1899. 2 Fliigge, "Die Microorg.," etc. zMigula, "System d. Bakt.," Jena, 1897. 4 Schneider, Arb. a. d. bakt. Inst., Karlsruhe, 1, vol. i, 1894. 5 Fischer, "Vorles. iiber die Bakt.," Jena, 1903. 6 Sternberg, "Textbook," etc., N. Y., 1901, p. 375. 32G PATHOGENIC MICROORGANISMS Desiccation is usually well borne, staphylococci remaining alive for six to fourteen weeks when dried upon paper or cloth.1 On slant agar, staphylococci may be safely left for three or four months without trans- plantation, and remain alive.2 The resistance of staphylococci to chemicals, a question of great surgical importance, has been made the subject of extensive researches, notably by Ltibbert,3 Abbott,4 Franzott,5 and many others. According to Lubbert, inhibition of staphylococcus growth is attained by the use of boric acid 1 in 327, salicylic acid 1 in 650, corrosive sublimate 1 in 80,000, carbolic acid 1 in 800, thymol 1 in 11,000. Staphylococci are killed by corrosive sublimate 1 in 1,000 in ten minutes, by carbolic acid 1 per cent in 35 minutes, 3 per cent in 2 minutes (Franzott). Ethyl alcohol,6 even wThen absolute, is not very efficient as a disinfectant. Nascent iodin, as split off from iodoform in wounds, is extremely power- ful in destroying staphylococci. Pathogenicity. — Separate strains of Staphylococcus pyogenes aureus show wide variations in relative virulence. The most highly virulent are usually those recently isolated from human suppurative lesions, but no definite rule can be formulated in this respect. The virulence of a given strain, furthermore, may be occasionally enhanced by re- peated passages through the body of a susceptible animal. Prolonged cultivation upon artificial media is liable to decrease the virulence of any given strain, though this is not regularly the case. There are, more- over, unquestionably, many staphylococci constantly present in the air, dust, and water, which although morphologically and culturally not unlike the pathogenically important species, may be regarded as harmless saprophytes. The susceptibility of animals to staphylococcus infection is, likewise, subject to extreme variations, depending both upon differ- ences between species and upon fortuitous individual differences in susceptibility among animals within the same species. Animals on the whole are less susceptible to staphylococcus than is man. Among the ordinary laboratory animals, rabbits are most sus- ceptible to this microorganism. Mice, and especially the white 1 Deslongchamps, Paris, 1897. 2Passet, Fort. d. Med., 2 and 3, 1885. 3 Ltibbert, "Biol. Untersuch.," Wurzburg, 1886. 4 Abbott, Medical News, Phila., 1886. 5 Franzott, Zeit. f. Hyg., 1893. 6 Hand, Beit. z. klin. Chir., xxvi. STAPHYLOCOCCUS PYOGENES AUREUS 327 Japanese mice, show considerable susceptibility. Guinea-pigs possess a relatively higher resistance.1 Subcutaneous or intramuscular inoculation of a susceptible animal usually results in the formation of a localized abscess with much pus formation and eventual recovery. Intraperitoneal inoculation is more often fatal. Intravenous inoculation of doses of 0.5 c.c, or more; of fresh broth cultures of virulent staphylococci usually leads to pyemia with the production of secondary abscesses, located chiefly in the kid- neys and the heart and voluntary muscles, but not infrequently in other organs as well. In the kidney they occur as small foci, situated most often in the cortex, composed of a central, necrotic pus cavity, surrounded by a zone of acute inflammatory exudation. Staphylo- coccus lesions form histologically the typical "acute abscess." Not infrequently the pyemic condition is accompanied by suppurative lesions in the joints. Intravenous injections of virulent staphylococci preceded by injury to a bone is often followed by the development of osteomyelitis. Mechanical or chemical injury of the heart valves preceding intravascular staphylococcus inoculation may result in localization of the infection on or about the heart valves, leading to " malignant endocarditis." The pyemic conditions following staphy- lococcus inoculation usually lead to chronic emaciation and death after an interval dependent upon the relative virulence of the micro- organism, the amount injected, and the resistance of the infected subject. Large doses of unusually virulent cultures cause death within twenty -four hours, or even less, without abscess formation. As above stated, the susceptibility of man to spontaneous staphy- lococcus infection is decidedly more marked than is that of animals. The form of infection most frequently observed is the common boil or furuncle. As Garre,2 Budinger,3 Schimmelbusch,4 and others have demonstrated by experiments upon their own bodies, energetic rubbing of the skin with virulent staphylococcus cultures may often be followed by the development of a furuncle. Subcutaneous inoculation of the human subject invariably gives rise to an abscess. The pathological lesions which may be produced in man by virulent staphylococci are naturally of great variety, depending upon the mode of inoculation, and 1 Terin, Ref. in Lubarsch und Ostertag, Ergebnisse, 1896; Lingelsheim, "Aetiol. d. Staph. Inf.," etc., Wien, 1900. 2 Garre, Beit. z. klin. Chir., x, 1893. 3 Budinger, Lubarsch und Ostertag, Ergebnisse, etc., 1896. 4 Schimmelbusch, Ref. by Budinger. 328 PATHOGENIC MICROORGANISMS upon the relation between the virulence of the incitant and the resist- ance of the subject. Apart from the formation of localized abscesses, staphylococci are common as the incitants of surgical suppurations and wound infections. The large majority of acute suppurative in- flammations of bone (osteomyelitis) are caused by staphylococci. Ab- scesses of the brain, of the liver, and of the lung may be due to this microorganism. It may give rise to ascending infections of the genito- urinary tract, leading to pyelonephritis. Empyema or peritonitis may be caused by its entrance into the serous cavities from the lung or bowel. When gaining access to the circulation from some localized focus, it gives rise to septicemia and may lead to malignant endocarditis and, by secondary localization in the viscera, to general pyemia. As the incitant of septicemia it can frequently be found by blood culture during the life of the patient. Puerperal sepsis is not infrequently a staphylococcus disease. Of recent years several authors have claimed direct etiological relationship for the Staphylococcus pyogenes aureus with acute articular rheumatism.1 While not unlikely, this claim is not, at present, substantiated by sufficiently exact evidence. Apart from the local inflammatory reactions called forth by staphylococcus invasion, all such infections, if severe or prolonged, give rise to profound toxic manifestations evidenced by characteris- tically irregular temperature (the so-called " septic type"), by head- ache, nausea, and general malaise, and not infrequently by chills. Prolonged chronic infection with staphylococci may give rise to the so-called amyloid changes in liver, spleen, and kidneys. Toxic Products. — Endotoxins. — The dead bodies of staphylococci injected into animals may occasionally give rise to abscess formation, and,2 if in sufficient quantity, may cause death. To obtain the latter result, however, large quantities are necessary, the endotoxic substances within the dead cell body of these microorganisms being probably neither very poisonous nor abundant.3 That dead cultures of Staphylococcus aureus exert a strong positive chemotaxis for leucocytes was shown beyond question by the experi- ments of Borissow.4 Hemolysins. — In 1900 Kraus5 noticed the hemolytic action of 1 A. H. Weis, Inaug.-Diss., Berlin, 1901. 2 Schattenfroh, Arch. f. Hyg., xxxi, 1887. 3 v. Lingelsheim, " Aetiol. u. Therap. d. Staph. Krank.," Wien, 1900. 4 Borissow Zieglers Beitr., xvi, 1894. 5 Kraus, Wien. klin. Woch., iii, 1900. STAPHYLOCOCCUS PYOGENES AUREUS 329 staphylococci growing upon blood- agar plate cultures. Neisser and Wechsberg x then showed that this hemolytic substance, secreted by the staphylococcus, could be demonstrated in filtrates of bouillon cultures. Such hemolysins are produced by Staphylococcus aureus, and, to a lesser degree, by Staphylococcus albus. The quantity produced varies enormously with different strains and seems to be roughly proportionate to the virulence of the particular microorganism, though exceptions to this rule are not uncommon. Absolutely avirulent races do not, so far as we know, produce hemolysins. The culture medium most favor- able to the formation of these substances is, according to Neisser and Wechsberg, a moderately alkaline beef bouillon. Cultivated at 37.5° C, the bouillon contains the maximum amount of hemolytic substance be- tween the eighth and fourteenth day, and this may be separated from the bacteria by filtration through Berkefeld or Chamberland filters. The hemolytic action may be observed by the general technique for determining hemolysis (given on page 259) . It is important to wash the red blood corpuscles used for the experiments, since many animals normally possess small quantities of antihemolysin in their blood-sera (man and horse especially) .2 The red blood corpuscles of rabbits, dogs, and guinea-pigs are extremely susceptible to the action of the staphylo- hemolysin. Those of man are less easily injured by it. The hemolytic action takes place, as Todd3 and others4 have shown, not only in vitro, but in the living animal as well. The staphylo-hemolysin is comparatively thermolabile. According to Neisser and Wechsberg, heating it to 56° C. for twenty minutes de- stroys it. According to some other authors, however, higher tempera- tures (60° to 80° C.) are required. Reactivation of a destroyed staphylo- hemolysin has so far been unsuccessful. The fact that antistaphylolysin is occasionally present in normal sera has been mentioned above. This antibody is most abundant in the blood of horses and of man. Arti- ficially antistaphylolysin formation is easily induced by subcutaneous inoculation of staphylolysin into rabbits. Leucocidin. — In 1894, Van de Velde5 discovered that the pleural exudate of rabbits following the injection of virulent staphylococci showed marked evidences of leucocyte destruction. He was subse- 1 Neisser und Wechsberg, Zeit. f. Hyg., xxxvi, 1901. 2 Neisser, Deut. med. Woch., 1900. s Todd, Trans. London Path. Soc, 1902. ±Kraus, Wien. klin. Woch., 1902. s Van de Velde, La Cellule, x, 1894. 330 PATHOGENIC MICROORGANISMS quently able to show that the substance causing the death and partial solution of the leucocytes was a soluble toxin formed by the staphylo- coccus, not only in vivo, but in vitro as well; for cultures of Staphylo- coccus pyogenes aureus, grown in mixtures of bouillon and blood serum, contained, within forty-eight hours, marked quantities of this "leucocidin." Other workers since Van de Velde have evolved various methods for obtaining potent leucocidin. Bail1 obtained it by growing virulent staphylococcus in mixtures of one-per-cent glycerin solutions and rab- bit serum. Neisser and Wechsberg2 advise the use of a carefully titrated alkaline bouillon. To obtain the leucocidin free from bacteria, the cultures are passed through Chamberlancl or Berkefeld filters, after about eight to eleven days' growth at 37° C, at which time the con- tents in leucocidin are usually at their highest point. The action of leucocidin upon leucocytes may be observed in. vivo by the simple method of Van de Velde, of injecting virulent staphylo- cocci intrapleurally into rabbits and examining the exudate. Bail advises the production of leucocytic intrapleural exudates by the use of aleuronat and following this after twenty-four hours by an injection of leucocidin-filtrate. In vitro the phenomenon may be observed by direct examination of mixtures of leucocytes and leucocidin in the hanging drop on a warmed stage, or by the " methylene-blue method" of Neisser and Wechsberg. This method is based upon the fact that living leucocytes will reduce methylene-blue solutions and render them colorless, while dead leucocytes have lost this power. Leucocidin and leucocytes are allowed to remain in contact for a given time and to them is then added an extremely dilute solution of methylene-blue. If the leucocytes have been actively attacked by leucocidin, no reduction takes place. This method is -particularly adapted for quantitative tests. All staphylococcus strains do not produce leucocidin to the same degree. Almost all true Staphylococcus pyogenes aureus cultures produce some of this toxin, but one strain may produce fifty- and a hundred-fold the quantity produced by another. Staphylococcus pyogenes albus gives rise to this substance but rarely, and then in small quantity. Leucocidin seems to be similar to the soluble toxins of other.bacteria. It is rapidly destroyed by heat at 58° C, and deteriorates quickly in *Bail, Arch. f. Hyg., xxxii, 1898. 2 Neisser und Wechsberg, Zeit. f. Hyg., xxxvi, 1901. STAPHYLOCOCCUS PYOGENES AUREUS 331 culture fluids at incubator temperatures. It is distinct from staphylo- hemolysin as shown by differences in thermostability. Soon after Van de Velde's discovery of leucocidin, Denys and Van de Velde1 produced an antileucocidin by treating rabbits with pleural exudate containing leucocidin. Neisser and Wechsberg 2 later confirmed these results and showed that among staphylococci, leucocidin is not specific, the toxin of all strains of Staphylococcus aureus and albus examined being neutralizable by the same antileucocidin. Antileuco- cidin is often found in the normal sera of horses and man.3 Leucocidin should not be confounded with " leucotoxin," a substance obtained in serum by treatment of animals with leucocytes, a true "cytotoxin," having no connection whatever with the staphylococcus. Staphylococci, besides the toxic substances already mentioned, give rise to gelatinase, spoken of in the section upon cultivation, and to a proteolytic ferment by means of which albuminous media (Loeffler's serum) may be slightly digested. Immunization. — Animals can be rendered actively immune by re- peated inoculations with carefully graded doses of living or dead staphylococcus cultures.4 The production of antistaphylolysin and of antileucocidin in the sera of animals so treated, has been alluded to in the preceding sections. The sera of such actively immunized animals possess distinct protective power when administered to other animals, slightly before or at the same time with an inoculation of staphylo- cocci. They do not, however, exhibit very high bactericidal value in vitro. The use of immune sera to combat staphylococcus infection has not so far given very encouraging results.5 Agglutinins have been demonstrated in staphylococcus immune sera by a number of authors, and have been shown to be of value in differ- entiating between the several groups of staphylococci.6 A rather sur- prising result of these researches has been the recognition that immune sera obtained with pathogenic staphylococci will agglutinate other pathogenic staphylococci, whether belonging to the group of Staphy- lococcus pyogenes aureus or that of Staphylococcus pyogenes albus, 1 Denys et Van de Velde, La Cellule, xi, 1895. 2 Loc. cit. 3 Van de Velde, Presse medicale, i, 1900. 4 Richet et Hericourt, Compt. rend, de Facad. des sci., cvii, 1888. &Kolle und Otto, Zeit. f. Hyg., xli, 1902. 6 Proscher, Cent. f. Bakt., xxxiv, 1903; v. Lingelsheim, " Aetiol. u. The rap. d Staphyl.," etc., Wien, 1900. 332 PATHOGENIC MICROORGANISMS but will not agglutinate any of the non-pathogenic members of either group.1 Active immunization of human beings suffering from staphylococcus infections has been extensively practiced by Wright, in connection with his work on opsonins. There can be no question about the fact that the opsonic substances in the blood are increased by the injection of dead staphylococci. The procedure is of therapeutic value in subacute and chronic cases. The work of Hiss on the use of leucocyte extracts in animals infected with Staphylococcus pyogenes aureus has given en- couragement for such treatment in human beings. A number of staphylococcus infections in man have been successfully treated with leucocyte extract by Hiss and Zinsser. STAPHYLOCOCCUS PYOGENES ALBUS This coccus differs from Staphylococcus pyogenes aureus simply in the absence of the golden yellow coloration of its cultures. Morpho- logically, culturally, and pathogenically, it is in every way identical with the staphylococcus described in the preceding section, but its toxin- and enzyme-producing powers in general are less developed than those of the aureus variety. Its close biological relationship to aureus is furthermore demonstrated by its agglutination in Staphylococcus pyogenes aureus immune sera. STAPHYLOCOCCUS EPIDERMIDIS ALBUS The Staphylococcus epidermidis albus described by Welch is merely one of the non-pathogenic varieties of Staphylococcus pyogenes albus and possibly does not deserve separate classification. It may give rise to unimportant stitch abscesses. STAPHYLOCOCCUS PYOGENES CITREUS Staphylococcus pyogenes citreus produces a bright yellow or lemon- colored pigment of distinctly different hue from that of Staphylococcus pyogenes aureus. It may be pyogenic and in every way similar to Staphylococcus pyogenes aureus, but is less often found in con- tion with pathological lesions than either of the preceding staphy- lococci. 1 Proscher, Deut. med. Woch., xi, 1903. STAPHYLOCOCCUS, PYOGENES CITREUS 333 A large number of staphylococci, differing from those described above in one or another detail, have been observed. They are of com- mon occurrence and are met with chiefly as contaminations in the course of bacteriological work. Few of these have any pathological significance and none of them are toxin-producers, so far as we know. Many of them differ, furthermore, in their inability to liquefy gelatin. All of them belong more strictly to the field of botany than to that of patho- logical bacteriology. Atypical pathogenic staphylococci have been described by a number of observers. Thus Weichselbaum * isolated a staphylococcus from a case of malignant endocarditis which could not be cultivated at room temperature, and grew only in very delicate colonies. Veillon,2 moreover, has described a strictly anaerobic staphylococcus cultivated from the pus of an intra-abdominal abscess. MICROCOCCUS TETRAGENUS In 1881, Gaffky 3 discovered a micrococcus which occurs regularly in groups of four or tetrads. He first isolated it from the pus discharged by tuberculous patients with pulmonary lesions. Observed in smear preparations from pus, the tetrads are slightly larger in size than the ordinary staphylococcus, flattened along their adjacent surfaces, and surrounded by a thick halo-like capsule. Preparations from cultures often lack these capsules. The micrococcus is easily stained by the usual basic anilin dyes. Stained by Gram's method, it is not decolor- ized, retaining the gentian-violet. Cultivation. — Micrococcus tetragenus grows on the ordinary labora- tory media, showing a rather more delicate growth than do the staphy- lococci. On agar, the colonies are at first transparent, later they become grayish-white, but are always more transparent than are staphylococcus cultures. On gelatin, growth is rather slow and no liquefaction takes place. Broth is evenly clouded. On potato there is a white, moist growth which shows a tendency to confluence. 1 Weichselbaum, Baumgarten Jahresb., 1899, Ref. 2 Veillon, Compt. rend. soc. de biol., 1893. 3 Gaffky, Mitteil. a. d. kais. Gesundheitsamt, i, 1881. 334 PATHOGENIC MICROORGANISMS Milk is coagulated and litmus milk indicates acid formation. Pathogenicity. — Micrococcus tetragenus is especially pathogenic for Japanese mice, which succumb within three or four days to subcuta- neous inoculation.1 Gray mice, rats, guinea-pigs, and rabbits are less susceptible, showing only a localized reaction at the point of inoculation. Fig. 71. — Micrococcus Tetragenus. (In spleen of infected mouse.) The organism has occasionally been isolated from spontaneous abscesses observed in domestic animals. In man, this microorganism is usually found without any particular pathogenic significance, in sputum or saliva. In isolated cases, how- ever, it has been described as the sole incitant of abscesses. 1 M Oiler, Wien. klin. Woch., 17, 1904. MICROCOCCUS TETRAGENUS 335 Bezancon * has isolated Micrococcus tetragenus from a case of menin- gitis. A single case of tetragenus septicemia is on record, reported in 1905 by Forneaca. 2 In America, this microorganism has not been frequently observed in connection with disease. It is often found, however, in considerable numbers, in smears of sputum which are being examined for pneumo- cocci or tubercle bacilli. 1 Bezangon, Semaine med., 1898. 2 Forneaca, Rif. med., 1903. CHAPTER XXII THE STREPTOCOCCI Among the pyogenic cocci, there is a large and important group of organisms which multiply by division in one plane of space only, and thus give rise to appearances not unlike chains or strings of beads. The term streptococcus or chain-coccus is, therefore, a purely morpho- logical one which includes within its limits microorganisms which may differ from each other considerably, both as to cultural and pathogenic properties. Thus, cocci which form chains may be isolated from water, milk, dust, and the feces of animals and man. These may have little but their morphological appearance in common with the pyogenic streptococci which are so important as the incitants of disease. The interrelationship between streptococci from different sources, how- ever, is by no means fully understood, and we are forced at present to content ourselves with the recognition of a large morphological group, in no individual case taking the pathogenic or more special cultural characteristics for granted. STREPTOCOCCUS PYOGENES Of paramount importance among the streptococci are those which possess the power of giving rise to disease processes in animals and in man, and which, because of their frequent association with suppura- tive inflammations, are roughly grouped under the heading of Strep- tococcus pyogenes. The same researches upon surgical infections which led to the dis- covery of the staphylococci, laid the basis for our knowledge of the streptococci. The fundamental studies of Pasteur and Koch1 were fol- lowed, in 1881, by the work of Ogston,2 who was the first to differentiate between the irregularly grouped staphylococci and the chain-cocci. 1 Koch, " Untersuch. iiber Wundinfektion," etc., 1878. 2 Ogston, Brit. Med. Jour., 1881. 336 STREPTOCOCCUS PYOGENES 337 Pure cultures of streptococci were first obtained by Fehleisen1 in 1883 and by Rosenbach2 in 1884. The thorough and systematic researches of the last-named authors, together with those of Passet,3 were of special i ' • Ai #* • # — \ * ' tsmmmttf . *® * * j^ * * f / 0 /• * f* x ** ' 0 ' - 1 M j> ■■ i M i a Fig. 72. — Streptococcus pyogenes. influence in placing our knowledge of the pathogenic properties of streptococci upon a scientific basis. Morphology and Staining.— The individual streptococcus is a spherical microorganism measuring from 0.5 micron to 1 micron in diameter. Since the line of cleavage of cocci, when in chains, is perpendicular to the 1 Fehleisen, " Aetiol. d. Erysipelas," Berlin, 1883. 2 Rosenbach, " Mikroorg. bei Wundinfektion," etc., Wiesbaden, 1884. 3 Passet, " Untersuch. iiber die eitrigen Phlegm.," etc., Berlin, 1885. 22 338 PATHOGENIC MICROORGANISMS long axis of the chain, adjacent cocci often show slight flattening of the contiguous surfaces, forming, as it were, a series of diplococci arranged end to end. As a general rule the streptococci pathogenic for man, when grown upon favorable media, have a tendency to form chains made up of at least eight or more individuals, while the more saprophy- tic, less pathogenic varieties are apt to be united in shorter groups. Upon this basis a rough morphological distinction has been made by v. Lingelsheim,1 who first employed the terms Streptococcus "longus" and "brevis." A differentiation of this kind can hardly be re- lied upon, however, since the length of chains is to some degree de- pendent upon cultural and other environmental conditions. Species which exhibit long and tortuous chains, when grown upon suitably alkaline bouillon, or ascitic broth, may appear in short groups of three or four, or even in the diplo form, when cultivated upon solid media or unfavorable fluid media. Stained specimens often show swelling and enlargement of individual cocci, giving the chains an irregularly beaded appearance. These swollen individuals are probably to be interpreted as involution forms and are seen with especial frequency in old cultures. Streptococci do not form spores, are non-motile, and do not possess flagella. There can be no doubt that certain species of true streptococci may possess capsules, though these are not so regularly demonstrable and are more delicately dependent upon cultural conditions than are the capsules of the pneumococci.2 The capsulated streptococci will be dis- cussed more comprehensively in the section upon the differentiation of pneumococcus from streptococcus (page 367). Streptococci are easily stained by the usual anilin dyes. Stained by the method of Gram, the pyogenic streptococci are not decolorized and invariably retain the gentian- violet. Certain species found in stools and described as Gram-negative, are rare and are non-pathogenic. Others of the " Streptococcus brevis" variety, and purely saprophytic, may stain irregularly by the Gram method. Cultivation. — The pyogenic streptococci are easily cultivated upon all the richer artificial media. While meat extract-pepton media may suffice for certain strains, it is usually better to employ those media which have the beef or veal infusion for a basis. For the cultivation of more delicate strains of streptococci, especially when 1v. Lingelsheim, " Aetiol. u. Therap. d. Streptok. Infek." Beit. z. Exp. Ther., Hft. 1, 1899. tPasquale, Zieglers Beit., xii; Bordet, Ann. de l'inst. Pasteur, 1887; Schottmiiller , Munch, med. Woch., xx; 1903; Hiss, Jour. Exp. Med., vi, 1905. STREPTOCOCCUS PYOGENES 339 taken directly from the animal or human body, it is well to add to the media animal albumin in the form of whole blood, blood serum, or asci- tic or pleural transudates. Glucose, added in proportions of one to two per cent, likewise renders media more favorable for streptococcus culti- vation. Prolonged cultivation of all races upon artificial media renders them less fastidious as to cultural requirements. The most favorable reaction of media for streptococcus cultivation is moderate alkalinity (two-tenths to five-tenths per cent alkalinity to phenolphthalein) . Growth may be readily obtained, however, in neutral media or even in those slightly acid. The optimum temperature for growth is at or about 37.5° C. Above 43° to 45° C., development ceases. At from 15° to 20° C., growth, while not energetic, still takes place, an important point in the differentiation of these microorganisms from pneumococci. While the free access of oxygen furnishes the most suitable environment for most, races of streptococci, complete anaerobiosis does not pre- vent development in favorable media. Strictly anaerobic streptococci have been cultivated from the human intestinal tract by Perrone1 and others. In alkaline bouillon at 37.5° C, pyogenic streptococci grow rapidly, form long and tortuous chains, and have a tendency to form flakes which rapidly sink to the bottom. Diffuse clouding occurs rarely and is a characteristic rather of the shorter so-called Streptococcus brevis. When sugar has been added to the broth the rapid formation of lactic acid soon interferes with extensive development. This may be obviated, especially when mass cultures are desired, without sacrifice of the growth- increasing influence of the glucose, by adding to the sugar-broth one per cent of sterile powdered CaC03.2 In milk, Streptococcus pyogenes grows readily with the formation of acid, followed, in most cases, by coagulation of the medium. On agar-plates at 37.5° C., growth appears within eighteen to twenty- four hours. The colonies are small, grayish, and delicately opalescent. They are round with smooth or very slightly corrugated or lace-like edges, and rise from the surface of the medium in regular arcs, like small droplets of fluid. Microscopically they appear finely granular and occasionally, under high magnification, may be seen to be composed of long intertwining loops of streptococcus chains, which form the lace-like edges. When ascitic fluid or blood serum has been added to agar, growth is more energetic and the colonies correspondingly more rapid in 1 Perrone, Ann. de l'inst. Pasteur, xix, 1905. 2 Hiss, Jour. Exp. Med., vi, 1905. 340 PATHOGENIC MICROORGANISMS appearance and luxuriant in development. In glucose-ascitic-agar, acid formation from the sugar causes coagulation of albumin with the consequent formation of flaky white precipitates throughout the medium.1 In gelatin stab-cultures growth takes place slowly, appearing after twenty-four to thirty-six hours as a very thin white line, or as discon- nected little spheres along the line of the stab. The colonies on gelatin plates are similar in form to those on agar, but are usually more opaque and more distinctly white. The gelatin is not liquefied by the pyogenic streptococci, though certain of the more saprophytic forms may occa- sionally bring about slow fluidification. On Loeffler's coagulated blood serum, growth isradip and luxu- Fig. 73. — Streptococcus Colonies, on Serum Agar riant, and may show a slight tendency to confluence if the medium is very moist. Good chain formation takes place on this medium. Upon potatoes, growth is said not to take place.2 On media containing red blood cells, most pathogenic streptococci cause hemolysis and decolorization (see Fig. 74, p. 346). It is useful to remember this when examining blood-culture plates, for here the yellow transparent halo of hemolysis and decolorization surrounding the colonies may aid in differentiating these bacteria from pneumococci. This is of especial importance since many streptococci, when cultivated directly out of the human blood, do not exhibit chain formation, but appear as diplococci. 1 Libman, Medical Record, lvii, 1900. 2 Frosch und Kolle, in Fliigge, "Die Mikroorganismen," 1891. STREPTOCOCCUS PYOGENES 341 In the inulin-serum media of Hiss,1 streptococci do not produce acid and coagulation. The so-called Streptococcus mucosus, a capsule- bearing, inulin-fermenting microorganism, is very probably a sub-species of the pneumococcus (see later section) . Resistance. — Streptococci on the ordinary culture media, without transplantation and kept at room temperature, usually die out within ten days to two weeks. They may be kept alive for much longer periods by the use of the calcium-carbonate-glucose bouillon, if the cultures are thoroughly shaken and the powdered marble thoroughly mixed with the bouillon from time to time.2 Preservation at low temperatures (1° to 2°C), in the ice chest, considerably prolongs the life of cul- tures. Virulence is preserved longest by frequent transplantation upon albuminous media. In sputum or animal excreta, streptococci may remain alive for several weeks. Streptococci are killed by exposure to a temperature of 54° C. for ten minutes.3 Low temperatures, and even freezing, do not destroy some races. The action of various chemical disinfectants has been thoroughly investigated by v. Lingelsheim,4 who reports among others the following results: Carbolic acid 1 : 200 kills streptococci in fifteen minutes. In the same time, bichlorid of mercury is efficient in a dilution of 1 : 1,500, lysol in a dilution of 1 : 200, peroxid of hydrogen 1 : 35, sulphuric acid 1 : 150, and hydrochloric acid 1 : 150. Inhibition is exerted by car- bolic acid 1 : 550, and by bichlorid of mercury 1 : 65,000. Exposure to direct sunlight kills streptococci in a few hours. Virulence and Pathogenicity. — Different races of pyogenic strepto- cocci show considerable variations in virulence and there are few organ- isms, pathogenic both for animals and man, which show such peculiari- ties in virulence. The character or severity of the lesion in man gives little evidence as to the virulence of the organism for animals. Such differences are, to a certain extent, dependent upon inherent individual characteristics, but are rather more likely to be the consequences of pre- vious environment or habitat. Prolonged cultivation upon artificial media usually results in the reduction of the virulence of a streptococcus, while an originally low or reduced virulence may often be much en- ^Hiss, Jour. Exp. Med., vi, 1905. 2 Hiss, loc. cit. 3 Sternberg, "Textbook of Bact.," 2d ed., 1901; Hartmann, Arch. f. Hyg., vii. 4 v. Lingelsheim, "Aetiol. u. Therap. d. Streptoc. Inf.," etc., Beit. z. Exper. Therap., Hft. 1, 1899. 342 PATHOGENIC MICROORGANISMS hanced by repeated passage of the streptococci through animals. It is noteworthy, however, that while the passage of a streptococcus through rabbits will usually enhance its virulence for susceptible animals in general, repeated passages through mice may increase the virulence for these animals only, even occasionally depressing the virulence for rabbits.1 Among the domestic animals, those most susceptible to experimental streptococcus infection are white mice and rabbits. Guinea-pigs and rats are less easily infected, and the larger domestic animals, cattle, horses, goats, cats, and dogs, are extremely refractory. Almost complete immunity towTard streptococcus infections prevails among birds. The nature of the lesions following animal inoculation depends upon the manner of inoculation, the size of the dose given, and most of all, upon the grade of virulence of the inoculated germ. Subcutaneous inoculations, according to the virulence of the inoculated material, may result in a simple localized abscess, differing from a staphylococcus abscess only in the more serous nature of the exudate and the frequent occurrence of edema, or in a severe general septicemia with a hardly noticeable local lesion. Subcutaneous inoculation of mice results almost invariably in general sepsis followed by death within thirty-six to forty- eight hours, or less, and the presence of streptococci in the heart's blood and the viscera. Intrapleural or intraperitoneal inoculation of suscep- tible animals with virulent streptococci leads usually to a peculiarly hemorrhagic form of exudate, due both to the diapedesis caused by the violent inflammatory process, and to the hemolysis of the red cells by the streptococcic hemolysins. Inoculation of rabbits at the base of the ear with virulent streptococci may result in the formation of a lesion indistinguishable histologically from erysipelas in man.2 Marbaix 3 has shown that such erysipeloid lesions could be produced in rabbits by streptococci from various and indifferent sources, provided that the virulence of each strain could be sufficiently enhanced. This marked variability of the resulting lesion as determined by the degree of virulence of the incitant, whatever its original source, forms a strong argument in favor of the opinion that all the pyogenic streptococci are members of a single species. # Intravenous inoculation of rabbits with virulent cultures usually results in a rapidly fatal septicemia. An animal which has died of a 1 Knorr, Zeit. f. Hyg., xiii. 2 Fehleisen, loc. cit.; Frankel, Cent. f. Bakt., vi. 3 Marbaix, La Cellule, 1892. STREPTOCOCCUS PYOGENES 343 streptococcus infection usually shows serosanguineous edema about the point of inoculation, multiple hemorrhagic spots upon the serous mem- branes, and congestion of the viscera. The microorganisms can almost invariably be found in the heart's blood, in the spleen, and in the exudate about the inoculated area. Microscopically, when the process has lasted sufficiently long, parenchymatous degeneration of all the organs may be observed. In the more chronic infections articular and periarticular lesions may occur.1 Spontaneous streptococcus disease seems to occur among some of the larger domestic animals. Thus, a contagious form of inflammation of the respiratory passages of horses has been attributed to streptococcus infection.2 Among cattle these microorganisms have been found to roduce purulent inflammation of the udder and occasionally post- partum uterine inflammation in cows. Among the smaller labora- tory animals, occasional streptococcus infections may be observed in rabbits. Recently an epidemic disease among white mice due to strep- tococcus was studied by Kutscher.3 As a rule, however,, streptococcus disease is by far more rare among animals than it is among human beings. In man, a large variety of pathological processes may be caused by streptococci and here again the nature of the infection, whether definitely localized or generally distributed, depends upon the relationship existing between the virulence of the incitant and the resistance of the subject. The first cultivation of streptococcus from human lesions was made by Fehleisen,4 who obtained them from cases of erysipelas. It was long believed that the so-called Streptococcus erysipelatis was a similar but essentially different species from the common Streptococcus pyogenes. The production of erysipelas in animals with streptococci from other sources, however, has shown definitely that the two groups can not be separated.5 Superficial cutaneous infections are frequently caused by streptococci and these in the milder cases may be similar to the localized abscesses caused by staphylococci. In severe cases, however, infection is followed by rapidly spreading edema, lymph- angitis, and severe systemic manifestations with the development of a grave cellulitis, often threatening life and requiring energetic surgical interference. Invasion of the respiratory organs by streptococci is not 1 Schiitz, Zeit. f. Hyg., iii; Hiss, Jour. Med. Res., xix, 1908. 2 Van de Velde, Monatsheft f. Bakt., Thierheilk., ii. 3 Kutscher, Cent. f. Bakt., xlvi. 4 Fehleisen, loc. cit. 5Marbaix, La Cellule, 1892; Petruschky, Zeit. f. Hyg., xxiii. 344 PATHOGENIC MICROORGANISMS rare, and may lead to bronchitis, pneumonia, or empyema. They are frequently present also as secondary invaders in pulmonary tubercu- losis.1 Streptococcus infections of the lungs and pleura not infrequently lead to pericardial involvement. Suppurations of bone may be caused by streptococci, and constitute a severe form of osteomyelitis. Such lesions when occurring in the mastoid bone are not infrequently secondary to streptococcus otitis and may lead to a form of meningitis which is in most cases fatal. In the mouth and throat streptococci may give rise to pharyngitis and are one of the most frequent causes of a form of tonsillitis often clinically indis- tinguishable from diphtheria. The throat inflammation accompanying scarlatina is, almost without exception, referable to streptococcus infec- tion.2 The occasional presence of the streptococcus in the blood of •scarlatina patients, moreover, has led some authors to suggest a pos- sible etiological connection between this microorganism and the disease.3 This, however, is at present merely conjectural. In diphtheric inflammations of the throat? a secondary streptococcus infection is a frequent and serious complication. As incitants of disease of the intestines, streptococci have been found in appendicular abscesses 4 and have been described as the cause of some forms of infantile diarrhea.5 From any of the local processes streptococci may pass into the circulation, causing sepsis. The septicemia occurring during the puerperium is most often caused by this microorganism. Secondary foci in the viscera may be established, leading to pyemia,6 or, if these localizations occur upon the heart valves, septic endocarditis may ensue. All such forms of general streptococcus infection, whether running acute or chronic courses, present a high rate of mortality The diagnosis in these cases is usually easy if blood cultures are taken upon suitable media. Toxic Products. — In spite of extensive researches by many inves- tigators upon the nature of the poisons produced by streptococci, our understanding of these substances is still very incomplete. The grave systemic symptoms so often accompanying comparatively 1 Cornet, " Die Tuberkulose," Wien, 1899. 2 Baginsky, Deut. med. Zeit., 1900. 3 Baginsky und Sommerfeldt, Berl. klin. Woch., xxvii, 1900. 4 Kelly, " Pathogenesis of Appendicitis." 5 Lanz and Tavel, Rev. de Chir., 1904; Perrone, Ann. de Tinst. Pasteur, 1905; Escherich, Jahrb. f. Kinderheilkunde, 1899. 6 Libman, Cent. f. Bakt., xxii. STREPTOCOCCUS PYOGENES 345 slight streptococcus lesions argue strongly for the production by these microorganisms of a powerful diffusible poison. Toxic filtrates of streptococcus cultures have indeed been obtained by Roger/ Marmier,2 Baginsky and Sommerfeld,3 Marmorek,4 and many others; but these have in no case been comparable in potency to the soluble toxins of diphtheria or of tetanus. When injected into young guinea-pigs in sufficient quantity, these filtrates produce rapid collapse and death. The inability to produce strong toxins is generally attributed to the difficulty of obtaining very abundant growth of these bacteria upon fluid media, development being self-limiting, either because of the ex- haustion of specific nutrit ve material (Marmorek5), or, more probably, because of the inhibitory effects of the products of growth, chiefly acid formation. This last factor can be partially overcome by the use of the glucose-calcium-carbonate broth mentioned above, in which acid neutral- ization is constantly taking place. For toxin production, Baginsky and Sommerfeld6 advise a strongly alkaline reaction of the media; Mar- morek 7 has used human blood-serum-bouillon with success. The toxins so produced are relatively thermostable. According to v. Lingelsheim, heating to 60° or 70° C. destroys them in part only. The endotoxins contained within the cell-bodies of streptococci them- selves have been found to possess but slight toxic qualities. Apart from these substances, some streptococci produce a hemolysin which has the power of bringing about destruction of red blood cor- puscles. The observation of this phenomenon for streptococci was first made by Marmorek 8 in 1895. According to this author, there is a direct relationship between virulence and hemolytic power. Other investigators, however, notably Schottmuller,9 believe the hemolytic power to be a constant characteristic of certain strains unchangeable by experimental enhancement or reduction of the virulence. Streptococcus hemolysins may be conveniently observed by cultivation of the organ- isms upon blood-agar plates. They may be produced in alkaline pepton- broth and obtained separate from the bacteria by filtration — a procedure, 1 Roger, Rev. de med., 1892. 2 Marmier, Ann. de l'inst. Pasteur, ix, 1895, p. 533. 3 Baginsky und Sommerfeld, Berl. klin. Woch., 1900. 4 Marmorek, Berl. klin. Woch., 1902. 5 Marmorek, Berl. klin. Woch., xiv, 1902. 6 Loc. cit. 7 Marmorek, Ann. de l'inst. Pasteur, 1895. 8 Marmorek, Ann. de l'inst. Pasteur, 1895. 9 Schottmuller, Munch, med. Woch., 1903. 346 PATHOGENIC MICROORGANISMS however, in which the quantities obtained are never large. Besredka l and Schlesinger 2 believe, for this reason, that the hemolytic substances are slosely attached to the bacterial bodies. The last-named author, furthermore, has determined that, in contradistinction to the othei toxic substances, streptococcus hemolysins are extremely labile, disap- Fig. 74. — Streptococcus Colonies from Blood Culture on Blood-Agar Plate. Showing areas of hemolysis about colonies. pearing from culture fluids after standing for from five to seven days at room temperature. Immunization. — For reasons not wholly understood at present, 1 Besredka, Ann. de l'inst. Pasteur, xv, 1901, p. 880. 2 Schlesinger , Zeit. f. Hyg., xxiv, 1903. STREPTOCOCCUS PYOGENES 347 recovery from streptococcus infection does not to any marked degree produce immunity against these bacteria. Active immunity may, however, be produced in rabbits, goats, horses, and other domestic animals by treatment with gradually increasing doses of streptococcus cultures.1 In carrying out such immunizations it is necessary to use for the first injection attenuated or dead bacteria. Attenuation may be accom- plished by moderate heating or by the addition of chemicals (terchlorid of iodin). Neufeld 2 advises, for the first injection, in immunizing rabbits, the use of ascitic-broth cultures killed by heating to 70° C. This is followed, after ten days, by a second injection of a small quantity of fully virulent cocci. Following this, injections are made at intervals of ten days with constantly increasing doses. Modifications of these general principles are employed in most laboratories. The sera of animals so treated contain no demonstrable antitoxic or antihemolytic substances.3 They exert, however, demonstrable bacteri- cidal power both in vivo and in vitro and distinctly enhance phagocytosis when brought into contact with leucocytes' and streptococci. This " opsonic" power has been noticed both intraperitoneally (Bordet 4) and in vitro (Denys and Leclef 5) . The protective value of streptococcus immune sera for infected animals is considerable, reaching often a potency hardly explicable by the demonstrable bactericidal or opsonic power, and thereby suggesting some other active factor not understood as yet.6 Aronson 7 has produced immune sera by the treatment of horses with a streptococcus derived from a case of scarlatina, 0.0004 cm. of which sufficed to protect mice from ten times the fatal dose of a streptococcus culture. These high protective values, however, are obtained only when the serum injections are given simultaneously with the bacteria. Given four or six hours after infection, much higher dosage must be employed and protective results are much less regular in occurrence.8 Other anti-streptococcic 1 Koch und Petruschky , Zeit. f. Hy^., xxiii, 1896. 2 Neufeld, Zeit. f. Hyg., xliv, 1903. 3 Lingelsheim, Zeit. f. Hyg., x, 1891. 4 Bordet, Ann. de l'inst. Pasteur, 1897. 5 Denys et Leclef, Cellule, t. ix. 6 Denys et Marchand, " Mecanisme de Fimmunite," etc., Brussels, 1896. 7 Aronson, Berl. klin. Woch., xxxii, 1896; ibid., xlii and xliii, 1902; ibid., viii and ix, 1905. 8 Denys, "Le Serum antistreptoe./' Louvain, 1896; Van de Velde, Ann. de l'inst. Pasteur, 1896. 348 PATHOGENIC MICROORGANISMS sera have been produced by Denys, Menger, Tavel, and others, all show- ing more or less marked potency in protecting animals.1 Since these sera, while in a general way potent against all streptococci, have been found protective chiefly against the specific microorganism em- ployed for their production, Van de Velde,2 Denys, Aronson, and others have advised the immunization of the animal with a large variety of streptococcus races, derived from many different human sources. The resulting " polyvalent " serum is more apt to exert equally high protective powers against all streptococcus infections. The therapeutic value of such sera in the treatment of human infections is still sub judice. Un- deniably favorable reports are published each year in increasing number, but are by no means regular or comparable to results such as those ob- tained in diphtheria with diphtheria antitoxin. Nevertheless, in mild cases or in those in which the lesions have been distinctly localized, the sera have seemed to be sufficiently useful to justify their use and to necessitate their standardization. Standardization is accomplished by the methods first devised by Marx 3 for the standardization of swine-plague serum, and depends upon the ability of the serum to protect animals against a measured dose of virulent streptococci. Aronson4 designates as a "normal serum" one of which 0.01 c.c. will protect a mouse against ten to one hundred times the fatal dose of virulent streptococci. One cubic centimeter of this serum equals one serum unit. Comparisons by animal experiment with this standard serum approximately determine the value of other sera. Leucocyte extracts have been employed by the writers and others, as advised by Hiss,5 in various forms of streptococcus infections of man with success in many cases. Very uniformly favorable results have been obtained with these extracts in cases of erysipelas. The agglutinins found in streptococcus immune sera are usually most active toward the race of bacteria employed in the immunization. Other streptococci, however, are also agglutinated, but in relatively higher concentration of the serum. Thus, while a specific group reaction is extremely useful in differentiating streptococci from other species, such as 1 Denys et Marchand. Bull, de l'acad. roy. de med. de Belgique, 1898; Menger, Berl. klin. Woch., 1902; Tavel, Corr.-Bl. f. Schw. Aerzte. 2 Van de Velde, Arch, de med. exper., 1897. 3 Marx, Deutsche thierarzt. Woch., vi, 1901. 4 Aronson, Berl. klin. Woch., xliii, 1902; Otto, Arb. a. d. konigl. Inst., etc., Frankfurt a. M., Heft 2, 1906. 5 Hiss, Jour. Med. Res., xix, 1908. STREPTOCOCCUS PYOGENES 349 pneumococci, agglutination can not be relied upon to differentiate in- dividual streptococci from one another (Hiss). It has even been found that a serum produced with a streptococcus from one source, con- tained a higher agglutinating value for some other streptococcus than for the one employed in its production. Agglutinins may be produced by treating animals with dead as well as with the living virulent streptococi. While the technique of the streptococcus agglutination tests is not difficult when we are dealing with strains which grow diffusely and with even clouding in fluid media, the frequency with which these micro- organisms clump spontaneously in broth cultures necessitates the use of a special technique. The most simple of these methods, and possibly the best, is the one in which calcium-carbonate-glucose broth is used for cultivation.1 Growing in this medium and thoroughly shaken once a day, the streptococci are usually found evenly divided in the supernatant fluid after the settling out of the heavier calcium-carbonate powder. Precipitins have been found by Aronson 2 in streptococcus immune horse serum. Special methods of extracting the bacteria were em- ployed. Classification. — Frequently observed differences in the minor cul- tural characteristics and in the virulence of streptococci obtained from various sources have given rise to much discussion as to the identity of all races of streptococci. The earliest observers were forced to abandon their separation of the streptococci of erysipelas from other streptococci because of the work of Marbaix3 and others, who produced erysipelas in rabbits with streptococci from non-erysipeiatous lesions, after en- hancement of their virulence, v. Lingelsheim 4 has proposed a purely morphological differentiation of "longus" and "brevis"; the former class including the streptococci most usually found in pyogenic le- sions and having a tendency to form chains of six or more links, the latter designating the short-chained varieties, including, as a rule, the less virulent streptococci. This classification, however, is not scientifically tenable because of the considerable dependence of chain- formation upon reaction, consistency, and nutritive qualities of the media employed for cultivation, and upon the influence of animal fluids if the microorganisms are taken direct from lesions. Schottmuller, 5 1Hiss, Jour. Exp. Med., vii, 1995. 2 Aronson, Deut. med. Woch., 25, 1903. 3 Marbaix, loc. cit. 4 v. Lingelsheim, " Aetiol. u. Therap. d. Streptokok. Krankh.," etc., Berlin, 1899. 5 Schottmuller, Munch, med. Woch.. 1903. 350 PATHOGENIC MICROORGANISMS who has made a careful study of streptococci, in 1903 proposed a classi- fication based both upon morphology and the appearance of cultures upon human blood agar. By this method he divided streptococci into two main groups as follows: I. Streptococcus longus seu erysipelatos, consisting of the most virulent varieties, having a tendency to form long chains, and regularly producing hemolysis upon blood media. II. Streptococcus mitior seu viridans, including less virulent strains, wTith usually shorter chain-formation, and producing green, non-hemolyzing colonies upon blood media. These are the streptococci which he usually obtained from milder or more chronic lesions. A third group which he adds, u Streptococcus mu^sus,^ will receive special considera- tion in a separate section, and is probably more closely related to the pneumococci than to the streptococcus groups. Attempts to separate the streptococci into subdivisions by their powers to ferment various carbohydrates have been made by Hiss, Gordon, and others. These attempts have, so far, been without practical result. Hiss * indicated a tentative division of streptococci into those which fermented monosaccharids alone, those which were also able to ferment disaccharids, and those in which the fermentative powers were extended to the polysaccharids, starch, dextrin, and glycogen. Gordon,2 after a thorough study of many strains upon seven carbo- hydrates, found ten different fermentation reactions among twenty pyogenic streptococci examined, and forty-eight different fermentation reactions among two hundred streptococci isolated from saliva. Other work by Andrewres and Horder and by Buerger3 confirms the irregu- larity of the fermentation reactions within this group. Probably the most reliable method of determining the interrelation- ships existing between bacteria, not only within this group, but in all bacterial classes, is that depending upon their reactions to immune sera. The wTork of Aronson,4 Marmorek,5 and others has showm that streptococcus immune sera produced with any one race of pyogenic streptococci exerted considerable, though variable, protective action against many other strains of streptococci. The same authors, as well as many others, working with the agglutination reaction, have shown that the agglutinins produced with one streptococcus strain were active 1 Hiss, Cent. f. Bakt., xxxi, 1902; Jour. Exp. Med., vi, 1905. 2 Gordon, Annual Report, Local Govern. Board, 33, London, 1903. 3 Avdrewes and Horder, Lancet, 1906; Buerger, Jour. Exp. Med., ix, 1907. 4 Aronson, Berl. klin. Woch., 1902; ibid., 1903. e Marmorek, Berl. klin. Woch., 1902. STREPTOCOCCUS MUCOSUS 351 against many other streptococci. While most active usually against the particular microorganism with which they were produced, this was by no means the rule, a serum produced with a streptococcus from a case of sepsis, in one case, agglutinating a streptococcus from a case of scarlatina more highly than its own microorganism. As with other "group agglutinations," the more highly immune the serum is, the more general is the agglutinating power over the whole group. Thus, while agglutination is practically useless in separating streptococci from one another, it is highly useful in differentiating these organisms from allied groups, such as the pneumococci. The immune reactions, therefore, seem to indicate a very close relationship between strepto- cocci as a class. Streptococcus mucosus. — This microorganism was first definitely described by Howard and Perkins 1 in 1901, and was subsequently care- fully studied by Schottmuller,2 who isolated it from cases of parame- tritis, peritonitis, meningitis, and phlebitis. The organism has since been described by many observers as the incitant of a variety of lesions and as an apparently harmless inhabitant of the normal mouth. Morphologically, though showing a marked tendency to form chains, on solid media it often appears in the diplococcus form. It is enclosed in an extensive capsule, which appears with much regularity and persist- ence. Though very similar in appearance, therefore, to pneumococci, these bacteria do not appear in the typical lancet shape. Upon solid media they show a tendency to grow in transparent moist masses. The regularity with which this microorganism ferments inulin medium, and its agglutinative characters, make it probable that it is more accurate to place it with the group of pneumococci than with that of streptococci.3 (For agglutinations see section on pneumococcus agglutination, p. 364.) 1 Howard and Perkins, Jour. Med. Res., 1901, N. S., i. 2 Schottmuller , Munch, med. Woch., xxi, 1903. 3 Hiss, Jour. Exp. Med., 1905; Buerger, Cent. f. Bakt., I, xli, 1906. CHAPTER XXIII DIPLOCOCCUS PNEUMONLE (Pneumococcus, Diplococcus lanceolatus) The opinion that lobar pneumonia is an infectious disease was held by many far-sighted clinicians long before the actual bacteriological facts had been ascertained. This idea, so well founded upon the nature of the clinical course of the disease, with its violent onset and equally rapid defervescence, led many of the earlier bacteriologists to make it the subject of their investigations — a subject made doubly difficult by the abundant bacterial flora found normally in the upper respiratory pas- sages, and by the fact, which is now recognized, that lobar and other pneumonias are by no means always caused by one and the same micro- organisms. Cocci of various descriptions and cultural characteristics were isolated from pneumonia cases by Klebs,1 Koch,2 Gunther,3 Talamon,4 and many others, which, however, owing to the insufficient differential methods at the command of these investigators, can not positively be identified with the microorganism now known to us as Diplococcus pneumonise or the pneumococcus. Although thus unsuccessful as to their initial object, these early investigations were by no means futile, in that they gave valuable information regarding the manifold bacterial factors involved in acute pulmonary disease and incidentally led to the dis- covery by Friedlander 5 of B. mucosus capsulatus. Communications upon lance-shaped cocci found in saliva, and capable of producing septicemia in rabbits, were published almost simul- taneously by Sternberg 6 and by Pasteur 7 in 1880. These workers * Klebs, Arch. f. exp. Path., 1873. 2 Koch, Mitt. a. d. kais. Gesundheitsamt, Bd. 1. s Gunther, Deut. med. Woch., 1882. 4 Talamon, Progr. med., 1883. 5 Friedlander, Virchow's Arch., lxxxvii. 6 Sternberg, Nat. Board of Health Bull., 1881. 7 Pasteur, Bull, de l'acad. de med., 1881. 352 DIPLOCOCCUS PNEUMONIA 353 beyond reasonable doubt were dealing with the true pneumococcus, but did not in any way associate the microorganisms they described with lobar pneumonia. The solution of this problem was reserved for the labors of A. Frankel * and Weichselbaum 2 who published their results, independently of each other, in 1886, demonstrating beyond question that the pneumococcus is the etiological factor in a large majority of cases of lobar pneumonia. Morphology and Staining. — The morphology of the pneumococcus is, in general, one of the most valuable guides to its identity. When typical, the pneumococcus is a rather large, lancet-shaped coc- cus, occurring in pairs, and surrounded by a definite and often wide capsule, which usually includes the two approximated cocci without a definite indentation opposite their lines of division. The pneumococci may, however, occur singly or in short chains, and even fairly long chains are not infrequently met with under artificial cultural conditions. This may be chiefly due to the cultural conditions or may be a promi- nent characteristic of certain strains. Apparently the capsules of or- ganisms making up the chains are continuous; wavy indentations are usually present, however, in the capsule of chains, and at times distinct divisions are observed. The chief variations from the typical morphology consist either in the assumption of a more distinctly spherical coccus type, or in an elongation approximating the bacillary form. Under certain conditions of artificial cultivation a distinct flattening of the organisms, particularly of those making up chains, may be seen, and even the impression of a longitudinal line of division, characteristic of many streptococcus cultures, is not infrequently gained. The capsules under certain conditions, especially in artificial media, may be absent or not demonstrable, and in certain strains capsules ap-. parently may not be present under any conditions. Practically any of the described variations may dominate one and the same culture under different or even apparently the same conditions of cultivation, and all grades may occur in capsule development, from its typical formation through all variations, to its total and apparently permanent absence. The presence or absence of capsules depends, to a large extent, upon the previous environment of the pneumococci under observation. The most favorable conditions for the development or preservation of the pneumococcus capsule are found in the body fluids of man and animals 23 i A. Frankel, Zeit. f. klin. Med., x, 1886. 2 Weichselbaum, Med. Jahrbiicher, Wien, 1886. 354 PATHOGENIC MICROORGANISMS suffering from pneumococcus infection. For instance, capsules may be demonstrated with ease by the usual capsule-staining methods in the blood, serum, and inflammatory exudate of the infected rabbit and white mouse. Capsules may be equally well marked in the fresh sputum of pneumonia patients, especially in the early stages of the disease and in the exudate accompanying such pneumococcus infections as menin- gitis, otitis media, and empyema. In sputum and the exudates of various localized infections, the organisms are, however, frequently degenerated or under chemical conditions unfavorable for capsule staining, and satisfactory results are not then easily obtained. The ■ff 9 Fig. 75. — Pneumococci, Grown on Loeffler's Serum. (Capsule stain by gentian- violet-potassium-carbonate method.) Fig. 76. — Pneumococci, from Rab- bit's Heart Blood. (Capsule stain by copper-sulphate method.) same is often true of the scrapings from lungs of patients dead of pneumonia, even in the stage of red hepatization. In artificial cultivation, if the nutrient medium is not milk or does not contain serum, capsules can not usually be demonstrated by the ordinary methods of preparing and staining. Capsules may, however, with much regularity be demonstrated on pneumococci, in agar, broth, or on almost all, if not all, artificial media, irrespective of the length of time the organ- isms have been under artificial cultivation if beef or rabbit serum is used as the diluent, when they are spread on the cover-glass for staining.1 The pneumococcus is non-motile and possesses no flagella. Spores are not formed. Swollen and irregular involution forms are common in cultures more than a dav old. 1 Hiss, Cent. f. Bakt,, xxxi. 1902; Jour. Exp. Med., vi, 1905. DIPLOCOCCUS PNEUMONIA 355 The pneumococcus is stained readily with all the usual aqueous anilin dyes. Stained by the method of Gram, it is not decolorized. Special methods of staining have been devised for demonstra- tion of the capsule. The ones most generally used are the glacial acetic-acid method of Welch 1 and the copper-sulphate method of Hiss.2 More recently Buerger 3 has devised a more complicated method for staining capsules, for which he claims differential value. (For methods see section on Technique, p. 98.) For simple staining of pneumococci in tissue sections, the Gram- Weigert technique is excellent. For demonstration of the capsules in tissue sections, Wadsworth 4 has described a simple method. Cultivation and Isolation. — The pneumococcus being more strictly parasitic than many other bacteria, presents greater difficulties in its cultivation. On meat-extract media growth does not take place with regularity. On those media, however, which have beef or veal infusion for their basis, growth can be obtained with considerable regularity, although such growth may be sparse and delicate. Growth takes place most regularly at a temperature of 37.5° C. Development does not usually occur below 25° nor above 41° C.;> At ordinary room temperature, 18-22° C, the temperature used for gelatin cultivation, growth either does not take place at all or is exceedingly slow and unenergetic. Aerobic and anaerobic conditions are equally favorable for pneumococcus cultivation, there being very little difference in speed or extent of growth along the course of deep stab cultures in favorable media. The most favorable reaction of media for the culti- vation of this microorganism is neutrality or moderate alkalinity (two- tenths to eight-tenths per cent alkalinity to phenolphthalein) . Slight acidity, however, if not exceeding eight-tenths per cent, does not materially hamper development. The growth of pneumococci on all media may be considerably enhanced by the addition to these media of animal or human serum or whole blood. Additional substances which, among others, unquestion- ably have a favorable influence upon pneumococcus growth, are glucose, nutrose, and glycerin. The addition of the latter substances to the media, however, probably because of acid formation, hastens the death 1 Welch, Johns Hopk. Hosp. Bull., xiii, 1892. 2 Hiss, Cent. f. Bakt., xxxi, 1902; Jour. Exp. Med., vi, 1905. 3 Buerger, Medical News, lxxxviii, 1904. 4 Wadsworth, " Studies by the Pupils of W. T. Sedgwick," Chicago, 1896. 5 A. Frankel, Dent. med. Woch., xiii, 1886. 356 PATHOGENIC MICROORGANISMS of pneumococcus cultures. An increase of the amount of pepton used for the preparation of media is desirable for the cultivation of this microorganism; two to four per cent of pepton may be found advantageous. In suitably alkaline, nutrient broth, growth is rapid, and within twenty-four hours leads to slight clouding of the fluid. This clouding, as a rule, eventually disappears as the microorganisms, sinking to the bottom of the tube or disintegrating, leave the fluid more or less clear. In broth, pneumococci have a tendency to form short chains. When glucose has been added to the broth, growth is more rapid and profuse, but considerable acid formation causes the cultures to die out rapidly. It is possible, however, to employ glucose as a growth-enhancing element in broth cultures without interfering with the viability of the cultures by adding small quantities (one per cent) of sterile, powdered calcium carbonate. This method of cultivation in broth is especially adapted to the production of mass cultures for purposes of immunization or agglutination.1 The addition of ascitic fluid or blood serum to broth, in the proportion of one to three, makes an extremely favorable medium in which growth is rapid and profuse. Upon agar plates, pneumococcus growth is not unlike that of strepto- coccus. The colonies are small, round, and slightly more transparent than those of the streptococci. They appear more moist than strepto- coccus colonies and often are more flat. Microscopically examined, the colonies are finely granular, with dark centers and slightly corrugated lighter-colored peripheral areas. Under high magnification no such in- tertwining convolutions can be seen as those noticed under similar magnification in streptococcus cultures. The addition of animal albu- min to agar results in the more rapid development, larger size, and deeper opacity of the colonies. Agar stab cultures show growth within twenty-four to thirty-six hours, which takes place with equal thickness along the entire course of the stab. There is nothing distinctive in these cultures to differentiate them from similar streptococcus cultures. In gelatin plate said stab cultures at 22° C, growth, as a rule, does not take place. This, however, is not true of all races of pneumococci. Occasionally strains are met with which will grow fairly abundantly in gelatin at a temperature of 22° C. When the gelatin is rendered suffi- ciently firm to bear 25° to 26° C. without melting, growth appears 1 Hiss, Jour. Exp. Med., vii, 1905. DIPLOCOCCUS PNEUMONIA 357 slowly and sparsely as minute, grayish-white, transparent colonies. The gelatin is not liquefied by the organisms. Growth upon milk is rapid and profuse, resulting usually in the production of acid and consequent coagulation of the medium. Ex- ceptionally, races are encountered in which this function is suppressed and coagulation in milk is absent or long delayed. Upon potato, a thin, grayish, moist growth occurs, hardly visible to the naked eye, and often indistinguishable from an increased moisture on the surface of the medium. Upon Loefjler's coagulated blood serum, the pneumococcus develops into moist, watery, discrete colonies wThich tend to disappear by a dr}dng out of the colonies after some days, differing in this from strep- tococcus colonies, which, though also discrete, are usually more opaque and whiter in appearance than those of the pneumococcus and remain unchanged for a longer time. This medium, as will be seen, is use- ful in differentiating pneumococci from the so-called Streptococcus mucosus. Upon a medium made up of mixtures of whole rabbit's blood and agar,, the pneumococcus grows with considerable luxuriance, and forms, after four or five days or longer, thick black surface colonies, not unlike small sun blisters on red paint. These colonies are easily distinguished from the hemolyzing colonies of most streptococci, and are in this respect of considerable differential value.1 Special media of various descriptions have been devised for pneu- mococcus cultivation. Thus, Guarnieri 2 has recommended a medium with a pepton-beef-infusion basis rendered semisolid by mixtures of agar and high percentages of gelatin. A modification of this medium has been described by Welch 3 and has been much employed in work with the pneumococcus. Cultivation within eggs and upon egg media4 has been advised and used by various observers. Wadsworth 5 has recommended a medium composed of ascitic fluid to which three-tenths per cent agar has been added — sufficient to give a soft jelly-like con- sistency to the medium. He observed prolonged viability and the preservation of the virulence in this medium. For the purpose of differentiating pneumococci from streptococci, 1 Hiss, loc. cit. 2 Guarnieri, Att. dell' Acad, di Roma, 1883. s Welch, Johns Hopk. Hosp. Bull., iii, 1892. 4 Sclavo, Riv. d'Igiene, 1894. 5 Wadsworth, Proc. N. Y. Path. Soc, 1903. 358 PATHOGENIC MICROORGANISMS Hiss l devised a medium composed of beef serum one part, and dis- tilled water two parts, to which is added one per cent of inulin (c. p.), and enough litmus to render the medium a clear, transparent blue. By fermentation of the inulin the pneumococcus acidifies this mixture, rendering the limus red and causing coagulation of the serum. Strep- tococci do not ferment inulin and the medium remains blue and fluid. (For the preparation of special media, see section on Media, p. 132.) For the isolation of pneumococci from mixed cultures or from material containing other species, such as sputum, the most reliable method is to make surface smears of the material containing the bac- teria upon plates of neutral glucose-agar or preferably of glucose-serum- agar. According to the number of bacteria present in the infected ma- terial from which the isolation is to be made, it may be smeared directly upon the plate, or diluted with sterile broth or salt solution before planting. After incubation for twenty-four hours, the pneumo- coccus colonies are easily differentiated from all but those of strepto- coccus. With practice, however, they may be distinguished from these also, by their smoother edges and greater transparency and flat- ness. Pour-plates, prepared in the usual way, can also be made but are less useful since deep colonies of pneumococci show no distinctive features. Another method for pneumococcus isolation, useful to eliminate other bacteria, is that of animal inoculation. White mice are inoc- ulated with 0.5 to 1 c.c. of the infectious material by subcutaneous injection, made most easily at the base of the tail. If virulent pneumo- cocci are present in the inoculated material, death from septicemia usually occurs within twenty-four to forty-eight hours. Surface smears should be made on glucose-agar plates with the heart's blood. By this method pure cultures may usually be obtained directly from the mouse blood. Resistance. — Kept upon artificial media, the viability of the pneu- mococcus is not great. Cultures upon agar or bouillon should, to be kept alive, be transplanted every third or fourth day, if the cultures are kept at incubator temperatures. In all media in which rapid acid formation takes place, such as glucose media, the death of cultures may occur even more rapidly. In media containing albumin and of a proper degree of alkalinity, preservation for one or even two weeks is possible. The longer the particular race has been kept upon artificial media, the Hiss, Jour. Exp. Med., vi, 1905. DIPLOCOCCUS PNEUMONIAE 359 more profuse is its growth, and the greater its viability, both qualities going hand in hand with its diminishing parasitism. The length of life of these bacteria may be much increased by their preservation at a low temperature, in the dark, and by the exclusion of air. By far the best medium for keeping pneumococci alive is the previously mentioned calcium-carbonate-infusion broth. Grown in this medium and kept in the ice-chest, cultures may often remain alive for months. In sputum the viability of pneumococci seems far to exceed that observed upon culture media. The studies of Guarnieri,1 Bordoni- Uffreduzzi,2 and others have shown that pneumococci slowly dried in sputum may remain not only alive but virulent, after from one to four months, when protected from light; and as long as nineteen days when exposed to diffused light at room temperature. Experiments by Ottolenghi 3 have, in the main, confirmed these results; the virulence seems, in Ottolenghi's experiments, to have become considerably attenu- ated before the death of the cocci occurred. More recent studies by Wood,4 whose attention was focused chiefly upon pneumococcus viabil- ity in finely divided sputum — in a condition, in other words, in which in- halation transmission would be possible — have shown that pneumococci in finely sprayed sputum survive for only about one and one-half hours, under ordinary conditions of light and temperature. Exposed to strong sunlight pneumococci die off within an hour, often within a few minutes. Low temperatures are well borne by pneumococci, temperatures slightly above zero being even conducive to the prolongation of life and the preservation of virulence. The resistance of the pneumococcus to heat, on the other hand, is low, 52° C. destroying it within ten minutes.5 To germicidal agents, carbolic acid, bichlorid of mercury, permanganate of potassium, etc., the pneumococcus is extremely sensitive, being destroyed by weak solu- tions after short exposures. The disinfection of sputum, offering considerable difficulties because of the protective coating of the secretions about the bacteria, has been recently made the subject of a spec'al study by Wadsworth.6 The con- clusions reached by this writer indicate that pneumococci in exudates 1 Guarnieri, Att. della R. Acad. Med. di Roma, iv, 1888. 2 Bordoni-Uffreduzzi, Arch. p. 1. sc. med., xv, 1891. 3 Ottolenghi, Cent. f. Bakt., xxv, 1889. 4 Wood, Jour. Exp. Med., vii, 1905. 5 Sternberg , Cent. f. Bakt., xii, 1891. 6 Wadsworth, Jour. Inf. Diseases, iii, 1906. 360 PATHOGENIC MICROORGANISMS are most rapidly destroyed by twenty per cent alcohol, other and stronger disinfectants being less efficient, probably because of slighter powers of diffusion. Virulence and Pathogenicity. — The virulence of pneumococci is subject to much variation, depending largely upon the length of time during which the microorganism has been cultivated under artificial conditions. It has been mentioned above that under certain conditions — such as those prevailing in dried sputum or blood l — the virulence of pneumococci may be preserved for several weeks. Ordinarily, however, the virulence diminishes gradually as the cocci adapt themselves more saprophytically to life upon artificial media. Upon media containing animal albumin, such as ascitic fluid or blood agar, this attenuation is less rapid than upon the simple meat-infusion preparations. In the blood of rabbits dead of a pneumococcus infection, taken directly into sterilized tubes, sealed and kept in the dark, Foa2 has been able to preserve the virulence of pneumococci for as long as forty-five days. Whether or not the virulence of pneumococci is attenuated by sojourn within the human body during disease is a question much dis- cussed but hardly settled. It is a matter of fact, however, that many pneumococci obtained by blood culture from more or less chronic cases of pneumococcus septicemia fail to kill susceptible test animals, even when injected in considerable doses. The attenuation of virulent pneumococci on artificial media may be hastened, according to Frankel,3 by cultivation of the organism at or above a temperature of 41° C. Freshly isolated from the human saliva or pneumonic lesions, the differences in virulence between various strains of pneumococci are not very marked, almost all such strains showing considerable pathogenic powers toward the usual test animals. The virulence of attenuated cultures may be rapidly enhanced by the passage of the organisms through the bodies of susceptible animals. The extreme virulence of some of these pneumococcus strains may be illustrated by citing the experiments of Eyre and Washburn 4 who possessed cultures of which one millionth of a loopful would kill a mouse within four days. Among the domestic animals those most susceptible to pneumococcus infection are white mice and rabbits. Guinea-pigs, dogs, rats, and cats 1 Guarnieri, loc. cit. 2 Foa, Zeit. f. Hyg., iv, 1888. 3 Frankel, Deut. med. Woch., 13, 1886. i Eyre and Washburn, Jour, of Path, and Bac, v. DIPLOCOCCUS PNEUMONIA 361 are more resistant, but still may be infected with large doses. Young animals are usually more susceptible than adults. Birds are practically immune. The results of pneumococcus inoculation into susceptible animals vary according to the size of the dose, the virulence of the introduced bacteria, the mode of administration, and the susceptibility of the subject of the inoculation. Subcutaneous inoculation of virulent pneumococci into mice and rabbits usually results in an edematous, often fibrinous exudation at the point of inoculation, which, in all cases n which the dose given has not been extremely small, leads to septicemia and death within twenty -four to seventy-two or more hours. When the dose has been extremely small or the culture unusually attenuated, a localized abscess may be the only result. Intravenous inoculation is usually more rapidly fatal in these animals than the subcutaneous method. Intraperitoneal inoculation in rabbits results in the formation of a rapidly spreading peritonitis in which the inflammatory exudate in many cases exhibits differences from similar exudates produced by the streptococcus. Pneumococcus exudates are apt to be thicker, to be accompanied by a deposit of fibrin, and to lack the transparent red color so often caused by the hemolyzing streptococci. With very virulent strains, these differences are less marked. In almost all of these infec- tions death is preceded by septicemia and the microorganisms can be recovered from the heart's blood of the victims. After such infections, the animals exhibit a rise of temperature, at times visible depression, and, rarely, diarrhea. General hyperemia of the organs with secondary effusions in the pleural cavities and often hemorrhages upon the serous surfaces may be found at autopsy. The production in animals of lesions comparable to the lobar pneu- monia of human subjects has been the aim of many investigators. Wadsworth,1 recognizing that such lesions probably depended upon the partial immunity which enabled the infected subjects to localize the pneumococcus processes in the lungs after infection by way of the respiratory passages, succeeded in producing typical lobar pneumonia in rabbits by partially immunizing these animals and inoculating them intratracheally with pneumococci of varying virulence. By this method he actually carried out, for the first time, Koch's postulates in regard to lobar pneumonia. In man, the most frequent lesion produced by the pneumococcus is 1 Wadsworth, Amer. Jour. Med. Sci., May, 1904. 362 PATHOGENIC MICROORGANISMS acute lobar pneumonia. About ninety per cent of all cases of this disease are caused by the pneumococcus,1 the remainder being due to streptococci, influenza bacilli, Friedlander's bacilli; and exceptionally to other microorganisms. Lobular pneumonia is caused by the pneu- mococcus with almost equal regularity. During the course of these diseases the cocci are found in large numbers within the pulmonary alveoli, and in the capillaries and lymph vessels of the lung. Whether or not the pneumococci enter the blood stream in all these cases is a question not yet definitely settled. Frankel 2 states it as his belief that in most, if not in all, cases, the diplococci at some time during the disease could be found in the circulating blood. Prochaska in a study of ten unselected cases obtained positive blood cultures in every one of them. A review of the literature upon the question indicates positive blood- culture findings in certainly over twenty-five per cent of the cases. In complications of pneumonia, pneumococci are found usually in the pleura where they may cause a simple dry pleurisy or even empy- ema. Less frequently they may cause pericarditis and endocarditis. Meningitis may be caused by pneumococci, either secondarily to pneu- monia or independently. Such cases are extremely grave, almost invariably ending in death. Other lesions which may be caused by pneumococci, either as post-pneumonic processes or without previous pneumonia, are otitis media, osteomyelitis, and arthritis. Cases of pneumococcus peritonitis occur sometimes secondary to appendicular inflammations, occasionally without traceable portal of entry. Severe catarrhal conjunctivitis may be caused by these diplococci, usually during the course of a pneumonia. Ulcerative endocarditis with pneu- mococcus septicemia, apparently independent of a pulmonary lesion, is not infrequent. Toxic Products of the Pneumococcus. — Our knowledge of pneumococcus poisons is still very imperfect. Attempts to obtain soluble toxins by the filtration of cultures have been practically unsuccessful in the hands of many careful workers. G. and F. Klemperer,3 Mennes,4 Pane,5 Foa and Carbone,6 and others failed to obtain pneumococcus filtrates of any marked degree of toxicity, though working with highly virulent x Netter, Compt. rend, de la soc. de biol., 1890. 2 Frankel, "v. Leyden Festschr.," 1902. 3 67. and F. Klemperer, Berl. klin. Woch., xxxiv and xxxv, 1891. 4 Mennes, Zeit. f. Hyg., xxv, 1897. sPane, Rif. med., xxi, 1898. 6 Foa und Carbone, Cent. f. Bakt., x, 1899. DIPLOCOCCUS PNEUMONIA 363 strains. Attempts to demonstrate by the production of antitoxin the specific nature of the feeble poisons obtained have also met with failure. Isaeff,1 though confirming the feeble toxicity of fluid cultures, made the interesting observation that a filtrate of the blood of pneumo- coccus-infected rabbits contained a poison often more potent than that obtained in culture filtrates. Carnot and Fournier 2 obtained a poison of distinct though feeble potency by dialysis of pneumococcus cultures. The general failure, however, to procure strong soluble poisons from cultures, gives weight to the assumption that the most potent toxic products of pneumococci are in the nature of endotoxins and closely bound to the cell-bodies themselves. This assumption is borne out by the more recent experiments of Macfadyen.3 This author obtained acutely poisonous substances from pneumococci by trituration of the organisms after freezing, and extracting them with a one 1 : 1,000 caustic potash solution. With the filtrates of these extracts he was able to cause rapid death in rabbits and guinea-pigs by the use of doses not exceeding 0.5 to 1 c.c. He found furthermore, a striking parallelism between the degree of toxicity and the virulence of the extracted culture. Immunization. — Recovery from a spontaneous pneumococcus in- fection confers immunity for only a short period. Two and three attacks of lobar pneumonia in the same individual are not unusual, and it is uncertain whether even a temporary immunity is acquired in such infections. Active immunization of laboratory animals may be carried out by various methods. The method usually followed is to begin by injecting attenuated 4 or dead bacteria or bacterial ex- tracts. Subsequent injections are then made with gradually increas- ing doses of living, virulent microorganisms. Great care in increasing the dosage should be exercised since the loss of an animal after two or three weeks' treatment by a carelessly high dose of pneumococci is not unusual. Wadsworth has recommended the following method for preparing pneumococci for the first injections in immunizing rabbits. Freshly grown pneumococcus cultures are centrifugalized, and the supernatant bouillon is thoroughly decanted. To the pneumococcic sediment a definite quantity of concentrated salt solution is added, and the mixture is allowed to stand over night. At the end of this time, the pneumococci are dead and considerable destruction of the cell-bodies 1 Isaeff, Ann. de l'inst. Pasteur, vii, 1893. 2 Carnot et Fournier, Arch, de riied. exper., 1900. 3 Macfadyen, Brit. Med. Jour., ii, 1906. * Radziewsky, Zeit. f. Hyg., xxxvii, 1901; Neufeld, Zeit. f. Hyg., xi, 1902. 364 PATHOGENIC MICROORGANISMS has taken place. Dilution with water until the solution equals 0.85 per cent NaCl now prepares the emulsion for inoculation. Whichever of the various methods is adopted, the intervals of injection should not be shorter than a week, preferably ten days. The animals so immunized will at the end of six or more wreeks withstand an inoculation with many times the fatal dose of virulent pneumococci. The sera of animals immunized with pneumococci contain active bacteriolytic and bacteri- cidal substances, easily demonstrable in vivo and in vitro. Specific agglutinins in pneumococcus immune sera wrere first thoroughly studied by Neufeld * and since then have been made the subject of ex- tensive studies by Wadsworth,2 Hiss,3 and many others. In the sera of normal animals and man, pneumococci are rarely agglutinated in dilu- tions higher than one in ten. In the serum of patients suffering from lobar pneumonia, pneumococci agglutinate in dilutions ranging any- where from one in ten to one in fifty. In the sera of immunized rab- bits, readings up to one in eight hundred are not rare. Such specific agglutinating sera are most reliable, therefore, in differentiating be- tween pneumoccoci and closely allied bacteria and in demonstrating the identity of all pneumococci whatever their source. The table which follows not only illustrates this uniformly high agglutinative power of various pneumococcus-immune sera upon several races of this microorganism, but shows, as well, the value of such sera for biological differentiation. The table, furthermore, records the peculiar fact that pneumococci are agglutinated in high dilution by sera obtained by immunization with Streptococcus mucosus, a fact which argues strongly in favor of classify- ing Streptococcus mucosus more intimately with the pneumococci than with the streptococci of the pyogenes group. The technique of performing agglutination tests with pneumococci is often made difficult by the tendency to clumping displayed spon- taneously by these bacteria in fluid media and by the relative sparsity of their growth. To overcome these difficulties, Wadsworth 4 has pro- posed centrifugalizing young broth cultures and shaking up the sedi- ment with small quantities of isotonic salt solution. Hiss recommends 5 cultivation in glucose-calcium-carbonate broth in small flasks containing 1 Neufeld, loc. cit. 2 Wadsworth, loc. cit. 3 Hiss, Jour. Exp. Med., vii, 1905. * Wadsworth, Jour. Med. Res., x, 1905. 5 Hiss, loc. cit. DIPLOCOCCUS PNEUMONIA 365 100 to 150 c.c. each. After three or four days at 37° C, the growth is usually at its optimum for agglutination work. The flasks should be thoroughly shaken at least once in twenty-four hours in order to dis- tribute the calcium carbonate thoroughly. About one hour before use Organism. IMMUNE SERA1 Pneum. 1. Pneum. 3. Pneum. 23. Strepto- coccus mucosus 7. Strepto- coccus mucosus 7 a. Strepto- coccus pyo- genes. Pneumo. 1. . .. Pneumo. 3. . . . 400-800 200-800 400-800 100-800 400-800 100-800 200-800 400-800 200-800 400 200-800 0-100 0-100 Pneumo. 23. . . 200-800 600-200 100-200 200-800 Pneumo. 45. . . 100-400 100-200 Pneum. El... Pneum. E 32. . 100 + 200-400 200-800 200-800 Pneum. E 55. . Pneum. N 7.. . Pneum. N 17 . Streptococcus pyog. 1 . . . . 100-400 200-800 200-800 400-800 800 800 200-800 200-800 200-800 0-10 0-10 800-6400 0-50 Streptococcus mucos. 7. . . Streptococcus mucos. 22. . 0-10 0-10 10-100 10-50 10-200 0-50 the flasks are again shaken and the calcium carbonate and larger clumps are allowed to settle. The sample used for the test should then be pi- petted from the upper layers of the fluid. Precipitins have been demonstrated in pneumo coccus immune sera 1 Hiss, Jour. Exp. Med., vii, 1905, p. 564. 366 PATHOGENIC MICROORGANISMS by Neufeld,1 Wadsworth,2 and others. Neufeld obtained precipitates with pneumococcus cultures in which lysis had been produced by the addition of bile. He found that normal rabbit's bile added to pneu- mococcus cultures (one drop to 1 c.c. of culture) caused the cultures to become perfectly clear and transparent, and no longer contain demon- strable pneumococcus cell bodies. The addition of pneumococcus im- mune sera to such cultures produced precipitates. Wadsworth elimi- nated all possibility of participation of the bile in this reaction by obtaining similar precipitates with pneumococcus cultures subjected to treatment with concentrated salt solution as described above (see p. 363). In addition to these antibodies, pneumococcus immune sera contain specific phagocyosis-stimulating substances identical with or similar to the opsonins of Wright and Douglas. The first investigators to de- scribe these substances for pneumococcus sera, Neufeld and Rimpau,3 separated them from the opsonins on the basis of their greater thermo- stability and named them bacteriotropins. It is doubtful whether, in view of the described thermostability of some immune opsonins, such differentiation is tenable. Great importance in the mechanism of pneumococcus immunity is attributed to these bodies by some authors. This question has been studied more recently by Park and Williams,4 however, who were unable to find any distinct parallelism between the opsonic power and the protective value of a serum. Passive immunization with pneumococcus immune sera has been extensively attempted. Washburn,5 Mennes,6 Pane,7 and many others have succeeded in protecting subsequently infected animals by treatment with pneumococcus immune sera. Occasionally favorable results have been reported when similar sera were used in pneumonia in human beings. All such results, however, have been too irregular to permit, at present, of a hope for the definite solution of the therapeutic problem along these lines. Encouraging results have been obtained by Hiss in the treatment of pneumococcus infection in animals and by Hiss and Zinsser 8 in the treatment of pneumonia in man with aqueous leucocyte extracts. 1 Neufeld, Zeit. f. Hyg., xi, 1902. 2 Wadsworth, loc. cit. 3 Neufeld und Rimpau, Deut. med. Woch., 1904. 4 Park and Williams, Jour. Exp. Med., vii, 1905. 5 Washburn, Brit, Med. Jour., 1897. fi Mennes, Zeit. f. Hyg., 1897. -Pane, Rif. med., 1897. 8 Hiss and Zinsser, Jour. Med, Res., xix, 1908. DIPLOCOCCUS PNEUMONIAE 367 Differentiation of Pneumococcus from Streptococcus. — Pneumococci and streptococci which do not differ in morphology from their classic types can usually be differentiated from each other and identified by their morphological characters without difficulty; but it is equally true that certain cultures of these organisms, either at the time of their isolation or after cultivation on artificial media, approach the type of the other so closely that it may be impossible to identify them by their mor- phology alone. When such morphological variations occur there are no constant and distinctive cultural or pathogenic characters as yet de- monstrated which can with certainty be depended upon as distinguish- ing marks between these organisms. This lack of distinct cultural differences between pneumococci and streptococci has not infrequently led to confusion, and that uncertainty should exist and mistakes be made in identification is not surprising when one considers the characters usually depended upon to distinguish pneumococci from streptococci. Chief among these, as has just been implied, are the morphological features which are, in the case of pneu- mococci, a slightly lancet or elongated form rather than the more typical coccus form characteristic of the streptococci, and an arrangement of such cocci in pairs rather than in chains ; added to these features is the possession of a more or less well-defined capsule. All of these char- acters are subject to variation or may be absent. Compared with the morphological, the cultural characters are of minor importance and are variable. They consist in a more moist and flatter appearance of the pneumococcus colonies on coagulated blood serum and on agar, and in the usual inability of the freshly isolated pneumococcus to develop readily or at all on gelatin at temperatures below 22° C. The distinctness of the capsule of the pneumococcus in the body fluids of man and an'mals, and at times when this organism is culti- vated artificially on blood serum, milk, or serum agar, has really been depended upon as the chief distinguishing and diagnostic character. Nevertheless, from time to time, instances have been reported of distinct capsule formation by organisms which had either been pre- viously identified as Streptococcus pyogenes, or at the time of their isolation could not be definitely identified by their discoverers as be- longing to either this group or to the pneumococci, but were considered intermediate in their character.1 1 Brief Description of Organisms Reported as Capsulated Streptococci. — Bordet (Bordet, Ann. de l'inst. Pasteur, 1897, xi, p. 177), working with an organism previously 36S PATHOGENIC MICROORGANISMS There are occasions, then, both within the animal body and in arti- ficial cultivations, when it is practically impossible to distinguish defi- nitely between some races of pneumococci and races of streptococci. This difficulty is especially heightened when the pneumococcus has become non-virulent, and at the same time no very typical morphology or capsule formation is to be determined and a tendency to chain-forma- tion is marked. Cultures of pneumococci in such condition can not readily be distinguished morphologically from streptococcus cultures. Under these circumstances recourse must be had to a careful bio- logical study of the organism in question. The following are the criteria mainly relied upon at present for the differentiation of these two groups. identified as Streptococcus pyogenes, described such capsule formation occurring in the peritoneal exudate of infected rabbits. Schuetz' (Schuetz, Cent, f. Bakt.,Ref. 1, 1887, p. 393) Diplokokkus der Brustseuche der Pferde, Poels and Nolen's (Poels und Nolen, Fort. d. Med., iv, 1886, p. 217) streptococcus of contagious pneumonia of cattle, and especially the organism de- scribed by Bonome (Bonome, Ziegler's Beit., viii, 1890, p. 377) as Streptococcus der meningitis cerebrospinalis epidemica, may all be looked upon as organisms differentiated on insecure grounds from either pneumococcus or streptococcus. The first two of these organisms, however, are said to be decolorized by Gram's method, and as suggested by Frosch and Kolle (Frosch und Kolle, Flugge's " Mikro- organis.," ii, 1896, p. 161), in the case of Schuetz' organism may belong to a group intermediate between FraenkePs diplococcus and the chicken-cholera group. Tavel and Krumbein (Tavel und Krumbein, Cent. f. Bakt., xviii, 1895, p. 547) describe a streptococcus with a capsule, which was isolated from a small abscess on the finger of a child. Capsules were also present in the artificial cultures, and although ordinarily remaining uncolored, could be stained by Loefner's flagella stain. This organism was said to be differentiated from FraenkePs diplococcus and also in general from streptococcus (pyogenes) by a rapid and rich growth on gelatin, agar, and potato. A pellicle was formed on broth. The organisms forming this pellicle possessed capsules, but those in the deeper portions of the broth generally lacked the capsule. In 1897, Binaghi (Binaghi, Cent. f. Bakt., xxii, 1897, p. 273) described a capsulated streptococcus isolated from a guinea-pig dead of a spontaneous peribron- chitis and multiple pulmonary abscesses. In the pus were found some diplococci and short chains (four to six) surrounded by a capsule, which could be made evident by staining with carbol fuchsin. This organism he proposes to call Streptococcus capsulatus. Le Roy des Barres and Weinberg in 1899 (Le Roy des Barres et Weinberg, Arch, d. med. exper., xi, 1899, p. 399) published an account of a streptococcus with a capsule. This was isolated from a man who had apparently been infected from a horse which had died of an acute intestinal disorder. The patient neglected the infection and died. Diplococci and short chains furnished with a capsule were found in the subcutaneous tissue at the area of infection. The blood, liver, and DAPLOCOCCUS PNEUMONIA 369 Pneumococci ferment inulin, if cultivated in inulin-serum-water medium. Acid formation from the inulin results within two days or more in coagulation of the serum and reddening of the litmus. Streptococci because of their inability to attack the inulin leave the medium unchanged.1 Cultivated on whole-blood-agar, streptococci usually cause hemol- ysis, pneumococci usually do not.2 In contradistinction to Streptococ- cus viridans which does not hemolyze, pneumococci have a tendency on these media to form the black, dry, paint-blister colonies.3 Neufeld,4 in 1900, noticed that normal rabbits' bile added in quan- spleen also contained these organisms. The capsule in all the preparations remained uncolored, but the authors say that its existence was not to be doubted. Ascitic broth inoculated from the peritoneal exudate of a rabbit dying from the infection gave streptococci in extremely long chains and surrounded by capsules. These were not so distinct as in the case of the organisms in the original smear preparations. All fluid media (bouillon, milk, and ascitic broth) were said to be strongly acid after twenty-four hours. These authors report that Achard and Marmorek have assured them that they have seen capsulated streptococci, and that Marmorek showed them some preparations in which one of his streptococci presented the same characters as that isolated by them. Although Le Roy des Barres and Weinberg have used the term encapsulated, they believe that it would perhaps be more prudent to call their organism strepto- coque aureole, since they were not able to put this capsule definitely in evidence by staining it. Howard and Perkins {Howard and Perkins, Jour. Med. Res., 1901, iv, p. 163) have lately described an organism, probably of the foregoing type, which was present in a tubo-ovarian abscess and in the peritoneal exudate, the blood, and some of the organs of a woman dying in the Lakeside Hospital, Cleveland, Ohio. The organisms were biscuit-shaped cocci in pairs, usually arranged in chains of four, six, eight, or twenty elements, and surrounded by a wide and sharply staining capsule. In the artificial cultures special capsule stains, it was noted, failed to stain any definite area, but numerous small deeply stained granules were to be seen within the halo, espe- cially near its outer border. Howard and Perkins propose for the group composed of the streptococci of Bonome, Binaghi, and their own organism, the name Streptococcus mucosus. Reference to the original descriptions of these various capsulated streptococci will show that, with the exception of a rather poorly staining capsule, the majority of these organisms are separated from the typical Streptococcus pyogenes or from the pneumococcus by exceedingly slight and unstable morphological and cultural characters. The same is true of the difference observed in their pathogenic action in animals. 1Hiss, Cent. f. Bakt., xxxi, 1902; Jour. Exp. Med., vi, 1905. 2 Schottmiiller , Munch, med. Woch. 3 Hiss, Jour. Exp. Med., vii, 1905. * Neufeld, Zeit. f. Hyg., 1901. 24 370 PATHOGENIC MICROORGANISMS tities of 0.1 c.c. to each one or two cubic centimeters of a pneumococcus broth culture caused lysis of the bacteria, rendering the culture fluid transparent and clear. This phenomenon does not occur with strep- tococci, and has been used to differentiate the two species. According to the recent studies of Libman and Rosenthal,1 great reliance may be placed upon this method. Finally, decisive differential importance may be attached to the agglutinations of these microorganisms in immune sera (see p. 364). 1 Libman and Rosenthal, Proc. N. Y. Path. Soc, March, 1908, CHAPTER XXIV MICROCOCCUS INTRACELLULARS MENINGITIDIS (MENINGOCOCCUS) Infectious processes in the meninges may be caused by many dif- ferent microorganisms. Meningitis may be primary or secondary. Secondary meningitis may often occur during the course of pneumonia, when pneumococci, carried to the meninges by the blood stream, .give rise to a usually fatal form of the disease. More rarely a similar process may occur as a secondary manifestation of typhoid fever or influenza. Meningitis may also result secondarily by direct extension from suppurative lesions about the skull, such as those occurring in diseases of the middle ear or frontal sinuses or after compound fractures. In such cases the invading or- ganisms are usually staphylococci, streptococci, or pneumococci. Isolated cases of meningeal infection with B. coli, B. paratyphosus, Bacillus pestis, and Bacillus mallei have been reported. A frequent, more chronic form of the disease is caused by Bacillus tuberculosis. Primary acute meningeal infection, however, is due chiefly to two microorganisms, Micrococcus intracellularis meningitidis, and the pneu- mococcus. A tabulation of the comparative frequency with which the various microorganisms are found in the meninges has been attempted by Marschal.1 This author estimates that about 69.2 per cent of all acute cases are due to the meningococcus, 20.8 per cent to Diplococcus pneumoniae, and the remaining 10 per cent to the other bacteria mentioned. The cases caused by the pneumococcus and the other less frequent incitants usually occur sporadically. When the disease occurs in epi- demic form, it is almost always due to the meningococcus. Diplococcus intracellularis meningitidis was first seen in menin- geal exudates by Marchiafava and Celli 2 in 1884. These authors not only described accurately the morphological characteristics now recog- 1 Marschal, Diss. Strassburg, 1901, Quoted from Weichselbaum, in Kolle und Wassermann, " Handbuch." 2 Marchiafava and Celli, Gaz. degli ospedali, 8, 1884. 371 372 PATHOGENIC MICROORGANISMS nized, but also called attention to the intracellular position of the micro- organism and to its gonococcus-like appearance. They failed, however, to cultivate it. Observations confirmatory of the Italian authors were, soon after, made by Leichtenstern.1 Cultivation and positive identification as a separate species was not accomplished, however, until Weichselbaum,2 in 1887, reported his observations upon six cases of epidemic cerebro- - * # « • * • • % ^ «* . % * • * • • • • • * % • * # #* • * «- 1 J* • • * • 4 Fig. 77. — Meningococcus, Pure Culture. (Very highly magnified.) spinal meningitis in which he not only found the cocci morphologically, but was able to study their biological characteristics in pure culture. The researches of Weichselbaum were soon confirmed and extended by elaborate studies 3 which left no doubt as to the specific relationship between the microorganism cultivated by him and the clinical condition. r 1 Leichtenstern, Deut. med. Woch., 1885. 2 Weichselbaum, Fort. d. Med., 1887. 3 Councilman, Mallory, and Wright, Special Rep. Mass. Board of Health, 1898; Albrecht und Ghon, Wien. klin. Woch., 1901. MICROCOCCUS INTRACELLULARS MENINGITIDIS 373 Morphology and Staining. — Stained in the spinal fluid from an in- fected patient, the meningococcus bears a striking similarity to the gon- ococcus. The microorganisms appear intra- and extraccllularly, usually in diplococcus groups, sometimes as tetrads, or even in larger agglomer- ations. The individual diplo-forms are flattened on the sides facing each other, presenting somewhat the biscuit-form of the gonococcus. The variation in size of the cocci in the same smear is a noticeable feature Fig. 78. — Meningococcus in Spinal Fluid. and of some diagnostic importance. This dissimilarity in size is notice- able also in cultures, which, especially when older than twenty-four hours, contain forms double or even triple the size of the average coccus. These may possibly be involution forms. The meningococcus is non-motile and non-spore forming. It stains easily with all the usual aqueous anilin dyes. Its behavior toward Gram's stain was long a subject of controversy, owing to the error of Jaeger,1 who claimed to have found it Gram-positive. There 1 Jaeger, Zeit. f. Hyg., xix, 1895. 374 PATHOGENIC MICROORGANISMS is no question now, however, that the cocci decolorize by Gram's method when this is carefully carried out. In spinal fluid very beautiful preparations may be obtained by staining in Jenner's blood stain. Councilman, Mallory, and Wright l were the first to notice that, when stained with Loeffler's methylene-blue, meningococcus stains irregularly, showing metachro- matic granules in the center of the cell bodies. These granules can be demonstrated more clearly with the Neisser stain employed for similar demonstration in the case of B. diphtheria? (see p. 107) and have some value in differentiating meningococcus from gonococcus. Cultivation. — Micrococcus intracellularis meningitidis grows readily upon all the meat-infusion culture media. It may even be culti- vated upon meat-extract media, but growth upon these is not profuse. Upon agar, colonies appear within eighteen to twenty-four hours as grayish, glistening spots with smooth edges and raised granular centers. These show a tendency to enlargement and eventual confluence. Growth is more luxuriant and rapid upon media to which animal proteid in the form of blood serum or ascitic fluid has been added. Co- agulated serum is not liquefied. For cultivation of the meningococcus directly from the human body it is wise to use the richer serum or blood media, ability to grow easily upon simple agar being occasionally acquired only after previous cultivation upon richer media. Agar to which whole rabbit's blood has been added forms an excellent medium, both for cul- tivation and for keeping the organism alive. Loeffler's blood serum is also very favorable. It is advisable, too, when cultivating directly from spinal fluid, to plant rather large quantities (1 to 2 c.c), since many of the cocci in the exudate will fail to develop colonies, possibly because of their prolonged exposure either to the body fluids or to their own products in a closed space. Upon broth, growth is slow and takes place chiefly upon the surface, the sediment consisting mainly of dead bacteria. Glucose added to agar or to broth renders the medium more favorable for rapid growth, but, owing to acid formation, tends to cause a more rapid death of the culture. In flasks of broth containing glucose one per cent, and CaC03 one per cent, however, cultures have been kept alive for as long as fourteen months (Hiss). On milk, growth takes place without coagulation of the casein. Potatoes are not a favorable medium, though growth occasionally takes place. 1 Councilman, Mallory, and Wright, Rep. Mass. State Bd. of Health, 1898. MICROCOCCUS INTRACELLULARS MENINGITIDIS 375 While slight alkalinity or acidity does not inhibit, the most favor- able reaction of media is neutrality. Oxygen is necessary for development. Complete anaerobiosis, while not absolutely inhibitory, is extremely unfavorable, unless proper carbohydrates be present. While growth may take place at temperatures ranging from 25° Fig. 79. — Meningococcus Culture. Streak cu'ture from spinal fluid on serum agar-plate. to 42° C, the optimum is 37.5° C. Apart from the remarkable viability displayed upon calcium-carbonate broth, the average length of time during which the meningococcus will remain alive without transplanta- tion is rather short. Recently isolated cultures grown on agar or serum- agar may die within two or three days. Accustomed to artificial cul- tivation through a number of generations, however, the cultures become 376 PATHOGENIC MICROORGANISMS more hardy and transplantation may safely be delayed for a week or even longer. Albrecht and Ghon x have kept a culture alive on agar for one hundred and eighty-five days. It is a strange fact that after prolonged artificial cultivation some strains of meningococcus may gradually lose their growth energy and finally be lost because of their refusal to develop in fresh transplants. Storage is best carried out at incubator temperatures. At room temperatures or in the ice chest, the diplococcus dies rapidly.2 Resistance. — The meningococcus is killed by exposure to sunligh tor to drying within twenty-four hours.3 It is extremely sensitive to heat and cold and by the common disinfectants is killed in high dilutions and by short exposures. At 0° C. it usually dies within two or three days. Pathogenicity. — As stated above, the form of meningitis caused by the diplococcus of Weichselbaum occurs usually in epidemics, though isolated sporadic cases are seen from time to time in all crowded com- munities. Epidemics have been numerous and widespread, and their records far antedate the discovery of their causative agent. As a rule, these epidemics have occurred during the winter and spring months, and have attacked chiefly that part of the population which is forced by poverty to live in crowded unhygienic surroundings. The manner in which the microorganism enters the human body is still a subject for investigation. Weichselbaum,4 Ghon and Pfeiffer,5 and, more recently, Goodwin and v. Sholly 6 of the New York Department of Health, have succeeded in demonstrating culturally the presence of the meningococ- cus in the nasal cavities, not only of patients suffering from the disease, but occasionally in those of healthy subjects as well. Similar findings have been reported by many others; but in many cases morphological examination only was made, which, owing to the danger of confusion with Micrococcus catarrhalis, a frequent inhabitant of the nose, renders such reports valueless. The careful work of the writers mentioned, how- ever, has given ground for the theory that meningeal infection, which is 1 Albrecht und Ghon, Wien. klin. Woch., 1901. 2 A very thorough biological study of meningococcus and related organisms has recently been made by Elser and Huntoon (Jour. Med. Res., N. S. vol. xv, 1999), which may be consulted for a more detailed description of cultural characteristics. 3 Councilman, Mallory, and Wright, Boston, 1898; Albrecht and Ghon, loc. cit. * Weichselbaum, Fort. d. Med., 1887. *Ghon und Pfeiffer, Zeit. f. klin. Med., xliv, 1901. 6 Goodwin und v. Sholly, Jour. Inf. Dis., Suppl. 2, Feb., 1906. MICROCOCCUS INTRACELLULARS MENINGITIDIS 377 often preceded by nasal catarrh, may take place along the paths of the lymphatics, passing out of the nose and its accessory cavities toward the base of the skull. These facts, together with the low resistance shown by the meningococcus against drying, and the general failure so far to demonstrate it in air, dust, or fomites, would seem to indicate that trans- mission usually occurs directly from one human being to another. The disease produced in man consists anatomically in a suppurative lesion of the meninges, involving the base and cortex of the brain and the surface of the spinal cord. The nature of the exudate may vary from a slightly turbid serous fluid to that of a thick fibrinous exudate. In chronic cases encephalitis and dilatation of the ventricles may take place. Apart from their presence in the meninges and in the naso- pharynx, meningococci have not been satisfactorily demonstrated in any of the complicating lesions of the disease. Reports of their presence in the conjunctivae, in the bronchial secretions from broncho- or lobar pneumonia, and in otitis media, have usually been based upon insuf- ficient bacteriological evidence. The occurrence of this microorganism in the circulating blood of men- ingitis cases has been definitely proved by Elser,1 who found it in ten cases. Animals are not very susceptible to infection with Diplococcus meningitidis. Subcutaneous inoculation is rarely followed by more than a local reaction unless large quantities are used. White mice are rather more susceptible than other species. Intraperitoneal and intra- venous inoculation of sufficient quantities usually results in the death of mice, rabbits, guinea-pigs, and dogs. Occasional strains have been found to possess a not inconsiderable degree of toxicity for rabbits, grave symptoms or even death following intravenous injection of but moderate quantities without any traceable development of the micro- organisms in the organs of the animals. Similar observations have been made by Albrecht and Ghon,2 who succeeded in killing white mice with dead cultures. It would seem therefore that the effect of this coccus upon animals depends chiefly upon the poisonous substances contained in the bacterial bodies (endo- toxins) . Lepierre 3 has obtained the meningococcus toxin by alcohol precipitation of broth cultures. Weichselbaum himself succeeded in producing meningeal suppura- 1 Elser, Jour. Med. Res., xiv, 1906. 2 Albrecht und Ghon., loc. cit. 3 Lepierre, Jour, de phys. et de path, gen., v, No. 3. 378 PATHOGENIC MICROORGANISMS tion and, in one case, brain abscess, by subdural inoculation of dogs. Councilman, Mallory, and Wright 1 produced a disease in many re- spects similar to the human disease by intraspinous inoculation of a goat. Recently, Flexner 3 has succeeded in producing in monkeys a condition entirely analogous to that occurring in human beings. Agglutination. — Immunization of animals by repeated inoculations of meningococcus 3 results in the formation in the blood serum of agglutinins. Kolle and Wassermann 4 obtained from horses a serum which had an agglutinating value of 1 : 3,000 for the homologous strain, and of as much as 1 : 500 for other true meningococcus strains. Similar experiments by Dunham 5 and others have proved the unquestionable value of agglutination for species identification of this group. Great differences may, however, exist between individual races in their agglutinability in the same immune serum. Kutscher has recently called attention to the fact that strains which can not be agglutinated in specific sera at 37° C. will often yield positive results when subjected to 55° C, a fact of some practical im- portance if confirmed. Elser and Huntoon 6 have shown that in the serum of infected human subjects agglutination of some strains takes place in dilutions as high as 1 : 400. Serum Therapy of Meningitis. — During recent years, many attempts have been made to treat epidemic cerebrospinal meningitis by injec- tions, subcutaneous and intraspinous, of meningococcus-immune serum. Wassermann,7 in 1907, reported the results obtained by such treatment in one hundred and two patients, with a recovery of 32.7 per cent. The serum, as manufactured by Wassermann and his associates, was obtained from horses immunized with pure cultures of meningococcus x and with toxic meningococcus extracts. More recently Flexner and Jobling 8 have used a similar serum in the United States with appa- rently excellent results. The serum, in Flexner's cases, is injected intraspinously after a quantity of spinal fluid had been withdrawn. The cases treated by Flexner and Jobling's method have now reached 1 Councilman, Mallory, and Wright, loc. cit. 2 Flexner, Jour. Exp. Med., 1906. 3 Albrecht and Ghon, Wien. klin. Woch., 1901. 4 Kolle und Wassermann, Deut. med. Woch., 15, 1906. 5 Dunham, Jour. Inf. Dis., 11, 1907. 6 Elser and Huntoon, loc. cit. 7 Wassermann, Deut. med. Woch., 39, 1907. 8 Flexner and Jobling, Jour. Exper. Med., x, 1908. MICROCOCCUS INTRACELLULARS MENINGITIDIS 379 large numbers, both in this and foreign countries and the value of the serum as a therapeutic agent seems firmly established. Hiss and Zinsser l have treated a number of meningitis patients with subcutaneous injections of leucocyte extracts and believe that they have favorably influenced the course of the disease. Pseudomeningococcus. — Elser and Huntoon 2 have described a diplo- coccus very similar to the meningococcus which they differentiated from it only by serum reactions. This diplococcus could be identified only by agglutinin absorption tests. They named it pseudomeningo- coccus. 1 Hiss and Zinsser, Jour. Med. Res., Nov., 1908. 2 Elser and Huntoon, Jour. Med. Res., xxi, 1909. CHAPTER XXV DIPLOCOCCUS GONORRHCE^ (GONOCOCCUS), MICROCOCCUS CATARRHALIS, AND OTHER GRAM-NEGATIVE COCCI DIPLOCOCCUS GONORRHOEA Neisser,1 in 1879, described diplococci which he had found regularly in the purulent secretions of acute cases of urethritis and vaginitis and in the acute conjunctivitis of the new-born. His researches were purely morphological, as were the numerous confirmatory investigations which rapidly followed his announcement. Cultivation of this diplococcus, now usually spoken of as gonococcus, was not definitely successful until 1885, when Bumm 2 obtained growth upon tubes of coagulated human blood serum. Bumm was not only able to keep the organisms alive by transplantation in pure culture, but produced the disease by inoculation of his cultures upon the healthy urethra. Morphology and Staining. — The gonococcus is usually seen in the diplococcus form, the pairs being characteristically flattened along the surfaces facing each other. This gives the cocci a peculiar coffee-bean or biscuit shape. The size of the diploforms is about 1.6 micra in the long diameter, about 0.8 micron in width. Stained directly in gonorrheal pus from acute cases, the microorganisms are found both intra- and extracellularly, a large number of them crowded characteristically within the leucocytes. They are never found within the nucleus. The phagocytosis which produces this picture has been shown by Scholtz 3 and others to take place in the free secretions, not in the depth of the tissues. The intracellular position-, which is of considerable diagnostic importance, is lost to a great extent in secretions from chronic cases. In smears made from pure cultures the arrangement in groups of two may often be less marked than in pus, clusters of eight or more being common. 1 Neisser, Cent. f. d. med. Wiss., 1879. 2 Bumm, " Beitr. z. Kenntniss des Gonococcus/' Wiesbaden, 1885. 3 Scholtz, Arch. f. Dermat., 1899. 330 DIPLOCOCCUS GONORRHOEAE 381 The gonococcus is non-motile and does not form spores. It is easily stained with the usual aqueous anilin dyes. Methylene-blue alone, or eosin followed by methylene-blue, or the neutral red stain of Plato,1 gives good results. Gram's method of staining, however, is the only one of differential value. With this method the gonococcus is rapidly decolorized and can be counterstained with fuchsin or Bismarck brown. The Gram stain applied to pus from the male urethra, while not absolutely reliable, is, for practical purposes, sufficiently so to make a diagnosis. In exudates from the vagina or from the eye the mor- phological picture is not so reliable, owing to the frequent presence Fig. 80. -Gonorrheal Pus from Urethra, showing the Cocci within a Leucocyte. in these regions of other Gram-negative cocci. The great scarcity of gonococci in very chronic discharges necessitates thorough cultural investigation; negative morphological examination in such cases can not be regarded as conclusive.2 Cultivation. — The gonococcus is extremely delicate and is difficult to cultivate. After many failures to grow it upon the ordinary media, Bumm 3 obtained his first growths upon human blood serum which had been heated to partial coagulation. The medium most commonly used at the present day was introduced i Plato, Berl. klin. Woch., 1894. 2 Heiman, Medical Record, 1896. 3 Bumm, Deut. med. Woch., 1885. 382 PATHOGENIC MICROORGANISMS by Wertheim,1 and consists of a mixture of two or three parts of meat infusion-agar with one part of uncoagulated human ascitic fluid, hydrocele fluid, or blood serum. The agar is melted and cooled to 45° before the serum is added. The mixture may then be slanted in the test tube or poured into a Petri plate. This medium may be improved by the addition to the agar of six per cent of glycerin or one per cent of *r w v r • * • " • .N • •• • •% • # * • i • * , «>». « * * • •% • . 9 \ • * " * • - ?•*.*•■ •; » w • • *• ■t * »% • .-#»■ * • • •• - .%- • • • * * • * 1 %* • * l • . • •♦* % • ••• «• #_ ■ «• t *.*'• •/ - 1 * • •. n %^% •• * * L 0 ■ .* * * , v . / # • % » * / •./ #• /* * <5 «• * N^ « i • 7- ^-"""^ Fig. 81. — Gonococcus. Smear from pure culture. glucose. Cultures in fluid media may be obtained by similar addi- tions of serum to meat-infusion-pepton-broth (pep ton, one to two per cent) . While human sera may be replaced by animal sera, these in general are not so favorable for growth of the gonococcus. They are useful chiefly in cases where it is difficult to obtain the human serum. Whole rabbit's blood added to agar, or the swine- Wertheim, Arch. f. Gynakol., 1892. DIPLOCOCCUS GONORRHCEiE 383 serum-nutrose medium of Wassermann * may occasionally be used with success. Plates may also be made by smearing for enrichment a drop of blood from the finger over the surface of agar in the manner of Pfeiffer's method for influenza-bacillus cultivation. Inoculations from gonorrheal material are best made by surface smearing upon plates, since the gonococcus grows best in the presence of free oxygen. Growth is more easily obtained and becomes more luxuriant after prolonged culti- Fig. 82. — Gonococcus Colony. Low power of magnification, and Wright.) (After Mallory vation upon artificial media. The most favorable reaction of media is neutrality or slight acidity. Whenever the gonococcus has been successfully cultivated from pus upon media without serum additions , the success has probably been due to the substances carried over in the pus. The gonococcus will develop a sparse growth under anaerobic con- ditions, but displays a very marked preference for aerobiosis. The op- timum temperature for growth is 37.5° C. Growth ceases above 38.5° and below 30°. ^Wassermann, Berl. klin. Woch., 1897. (Fifteen c.c. swine-serum, 35 c.c. of water, 3 c.c. glycerin, with two per cent nutrose. The nutrose is dissolved by boiling and the solution sterilized. This is then added to agar, in equal parts, and used in plates.) 384 PATHOGENIC MICROORGANISMS Upon suitable media colonies appear as extremely delicate, grayish, opalescent spots, at the end of twenty to twenty-four hours. The sepa- rate colonies do not tend to confluence and have slightly undulated margins. Touched with a platinum loop their consistency is found to be slimy or sticky. In fluid media, growth takes place chiefly at the surface and there is no general clouding. Resistance. — Cultures of gonococcus, if not transplanted, usually die out within five or six days at incubator temperature. At room temperature they die more rapidly. In the ice chest they may be kept alive for somewhat longer periods. In this they differ from meningo- cocci, which are always killed by temperatures approximating zero C. In these respects, however, individual strains show much variation, some cultures dying out after but a few hours' removal from the incubator. The resistance of the gonococcus to light and heat is very slight. A temperature of 41° to 42° kills it after a brief exposure. Complete drying destroys it in a short time. Incompletely dried, however, and protected from light (gonorrheal pus) it may live, on sheets and cloth- ing, for as long as eighteen to twenty -four hours.1 It is easily killed by most disinfectant solutions 2 even when these are highly diluted and seems to be almost specifically sensitive to the various silver salts, a fact of therapeutic importance. Pathogenicity. — Gonorrheal infection occurs spontaneously only in man. True gonorrheal urethritis has never been experimentally pro- duced in animals. In human beings, apart from the common seats of the infection in the male and female genital tracts, and in the conjunc- tivae, the gonococcus may produce cystitis, proctitis, and stomatitis. It may enter the general circulation, giving rise to septicemia 3 and, secondarily, to endocarditis and arthritis. Isolated cases of gonorrheal periostitis and osteomyelitis have been reported.4 The common acute infections of the genito-urinary passages in man are often followed by an indefinitely prolonged chronic infection, which, though quiescent, may for many years be a source of social danger. In children, especially females, the infection is not rare, and may assume epidemic characters, traveling from bed to bed in institu- tions. Such hospital epidemics can be stopped only by the most rigid isolation. It is advisable, in view of this danger, to examine all 1 Heiman, Medical Record, 1896. 2 Schaeffer und Steinschneider , Kong. Deut. Dermat. Gesells., Breslau, 1894. 3 Review of cases of Gon. Septicemia, Faure-Beaulieu, Thesis, Paris, 1906. * Ullmann, Wien. med. Presse, 1900. MICROCOCCUS CATARRHALIS 385 female children applying for admission to a hospital, by vaginal smear and, if possible, to keep them in a receiving ward for twenty- four hours in order that the examination may be repeated before admission to the general wards. In the best-equipped institutions, furthermore, separate thermometers, bed linen, and diapers are set aside for each child in order to preclude any possibility of accidental transmission from cases which may have escaped detection by smear examination. While inoculation of animals has never resulted in active prolifera- tion of the gonococcus upon the new host, local necrosis, suppuration, and temporary systemic reactions have been produced by subcutaneous and intraperitoneal inoculation. These are probably referable to the. endotoxin contained in the bodies of the gonococci. This toxin has been isolated by Nikolaysen l by extraction from the bacterial bodies with distilled water or sodium hydrate solutions. It was found to be resist- ant to a temperature of 120° and to remain potent after complete drying. The same author found that the isolated toxin and dead cultures were fully as toxic for animals as living cultures, 0.01 gram killing a white mouse. Specific injury to the nervous system by injections of gonococcus toxin has been reported by Moltschanoff.2 The secretion of a true soluble toxin by the gonococcus, asserted by Christmas,3 is denied by Wassermann,4 Nikolaysen,5 and others. The two authors last named, furthermore, do not believe that a general immunity is developed in subjects infected with gonococcus. Christ- mas 6 on the other hand, and, more recently, Torrey,7 have reported successful active immunization of animals by repeated injections of whole bacteria. Torrey and others apparantly have successfully treated human cases by injections of the serum of immunized animals. MICROCOCCUS CATARRHALIS Micrococcus catarrhalis is a diplococcus described first by R. Pfeiffer,8 who found it in the sputum of patients suffering from catarrhal in- 1 Nikolaysen, Cent. f. Bakt., 1897. 2 Moltschanoff, Munch, med. Woch., 1899. 3 Christmas, Ann. de l'inst. Pasteur, 1897. 4 Wassermann, Zeit. f. Hyg., xxvii, 1897. 5 Nikolaysen, Fort. d. Med., xxi, 1897. c Christmas, loc. cit. 7 Torrey, Jour. Amer. Med. Assn., xlvi, 1906. *FMgge, " Die Mikroorg.," 3d ed., 1896. 25 386 PATHOGENIC MICROORGANISMS flammations of the upper respiratory tract. It was subsequently care- fully studied by Ghon and H. Pfeiffer.1 According to these authors the pathogenic significance of the micrococcus is slight, though occasion- ally it may be regarded as the causative factor in catarrhal inflammations. Its chief claim to attention, however, lies in its similarity to the meningo- coccus and the gonococcus, from neither of which it can. be morphologi- cally distinguished. It is decolorized by Gram's stain, appears often in the diplococcus form, and has a tendency, in exudates, to be located intracellularly. Not unlike the two microorganisms mentioned, too, it shows but slight pathogenicity for animals. Differentiation from gonococcus is extremely simple in that Micro- coccus catarrhalis grows easily on simple culture media and shows none of the fastidious cultural requirements of the gonococcus. From meningococcus the differentiation is less simple and, because of the presence of both microorganisms in the nose, is of great impor- tance. Distinction between the two is made entirely upon cultural charac- teristics and agglutination reactions. Culturally, Micrococcus catar- rhalis grows more heavily than meningococcus upon .the ordinary culture media. The colonies of Micrococcus catarrhalis are coarsely granular and distinctly white in contradistinction to the finely granu- lar, grayish meningococcus colonies.2 Micrococcus catarrhalis will develop at temperatures below 20° C, while meningococcus will not grow at temperatures below 25° C.3 Dunham,4 who has recently made a comparative study of meningo- coccus and other Gram-negative diplococci from the nose and throat, states that while some of the supposed Micrococcus catarrhalis cul- tures are easily distinguished from meningococcus simply by the char- acteristics of their growths upon two-per-cent glucose agar, others offer great difficulties to differentiation. He recommends as a differential medium a mixture of sheep serum and bouillon containing one per cent of glucose. Upon this medium all true meningococci produce acid, but no coagulation, with twenty-four hours. Cultures from the nose and throat, however, produce acid and coagulation, or else pro- duce an alkaline reaction. 1 Ghon und H. Pfeiffer, Zeit. f. klin. Med., 1902. 2 Ghon und Pfeiffer, loc. cit. 3 Weichselbaum, in Kolle und Wassermann, Bd. iii, p. 269. * Dunham, Jour. Inf. Dis., 1907. GRAM-NEGATIVE COCCI 387 OTHER GRAM-NEGATIVE COCCI Micrococcus pharyngis siccus. — This organism was first described by von Lingelsheim 1 in 1906. It is described by Elser and Huntoon as readily differentiable from meningococcus and other Gram-negative cocci by the firm adherence and dryness of its colonies. It is similar to Micrococcus catarrhalis from which it may, however, be differentiated by fermentation tests. Diplococcus mucosus. — This organism was also described by von Lingelsheim together with the preceding one. Its colony formation is similar to that of meningococcus, but slightly more sticky and mucoid. Stained by the capsule methods, it is seen to possess a distinct capsule. Chromogenic Gram-negative Cocci. — These microorganisms all pro- duce a greenish-yellow pigment on the ordinary culture media. When pigment is absent, as is frequently the case when grown upon sugar-free media, these microorganisms can be distinguished from meningococcus only by sugar fermentation and serum reactions. An exhaustive study of Gram-negative micrococci has recently been made by Elser and Huntoon.2 These authors, in studying the differential value of sugar fermentation in the diagnosis of these bacteria, have constructed the following table: Strains Tested. Meningococcus Pseudomeningococcus Gonococcus Micrococcus catarrhalis Micrococcus pharyngis siccus Chromogenic group I Chromogenic group II Chromogenic group III Jaeger meningococcus, Krai. Diplococcus crassus, Krai . . . oi w 03 h! o o m o Hi 200 + + 0 0 0 6 + 4- 0 0 0 15 + 0 0 0 0 64 0 0 0 0 0 2 + + + + 0 28 + + + + 0 11 + + + 0 0 9 + + 0 0 0 1 + + + + + 1 + + + + + c3 o 0 0 0 0 0 0 0 0 + + v. Lingelsheim, Klin. Jahrb., 15, 1906. 2 Elser and Huntoon, loc. cit. CHAPTER XXVI BACILLI OF THE COLON-TYPHOID-DYSENTERY GROUP The bacilli belonging to this group of microorganisms, while present- ing great differences in their pathogenic characteristics, possess many points of morphological and biological similarity which have made their differentiation extremely difficult. Among pathogenic bacilli, they are probably the ones most commonly encountered and because of the fact that some of them are specifically pathogenic, while others are essen- tially saprophytic and are pathogenic only under exceptional conditions, the necessity of accurate differentiation is a daily occurrence in bacteri- ological laboratories. It has been through the study of this group par- ticularly that many of the modern differential methods of bacteriology have been developed. The group includes the colon bacillus and its allies, the typhoid bacillus, the paratyphoid organisms, the several varieties of dysentery bacillus and numerous closely related species, and Bacillus fecalis alka- ligenes. Closely related to the group though not properly within it, are Bacillus lactis aerogenes, bacilli of the Friedlander or mucosus capsulatus group, and a number of less important subdivisions of this last group. All bacilli of the group possess morphological characteristics which, although exhibiting slight differences, are insufficient to permit accurate morphological diagnosis. They are none of them spore-bearing. Stained by Gram's method they are decolorized. Cultivated upon artificial media, they grow readily both at room and at incubator temperatures. None of them liquefies gelatin. Though showing, often, distinct differences in the speed and luxuriance of growth upon ordinary media, these differences are, nevertheless, too slight to become the basis of differentiation. In order to distinguish between the individual members of this group, therefore, we are forced to a careful biological and cultural study. This is carried out by the observation of the cultural character- istics upon special media and by the study of Serum reactions in speci- fic immune sera. Our mainstays in the accurate differentiation of these 388 BACILLUS COLI COMMUNIS 389 bacilli are the observation of their fermentative action upon carbohy- drate media, and their agglutinating reactions in immune sera. These points will be alluded to in the description of the individual microor- ganisms, and will be again summarized in the differential tables given at the end of the chapters dealing with this group. BACILLUS COLI COMMUNIS AND MEMBERS OF THE COLON BACILLUS GROUP Under the name of " colon bacilli " are grouped a number of bac- terial varieties differing from one another somewhat in minor character- istics, but corresponding in certain cardinal points which stamp them as close relatives and amply warrant their consideration under one heading. While usually living as harmless parasites upon the animal and human body, and capable of leading a purely saprophytic existence, they may, nevertheless, under certain circumstances become pathogenic and thus, both culturally and in their pathological significance, form a link between pure saprophytes like Bacillus lactis aerogenes, on the one hand, and the more strictly pathogenic Gram-negative bacilli of the paratyphoid, typhoid, and dysentery groups, on the other. As a type of the group we may consider in detail its most prominent and thoroughly studied member, Bacillus coli communis. BACILLUS COLI COMMUNIS This microorganism was seen and described by Buchner * in 1885. It was thoroughly studied in the years immediately following, especially by Escherich,2 in connection with the intestinal contents of infants. Morphology. — Bacillus coli communis is a short, plump rod about 1-3 micra long, and varying in thickness from one-third to one-fifth of its length. Under varying conditions of cultivation, it may appear to be more slender than this or shorter and even coccoid in form. In stained preparations, it usually appears singly, but occasionally may be seen in short chains. It stains readily with the usual anilin dyes and decolorizes by Gram's method. Spores are not formed. It is motile, and flagella staining reveals eight or more flagella peripherally arranged. Its motility is subject to wide variations. Young cultures, in the first gen- i Buchner, Arch. f. Hyg., 3, 1885. 2 Escherich, "Die Darmbakt. des Sauglings," Stuttgart, 1886; Cent. f. Bakt., 1, 1887. 390 PATHOGENIC MICROORGANISMS eration, after isolation from the body, may be extremely motile, while old laboratory strains may show almost no motility. Independent of these modifying conditions, however, separate races may show individual characteristics as to motility, varying in range between a motility hardly distinguishable from Brownian movement and one which is so active as to be but little less than that of the typhoid bacillus. Ordi- narily, the colon bacillus possesses a motility intermediate between these two extremes. Cultivation. — The bacillus is an aerobe capable of anaerobic growth under suitable cultural conditions. It grows well on the simplest media Fig. 83. — Bacillus coli communis. at temperatures ranging from 20° to 40° C, but finds its optimum growth at about 37.5° C. Upon broth it grows rapidly, giving rise to general clouding; later to a pellicle and a light, slightly slimy sediment. Within moderate ranges, it is not delicately susceptible to reaction, growing equally well on media slightly acid and on those of a moderate alkalinity. Upon agar, it forms grayish colonies which become visible within twelve to eighteen hours, gradually becoming more and more opaque as they grow older. The deep colonies are dense, evenly granular, oval; BACILLUS COLI COMMUNIS 391" or round. Surface colonies often show a characteristic grape-leaf structure, or may be round and flat, and show a definitely raised, glisten- ing surface. Upon agar slants, growth occurs in a uniform layer. On gelatin the colon bacillus grows rapidly, causing no liquefaction. Surface colonies are apt to show the typical grape-leaf formation. Deep colonies are round, oblong, and glistening. In gelatin stabs growth takes place along the entire line of inoculation, spreading in a thin layer over the surface of the medium. On potato, growth is abundant and easily visible within eighteen to twenty-four hours, as a grayish-white, glistening layer which later turns to a yellowish-brown, and in old cultures often to a dirty green- ish-brown color. In pepton solution indol is formed. In milk there is acidity and co- agulation. In lactose-litmus-agar acid is formed, the medium becom- ing red, and gas-bubbles appear along the line of the stab inoculation. In carbohydrate broth, gas is formed in dextrose, lactose, and mannit, but not in saccharose. Levulose, galactose, and maltose are also fer- mented with the formation of acid and gas. Cultures of the colon bacillus are characterized by a peculiar fetid odor which is not unlike that of diluted feces. The acids formed by the colon bacillus from sugars are chiefly lactic, acetic, and formic acids. The gas it produces consists chiefly of C02 and hydrogen. The bacillus grows well on media containing urine and on those containing bile. Upon the latter fact some methods for the isolation of the colon bacillus from water and feces have been based. Isolation of the colon bacillus from mixed cultures is most easily accomplished by plating upon lactose-litmus-agar, the Conradi-Drigal- ski medium, or the Endo medium after preliminary enrichment of the specimen to be tested in bile or malachite-green broth. (In the case of feces such enrichment is superfluous.) Distribution. — The colon bacillus is a constant inhabitant of the intestinal canal of human beings and animals. It is also found occasion- ally in soil, in air, in water, and in milk and is practically ubiquitous in all neighborhoods which are thickly inhabited. When found in nature its presence is generally taken to be an indication of contamination from human or animal sources. Thus, when found in water or milk, much hygienic importance is attached to it. Recently, Papasotiriu x and, independently of him, Prescott,2 have reported finding bacilli apparently 1 Papasotiriu, Arch. f. Hyg., xli. 2 Prescott, Cent. f. Bakt., Ref., xxxiii, 1903. 392 PATHOGENIC MICROORGANISMS identical with Bacillus coli upon rye, barley, and other grains. They believe, upon the basis of this discovery, that Bacillus coli is widely distributed in nature and that its presence, unless it appears in large numbers, does not necessarily indicate recent fecal contamination. These reports, however, have not found confirmation by the work of others, and can not, therefore, be as yet accepted. In man, Bacillus coli appears in the intestine normally soon after birth, at about the time of taking the first nourishment.1 From this time on, throughout life, the bacillus is a constant intestinal inhabitant ap- parently without dependence upon the diet. Its distribution within the intestine, according to Gushing and Livingood,2 is not uniform, it being ( found in the greatest numbers at or about the ileocecal valve, diminish- ing from this point upward to the duodenum and downward as far as the rectum. Adami 3 and others claim that, under normal conditions, the bacillus may invade the portal circulation, possibly by the inter- mediation of leucocytic emigration during digestion. After death, at autopsy, Bacillus coli is often found in the tissues and the blood with- out there being visible lesions of the intestinal mucous membrane.4 It is probable, also, that it may enter and live in the circulation a few hours before death during the agonal stages. Extensive investigations have been carried out to determine wheth- er or not the constant presence of this microorganism in the intestinal tract is an indication of its possessing a definite physiological function of advantage to its host. It has been argued that it may aid in the fermen- tation of carbohydrates. The question has been approached experiment- ally by a number of investigators. Nuttall and Thierfelder 5 delivered guinea-pigs from the mother by Cesarean section and succeeded in keeping them without infection of the intestinal canal for thirteen days. Although no microorganisms of any kind were found in the feces of these animals, no harm seemed to accrue to them, and some of them even gained in weight. Schottelius,6 on the other hand, obtained con- tradictory results when working with chicks. Allowing eggs to hatch in an especially constructed glass compartment, he succeeded in keeping the 1 Schild, Zeit. f. Hyg., xix, 1895; Lembke, Arch. f. Hyg., xxvi, 1896. 2 Cushing and Livingood, " Contributions to Med. Sci. by Pupils of Wm. Welch," Johns Hopk. Press, 1900. 3 Adami, Jour, of Amer. Med. Assn., Dec, 1899. * Birch-Hirschfeld, Ziegler's Beitr., 24, 1898. 5 Nuttall und Thierfelder, Zeit. f. Physiol. Chemie, xxi and xxii. 6 Schottelius, Arch. f. Hyg., xxxiv, 1889. BACILLUS COLI COMMUNIS 393 chicks and their entire environment sterile for seventeen days. During this time they lost weight, did not thrive, and some of them were mori- bund at the end of the second week, in marked contrast to the healthy, well-nourished controls, fed in the same way, but under ordinary environmental conditions. Although insufficient work has been done upon this important question, and no definite statement can be made, it is more than likely that the function of the Bacillus coli in the intes- tine is not inconsiderable if only because of its possible antagonism to certain putrefactive bacteria, a fact which has been demonstrated in interesting studies by Bienstock 1 and others.2 Pathogenicity. — The pathogenicity of the colon bacillus for animals is slight and varies greatly with different strains. Intraperitoneal in- jections of 1 c.c. or more of a broth culture will often cause death in guinea-pigs. Large doses intravenously administered to rabbits may frequently cause a rapid sinking of the temperature and death with^ symptoms of violent intoxication within twenty -four to forty-eight hours. Subcutaneous inoculation of moderate doses usually results in nothing more than a localized abscess from which the animals recover. It is likely that, even in fatal cases, death results chiefly from the action of poisons liberated from the disintegrating bacteria and not from the multipli- cation of the bacilli themselves, for often no living organism can be found unless large doses are given. In man, a large variety of lesions produced by Bacillus coli have been described. It is a surprising fact that disease should be caused at all, in man, by a microorganism which is so constantly present in large numbers in the intestine and against which, therefore, it is to be expected that a certain amount of immunity should be developed. A number of explanations for this state of affairs have been advanced, none of them entirely satisfactory. It is probable that none of the poi- sonous products of the colon bacillus is absorbed unchanged by the healthy unbroken mucosa and that, therefore, the microorganisms are, strictly speaking, at all times, outside of the body proper. Under these circumstances, no process of immunization would be anticipated. It is also possible that, whenever an infection with Bacillus coli does occur, the infecting organism is one which has been recently acquired from another host, having no specific adaptation to the infected body. Viru- lence may possibly be enhanced by inflammatory processes caused by other organisms. Considering the subject from another point of view, 1 Bienstock, Arch. f. Hyg., xxxix, 1901. 2 Tissier and Martelly, Ann. de l'inst. Pasteur, 1902. 394 PATHOGENIC MICROORGANISMS colon-bacillus infection may possibly take place simply because of unu- sual temporary reduction of the resistance of the host. Whether or not altered cultural conditions in the intestine may lead to marked enhance- ment in the virulence of the colon bacilli can not at present be decided. The opinion has been frequently advanced, however, without adequate experimental support. Septicemia, due to the colon bacillus, has been described by a large number of observers. It is doubtful, however, whether many of these cases represent an actual primary invasion of the circulation by the bacilli, or whether their entrance was not simply a secondary phenomenon occurring during the agonal stages of another condition. A few unques- tionable cases, however, have been reported, and there can be no doubt about the occurrence of the condition, although it is probably less frequent than formerly supposed. The writers have observed it on two occasions in cases during the lethal stages of severe systemic disease due to other causes. An extremely interesting group of such cases are those occurring in new-born infants, in which generalized colon-bacillus infection may lead to a fatal condition known as WinckeFs disease or hemorrhagic septicemia.1 Prominent among disease processes attributed to these microorganisms are various diar- rheal conditions, such as cholera nostras and cholera infantum. The relation of these maladies to the colon bacillus has been studied es- pecially by Escherich,2 but satisfactory evidence that these bacilli may specifically cause such conditions has not been brought. While it is not unlikely that under conditions of an excessive carbohydrate diet, colon bacilli may aggravate morbid processes by a voluminous formation of gas, they do not, of themselves, take part in actual putrefactive proc- esses. It is likely, therefore, that in most of the intestinal diseases formerly attributed purely to bacilli of the colon group, these micro- organisms actually play but a secondary part.3 It is equally difficult to decide whether or not these bacilli may be regarded as the primary cause of peritonitis following perforation of the gut. Although regularly found in such conditions, they are hardly ever found in pure culture, being accompanied usually by staphylococci, streptococci, or other microorganisms, whose relationship to disease is far more definitely established. Isolated cases have been reported, however, one of them by Welch,1 in which Bacillus coli was present in 1Kamen, Ziegler's Beitr., 14, 1896. 2 Escherich, loc. cit. sHerter, " Bact. Infec. of Digest. Tract/' N. Y., 1907. BACILLUS COLI COMMUNIS 395 the peritoneum in pure culture without there having been any intestinal perforation. Granting that the bacillus is able to proliferate within the peritoneum, there is no reason for doubting its ability of giving rise to a mild suppurative process. Inflammatory conditions in the liver and gall-bladder have fre- quently been attributed to the colon bacillus. It has been isolated from liver abscesses, from the bile, and from the center of gall-stones. Welch has reported a case of acute hemorrhagic pancreatitis in which the bacillus was isolated from the gall-bladder and from the pancreas. In the bladder, Bacillus coli frequently gives rise to cystitis and oc- casionally to ascending pyonephrosis. No other microorganism, in fact, is found so frequently in the urine as this one. It may be present, often, in individuals in whom all morbid processes are absent. The condition is frequently observed during the convalescence from t}^phoid fever. It may disappear spontaneously, or cystitis, usually of a mild, chronic variety, may supervene. - ' Localized suppurations due to this bacillus may take place in all parts of the bod}^. They are most frequently localized about the anus and the genitals. They are usually mild and easily amenable to the ■ simplest surgical treatment. Poisonous Products of the Colon Bacillus.— The colon bacillus belongs essentially to that group of bacteria whose toxic action is supposed to be due to the poisonous substances contained within the bacillary body. Culture filtrates of the colon bacillus show very little toxicity when in- jected into animals; whereas the injection of dead bacilli produces symptoms almost equal in severity to those induced by injection of the live microorganisms. Corroborative of the assumption of this endotoxic nature of the colon-bacillus poison is the fact that, so far, no antitoxic bodies have been demonstrated in serum as resulting from immuniza- tion. Immunization with the Colon Bacillus. — The injection into animals of gradually increasing doses of living or dead colon bacilli gives rise to srjecific bacteriolytic, agglutinating, and precipitating substances. The bacteriolytic substances may be easily demonstrated by the technique of the Pfeiffer reaction. In vitro bacteriolysis is less marked than in the case of some other microorganisms such as the cholera spiril- lum or the typhoid bacillus. Owing probably to the habitual presence of colon bacilli in the intestinal tracts of animals and man, considerable 1 Welch, Med. News, 59, 1891. 396 PATHOGENIC MICROORGANISMS bacteriolysis may occasionally be demonstrated in the serum of normal individuals. Agglutinins for the colon bacillus have often been produced in the sera of immunized animals in concentration sufficient to be active in dilu- tions of 1 : 5,000 and over. The agglutinins are produced equally well by the injection of live cultures and of those killed by heat, if the tem- perature used for sterilization does not exceed 100° C. It is 1 a notice- 1 2 3 Fig. 84. — Bacillus coli communis. Grown in: 1. Dextrose, 2. Lactose, 3. Saccharose broth. The bacillus forms acid and gas from dextrose and lactose, not from saccharose. Note the absence of growth in the closed arm of the sac- charose tube, in which no acid or gas is formed. able fact that the injection of any specific race of colon bacilli produces, in the immunized animal, high agglutination values only for the individual culture used for immunization, while other strains of colon bacilli, although agglutinated by the serum in higher dilution than are paratyphoid or typhoid bacilli, require much higher concen- tration than does the original strain. The subject has been extensively studied by a number of observers and illustrates the extreme individual i Wolff, Cent. f. Bakt., xxv, 1899. BACILLUS COLI COMMUNIS 397 specificity of the agglutination reaction. Thus a serum which will agglutinate its homologous strains in dilutions of one 1: 1,000 will often fail to agglutinate other races of Bacillus coli in dilutions of 1 : 500 or 1 : 600. The normal serum of adult animals and man will often agglutinate this bacillus in dilutions as high as 1 : 10 or 1 : 20 — a phenomenon pos- sibly referable to its habitual presence within the body. Corrobo- rating this assumption is the observation of Kraus and Low/ that the serum of new-born animals possesses no such agglutinating powers. The fact that agglutinins for the colon bacillus are increased 12 3 Fig. 85. — Bacillus coli communior. Grown in: 1. Dextrose, 2. Lactose, 3. Saccharose broth. in the serum of patients convalescing from typhoid fever or dysentery is probably to be explained, partly by the increase of the group agglutinins produced by the specific infecting agent, and partly by the invasion of colon bacilli, or the absorption of its products induced by the diseased state of the intestinal mucous membrane. Varieties of the Colon Bacillus. — During the earlier days of bacteriolog- ical investigations, a large number of distinct varieties of colon bacilli were described, many of which may now be dismissed as based simply 1 Kraus und Low, Wien. klin. Woch., 1899. 398 PATHOGENIC MICROORGANISMS upon a temporary depression of one or another cultural characteristic of Bacillus coli communis, while others can be definitely included within other closely related, but distinct groups. That secondary features, such as dimensions, motility, and luxuri- ance of growth upon various media, may be markedly altered by arti- ficial cultivation is a common observation. It has not, however, been satisfactorily shown that cardinal characteristics, such as the forma- tion of indol from pepton, or the power to produce gas from dextrose and lactose, can be permanently suppressed without actual injury or inhibition of the normal vitality of the microorganism. . Such alter- ation is, in fact, contrary to experience, which demonstrates that whenever such changes do occur, they are purely temporary and a few generations of cultivation under favorable environmental conditions will regularly restore the organism to its normal activity. Bacillus coli communior. — Distinct and constant varieties of Bacil- lus coli, however, do occur. The most common of these is one which Dunham has named Bacillus coli communior, because of the fact that he believes it to be more abundant in the human and animal intestine than is coli communis itself. This bacillus possesses all the cardinal characteristics of the colon group. It is a Gram-negative bacillus, moderately motile, non-sporulating, and morphologically indistinguish- able from the communis variety. It does not liquefy gelatin, it produces indol from pepton, coagulates and acidifies milk, and grows characteristically upon agar and potato. It differs from Bacillus coli communis in that it produces acid and gas from saccharose as well as from dextrose and lactose, whereas the former does not form acid or gas from saccharose. CHAPTER XXVII BACILLI OF THE COLON-TYPHOID-DYSENTERY GROUP {Continued) THE BACILLUS OF TYPHOID FEVER {Bacillus typhosus, Bacillus typhi abdominalis) Typhoid fever, because of its wide distribution and almost con- stant presence in most communities, has from the earliest days been the subject of much etiological inquiry. A definite conception as to its infectiousness and transmission from case to case was formed as early as 1856 by Budd.1 < But it was not until 1880 that Eberth 2 discovered in, the spleen and mesenteric glands of typhoid-fever patients who had come to autopsy, a bacillus which we now know to be the cause of the disease. Final proof of such an etiological connection was then brought by Gaffky,3 who not only saw the bacteria referred to by Eberth, but succeeded in obtaining them in pure culture and studying their growth characteristics. Morphology and Staining. — The typhoid bacillus is a short rod from 1-3.5 fi in length with a varying width of from .5 to .8 fi. In appear- ance it has nothing absolutely distinctive which could serve to differen- tiate it from other bacilli of the typhoid-colon group, except that it has a general tendency to greater slenderness. Its ends are rounded without ever being club-shaped. Contrary to the descriptions of the earliest observers, typhoid bacilli do not form spores. They are actively motile and have twelve or more flagella peripherally arranged. The bacilli stain readily with the usual anilin dyes. Stained by Gram's method, they are decolorized. Cultivation. — Bacillus typhosus is easily cultivated upon the usual laboratory media. It is not delicately susceptible to reaction, but will grow well upon media moderately alkaline or acid. It is an aerobic and facultative anaerobic organism, when the proper nutriment is present. Upon agar plates growth appears within eighteen to twenty-four hours lBudd, " Intestinal Fever," Lancet, 1856. 2 Eberth, Virch. Archiv, 81, 1880, and 83, 1881. ? Gaffky, Mitt. a. d. kais. Gesundheitsamt, 2, 1884. 399 400 PATHOGENIC MICROORGANISMS as small grayish colonies at first transparent, later opaque. Upon agar slants growth takes place in a uniform layer. There is nothing charac- teristic about this growth to aid in differentiation. In broth, the typhoid bacillus grows rapidly, giving rise to an even clouding, rarely to a pellicle. Upon gelatin, the typhoid bacillus grows readily and does not liquefy the medium. In stabs, growth takes place along the entire extent of the stab and over the surface of the gelatin in a thin layer. In gela- tin plates the growth may show some distinction from that of other mem- bers of this group, and this medium was formerly much used for isolation Fig. 86. — Bacillus typhosus, from twenty-four-hour culture on agar. of the bacillus from mixed cultures. Growth appears within twenty- four hours as small, transparent, oval, round, or occasionally leaf-shaped colonies which are smaller, more delicate, and more transparent than contemporary colonies of the colon bacillus. They do not, however, show any reliable differential features from bacilli of the dysentery group. As the colonies grow older they grow heavier, more opaque, and lose much of their early differential value. On potato the growth of typhoid bacilli is distinctive, and this medium was recommended by Gaffky l in his early researches for purposes of 1 Gaffky, loc. cit. BACILLUS OF TYPHOID FEVER 401 identification. On it typhoid bacilli, after twenty-four to forty-eight hours, produce a hardly visible growth, evident to the naked eye only by a slight moist glistening, an appearance which is in marked contrast to the grayish-yellow or even brown and abundant growth of the colon bacilli. If the potato medium is rendered neutral or alkaline, this distinction disappears, the typhoid bacillus growing more abundantly. In milk } typhoid bacilli do not produce coagulation. In litmus-milk, during the first twenty-four hours, the color is changed to a reddish or violet tinge by the formation of acid from the small quantities of mono- Fig. 87. — Bacillus typhosus, showing flagella. (After Frankel and Pfeiffer.) saccharid present. Later the color becomes deep blue from the forma- tion of alkali. In Dunham's pepton solution no indol is produced. According to Peckham, however, continuous cultivation in rich pepton media may lead to eventual indol formation by typhoid bacilli. This fact appears to have no bearing on the value of the indol test, as indol is never formed under the usual cultural conditions. In dextrose, mann t, lactose, and saccharose broth, the typhoid bacil- lus produces no gas. A comparative summary of the action of other bacilli of this group in these sugar media will be given in the final dif- ferential table on page 443. 26 402 PATHOGENIC MICROORGANISMS Tested for its power to form acid from sugars commonly used in differential tests, typhoid bacilli give the following reactions: Acid formation Dextrose + Levulose + Galactose + Mannit + Acid formation Maltose + Lactose — Saccharose — Dextrin + In the Hiss tube medium (see section on Media, page 133), the typhoid bacillus within eighteen to twenty -four hours produces an even clouding by virtue of its motility, but does not form gas. In contradis- tinction to this, dysentery bacilli grow only along the line of inocula- A Fig. 88. — Surface Colony of Bacillus typhosus on Geiatin. (After Heim.) tion, while bacilli of the colon group move in irregular sky-rocket-like figures away from the stab, at the same time breaking up the medium by the formation of gas-bubbles. Some actively motile colon bacilli cloud the medium, but the ruptures caused by the gas are always evident. The differentiation of the typhoid bacillus in pure culture from similar microorganisms by means of its growth upon media has been the sub- ject of many investigations. It is neither practicable nor desirable to enumerate all the various media which have been devised and reported. BACILLUS OF TYPHOID FEVER 403 The aim has been chiefly the differentiation of typhoid bacilli from the bacilli of the colon group, and most of the media have been devised with this end in view. (See section on Media.) Rothberger l has devised a mixture of glucose agar to which is added one per cent of a saturated aqueous solution of neutral-red. Shake-cul- tures or stab-cultures are made in tubes of this medium. The typhoid bacillus causes no changes in it, while members of the colon group, by reduction of the neutral-red, decolorize the medium and produce gas by fermentation of the sugar. Utilizing the fact that bile-salts are precipitated in the presence of acids, Macconkey devised a medium composed of sodium glycocholate, pepton, lactose, and agar (the composition of this medium is given on page 138), in which Bacillus typhosus grows without causing much change, but distinct clouding results from the growth of the colon bacillus which, producing acid from the lactose, causes precipitation of the bile- salts. On Wurtz's lactose-litmus-agar (see page 129) the typhoid bacillus produces no acid, but eventually deepens the purple color to blue; the colon bacillus produces acid and in stab-cultures gas bubbles and the color changes to red. In Barsiekow's (see page 139) lactose-nutrose-litmus mixture the typhoid bacillus causes no change, while the colon bacillus produces coagulation and an acid reaction. Especially designed for the isolation of typhoid bacilli from the feces, are the media of Drigalski and Conradi, the agar-gelatin media of Hiss, the medium of Hesse, the fuchsin medium of Endo, and the malachite-green media of Loeffler, and others. These media have all been described in detail in the section on the preparation of media, pages 133-138. Biological Considerations. — The typhoid bacillus is an aerobic and facultatively anaerobic organism growing well both in the presence and in the absence of oxygen when certain sugars are present, showing a slight preference, however, for well aerated conditions. It grows most luxu- riantly at temperatures about 37.5° C, but continues to grow within a range of temperature lying between 15° and 41° C. Its thermal death point, according to Sternberg, is 56° C. in ten minutes. It remains alive in artificial cultures for several months or even years if moisture is sup- plied. In carefully sealed agar tubes Hiss has found the organisms 1 Rothberger, Cent. f. Bakt., xxiv, 1898. 404 PATHOGENIC MICROORGANISMS alive after thirteen years. In natural waters it may remain alive as long as thirty-six days, according to Klein.1 In ice, according to Prud- den,2 it may remain alive for three months or over. Against the ordi- nary disinfectants, the typhoid bacillus is comparatively more resistant than some other vegetative forms. It is killed, however, by 1 : 500 bichlorid or five-per-cent carbolic acid within five minutes. Pathogenicity. — In animals, some early investigators to the contrary, typhoidal infection does not occur spontaneously and artificial inocula- tion with the typhoid bacillus does not produce a disease analogous to typhoid fever in the human being. Frankel 3 was able to produce intes- tinal lesions in guinea-pigs by injection of the bacilli into the duodenum, and recovered the bacteria from the spleens of the animals after death, but the disease produced was in no other respect analogous to typhoid fever in the human being. It is probable that typhoid bacilli injected into animals do not multiply extensively and that most of the symp- toms produced are due to the endotoxins liberated from the dead bac- teria. In corroboration of this view is the observation that inoculation with dead cultures is followed by essentially the same train of symp- toms as inoculation with live cultures.4 The injection of large doses into rabbits or guinea-pigs intravenously or intraperitoneally is usually followed by a rapid drop in temperature, often by respiratory em- barrassment and diarrhea. Occasionally blood may be present in the stools. According to the size of the dose or the weight of the ani- mal, death may ensue within a few hours, or, with progressive emacia- tion, after a number of days, or the animal may gradually recover. Welch and Blachstein 5 have shown that typhoid bacilli injected into the ear vein of a rabbit appear in the bile and may persist in the gall- bladder for weeks. Typhoid bacilli isolated from different sources may show considerable variations in virulence and toxicity. In man the overwhelming majority of typhoid infections take the form of the disease clinically known as typhoid fever. For a descrip- tion of the clinical course and pathological lesions of the disease, the reader is referred to the standard text-books of medicine and pathology. During the course of the disease, and during convalescence, the bacilli may be cultivated from the circulating blood, the rose spots, the feces, 1 Klein, Med. Officers' Report, Local Govern. Bd., London, 1894. 2 Prudden, Med. Rec, 1887. * Frankel, Cent. f. klin. Med., 10, 1886. *Petruschky, Zeit. f. Hyg., xii, 1892. 5 Welch and Blachstein, Bull. Johns Hop. Hosp., ii, 1891. BACILLUS OF TYPHOID FEVER 405 the urine, and in exceptional cases from the sputum. At autopsy the bacilli may be obtained from these sources as well as from the lesions in the intestine, the spleen, and often from the liver, kidneys, and from the gall-bladder. Typhoid Bacilli in the Blood during the Disease. — The investigations of many workers have shown that typhoid bacilli are present in the circulating blood of practically all patients during the early weeks of the disease. Series of cases have been studied by Castellani,1 Schottmuel- ler,2 and many others. More recently Coleman and Buxton 3 have reported their researches upon 123 cases, and have at the same time analyzed all cases previously reported. Their analysis of blood cultures taken at different stages in the disease is as follows: Of 224 cases during first week, 89 per cent were positive. Of 484 cases during second week, 73 per cent were positive. Of 268 cases during third week, 60 per cent were positive. Of 103 cases during fourth week, 38 per cent were positive. Of 58 cases after fourth week, 26 per cent were positive. The technique recommended by Coleman and Buxton for obtaining blood cultures is that recommended by Conradi,4 slightly modified. The blood is taken into flasks each containing about 20 c.c. of the following mixture : Ox bile 900 c.c. Glycerin 100 c.c. Pepton 20 grams. About 3 c.c. of blood are put into each flask. The ox-bile, besides preventing coagulation, may possibly neutralize the bactericidal sub- stances present in the drawn blood. The flasks are incubated for eigh- teen to twenty -four hours, at the end of which time streaks are made upon plates of lactose-litmus-agar and the organisms identified by agglutination or by cultural tests. European workers have generally preferred to make high dilution of the blood in flasks of bouillon, small quantities of blood, 1 to 2 c.c, being mixed with 100 to 150 c.c. of nutrient broth. Epstein 5 has reported excellent results from mixing the blood in 1 Castellani, Riforma medica, 1900. 2 Schottmueller,Deut. med. Woch., xxxii, 1900, and Zeit. f. Hyg., xxxvi, 1901. 3 Coleman and Buxton, Am. Jour, of Med. Sci., 133, 1907. * Conradi, Deut. med. Woch., xxxii, 1906. s Epstein, Proc. N. Y. Path. Soc, N. S., vi„ 1906. 406 PATHOGENIC MICROORGANISMS considerable concentration with two-per-cent glucose agar and pouring plates. The writers in hospital work have had equally good results with the bile medium and with broth in flasks, rather less uniform but still satis- factory results with the plating method. In general it may be said that any one of these methods carried out with reasonable accuracy may be satisfactorily employed. Typhoid Bacilli in the Stools. — The examination of the stools for typhoid bacilli is performed for diagnostic purposes chiefly in obscure cases. It may, furthermore, furnish information of extreme hygienic importance. Thus Drigalski 1 and Conradi have succeeded in Fig. 89. — Bacillus coli. Deep colonies on Hiss plate medium. isolating typhoid bacilli from the stools of ambulant cases so mild that they were not clinically suspected. It is by means of such examina- tions that the so-called t}rphoid-carriers are detected, cases which, though perfectly well themselves, may be a means of spreading the disease. Such cases have been known to harbor the bacilli for periods as long as several years. The examination itself is fraught with great difficulties, owing to the preponderating numbers of colon bacilli found in all feces and the difficulty of isolating the typhoid bacilli from such mixtures. Reviewing the data collected by a number of investigators, it seems probable that the bacilli do not appear in the stools, at least in 1 Drigalski and Conradi, Zeit. f. Hyg., xxxix, 1902. BACILLUS OF TYPHOID FEVER 407 numbers sufficient for recognition, much before the middle of the sec- ond week, or, in other words, as pointed out by Hiss, about the time that the intestinal lesions are well advanced and ulceration is occur- ring. Thus Wiltschour l could not determine their presence before the tenth day; Redtenbacher,2 in reviewing the statistics, states that in a majority of cases the bacilli first appear toward the end of the second week, and Horton-Smith 3 could not find the bacilli before the eleventh Fig. 90. — Bacillus typhosus. Deep colonies in Hiss plate medium. day. Hiss,4 in an investigation of the same subject, obtained the fol- lowing results: First to tenth day, inclusive, twenty-eight cases examined; typhoid bacilli isolated from three; percentage of positive cases 10.7 per cent. Eleventh to twentieth day, inclusive, forty-four cases examined; ty- phoid bacilli from twenty-two; percentage of positive cases 50 per cent. Twenty-first day to convalescence, sixteen cases examined; typhoid 1 Wiltschour, Cent. f. Bakt., 1890. 2 Redtenbacher, Zeit. f. klin. Med., xix, 1891. 3 Horton-Smith, Lancet, May, 1899. 4 Hiss, Med. News, May, 1901. 40S PATHOGENIC MICROORGANISMS bacilli isolated from thirteen; percentage of positive cases 81.2 per cent. The difficulties encountered in such examinations have led to the development of a large number of methods. The first method which yielded successful results was that of Eisner/ who employed a potato- extract gelatin containing one per cent of potassium iodid, a medium which prevented the growth of many intestinal bacteria, allowing only colon, typhoid, and a few others to develop. This medium is at present rarely used. Hiss 2 has employed with success an agar-gelatin mixture containing one per cent of glucose, the preparation of which has been described in detail in the section on media. The actual technique of the test is as follows: One to two loopfuls of feces are transferred to a tube of broth, making the broth fairly cloudy. From this emulsion five or six plates are made by transferring in series one to five loopfuls of the emulsion to tubes containing the melted plate medium, and then pouring the con- tents of these tubes into Petri dishes. These dishes, after the medium has hardened, are placed in an incubator at 37° C, and allowed to re- main for eighteen to twenty-four hours, when they are ready for examina- tion. If typhoid bacilli are present they will be found as small, usually glistening colonies with a fringe of threads growing out like flagella from their peripheries (see Figs. 90 and 91). The colonies are smaller and quite distinct from those of colon bacilli, which are heavier and darker and do not display the fringing threads. Suspicious colonies may be fished and transferred to the Hiss tube medium (see page 133) or iden- tified by other reliable methods. A method which has been found useful, especially in Europe, is that in which smears of diluted feces are made upon large plates of the Conradi-Drigalski medium. (For preparation see page 135.) The principles underlying the use of this medium are the formation of acid from the lactose bj^ the colon bacilli and the inhibition of cocci and many other other bacteria by the crystal- violet. In practice, an emulsion is made of a loopful of feces in a tube of broth. Into this is dipped a bent glass smearing rod, the excess of fluid is allowed to drip off, and smears are made upon plates of the medium, several plates being smeared without redipping the rod. Colonies of the colon bacillus on these plates will appear opaque, comparatively large, and will produce an 1 Eisner, Zeit. f. Hyg., xxi, 1895. 2 Hiss, Jour, of Exp. Med., ii, 1897; Med. News, May, 1901; and Jour. Med. Res., N. S..iii. 1902. BACILLUS OF TYPHOID FEVER 409 acid reaction with consequent reddening of the medium. Typhoid col- onies will be smaller, transparent,- and without acid formation. These colonies are fished and the microorganisms may be identified by agglu- tination or by stab cultures in the Hiss tube medium. The malachite-green media of Loefner and others have found less general use than was originally expected, because of the difficulty in obtaining uniform preparations of malachite- green. Peabody and Pratt 1 have applied the principle of colon-bacillus inhibition by mala- chite-green, by adding this dye to broth in the manner described in the section on media (page 137), planting the feces directly into this Fig. 91. — Bacillus typhosus. Colony in Hiss plate medium, highly magnified. broth, and, after incubation for several hours, making smears from these tubes upon plates of the Conradi-Drigalski medium. Marked success has been reported in the isolation of typhoid bacilli from the feces by the use of the Endo fuchsin-agar. Emulsions of feces are made in tubes of ordinary broth in the manner described in the Con- radi-Drigalski method, and smears of this emulsion are made upon plates of the fuchsin-agar by means of a glass smearing rod. The colonies of Bacillus coli, after eighteen or more hours of incubation, will be found to have brought back a deep red color to the medium, whereas the typhoid colonies are smaller, more transparent, and have left the medium uncolored. 1 Peabody and Pratt, Boston Med. and Surg. Jour., 1908. 410 PATHOGENIC MICROORGANISMS In all cases where plates are prepared from broth emulsions of feces, it is desirable to allow the emulsion to stand at incubator temperature for several hours, or, better, to centrifugalize the emulsion and then allow it to stand without agitation. Subsequent removal of fluid from the upper layers of the medium is likely to bring away a comparatively larger number of the motile organisms. The methods of isolating typhoid bacilli given above do not ex- haust the records of work done upon this problem. Other methods have Fig. 92. — Colon and Typhoid Colonies in Hiss Plate Medium. (Planted from stool. Note the small thread-forming typhoid colonies.) been devised, but those given are the ones most generally in use. It is not satisfactory to compare any two of these methods as to practical value, since all of them require a considerable amount of working famil- iarity with organisms and media. In fact, it may be said that all of the methods given are satisfactory if consistently employed by a worker who has become thoroughly accustomed to the peculiarities and varia- tions of the typhoid colonies upon the medium with which he is working. BACILLUS OF TYPHOID FEVER 411 Typhoid Bacilli in the Urine. — Careful investigation by a number of workers has revealed typhoid bacilli in the urine in about twenty-five per cent of all patients. Neumann1 discovered the bacilli in eleven out of forty -six cases and Karlinski 2 in twenty-one out of forty-four cases. Investigations by Petruschy,3 Richardson,4 Horton-Smith,5 Hiss,6 and others have confirmed these results. In general the bacilli have not been found before the fifteenth day of the disease, and examination of the urine, therefore, can be of little early diagnostic value. A series of seventy-five cases examined by Hiss before the fourteenth day of the disease did not once reveal typhoid bacilli in the urine. On the other hand, they have been found to be present for weeks, months, and, in isolated cases, for years after convalescence, the examination thus hav- ing much hygienic importance. They are probably present in about twelve per cent of cases during the early days of convalescence. In most of these cases where typhoid bacilli are found, albumin is present in the urine in considerable quantities. The bacilli usually appear and disappear with the albuminuria. It is not infrequent that an obstinate cystitis caused by typhoid ba- cilli may follow in the path of typhoid fever. Such cases have been re- ported by Blumer,7 Richardson,8 and others. Suppurative processes in the kidneys are less frequent. It is noteworthy, also, that in the course of, and following, typhoid fever there often occurs voiding of Bacillus coli with the urine. This may obstinately persist for considerabe periods after convalescence. The reasons for this are not entirely clear. Typhoid Bacilli in the G all-Bladder . — Typhoid bacilli have been frequently observed in the gall-bladder at autopsy. They have also been found present in this organ, at operations for cholecystitis, months and years after the occurrence of typhoid fever. Miller 9 has reported a case in which typhoid bacilli were present in the gall-bladder seven years after the disease; v. Dungern 10 has cultivated them from an in- flamed gall-bladder fifteen years after the disease. Zinsser has had 1 Neumann, Berl. klin. Woch.; xxvii, 1890. 2Karlinsky, Prag. med. Woch., xv, 1890. 3Petruschy, Cent. f. Hyg., xxiii, 1898. 4 Richardson, Jour. Exp. Med., 3, 1898. 5 Horton-Smith, Lancet, May, 1899. "Hiss, Med. News, May, 1901. 7 Blumer, Johns Hopkins Hosp. Reports, 5, 1895. 8 Richardson, loc. cit. 9 Miller, Johns Hopkins Hosp. Bull., 1898. 10 v. Dungern, Munch, med. Woch., 1897. 412 PATHOGENIC MICROORGANISMS occasion * to observe acase in which an operation for gall-stone seven- teen years after the occurrence of typhoid fever revealed the pres- ence of the bacilli in the gall-bladder. In such cases typhoid bacilli may be constantly discharged from the intestine with the feces and prove a menace to the health of the community. An extremely interesting example of such a typhoid carrier has been carefully studied and re- ported by Park.2 Typhoid Bacilli in the Rose Spots. — A number of observers have succeeded in isolating typhoid bacilli from the rose spots. Neufeld,3 who made an extensive " investigation of this question, obtained positive results in thirteen out of fourteen cases. According to his researches and those of Frankel,4 the bacilli are localized not in the blood, which is taken when the rose spots are incised, but are crowded in large numbers within the lymph spaces. Typhoid Bacilli in the Sputum. — In rare cases typhoid bacilli have been found in the sputum of cases complicated by bronchitis, broncho- pneumonia, and pleurisy. Such cases have been reported by Chantemesse and Widal,0 Frankel,6 and a number of others. Empyema, when it occurs in connection with such cases, is usually accompanied by a mixed infection. From a hygienic point of view the spread of typhoid fever by means of the sputum must be considered, but is probably of rare occurrence. Suppurative Lesions Due to Typhoid Bacillus. — In the course of typhoid convalescence or during the latter weeks of the disease, suppurative lesions may occur in various parts of the body. The most frequent locali- zation of these is in the periosteum, especially of the long bones, and in the joints. A large number of such lesions have been described by Welch, Richardson,7 and others. They usually take the form of periosteal ab- scesses, often located upon the tibia, occurring either late in the disease or even months after convalescence, and are characterized by very severe pain. Osteomyelitis may also occur, but is comparatively rare. Subcutaneous abscesses and deep abscesses in the muscles, due to this bacillus, have been described by Pratt.8 Synovitis may also occur. 1 Zinsser, Proc. N. Y. Pathol. Soc, 1908. 2 Park, " Pathogenic Bacteria," N. Y„ 1908. s Neufeld, Zeit. f. Hyg., xxx, 1899. 4 Frankel, Zeit. f. Hyg., xxxiv, 1900. 5 Chantemesse and Widal, Arch, de physiol. norm, et path., 1887. 6 Frankel, Deut. med. Woch., xv and xvi, 1899. 7 Richardson, Jour. Boston Soc. Med. Sci., 5, 1900. * Pratt, Jour. Boston Soc. Med. Sci., 3, 1899. BACILLUS OF TYPHOID FEVER 413 Meningitis, due to the typhoid bacillus, occurs not infrequently, usually during convalescence from typhoid fever. A case of primary typhoid meningitis has been reported by Farnet.1 Peritoneal abscesses, due to the typhoid bacillus, have been re- ported. Zinsser 2 has reported a case in which typhoid bacilli were found free in the peritoneal cavity during typhoid fever without per- foration of the gut. Isolated instances of typhoid bacilli in abscesses of the thyroid and parotid glands and in brain abscesses have been observed. Typhoid Fever without Intestinal Lesions. — A considerable number of cases have been reported in which typhoid bacilli have been isolated from the organs after death or from the secretions during life of pa- tients in whom the characteristic lesions of typhoid fever have been lack- ing. Most of these cases must be regarded as true typhoid septicemias. In some cases the bacilli were isolated from the spleen, liver, or kidneys; in others, from the urine or the gall-bladder. In a case observed by Zinsser the bacilli were isolated from an infarct of the kidney removed by operation. In this case the clinical course of the disease had pointed only toward the existence of an indefinite fever accompanied by symp- tom's referable to the kidneys. The Widal test, however, was positive. An excellent summary of such cases, together with several personally observed, has been given by Flexner.3 Hygienic Considerations. — Although typhoid fever is frequently spoken of as an epidemic disease, it is, more truly, endemic in character in almost all parts of the world, but subject to occasional epidemic ex- acerbations. In the larger communities of the temperate zones these epidemic increases take place chiefly in the autumn and, unlike epidemics of diseases such as influenza, are usually distinctly circumscribed — limited usually by the distribution of a particular water-supply. Since the disease never occurs except by transmission, directly or indirectly, from a previous case, it is amenable more than most other maladies to sanitary regulation, and it may be said without exaggera- tion, in the light of our present knowledge, that any extensive prevalence of typhoid fever in a large community is a direct consequence of some defect in the system of sanitation. The disease is acquired by ingestion of the specific bacteria. Infection by any other channel than that of the alimentary tract has not, so far, been satisfactorily demonstrated. 1 Farnet, Bull, de la soc. med. des hop. de P., 3, 1891. * Zinsser, Proc. N. Y. Path. Soc, 1907. 3 Flexner, Johns Hopkkins Rep , 5, 1896. 414 PATHOGENIC MICROORGANISMS Prophylactic measures in typhoid fever, therefore, should begin with the isolation of the patient and the disinfection of excreta, dis- charges, linen, and all utensils which have been in contact with the patient. The bacilli leave the body chiefly in the feces and the urine and the dangers of contamination, by these substances, of all objects in immediate contact with the patient are considerable. Excreta should therefore be either mixed with boiling water or chemically disinfected, preferably by means of thoroughly mixing with carbolic acid, lysol, or a solution of freshly slaked lime, and, if possible, destroyed by burning. Linen, tableware, and eating utensils should be soaked in similar solutions and boiled. The observance of such measures, furthermore, should not be discontinued until bacteriological examination has demonstrated the absence of the bacilli from feces and urine. Disre- gard of this last precaution may well be one of the main causes of the endemic persistence of the disease in large cities — especially considered in the light of our recent knowledge of "typhoid carriers" in whom chronic infection of the gall-bladder leads tq the discharge of the bacilli in the feces for months and even years after the cessation of symptoms. It can hardly be doubted, at the present day, that typhoid fever, in the large majority of cases, is transmitted by the agency of water. In an analysis of six hundred and fifty typhoid epidemics Schuder 1 found four hundred and sixty-two reported, upon reasonable evidence , as orig- inating from water. The technical difficulties attending the isolation of typhoid bacilli from contaminated water have prevented actual bacteriological proof in most epidemics; nevertheless, indirect evi- dence of pollution of the suspected water-supply, correspondence of the distribution of this supply with that of the disease, and reduction of typhoid morbidity upon the substitution of an uncontaminated supply are sufficiently convincing to remove reasonable doubt. Added to this n our knowledge, from the experiments of Jordan, Russell, and Zeit 2 and others, that typhoid bacilli may remain alive in natural waters for as long as five days. That the bacilli may survive freezing for as long as three months has been demonstrated by Prudden, and dangers of in- fection from this source are therefore considerable. Next to water, the most important source of typhoid fever is found in contaminated milk. In the statistical summary by Schuder,3 quoted above, one hundred and ten of the four hundred and sixty epidemics 1 Schilder, Zeit. f. Hyg., xxxviii, 1901. 2 Jordan, Russell, and Zeit, Jour, of Inf. Dis., 1, 1904. 3 Schuder, loc. cit. BACILLUS OF TYPHOID FEVER 415 recorded, were attributable to milk. Actual discovery of Bacillus ty- phosus in milk by Vaughan, Conradi, and others has been discussed in another section (see page 685). The fact that this bacillus causes no visible modifications in milk makes this source especially insidious. When contamination of milk has occurred, it has often been traceable to the water used in washing the cans or to attendants employed at the dairies, who had been in contact with typhoid cases, or who are convalescing from, or actually suffering from, the infection them- selves. Excluding water and milk, all remaining causes of typhoid dissemi- nation constitute about twelve per cent and are found chiefly in the use of vegetables contaminated from infected soil, and other food prod- ucts. Recently Conn has called attention to the fact that oysters grown in waters close to sewage discharges may be the means of typhoid transmission. An epidemic occurring at Wellesley College was at- tributed by him to this cause. Experiments by Foote 2 have actually demonstrated that typhoid bacilli may be found alive within oysters for three weeks or more after they have disappeared from the sur- rounding water. Indirect contamination of food and water by the intermediation of flies and other insects has been emphasized by Veeder 3 as one of the methods of typhoid transmission. This observer called attention to the fact that in camps during the Spanish- American War flies in large numbers traveled to and fro between the sinks and the cook-tents, and it is not unlikely that at least some of the typhoid fever occurring at that time may have been caused in this way. Poisons of the Typhoid Bacillus. — The investigation of the toxic products of the typhoid bacillus has occupied the attention of a large number of workers. The first to do experimental work upon the sub- ject was Brieger 4 soon after the discovery and cultivation of the micro- organism. That toxic substances can be obtained from typhoid cultures is beyond question. There is, however, a definite difference of opinion as to whether these poisons are so-called endotoxins only, or whether they are in part composed of soluble toxins comparable to those of diphtheria and tetanus, following the injection of which antitoxic sub- stances may be formed. The evidence so far seems to bear out the original contention of 1 Conn, Med, Record, Dec, 1894. 2 Foote, Med. News, 1895. s Veeder, Med. Record, 45, 1898. i Brieger, Deut. med. Woch., xxvii, 1902. 416 PATHOGENIC MICROORGANISMS Pfeiffer,1 who first advanced the opinion that the poisonous substances are products of the bacterial body set free by destruction of the bacteria by the lytic substances of the invaded animal or human' being. These poisons, when injected into animals for purposes of immunization, in Pfeiffer's experiments, did not incite the production of neutralizing or antitoxic bodies, but of bactericidal and lytic substances. That these endotoxins constitute by far the greater part of the toxic products of the typhoid bacillus can be easily demonstrated in the laboratory, by the simple experiment of filtering a young typhoid culture (eight or nine days old) and injecting into separate animals the residue of bacilli and the clear filtrate respectively. In such an experiment there will be little question as to the overwhelmingly greater toxicity of the bacillary bodies as compared with that of the culture filtrate. On the other hand, if such cultures, especially in alkaline media, are allowed to stand for several months and the bacilli thus thoroughly extracted by the broth, the toxicity of the filtrate is found to be greatly increased. Nevertheless, more recent experiments by Besredka,2 Macfadyen,3 Kraus and Stenitzer,4 and others have tended to show that, together with such endotoxic substances, typhoid bacilli may produce a true toxin which is not only obtainable by proper methods from compara- tively young typhoid cultures, but which fulfils the necessary require- ment of this class of poisons by producing in treated animals a true antitoxic neutralizing body. The typhoid endotoxins may be obtained by a variety of methods. Halm5 has obtained what he calls " typhoplasmin " by subjecting them to a pressure of about four hundred atmospheres in a Buchner press. The cell juices so obtained are cleared by filtration. Macfadyen has obtained typhoid endotoxins by triturating the bacilli after freezing them with liquid air and extracting in 1 : 1,000 potassium hydrate. Besredka obtained toxic substances by emulsifying agar cultures of bacilli in salt solution, sterilizing them by heating to 60° C. for about one hour, and drying in vacuo. The dried bacillary mass was then ground in a mortar and washed in sterile salt solution which was again heated to 60° C. for two hours. The remnants of the bacterial i Pfeiffer, Deut. med.Woch., xlviii, 1894; Pfeiffer und Kolle, Zeit. f. Hyg., xxi, 1896. 2 Besredka, Ann. de l'inst. Pasteur, 1895, 1896. 3 Macfadyen and Rowland, Cent. f. Bakt., I, xxx, 1901; Macfadyen, Cent. f. Bakt., I, 1906. 4 Kraus und Stenitzer, Quoted from " Handb. d. Tech. /'etc., 1, Fischer, Jena, 1907. 5 Hahn, Miinch. med. Woch., xxiii, 1906. BACILLUS OF TYPHOID FEVER 417 bodies settle out and the slightly turbid supernatant fluid contains the toxic substances. Vaughan l has obtained poisons from typhoid bacilli by extracting at 78° C. with a two-per-cent solution of sodium hydrate in absolute alcohol. In this way he claims to separate by hydrolysis a poisonous and a non-poisonous fraction. He claims, moreover, that this poison- ous fraction is similar to the poisons obtained in the same way from Bacillus coli and the tubercle bacillus, and other proteid substances, believing that the specific nature of such proteids depends upon the non-toxic fraction. A simple method of obtaining toxins from typhoid bacilli is carried out by cultivating the microorganisms in meat-infusion broth, rendered alkaline with sodium hydrate to the extent of about one per cent. The cultures are allowed to grow for two or three weeks and then steril- ized by heating to 60° C. for one hour, and allowed to stand for three or four weeks at room temperature. At the end of this time the cul- tures may be filtered through a Berkefeld or Pasteur-Chamberland filter and will be found to contain strong toxic substances. The accounts concerning the thermostability of the various toxins obtained are considerably at variance. In general, corresponding with other endotoxins, observers agree in considering them moderately re- sistant to heat, rarely being destroyed at temperatures below 70° C. Intravenous inoculation of rabbits with typhoid endotoxins, if in sufficient quantity, produces, usually within a few hours, a very marked drop in temperature, diarrhea, respiratory embarrassment, and death. If given in smaller doses or by other methods of inoculation — subcutaneous or intraperitoneal — rabbits are rendered extremely ill, with a primary drop in temperature, but may live for a week or ten days, and die with marked progressive emaciation, or may survive. Guinea- pigs and mice are susceptible to the endotoxins, though somewhat less so than rabbits. Immunity in Typhoid Fever. — As a rule, one attack of typhoid fever protects against subsequent ones. Although exceptions to this rule may occur, they are so rare that the history of a previous attack of this disease practically excludes its consideration in the diagnosis of any obscure condition. Animals may be actively immunized by the injection of typhoid bacilli in gradually increasing doses. In actual practice, this is best 1 Vaughan, Am. Jour, of Med. Sci., 136, No. 3, 1908. 27 418 PATHOGENIC MICROORGANISMS accomplished by beginning with an injection of about 1 c.c. of broth culture heated for ten minutes at 60° in order to kill the bacilli. After five or six days, a second injection of a larger dose of dead bacilli is administered; at similar intervals, gradually increasing doses of dead bacilli are given and finally considerable quantities of a living and fully virulent culture may be injected without serious consequences to the animal. While this method is convenient and usually successful, it is also possible to obtain satisfactory immunization by beginning with very small doses of living microorganisms, according to the early method of Chantemesse and Widal,1 and others. Such active immunization, successfully carried out upon rabbits and guinea-pigs, within a short time after the discovery of the typhoid bacil- lus, was believed to depend upon the development of antitoxic sub- stances in immunized animals. This point of view, however, was not long tenable, and was definitely disproven by the investigations of Pfeif- fer and Kolle 2 in 1896. These investigators, as well as a large number of others working subsequently, have shown satisfactorily that there are present in the blood serum of typhoid -immune animals and human beings, bacteriolytic, bactericidal, and agglutinating substances, and to a lesser extent, precipitating and opsonic bodies. Bactericidal and Bacteriolytic Substances. — The bacteriolytic sub- stances in typhoid-immune serum may be demonstrated either by the intraperitoneal technique of Pfeiffer or in vitro. In the former experi- ment a small quantity of a fresh culture of typhoid bacilli is mixed with the diluted immune serum and the emulsion injected into the peritoneal cavity of a guinea-pig. Removal of peritoneal exudate with a capillary pipette and examination in the hanging drop will reveal, within a short time, a swelling and granulation of the bacteria — the so-called Pfeiffer phenomenon. The test in vitro, as recommended by Stern and Korte,3 may be carried out by adding definite quantities of a fresh agar culture of typhoid bacilli to progressively increasing dilu- tions of inactivated immune serum together with definite quantities of complement in the form of fresh normal rabbit or guinea-pig serum. At the end of several hours' incubation at 37.5° C. definite quantities of the fluid from the various tubes are inoculated into melted agar and plates are poured to determine the bactericidal action. Careful colony counting in these plates and comparison with proper controls 1 Chantemesse and Widal, Ann. de l'inst. Pasteur, 1892. 2 Pfeiffer und Kolle, Zeit. f. Hyg., xxi, 1896. 3 Stern und Korte, Berl. klin. Woch., x., 1904. BACILLUS OF TYPHOID FEVER 419 will not only definitely demonstrate the presence of bactericidal sub- stances in the immune serum, but will furnish a reasonably accurate quantitative estimation. (For these tests see p. 255.) Although normal human serum contains in small quantity substances bactericidal to typhoid bacilli, moderate dilution, 1 : 10 or 1 : 20, of such serum will usually suffice to eliminate any appreciable bactericidal action. The bactericidal powers of immune serum, on the other hand, are often active, according to Stern and Korte, in dilutions of over 1 : 4,000 and in one case even of 1 : 4,000,000. The specificity of such reactions gives them a considerable degree of practical value, both in the biological identification of a suspected typhoid bacillus in known serum and in the diagnosis of typhoid fever in the human patient by the action of the patient's serum on known typhoid bacilli. In the publication of Stern and Korte, quoted above, it was found that typhoid patients during the second week often possess a bactericidal power exceeding 1 : 1,000, whereas the blood of normal human beings was rarely active in dilu- tions exceeding 1 : 50 or 1 : 100. While scientifically accurate, the prac- tical application of bactericidal determinations for diagnosis presents considerable technical difficulties, and gives way to the no less accurate method of agglutination. Agglutinins. — Agglutinins are formed in animals and man inoculated with typhoid bacilli, and in the course of typhoid fever. It was, in fact, while studying the typhoid bacillus that the agglutinins were first dis- covered by Gruber and Durham. In animals, by careful immunization, specific typhoid agglutinins may easily be produced in sufficient quantity to be active in dilution of 1 : 10,000, and occasionally even 1 : 50,000 or over. In the blood of typhoid patients, the agglutinins may often be found in dilu- tions of 1 : 100 and over. It is interesting to note that irrespec- tive of the agglutinin contents of any given serum, there may occasionally be noted differences in the agglutinability of various typhoid cultures, a point which is practically important in the choice of a typhoid culture for routine diagnosis work. Weeny 1 has called attention to the fact that bacilli which do not readily agglutinate when directly cultivated from the body, may often be rendered more sensitive to this reaction by several generations of cultivation upon artificial media. Walker has noted 2 a loss of agglutinability if the bacilli i Weeny, Brit. Med. Jour., 1889. 2 Walker, Jour, of Path, and Bact., 1892; Totsuka, Zeit. f. Hyg., xlv, 1903. 420 PATHOGENIC MICROORGANISMS are cultivated in immune serum. A similar alteration in the agglutin- ability of typhoid bacilli was noted by Eisenberg and Volk 1 when they subjected the microorganism to moderate heat or to weak acids such as f HC1. The practical application of agglutination to bacteriological work is found, as in the case of the bactericidal substances, in the identification of suspected typhoid bacilli, and in the diagnosis of typhoid fever. When it is desired to determine by means of agglutination whether or not a given bacillus is a typhoid bacillus, mixtures may be made of young broth cultures, or preferably of emulsions of young agar cul- tures in salt solution, with dilutions of immune serum. The tests are made microscopically in the hanging-drop preparation or, preferably, macroscopically in small test tubes. In all cases it is desirable first to determine the agglutinating power of the serum when tested against a known typhoid culture. (For detailed technique, see chapter on Technique of Serum Reactions, p. 250.) In scientific investigations, specific agglutinations in high dilutions of immune serum constitute very strong proof of the species of the micro- organism and may often furnish much information as to the biological relationships between similar species. It is found in immunizing ani- mals with any given strain of typhoid bacilli, that there are formed the "chief" or "major" agglutinins which are specific and active against the species used in immunization, and the "group" or " minor " agglutinins, active also against closely related microorganisms. The following extract from a table will serve to illustrate this point in the case of typhoid and allied bacilli. Highly Immune Typhoid Serum. 1 : 100 1 :250 1 : 500 1 : 1,000 1 : 2,500 B. typh B. paratyph. (Schottmuller) + + + + + + + + + + B. enteritidis B. coli communis — The sera of most adult normal animals and human beings usually contain a small amount of agglutinin for these bacilli. Immuniza- tion with the typhoid bacillus, while increasing chiefly the agglutinin 1 Eisenberg und Volk, Zeit. f. Hyg., xlv, 1903. BACILLUS OF TYPHOID FEVER 421 for this bacillus itself, also to a slighter extent increases the group ag- glutinins for other closely allied species. That these group agglutinins are separate substances and not merely a weaker manifestation of the action of the typhoid agglutinin itself upon these other microorganisms, may be demonstrated by the experiments of agglutinin absorption. (See section on Agglutinins, page 234.) Immune serum obtained by immunization with one particular ty- phoid culture usually agglutinates this culture in higher dilutions than it will agglutinate other typhoid strains. This has been noticed in a large number of investigations, but is not always the case. In the clinical diagnosis of typhoid fever, the phenomenon of agglu- tination was first utilized by Widal.1 This observer called attention to the fact that during the last part of the first or the earlier days of the second week of typhoid fever, as well as later in the disease and in con- valescence, the blood serum of patients would cause agglutination of typhoid bacilli in dilutions of 1 : 10, or over, whereas the serum of normal individuals usually exerted no such influence. Upon this basis he recommended, for the diagnosis of the disease, the employment of a microscopic agglutination test carried out by the usual hanging-drop technique. The reaction of Widal is, at present, widely depended upon for diagnostic purposes and although not universally successful, owing to irregularities in agglutinin formation in some patients, and because of differences in agglutin ability of the cultures employed, it is nevertheless of much value. The original conclusions as to the dilutions of the serum which must be employed, have, however, necessarily been modi- fied. Owing to the fact that Gruber,2 Stern,3 Frankel,4 and a number of others have found that occasionally normal serum will give rise to ag- glutination of typhoid bacilli in dilutions exceeding 1 : 10, it has been found necessary, whenever making a diagnostic test, to make several dilutions, the ones most commonly employed being 1 : 20, 1 : 40, 1 : CO, and 1 : 80. The wide application of the method has given rise to the development of a number of technical procedures, all of them devised with a view toward simplification. In ordinary hospital work, it is most convenient to keep on hand upon slant agar, a stock typhoid culture, the agglutinability of which is well known. From this stock culture, fresh 1 Widal, Bull, de la soc. med. des hdpit., vi, 1896; Widal et Sicard, Ann. de Pinst. Pasteur, xi, 1897. • 2 Gruber, Verhand. Congr. f. inn. Med., Wiesbaden, 1896. * Stern, Cent. f. inn. Med., xlix, 1896. * Frankel, Deut. med. Woch., ii, 1897. i 422 PATHOGENIC MIRCOORGANISMS inoculations upon neutral bouillon should be made each day, so that a young broth culture may always be on hand to furnish actively motile, evenly distributed bacteria. These bouillon cultures may be grown for from six to eight hours at incubator temperature or for from twelve to eighteen hours at room temperature. The temperatures at which the broth cultures are kept must depend, to a certain extent, upon the peculiarities of the typhoid bacillus employed, since some strains are rather more actively motile and furnish a more suitable emulsion if kej)t at a temperature lower than 37.5° C. A false clumping in the broth cultures due to a too high acidity of the bouillon or a too prolonged incubation, must be carefully guarded against. It is also possible to use for this test an emulsion of typhoid bacilli prepared by rubbing up a small quantity of a young agar culture in salt solution. Uniformity in the preparation of broth cultures or of emulsions should be observed, since the quantitative relationship between typhoid bacilli and agglu- tinins will markedly affect the completeness or incompleteness of the reaction. In high dilutions an excess of typhoid bacilli may bring about complete absorption of all the agglutinins present, without agglutinat- ing all the microorganisms. The blood of the patient to be used for a Widal test may be obtained in a number of ways. The most convenient method is to bleed the pa- tient from the ear or finger into a small glass capsule, in the form of that used in obtaining blood for the opsonin test, or into a small centrifuge tube. About 0.5 to 1 c.c. is amply sufficient. These capsules or tubes, after clotting of the blood, may be placed in the centrifuge which in a few revolutions will separate clear serum from clot. The dilutions of the serum are then made. It is best to use sterile physiological salt solu- tion as a diluent, but neutral broth may be used. The dilutions may be made either by means of an ordinary blood-counting pipette or by means of a capillary pipette upon which a mark with a grease pencil, made about an inch from the tip, furnishes a unit of measure, and upon which suction is made by means of a rubber nipple. It is convenient to have at hand a small porcelain palette such as that used by painters, in which the various cup-like impressions may be utilized to contain the various dilutions. Dilutions of the serum are made, ranging from 1 : 10 to 1 : 50. A drop of each of these dilutions is mixed with a drop of the typhoid culture or emulsion upon the center of a cover-slip and the cover- slip inverted over a hollow slide. A control with normal serum and the same culture should always be made and also one with the culture alone to exclude the possibility of spontaneous clumping. Mixture BACILLUS OF TYPHOID FEVER 423 with the typhoid culture, of course, each time doubles the dilutions so that, for instance, a drop of serum dilution 1 : 10, plus a drop of the typhoid culture, gives the final dilution of 1 : 20. The prepara- tions may be examined with a high power dry lens or an oil im- mersion lens. In a positive reaction, the bacilli, which at first swim about actively, singly or in short chains, soon begin to gather in small groups and lose much of their activity. Within one-half to one hour, they will be gathered in dense clumps between which the fluid is clear and free from bacteria, and only upon the edges of the agglutinated masses may slight motility be observed. The degree of dilution and the time of exposure at which such a reaction may be regarded as of specific diagnostic value, have been largely a matter of empirical de- termination. It is generally accepted at present that complete agglu- tination within one hour in dilutions from 1 : 40 to 1 : 60 is definite proof of the existence of typhoid infection. Exceptions, however, to this rule may occur. Agglutinations of typhoid bacilli in dilutions of 1 : 40, and over, have occasionally been observed in cases of jaundice and of tuberculosis, and these conditions must occasionally be consid- ered, though their importance was formerly exaggerated. The method of making the Widal test from a drop of whole blood, dried upon a slide, is not to be recommended, as accuracy in dilution by this method is practically impossible. As stated above, the agglutinin reaction rarely appears in typhoid fever before the beginning of the second week. It may continue during convalescence for as long as six to eight weeks and occasionally, in cases where there is a chronic infection of the gall-bladder, a Widal reac- tion may be present for years after an attack. For very exact work, even in clinical cases, the microscopic agglu- tination method may be replaced by macroscopic agglutination, ac- cording to the technique described in another section (page 229) . In order to avoid both the necessity of keeping alive typhoid cultures for routine agglutination tests and also to preclude the danger of in- fection by the use of living culture, Ficker l has recommended the use of typhoid bacilli killed by formalin. This method has no advan- tages for practical purposes and in scientific bacteriological work it is, of course, not to be considered in comparison with the other exact methods. Precipitins. — The investigations of Kraus 2 in 1897, by which the 1 Ficker, Berl. klin. Woch., xlviii, 1903. 2 Kraus, Wien. klin. Woch., xxxii, 1897. 424 PATHOGENIC MICROORGANISMS precipitins were discovered, revealed specific precipitating substances, among others, also in typhoid immune sera. Since Kraus' original in- vestigation, these substances have been studied by Norris l and others.2 Opsonins. — A number of observers have shown that opsonins specific for the typhoid bacillus are formed in animals immunized with these organisms. Opsonins are formed also in patients suffering from typhoid fever, but exact opsonic estimations in all these cases are extremely difficult because of the rapid lysis which these bacteria may undergo both in the serum, and intracellularly after ingestion by the leucocytes. Klein 3 has attempted in part to overcome this difficulty by working with dilutions of serum and at the same time using comparatively thick bacterial emulsions and exposures to the phagocytic action not exceed- ing ten minutes. Chantemesse 4 has claimed that the opsonic index of typhoid patients was increased after treatment with a serum obtained by him from immunized horses, and Harrison 5 has reported similar results in patients treated by a modification of Wright's method of active immunization. Klein claims to have demonstrated that in typhoid-immune rabbits, after five injections, the opsonic contents of the blood were increased to an equal extent with the bactericidal sub- stances. He concludes from this interesting observation that it may well be that the opsonins are quite as important in typhoid immunity as are the latter substances. For diagnostic purposes in typhoid fever the estimation of the opsonic index, so far, has not been proven to be of great value. Specific Therapy in Typhoid Fever. — The failure to produce a soluble toxin from typhoid cultures has naturally so far precluded the possibilit)^ of an antitoxic therapy, such as that which has been successful in diph- theria. In the light of our present knowledge of the poisonous products of the typhoid bacillus it seems but natural that attempts by earlier investigators to apply the principles of Behring's work to typhoid fever were doomed to fail. Attempts to employ specific bactericidal and bac- teriolytic sera for therapeutic purposes in this disease have also been without favorable result. Active Immunization. — We have seen that work by PfeifTer and Kolle and subsequently by a large number of others has shown that it is com- i Norris, Jour, of Inf. Dis., I, 3, 1904. 2 Barker and Cole, 22d Ann. Session, Assn. of Amer. Phys., Wash., 1897. 3 Klein, Bull. Johns Hopkins Hosp., 1907. * Chantemesse, 14th Internatl. Cong, for Hyg., Berlin, 1907. 5 Harrison, Jour. Royal Army Med. Corps, 8, 1907. BACILLUS OF TYPHOID FEVER 425 paratively easy to immunize animals actively against typhoid infection by the systematic injection of graded doses, at first of dead bacilli, later of fully virulent live cultures. Attempts to apply these principles pro- phylactically have been made recently on a large scale by Wright and his associates upon English soldiers in South Africa, and by German observers in German East Africa. The first recorded experiment of this sort which was done upon human beings was that of Pfeiffer and Kolle,1 who in 1896 treated two in- dividuals with subcutaneous injections of an agar culture of typhoid bacilli which had been sterilized at 56° C. The first injection was made with two milligrams of this culture. Three or four hours after the in- jection the patient suffered from a chill, his temperature gradually rose to 105° F., and there was great prostration and headache, but within twenty-four hours the temperature had returned to normal. This experiment showed that such injections could be practiced upon human beings without great danger. Simultaneously with the work of Pfeiffer and Kolle, Wright 2 con- ducted similar experiments on officers and privates in the English army. The actual number of persons treated directly or indirectly under Wright's 3 supervision in an investigation covering a period of over four years comprised almost one hundred thousand cases. The methods employed by Wright have been modified several times by him and his collaborators in minor details; the principles, however, have remained consistently the same. In the first experiments Wright employed an agar culture three weeks old, grown at 37° C, then sterilized at a tem- perature below 60° C, and protected from contamination by the ad- dition of five-tenths per cent of carbolic acid. Later, Wright 4 employed bacilli grown in a neutral one-per-cent pepton bouillon in shallow layers or flasks. Great importance is attached both to the virulence of the ty- phoid strain, which may to a moderate extent be standardized by pas- sage through guinea-pigs, and to care in using low temperatures for final sterilization. The temperature recommended by Harrison,5 working with Wright's method, is 52° C, after which the cultures are carbolized. For the first dose in a human being, Wright recommends 1 Pfeiffer und Kolle, Deut. med. Woch., xxii, 1896; xxiv, 1898. 2 Wright, Lancet, Sept., 1896. 3 Wright and Semple, Brit. Med. Jour., 1897; Wright and Leishman, Brit. Med. Jour., Jan., 1900. * Wright, Brit. Med. Jour., 1901; Lancet, Sept., 1902; Brit. Med. Jour., Oct., 1903. 5 Harrison, Jour. Royal Army Medical Corps, 1907. 426 PATHOGENIC MICROORGANISMS the quantity of bacilli fatal for 100 grams of guinea-pig. The dose may also, according to Wright, be regulated by making numerical counts of the emulsions used, by his usual method of counting against red blood corpuscles, and using for the first injection 750 to 1,000 millions of dead bacteria. The second injection, given after eleven days, should be double this quantity. Usually the first dose is followed by local inflammatory symptoms and the general systemic symptoms of toxemia. These, however, usually disappear after forty-eight hours. Although the observations of Wright are extensive, it is nevertheless extremely difficult to tabulate satisfactory statistics from a mass of experiments which must of necessity be observed by a large number of individuals, in all of whom the personal equation modifies the results of the observations. On the whole, however, it seems fair to state that distinctly advantageous results followed the active immunization prac- ticed by Wright. Wright's own estimation, in a careful attempt to present the subject fairly, gives a reduction of the morbidity from typhoid fever in the British army of fifty per cent, and a reduction of the mortality of those who became infected in spite of inoculations of fifty per cent also. Combining these two results, the actual reduction of the death rate by the method of vaccination would appear to amount to at least seventy-five per cent. The method of Pfeiffer and Kolle, originally used by them in their experiments, has been extensively carried out by German observers upon the army taking part in the late East African campaign. Roughly, the method consists in the injection of salt-solution emul- sions of fresh agar cultures sterilized at 60° C. The results reported from a large material were in general favorable, indicating that the morbidity of all the troops taking part was reduced by the inoculation and that the death rate among the inoculated persons was lower than that among normal individuals. On the whole, it can hardly be doubted that typhoid vaccination has shown definitely favorable results. The degree to which this is true, and the question as to whether the methods in use at present are fully adequate, can not yet be decided. BACILLUS FEOALIS ALKALIGENES In 1896 Petruschky 1 described a bacillus which is a not infrequent inhabitant of the human intestine, being found chiefly in the lower part of the small intestine and the large intestine. This organism, 1 Petruschky , Cent. f. Bakt.; I, xix, 1896. BACILLUS OF TYPHOID FEVER 427 which he called Bacillus fecalis alkaligenes, is of little pathogenic im- portance, although Neufeld states that he has seen a case of severe gastroenteritis in which the watery defecations contained this bacillus in almost pure culture. As a rule, however, this organism can not be regarded as pathogenic, and is important chiefly because of the ease with which it may be mistaken for Bacillus typhosus. Bacillus fecalis alkaligenes is an actively motile, Gram-negative bacillus, possessing, like the typhoid bacillus, numerous peritrichal fla- gella. On the ordinary culture media it grows like the typhoid bacillus. It does not coagulate milk. It produces no indol, and on sugar media in fermentation tubes produces no acid or gas. On potato, its growth, while somewhat heavier than that of the typhoid bacillus, is not suf- ficiently so to permit easy differentiation. It differs from Bacillus typhosus in that it produces no acid on any of the sugar media, and is therefore easily differentiated by cultivation upon Hiss serum-water media or on pepton waters containing sugars. On the Hiss semi- solid tube-medium Bacillus fecalis alkaligenes, while clouding the medium throughout, grows most heavily on the surface where, eventu- ally, it forms a pellicle. CHAPTER XXVIII BACILLI OF THE COLON-TYPHOID-DYSENTERY GROUP {Continued) BAtflLLI INTERMEDIATE BETWEEN THE TYPHOID AND COLON ORGANISMS {Bacilli of Meat Poisoning and Paratyphoid Fever) There is an extensive group of Gram-negative bacilli which be- cause of their morphology, cultural behavior, and pathogenic properties, are classified as intermediate between the colon and the typhoid types. The microorganisms belonging to this group have been described, most of them, within the last fifteen years, but few of them have been fully identified with one another. They have been variously designated as thp "hog-cholera group," "the enteritidis group," the "paracolon group" or "paratyphoid group," because of the pathological conditions with which the chief members under investigation have been found associated. Attempts to systematize the group by the comparative study of a large number of its members have been made, notably by Buxton1 and b}T Durham,2 and the work of these writers, based on cultural and agglutinative studies, has added materially to our knowledge of these organisms. The microorganisms of this group are morphologically indistinguish- able from the colon and typhoid bacilli. They are Gram-negative and possess flagella. Their motility is variable, but usually approaches that of the typhoid bacilli in activity. They correspond, furthermore, to the two other groups in their cultural characteristics upon broth, agar, and gelatin. On potato, they vary, some of them approaching in deli- cacy the typhoid growth upon this medium, others more closely approximating the heavy brownish growth of B. coli. Indol is rarely formed by them, though this has not been absolutely constant in all descriptions. As a group, they are easily distinguished from Bacillus 1 Buxton, Jour. Med. Res., N. S., iii, 1900. 2 Durham, Jour. Exper. Med., v, 1901. 428 BACILLI BETWEEN TYPHOID AND COLON ORGANISMS 429 typhosus on the one hand, and from Bacillus coli on the other, by the following simple reactions tabulated by Buxton.1 Coagulation of milk Production of indol Fermentation of lactose with gas Fermentation of dextrose with gas Agglutination in typhoid- immune serum B. coli. Intermediates. B. typhosus. + — — + — — + — — + + — — — + The characteristics of the three groups as shown by fermentation tests may be tabulated as follows: Gas upon Dextrose. Gas upon Lactose. Gas upon Saccharose. B. typhosus Intermediates B. coli communis B. coli communior + + + + + + Pathogenically, the bacilli of this "intermediate group" have attracted attention chiefly in connection with meat poisoning, and with protracted fevers indistinguishable from mild typhoidal infections. In 1888, Gartner 2 described a bacillus which he isolated from the meat of a cow, the ingestion of which had produced the symptoms of acute gastrointestinal catarrh in fifty -seven people. One of these died of the disease and the bacilli could be demonstrated in the spleen and in the blood of the patient. This bacillus, called Bacillus enteritidis by Gartner, was actively motile, formed no indol, but produced gas in dextrose media. Acute gastrointestinal symptoms could be induced by feeding the organisms to mice, guinea-pigs, rabbits, and sheep, and the bacilli could be re- covered from the infected animals. An interesting observation, which has since become important in characterizing the group of these bacilli concerned in meat poisoning, was the fact that the bacterial bodies themselves were found by Gartner to be extremely toxic, containing a poison which, in contradistinction to the endotoxins of many other microorganisms, was extremely resistant to heat. Sterilized cultures showed the same pathogenic effects as the living bacilli. Epidemics 1 Buxton, loc. cit. 2 Gartner, Corresp. Bl. d. Aerzt. Vereins, Turingen, 1888. 430 PATHOGENIC MICROORGANISMS of meat poisoning similar to the one described by Gartner, in which similar bacteria were isolated, were those described by Van Ermengem,1 occurring at Morseele in 1891, the one described by Hoist,2 the Rotter- dam epidemic described by Poels and Dhont,3 the one described by Basenau, and many others. Bacillus Morseele of Van Ermengem, Bacillus bovis morbiftcans of Basenau,4 and the bacilli isolated in similar epidemics by other ob- servers, are, except for slight differences in minor characteristics, almost identical with Gartner's microorganism. In 1893, Theobald Smith and Moore,5 studying the diseases of swine, noted a great similarity between the so-called hog-cholera bacillus, the bacilli of the Gartner group, and Bacillus typhi murium isolated by Loeffler. These observers first used the term " hog-cholera " group for the organisms under discussion. In 1899 Reed and Carroll 6 called attention to the fact that Bacillus icteroides, associated by Sanarelli with yellow fever, was culturally closely similar to the bacillus of hog cholera. , Meanwhile, other observers had been isolating bacilli, similar to those spoken of above, from cases of protracted fevers in human beings, often closely simulating typhoid infections. The first cases of this kind on record were those of Achard and Bensaude.7 In 1897, Widal and Nobecourt 8 described a bacillus which they had isolated from an esophageal abscess following typhoid fever, which closely resembled Bacillus psittacosis of Nocard,9 and which, following a nomenclature previously suggested by Gilbert,10 they designated the paracolon bacillus. This microorganism, as well as Bacillus psittacosis, isolated from a parrot by Nocard, showed a close resemblance to bacilli of the Gartner group. In 1898, Gwyn u reported a case occurring at the Johns Hopkins 1 Van Ermengem, Bull. Acad. d. med. de Belgique, 1892; " Trav. de lab. de l'univ. de Gand," 1892. 2 Hoist, Ref. Cent. f. Bakt., xvii, 1895. 3 Poels und Dhont, Holland Zeit. f. Tierheilkunde, xxiii, 1894. 4 Basenau, Arch. f. Hyg., xx, 1894. 5 Th. Smith and Moore, U. S. Bureau of Animal Industry Bull, vi, 1894. 6 Reed and Carroll, Medical News, lxxiv, 1899. 7 Achard and Bensaude, Bull, de la soc. d. hopitaux de Paris, Nov., 1906. 8 Widal et Nobecourt, Semaine med., Aug., 1897. 9 Nocard, Ref. Baumgarten's Jahresb., 1896. 10 Gilbert, Semaine med., 1895. 11 Gwyn, Johns Hopkins Hosp. Bull., 1898. BACILLI BETWEEN TYPHOID AND COLON ORGANISMS 431 Hospital, which presented all the symptoms of typhoid fever, but lacked serum agglutinating power for Bacillus typhosus. From the blood of the patient, Gwyn isolated an organism, with cultural characteristics similar to those of the Gartner bacillus, which he called a " paracolon bacillus." This bacillus was agglutinated specifically by the serum of the patient. dishing,1 in 1900, isolated a similar microorganism from a cos- tochondral abscess, appearing during convalescence from typhoid fever. In the same year, Schottmuller 2 reported five cases from which bacilli similar to those previously described were isolated. Careful cultural and agglutination studies of the microorganisms obtained from these cases showed that they could be divided into two similar, yet distinctly different types, one of them, the "Miiller" organism, approaching closely to the typhoid type, especially in its growth upon potato; the other, the "Seeman" type, corresponding more closely to the Gartner enteritidis bacilli. Similar cases were soon after reported by Kurth,3 Buxton and Coleman,4 Libman,D and others. Clinically, the diseases caused by the bacteria of this class may be divided into two main groups. I. Those which fall into the category of meat poisoning, having a sudden and violent onset of gastroenteric symptoms directly following the ingestion of meat, and characterized by profound toxemia; and II. Those in which the disease simulates a mild form of typhoid fever, differing from this only by the absence of the specific agglutination re- action for typhoid bacilli. Most of these microorganisms possess pathogenicity for mice, guinea- pigs, and rabbits, which exceeds that of the colon or typhoid bacilli. A number of the bacilli of this group, furthermore, especially those most closely similar to the original B. enteritidis of Gartner, contain an endo- toxin which shows a high resistance to heat, which may explain the fact that illness has occasionally followed the ingestion of infected meat even after preparation by cooking. Bacteriological correlation of these bacilli has been attempted, as stated above, by Durham and by Buxton, and more recently by 1 Cushing, Johns Hopkins Hosp. Bull., 1900. 2 Schottmuller, Deut. med. Woch., 1900; Zeit. f. Hyg., xxvi. 3 Kurth, Deut. med. Woch., 1901. 4 Buxton and Coleman, Proc. N. Y. Pathol. Soc, Feb., 1902. 5 Libman, Jour. Med. Res., N, S., iii, 1902. 432 PATHOGENIC MICROORGANISMS Kutscher and Meinicke.1 The subject is a difficult one and for ultimate clearness will require much further work. Durham,2 on the basis of cul- tural and agglutinative studies, has formulated a classification of the Gram-negative bacilli of the typhoid-colon and allied groups, which, though hardly final, aids considerably in throwing light upon the in- terrelationships of the various species. Durham's divisions are as follows : Division I. Typhoid-like Morphology (motile). A. No sugars fermented. Type B. fecalis alkaligenes. B. Acid in dextrose, but no gas. Type B. typhosus. Agglutination in typhoid serum. C. Acid in dextrose, but gas only when other constituents are favor- able. No acid or gas from lactose or saccharose. No agglutination in typhoid serum. Includes Bacillus " Gwyn " and Bacillus " O " of Cushing. D. Acid and gas from dextrose. No acid or gas from lactose or saccharose. Grows more rapidly than typhoid. No agglutination in colon-immune serum. Slight reaction with some typhoid sera. Includes Gartner's B. enteritidis, B. Morseele, Giinther's meat-poisoning bacillus, hog cholera bacillus, B. psittacosis, B. morbificans bovis, Durham's Bacillus "A," B. typhi murium. Division II. Colon-like Morphology (motile). E. Acid and gas from dextrose, none from lactose or saccharose. Rate of growth and colony appearance more like colon than typhoid. F. Acid and gas from dextrose, and no gas from lactose. Types isolated b}r Durham. G. Acid and gas from dextrose; acid, no gas, from lactose. Differ from F in serum reactions. H. B. coli communis. Acid and gas from dextrose and lactose; none from saccharose. /. B. coli communior. Acid and gas from dextrose, lactose, and saccharose. Division III. Non-motile. Polysaccharide splitters (starch). Type B. lactis aerogenes. Includes bacilli of mucosus capsulatus group, and Friedlander's bacillus. 1 Kutscher und Meinicke, Zeit. f. Hyg., lii, 1906. 2 Durham, loc. cit. CHAPTER XXIX BACILLI OF THE COLON-TYPHOID-DYSENTERY GROUP {Continued) THE DYSENTERY BACILLI Although acute dysentery has been an extremely prevalent disease, occurring almost annually in epidemic form in some of the Eastern coun- tries and appearing sporadically all over the world, its etiology was obscure until 1898 when Shiga i described a bacillus which he isolated from the stools of patients suffering from this disease in Japan, and es- tablished with scientific accuracy its etiological significance. Since the discovery of Shiga's bacillus a number of other bacilli have been de- scribed by various workers, all of which, while showing slight biological differences from Shiga's microorganism, are sufficiently similar to it culturally and pathogenically to warrant their being classified together with it in a definite group under the heading of the "dysentery bacilli." The manner in which Shiga made his discovery furnishes an in- structive example of the successful application of modern bacteriological methods to etiological investigation. Many workers preceding Shiga had attempted to throw light upon this subject by isolations of bacilli from dysenteric stools, and by extensive animal inoculation. Shiga, following a suggestion made by Kitasato, approached the problem by searching for a microorganism in the stools of dysentery patients which would specifically agglutinate with the serum of these patients. His labors were crowned with success in that he found, in thirty-six cases, one and the same microorganism which showed uniform serum agglu- tinations. Further, he found that this bacillus was not present in the dejections of patients suffering from other diseases nor in those of normal men, and that when tested against the blood serum of such people it was not agglutinated. Morphology. — Shiga's bacillus is a short rod, rounded at the ends, 1 Shiga, Cent. f. Bakt., xxiii, 1898; ibid., xxiv, 1898; Deut. med. Woch., xliii, xliv, and xlv, 1901. 28 , 433 434 PATHOGENIC MICROORGANISMS morphologically very similar to the typhoid bacillus, and, like it, inclined to involution forms. The organism generally occurs singly, more seldom in pairs. It is decolorized by Gram's method of staining. With the ordinary anilin dyes it stains easily, showing a tendency to stain with slightly greater intensity at the ends. The organism is an aerobe and facultative anaerobe. Although described at first by Shiga as being motile, its motility has not been satisfactorily proven, and most observers agree in denying the presence of flagella and affirming the complete absence of motility. Cultural Characteristics. — On agar the colonies are not characteristic, resembling those of the typhoid bacillus. On gelatin, the colonies appear very much like typhoid colonies and the gelatin is not liquefied. On potato, the growth, like that of typhoid, is at first not visible, but after about a week turns reddish brown. In broth, there is clouding, with moderate deposits after some days. No pellicle is formed. Milk is not coagulated. Litmus milk shows a slight primary acidity, later again becoming alkaline and taking on a progressively deeper blue color. Indol is not formed in pepton water by all varieties. . No gas is formed in media containing dextrose, lactose, saccharose, or other carbohydrate. While not delicately susceptible to reaction, the bacillus prefers slightly alkaline media. Shiga differentiated his organism from the typhoid bacillus chiefly by supposed differences in colony characters and by the agglutination reaction. Following the work of Shiga, a large number of investigators turned their attention to the subject of d}^sentery, with the result that many new forms were discovered and at first a considerable amount of con- fusion prevailed. Flexner * in 1899 investigated dysentery in the Philippines, and isolated a bacillus which, he considered, corresponded to Shiga's organism. Strong and Musgrave 2 in 1900 described a bacillus isolated from 1 Flexner, Phila. Med. Jour., vi, 1900, and Bull. Johns Hopkins Hosp., xi, 1900. 8 Strong and Musgrave, Report Surg. Gen. of Army, Washington, 1900. THE DYSENTERY BACILLI 435 dysentery cases in the Philippines which was essentially like that of Flexner. Nearly simultaneously with the papers of Flexner and of Strong and Musgrave, Kruse i published investigations of an epidemic of dysentery occurring in Germany. His observations were of the greatest importance and largely formed the starting point of the further advances which have been made in the etiology of dysentery. Kruse's organism was described as forming colonies on gelatin and agar, practically like those of Bacillus typhosus. Like this bacillus, no gas was formed from grape sugar, and the growth in milk and on potato, and even in Piorkowski's urine gelatin, resembled that of Bacillus typhosus. According to Kruse, this organism was absolutely with- out motility. In 1901 Kruse 2 contributed a second paper. In this, besides con- firming his previous observations, he described another class of organ- ism coming from cases which he designated as " pseudo-dysentery of insane asylums. " In the case of one patient, and at two autopsies, he isolated organisms which he could not distinguish morphologically or cul- turally from the true dysentery bacillus, but which showed differences in their serum reaction. By careful study of the behavior of these bacilli in the serum of patients and in immune serum from animals, he not only showed that they were different from his original cultures from cases of epidemic dysentery which, no matter what their source, were found to be alike, but that they showed differences among themselves and apparently fell into two or more varieties. One of these organisms culturally and by its serum reactions showed itself practically identical with one of the cultures he had received from Flexner. Spronck 3 in 1901 described an organism isolated in Utrecht from dysentery cases, which showed great similarity to the Shiga-Kruse organism; but, when tested in the serum of a horse immunized against true dysentery bacillus, showed practically no agglutination. He placed this organism in the group designated by Kruse as the " pseudo-dysentery bacilli." His communication is of importance, since it is the first re- ported instance in which any investigator had recognized and associated the so-called pseudo-dysentery bacilli with dysentery approaching the acute epidemic form in type. Following this work a number of investigators, including Vedder 1 Kruse, Deut. med. Woch., xxvi, 1900. 2 Kruse, Deut. med. Woch., xxvii, 1901. 3 Spronck, Ref. Baumgarten's Jahresber., 1901. 436 PATHOGENIC MICROORGANISMS and Duval,1 Flexner, and Shiga 2 himself, published communications in which they claimed identity for the various forms previously described. In 1902 Park3 and Dunham described an organism which they found in a small outbreak of dysentery occurring in Maine. This organism differed from most of those previously described in that it was found to produce indol in pepton solutions. In the same year Martini 4 and Lentz published an article in which they attempted to differentiate various dysentery bacilli by means of agglutination. This research is of importance in that it supported the work of Kruse and of Spronck, indicating a difference between the ag- glutinative character of the Kruse organism and the so-called " pseudo- dysentery " type, in which Flexner's organisms were included. It is of further interest, since it indicated a marked difference between Flexner's Philippine cultures and the Philippine culture of Strong, the Strong organism refusing to agglutinate not only in "Shiga" immune serum, but also in " Flexner " immune serum. Simultaneously with this article Lentz 5 published the results of com- parative cultural researches with dysentery and " pseudo-dysentery " bacilli, in which he made the important observation that the original Shiga-Kruse bacilli did not affect mannit, while the "pseudo-dysentery " bacilli, including Flexner's and Strong's Philippine cultures, fermented mannit, giving rise to a distinct acid reaction in the medium. The Flexner organisms and others of the "pseudo-dysentery" bacilli, how- ever, fermented maltose, while the Shiga-Kruse type, as well as Strong's bacillus, left it unchanged at the end of fortjr-eight hours. In January, 1903, Hiss and Russell 6 described a bacillus (" Y") from a case of fatal diarrhea in a child, which by ordinary cultural test and absence of motility was found to resemble the Shiga-Kruse and Flexner bacilli. Immediately upon its isolation, it was found, however, to differ from the Kruse culture by its ability to ferment mannit. This observa- tion was made independently of Lentz's work, which, at that time, had not become known in America. In the comparative study of Hiss and Russell on the fermentative abilities of various dysentery cultures, the serum water media (described on page 132) were used. By the use of 1 Vedder and Duval, Jour. Exp. Med., vi, 1902. 2 Shiga, Zeit. f. Hyg., 41, 1902. a Park and Dunham, N. Y. Univ. Bull, of Med. Sci., 1902. 4 Martini und Lentz, Zeit. f. Hyg., xli, 1902. 5 Lentz, Zeit. f. Hyg., xli, 1902. 6 Hiss and Russell, Med. News, Feb., 1903. THE DYSENTERY BACILLI 437 these media, it was found that the Kruse culture, a culture of Flexner's bacillus from the Philippines, and Duval's "New Haven" culture fer- mented dextrose with the production of a solid acid coagulum, but did not affect mannit, maltose, saccharose, or dextrin. The culture of Hiss and Russell, on the other hand, fermented not only dextrose but also mannit with the production of acid and coagulation of the medium. Maltose, saccharose, and dextrin were not fermented. The " Y " bacillus, furthermore, was shown to differ entirely from the cultures of Shiga, Kruse, and "New Haven" in the serum of immunized animals. This serum had for bacillus " Y " a titer of 1 : 500 while the three other above- named organisms did not agglutinate in it at any dilution. In normal beef serum, the Hiss-Russell organism was found to agglutinate as highly at 1 : 320, while the other three cultures gave no reaction in dilutions of over 1 : 10 or 20. Park and Carey,1 in March, 1903, described an epidemic of dysen- tery occurring in the town of Tuckahoe, near New York City, and isolated an organism which resembled the Shiga-Kruse bacilli in not fermenting mannit, but produced indol in pepton solution after five days. It corresponded in agglutination with the cultures " New Haven " and "Shiga" when tested in the serum of a goat immunized against the mannit-fermenting culture "Baltimore," i.e., did not react at .1:50, whereas Flexner's "Manila" and "Baltimore" cultures, Park and Dun- ham's "Seal Harbor" culture, and some New York cultures, all fer- menting mannit, agglutinated up to two thousand dilution in the "Bal- timore" serum. The preceding review of a part of the literature, by which our knowl- edge of the dysentery bacilli was developed, demonstrates sufficiently that we have to deal in this group with a number of different micro- organisms. This, as we have seen, was a fact first recognized by Kruse when he spoke of his true dysentery and his pseudo-dysentery strains. In spite of much confusion at first, the careful study of fermentation phenomena, of specific agglutinations, and, more recently, by Ohno 2 and others, of the bacteriolytic phenomena in immune sera, has made it possible to distinguish sharply between a number of groups. Basing the grouping of these microorganisms upon a careful study of fermentations, Hiss 3 has divided them as follows: 1 Park and Carey, Jour. Med. Res., ix, 1903. 2 Ohno, Philippine Jour, of Sci., 1, ix., 1906. 3 Hiss, Jour. Med. Res., N. S., viii, 1904. 438 PATHOGENIC MICROORGANISMS 'Shiga" 'Kruse," Ferment dextrose. Group I. 'New Haven" 'Y" (Hiss and Russell type) 'Seal Harbor" 'Diamond" 'Ferra" 'Strong" (type) 'Harris" (type) 'Gray" ' Baltimore " 'Wollstein" Ferment dextrose and mannit. Group II. Ferments dextrose, mannit, saccharose Group III. Ferment dextrose, mannit, maltose, saccha- rose, dextrin. Group IV. It was noticed, it should be mentioned, however, that in the case of the "Y," "Diamond," and "Ferra" there was usually delayed acid fermen- tation of maltose, never any of dextrin. In studying the agglutinative characters of these groups, furthermore, it was found that fermentation tests and agglutinations went hand in hand. The following table will illustrate this point:1 Serum of Rabbit immunized against Group I. (Shiga's culture). Bacilli of Group I.: " Shiga " (homologous) 20,000 "Kruse" 20,000 "New Haven " 20,000 Bacilli of Group II.: "Y" 200 "Ferra" 200 "Seal Harbor" 200 Bacilli of Group IV.: "Baltimore" 800 "Harris" 800 "Gray" 800 "Wollstein" 800 Serum of Rabbit immunized against Group II. ("Y,; culture, Hiss and Russell). Bacilli of Group I.: "Shiga" less than 100 "Kruse" 100 " New Haven " 100 1 Hiss, Jour, of Med. Research, 13, N. S., viii, 1904. THE DYSENTERY BACILLI 439 Bacilli of Group II.: "Y" (homologous) 6,400 "Ferra" 6,400 "Seal Harbor 6,400 Bacilli of Group IV.: "Baltimore " 1,600 "Gray" 1,600 "Harris" 1,600 "Wollstein" 1,600 Serum of Rabbit immunized against Group IV. ("Baltimore" culture) . Bacilli of Group I.: "Shiga" less than 100 "Kruse" 100 "New Haven " 100 Bacilli of Group II.: "Y" 400 "Ferra" 400 "Seal Harbor" 400 Bacilli of Group IV.: "Baltimore " (homologous) 3,200 "Harris" 3,200 "Gray" 3,200 "Wollstein" 3,200 In common, all these groups possess an identical morphology, the Gram -negative staining characteristics, the lack of motility with close adherence to the line of inoculation in the Hiss tube medium, the in- ability to liquefy gelatin, the inability to form acid from lactose, and the inability to produce gas from any carbohydrate media. Biological Considerations. — The dysentery bacilli in neutral broth or upon agar slants may remain alive without transplantation for periods of several months. They are aerobes and facultative anaerobes when proper sugars are present, preferring, however, the aerobic environ- ment. They are easily destroyed by heafy an exposure to 60° C. killing them usually in a short time (ten minutes). Against cold they show considerable resistance, surviving freezing for a period of several weeks. They show little resistance to the usual strengths of the common chem- ical disinfectants. Pathogenicity. — There is practically no doubt at the present time as to the etiological connection between the bacilli of this group and the dis- eases clinically classified as acute dysentery. A more chronic form of 440 PATHOGENIC MICROORGANISMS dysentery due to a protozoan, the Amoeba coli, though presenting much clinical resemblance to the bacillary dysenteries is, nevertheless, an entirely distinct disease. Infection takes place, probably, entirely by ingestion of the bacteria with infected water or food contaminated from the feces of dysentery patients. A small epidemic occurring in a hospital in New York City and caused by the bacillus "Y" of Hiss and Russell was indirectly traced to milk by Zinsser.1 Endemic in a large part of the world, especially in the warmer climates, the disease most frequently occurs in epidemics of more or less definite localization, usually under conditions which accompany the massing of a large number of human beings in one place, such as those which occur in the crowded quarters of unsanitary towns, in insti- tutions such as insane asylums, or in military camps. The mortality of such epidemics may be very large. According to Shiga,2 the disease in Japan frequently shows a mortality of over twenty per cent. The disease in human beings usually begins as an acute gastro- enteritis which is accompanied by abdominal pain and diarrhea. As it becomes more severe, the colicky pains and diarrhea increase, the stools lose their fecal character, becoming small in quantity and filled with mucus and flakes of blood. There is often severe tenesmus at this stage, and the bacilli are present in large numbers in the dejecta. Owing to the absorption of toxic products, symptoms referable to the nervous system, such as muscular twitching, may supervene, and if the disease is at all prolonged, there are marked inanition and prostration. At autopsy in early stages there may be found only a severe catar- rhal inflammation of the mucous membrane of the large intestine. In the later stages there are extensive ulcerations, and the bacteria are histologically found lodged within the depths of the mucosa and sub- mucosa. Occasionally they may penetrate to the mesenteric glands, but as far as we know there is no penetration into the general circulation. Poisonous Products of the Dysentery Bacilli. — The separate types of dysentery bacilli vary exceedingly in their powers to pro luce toxic substances. Of all the various types which have been described, the strongest poisons have been produced with bacilli of the Shiga-Kruse variety, less regularly active ones with bacilli of the Flexner and of the " Y " type. In fact, investigations carried out with the Shiga bacillus have tended to show that the disease itself is probably a true toxemia, 'Zinsser, Proc. N. Y. Path. Soc, 1907. 2 Shiga, Cent. f. Bakt., xxiii, 1898. THE DYSENTERY BACILLI 441 its symptoms being referable almost entirely to the absorption of the poisonous products of the bacillus from the intestine. The earliest investigations, carried on chiefly upon rabbits, which are more susceptible to this poison than any other animals, showed that even small doses of cultures, of this bacillus administered intravenously or subcutaneously would produce death within a very short time. Conradi,1 Vaillarcl 2 and Dopter, and others, finding that toxic symptoms were almost as pronounced when dead cultures were given as when the living bacilli were administered, came to the conclusion that the poisons of this bacillus were chiefly of the endotoxin type. More recently Todd,3 Kraus,4 and Rosenthal 5 have claimed independently that they were able to demonstrate strong soluble toxins, similar in every way to diph- theria toxin. Kraus and Doerr,6 moreover, claim to have further cor- roborated this by producing specific antitoxins with these substances. It is easy to obtain poisonous substances from dysentery cultures in considerable strength, both by extracting the bacilli themselves and by filtration of properly prepared cultures. It is therefore not unlikely that both types of poison are produced by the bacilli. Neisser and Shiga 7 obtained toxins by emulsifying agar cultures in sterile salt solution, killing the bacilli at 60° C, and allowing them to extract at 37.5° C. for three days or more. The filtrates from such emul- sions were extremely toxic. The simplest method of obtaining poisons from these bacilli is to cultivate them for a week or longer upon moder- ately alkaline meat-infusion broth. At the end of this time, the micro- organisms themselves may be killed by heating to 60° and the cultures filtered. According to Doerr,8 the toxins may be obtained in the dry state by precipitation with ammonium sulphate and re-solution of the precipitate in water. The action of the dysentery toxin upon animals is extremely characteristic and throws much light upon the disease in man. The injection of a large dose intravenously into rabbits causes a rapid fall in temperature, marked respiratory embarrassment, and a violent 1 Conradi, Deut. med. Woch., 1903. 2 Vaillard et Dopter, Ann. de Finst. Pasteur, 1903. s Todd, Brit. Med. Jour., Dec., 1903, and Jour, of Hyg., 4, 1904. * Kraus, Monatschr. f. Gesundheit, Suppl. 11, 1904. 5 Rosenthal, Deut. med. Woch., 1904. 6 Kraus und Doerr, Wien. klin. Woch., xlii, 1905. 7 Neisser and Shiga, Deut. med. Woch., 1903. 8 Doerr, " Das Dysenterietoxin," Jena, 1907. 442 PATHOGENIC MICROORGANISMS diarrhea. This is at first watery, later contains large amounts of blood. If the animals live a sufficient length of time, paralysis may occur, the animal may fall to one side or may drag its posterior extremities. It is a remarkable fact that intravenous inoculation gives rise to intestinal inflammation of a severe nature, unquestionably due to the excretion of the poison by the intestinal mucosa and limited, usually, to the ce- cum and colon, rarely attacking the small intestine. Flexner,1 who has experimented extensively upon this question, believes it probable that most of the pathological lesions occurring in the intestinal canal of dysen- tery patients are referable to this excretion of dysentery toxin, rather than to the direct local action of the bacilli. Toxins from the Shiga-Kruse type are the most potent and those which cause paralysis. Immunization with Dysentery Bacilli. — The immunization of small animals, such as rabbits and guinea-pigs, against dysentery bacilli, especially those of the Shiga type, is attended with much difficulty, owing to the great toxicity of the cultures. Nevertheless, successful results may be accomplished by the administration of extremely small doses of living or dead bacilli, increased very gradually and at sufficient intervals. Horses may be more easily immunized. The serum of such actively immunized animals contains agglutinins in considerable con- centration and of a specificity sufficiently illustrated in the preceding- section dealing with the identification of the various species. For diagnostic purposes in human beings, the agglutination reaction, accord- ing to the technique of the Widal reaction for typhoid fever, has been utilized by Kruse 2 and others. According to most observers, normal human serum never agglutinates dysentery bacilli in dilutions greater than one in twenty, while the serum of dysentery patients will often be active in dilutions as high as one in fifty. Bactericidal substances have been demonstrated in the serum of im- munized animals as well as in the serum of diseased human beings. These have been determined, in vitro, by Shiga,3 and by the intraperito- neal technique of Pfeiffer by Kruse.4 Bacteriolysis may take place in high dilutions of the serum, and has recently been used for the differen- tiation of the types of the dysentery bacilli by Ohno.5 True antitoxins in immune sera have been recently described by Kraus and Doerr.6 1 Flexner, Jour. Exp. Med., 8, 1906. 2 Kruse, Deut. med. Woch., 1901 3 Shiga, Zeit. f. Hyg.; xli. 4 Kruse, Deut. med. Woch., 1903 5 Ohno, Philippine Jour, of Sci., vol. i, 1906. 6 Krcms und Doerr, loc. cit. THE DYSENTERY BACILLI 443 PQ fa O fa P o tf O o o o & g p p fa fa En 1° H fa H fa O u CO OS 2 IS 4) 3 w E* J* £ o 0 1- b S 3 « i b b 2 E 41 4) b b •i ^ E £ £ t £ v> 5 53 * n c Cv ^ g s! 2 0 o a o d o d £ 1 0 2 0 2 C7> s So go TO CO D D 0 ED 1 ) D d 0 0 t 't 1 n □ □ D a a ■ ■ g] a s a x 8 D ■■:■•: .- : a □ i — o b 0 -J D □ a a □ □ ■ 13 (U 4l to 0 "3 □ □ □ n D n 0 o 0 1 D □ a a □ a m 0 m e 3 □ □ a a o o o w □ □ □ a □ a 0 0 [H 01 n 0 £ ti Q □ □ □ a a a m 9 [ol ^^ u Q. a 3> b U (A "5 a. 3> £ 3 3 V) +< /> h u « L 3> b n c V 1 a a a i 10 in X 0 u tZ "0 E 0 w it- (0 3 e 1 o *5 >-/ *"* a) 's 0 E ? X (0 0 *E 4) 0 "£ 4) a c a; 4i *> 0 M 3 10 0 X a. 4m 0 0 0 (1 "3 0 c 4) n • m § • ■ w m i ** & 0 m • % % • * * % m %* * * # • i • % * 1 ^> 0 *0 ■ - <• 0* m • * % % . ■ • % * | * #' .% • • *» i •» m 1 % % % <•> in 0 m i %> 0% « * % 1 * % * % > \ i Fig. 94. — Bacillus mlcosus capsulatus. readily on all the usual culture media, both on those having a meat- infusion basis and on those made with meat extract. Growth takes place at room temperature (18° to 20°) and more rapidly at 37.5° C. A temperature of 60° C. and over kills the bacilli in a short time. The ther- mal death-point according to Sternberg is 56° C. Growth ceases below 10° to 12° C. Kept at room temperature and protected from drying, the bacillus may remain alive, in cultures, for several months. The bacillus is not very fastidious as to reaction of media, growing BACILLUS MUCOSUS CAPSULATUS 447 equally well on moderately alkaline or acid media. It is aerobic and facultatively anaerobic; growth under anaerobic conditions, however, is not luxuriant. On agar, growth appears in the form of grayish-white mucus-like colonies, having a characteristically slimy and semi-fluid appearance. Colonies have a tendency to confluence, so that on plates, after three or four days, a large part of the surface appears as if covered with a film of glistening, sticky exudate, which, if fished, comes off in a tenacious, stringy manner. It is often possible to make a tentative diagnosis of the bacillus from the appearance of this growth. In broth, there is rapid and abundant growth, with the formation of a pellicle, general clouding, and later the development of a profuse, stringy sediment. Stab cultures in gelatin show, at first, a white, thin line of growth along the course of the puncture. Soon, however, rapid growth at the top results in the formation of a grayish mucoid droplet on the surface, which, enlarging, gives the growth a nail-like appearance. This nail-shape was originally described by Friedlander and regarded as diag- nostic for the bacillus. The gelatin is not fluidified. As the culture grows older the entire surface of the gelatin tube may be covered with growth, flowing out from the edges of the nail-head. The gelatin acquires a darker color and there may be a few gas bubbles below the surface. Micro- scopically, colonies on gelatin plates have a smooth outline and a finely granular or even homogeneous consistency. On blood serum, a confluent mucus-like growth appears. On potato, abundant growth appears, slightly more brownish in color than that on other media. In pepton solutions, there is no indol formation. In milk, there is abundant growth and marked capsule develop- ment. Coagulation occurs irregularly. In considering the general cultural characteristics of the Fried- lander bacillus, it must not be forgotten that we are dealing with a rather heterogeneous group, the individuals of which are subject to many minor variations. Capsule development, lack of motility, in- ability to fluidify gelatin, failure to form indol, and absence of spores, are characteristics common to all. In size, general appearance, gas forma- tion, and pathogenicity, individual strains may vary much, one from the other. Strong * has studied various races as to gas formation and 1 Strong, Cent. f. Bakt., xxv, 1899. 448 PATHOGENIC MICROORGANISMS concludes that most strains form gas from dextrose and levulose, but that lactose is fermented by some only, other strains forming no gas from this sugar. About two-thirds of the gas formed is hydrogen, the rest C02. Acid formation, according to Strong, is also subject to much varia- tion among different races. Similar studies, made by Perkins,1 have shown that most of the ordinary cultural characteristics of bacilli of this group are extremely variable and can not serve as a basis for differentiation. Reactions on sugars, however, are more constant and Perkins suggests a tentative division into three classes on this basis, as follows: I. All carbohydrates fermented with the formation of gas. II. All carbohydrates, except lactose, fermented with the formation of gas. III. All carbohydrates, except saccharose, fermented with the forma- tion of gas. Type I. corresponds to B. aerogenes (Migula), Type II. to B. Friedlander or Bacterium pneumonia) (Migula), and Type III. to Bacillus lactis aerogenes. Differentiation by means of serum reactions has not proved satis- factory. Pathogenicity. — When Friedlander first described this microorganism, he assumed it to be the incitant of lobar pneumonia. Subsequent re- searches by Weichselbaum 2 and others have shown it to be etiologically associated with pneumonia in about seven or eight per cent of all cases. The percentage in this country is probably lower. In such cases the diagnosis can often be made by the presence of the bacilli in the sputum, which has a peculiarly sticky and stringy character. Cases of Friedlander pneumonia are extremely severe and usually end in death. The bacillus has been found in connection with ulcerative stomatitis and nasal catarrh, and by Zinsser in two cases of severe tonsillitis in children. Frankel and others have frequently found the bacilli in the pus from suppurations in the antrum of Highmore and the nasal sinuses. It has also been demonstrated in cases of fetid coryza (ozena) , of which disease it is supposed by Abel 3 and others to be the specific cause. Whether the ozena bacillus represents a separate species or not, can not at present be decided. The bacillus of Friedlander has been found in empyema fluid, in pericardial exudate (after pneumonia) , and 1 Perkins, Jour, of Infect. Dis., I, No. 2, 1904. 2 Weichselbaum, loc. cit. 3 Abel, Zeit. f. Hyg., xxi . BACILLUS OF RHINOSCLEROMA 449 in spinal fluid.1 Isolated cases of Friedlander bacillus septicemia have been described.2 Being occasionally a saprophytic inhabitant of the normal intestine, it has been believed to be etiologically associated with some forms of diarrheal enteritis. B. mucosus capsulatus is pathogenic for mice and guinea-pigs, less so for rabbits. Inoculation of susceptible animals is followed by local in- flammation and death by septicemia. If inoculation is intraperitoneal, there is formed a characteristically mucoid, stringy exudate. The question of immunization against bacilli of the Friedlander group is still in the stage of experimentation. Immunization with carefully graded doses of dead bacilli has been successful in isolated cases. Specific agglutinins in immune serum have been found by Clairmont,3 but irregularly and potent only against the particular strain used for the immunization. OTHER BACILLI OF THE FRIEDLANDER GROUP Bacillus of Rhinoscleroma. — This bacillus was discovered by v. Frisch 4 in 1882. It is a plump, short rod, with rounded ends, morpho- logically almost identical with Friedlander's bacillus; it is non-motile and possesses a distinct capsule. Although at first described as Gram- positive, it has been shown to be decolorized when this method of stain- ing is used. Its cultural characteristics are almost identical with those of B. mucosus capsulatus. It forms similar slimy colonies, has a nail- like appearance in gelatin stab cultures, and in pepton solutions pro- duces no indol. According to Wilde,5 it differs from B. mucosus cap- sulatus in forming no gas in dextrose bouillon, in producing no acid in lactose bouillon, and in never coagulating milk. Pathogenicity. — The bacillus of rhinoscleroma is but moderately pathogenic for animals delicately susceptible to the bacillus of Fried- lander. Rhinoscleroma, the disease produced by this bacillus in man, consists of a slowly growing granulomatous inflammation, located usu- ally at the external nares or upon the mucosa of the nose, mouth, pharynx, or larynx. It is composed of a number of chronic, hard, nodular swellings, which, on histological examination, show granulation tissue and productive inflammation. In the meshes of the abundant 1 Jdger, Zeit. f. Hyg., xix. 2 Howard, Johns Hopkins Hosp. Bull., 1899. 3 Clairmont, Zeit. f. Hyg., xxxix. 4 v. Frisch, Wien. med. Woch., 1882. 5 Wilde, Cent. f. Bakt., xx, 1896. 29 450 - PATHOGENIC MICROORGANISMS connective tissue lie many large swollen cells, the so-called " Mikulicz cells." * The rhinoscleroma bacilli lie within these cells and in the intercellular spaces. They can be demonstrated in histological sections and can be cultivated from the lesions, usually in pure culture. Rhino- scleroma is rare in America. It is most prevalent in Southeastern Europe. The disease is slowly progressive and comparatively intract- able to surgical treatment, but hardly ever affects the general health unless by mechanical obstruction of the air passages. B. Ozaenae. — The work of Abel 2 and others has shown that ozena, or Fig. 95. — Bacillus of Rhinoscleroma. Section of tissue showing the micro- organisms within Mikulicz cells. (After Frankel and Pfeiffer.) fetid nasal catarrh> is almost always associated with a bacillus morpho- logically and culturally almost identical with B. mucosus capsulatus. The bacillus can not be definitely separated from the latter. According to Wilde 3 it forms no gas in dextrose bouillon and is less pathogenic for mice than B. Friedlander. Whether it is a separate species, or merely an atypical form changed by environment, can not be stated at present. 1 Mikulicz, Arch. f. Chir., xx, 1876. 2 Abel, Zeit. f. Hyg., xxi 3 Wilde, loc. cit. BACILLUS LACTIS AEROGENES 451 BACILLUS LACTIS AEROGENES Bacillus lactis aerogenes is the type of a group which is closely similar to the colon group and often distinguished from it with difficulty. It was first described by Escherich l in 1885 who isolated it from the feces of infants. Since then it has been learned that this bacillus is almost constantly present in milk, and, together with one or two other micro- organisms, is the chief cause of the ordinary souring of milk. Apart from its occurrence in milk, moreover, the bacillus is widely distributed in nature, being found in feces, in water, and in sewage. It is distinguish- able from the colon bacillus chiefly by the fact that it is non-motile, possesses no flagella, hardly ever forms chains, and, when cultivated upon suitable media, especially milk, it possesses a distinct capsule. It differs from the colon bacillus, furthermore, in that it is capable of fermenting polysaccharids, such as starch, and does not form indol upon pep- ton media. It is distinguishable from the bacillus of Friedlander (B. mucosus capsulatus), according to Wilde,2 by its more energetic gas formation in dextrose broth, its ability to produce acid on lactose media, and its invariable coagulation of milk. The bacillus is about 0.5 to 1 micron in width and 2 to 4 micra in Length. It grows easily upon the simplest media, is a facultative anae- robe, and grows most abundantly at a temperature between 25° and 30° C. Upon agar and gelatin it grows readily with a heavy white growth, the colonies of which have a tendency to confluence and are distinctly more mucoid in appearance than are those of Bacillus coli. In broth, it causes a general clouding and a pellicle. The cultures have a slightly sour or cheesy odor. On potato, the growth is heavy and gas is formed. On milk, there is rapid coagulation and acid formation. It is charac- teristic of this bacillus that it is capable of producing a large amount of acid, chiefly lactic, and of being able to withstand these large amounts of acid without being injured by them. The pathogenicity of Bacillus lactis aerogenes for man is slight. Its chief claims to importance lie in its milk-coagulating properties and its almost constant presence in the human intestine. In infants, it may give rise to flatulence and it has been occasionally observed as the i Escherich, Fort. d. Med., 16, 17, 1885. 2 Wilde, Cent. f. Bakt., xx, 1896. 452 PATHOGENIC MICROORGANISMS sole incitant of cystitis. Among such cases rare instances have been observed in which it has formed gas in the bladder (pneumaturia) . When this occurs the urine is not ammoniacal but remains acid. Different strains of this bacillus vary much in their pathogenicity for animals. Wilde claims that it is more pathogenic for white mice and guinea-pigs than is the bacillus of Friedlander. He speaks of it as the most virulent member of this group. Kraus, writing in Fluegge's "Mikroorganismen," rates its pathogenicity less high. Closely related to this bacillus, as well as to those of the Friedlander group, is an encapsulated bacillus isolated from a case of broncho- pneumonia by Mallory and Wright/ which is strongly pathogenic for mice, guinea-pigs, and rabbits. BACILLI OF THE PROTEUS GROUP The bacilli of this group have little pathological interest, but are im- portant because of the frequency with which they are encountered in routine bacteriological work. They may confuse the inexperienced because of a superficial similarity to bacilli of the colon-typhoid group. In form they may be short and plump or long and slender, staining easily with anilin dyes and decolorizing with Gram's method. They are actively motile and possess many flagella. The individuals stain irreg- ularly, often showing unstained areas near the center. The type of the group is found in the so-called Bacillus proteus vulgaris described by Hauser 2 in 1885. Bacilli of this group are widely distributed, being found in water, soil, air, and wherever putrefaction takes place. In fact, proteus is one of the true putrefactive bacteria possessing the power to cause the cleav- age of proteids into their simplest radicles. Bacillus proteus vulgaris grows best at temperatures at or about 25° C. and develops upon the simplest media. It is a facultative anae- robe and forms no spores. In broth, it produces rapid clouding with a pellicle and the forma- tion of a mucoid sediment. In gelatin, the colonies are characteristically irregular, giving the name to this group. Gelatin is rapidly liquefied. Liquefaction, however, is diminished or even inhibited under anaerobic conditions. 1 Mallory and Wright, Zeit. f. Hyg., xx, 1895. 2 Hauser, "Ueber Faulniss-Bakt./' Leipzig, 1885. BACILLUS PROTEUS 453 On agar and other solid media, as well as upon gelatin before lique- faction has taken place, characteristic colonies are produced. From the central flat, grayish-white colony nucleus, numerous irregular streamers grow out over the surrounding media, giving the colony a stellate appearance. On potato, it forms a dirty, yellowish growth. In milk, there is coagulation and an acid reaction at first; later the casein is redissolved by proteolysis. Blood serum is often liquefied, but not by all races. The pathogenic powers of proteus bacilli are usually slight. Large doses injected into animals may give rise to localized abscesses. In man proteus infections have been described as occurring in the bladder; in most cases, however, in combination with some other microorganism. The so-called Urobacillus liquefaciens septicus described by Krogius was probably a variety of this group. Epidemics 1 of meat poisoning- have been attributed to members of the proteus family by some ob- servers. Thus Wesenberg 2 was able to cultivate a proteus bacillus from putrid meat which had caused acute gastroenteritis in sixty -three individuals. Similar epidemics have been reported by Silberschmidt,3 Pfuhl,4 and others. In some of these the bacilli proved to be unusually toxic when injected into animals, but could not be recovered from the organs after death. 1 Schnitzler, Cent. f. Bakt., viii, 1890. 2 Wesenberg, Zeit. f. Hyg., xxviii, 1898. 3 Silberschmidt, Zeit. f. Hyg., xxx, 1899. 4 Pfuhl, Zeit. f. Hyg., xxxv, 1900. CHAPTER XXXI BACILLUS TETANI Lockjaw or tetanus, though a comparatively infrequent disease, has been recognized as a distinct clinical entity for many centuries. The infectious nature of the disease, however, was not demonstrated until 1884, when Carlo x and Rattone succeeded in producing tetanus in rabbits by the inoculation of pus from the cutaneous lesion of a human case. Nicolaier,2 not long after, succeeded in producing tetanic symp- toms in mice and rabbits by inoculating them with soil. In connec- tion with the lesions produced at the point of inoculation, Nicolaier described a bacillus which may have been Bacillus tetani, but which he was unable to cultivate in pure culture. Kitasato,3 in 1889, definitely solved the etiological problem by obtaining from cases of tetanus pure cultures of bacilli with which he was able again to produce the disease in animals. Kitasato succeeded where others had failed because of his use of anaerobic methods and his elimination of non-spore-bearing con- taminating organisms by means of heat. His method of isolation was as follows: The material containing tetanus bacilli was smeared upon the surface of agar slants. These were permitted to develop at incubator temperature for twenty-four to forty-eight hours. At the end of this time the cultures were subjected to a temperature of 80° C. for one hour. The purpose of this was to destroy all non-sporulating bacteria, as well as aerobic spore-bearers which had developed into the vegetative form. Agar plates were then inoculated from the slants and exposed to an atmosphere from which oxygen had been com- pletely eliminated and hydrogen substituted. On these plates colonies of tetanus bacilli developed. Morphology and Staining. — The bacillus of tetanus is a slender bacil- lus, 2 to 5 micra in length, and 0.3 to 0.8 in breadth. The vegetative forms which occur chiefly in young cultures are slightly motile and are 1 Carlo e Rattone, Giornale d. R. Acad. d. Torino, 1884. 2 Nicolaier, Inaug. Diss., Gottingen, 1885. 3 Kitasato, Deut. med. Woch., No. xxxi, 1889. 454 BACILLUS TETANI 455 seen to possess x numerous peritrichal flagella, when stained by special methods. After twenty-four to forty-eight hours of incubation, the length of time depending somewhat on the nature of the medium and the degree of anaerobiosis, the bacilli develop spores which are char- acteristically located at one end, giving the bacterium the diagnostic drumstick appearance. As the cultures grow older the spore-bearing forms completely super- t * \ # V f 4 1 % * i 4j "Jh , Y t •• i % '? . *» * HBI ^v $f? -v«* * t X <•* : ^H^V t » 1 ^ % jt _ . $ • ^l .>"*.' V . #« * ' 1 * ' , 1 *> # » * V ' ; ' , V' "^* ■ -^ % % * . ■. * t .«*« ■ # '■ * * .„ , ■ ♦?* * t i «* vy « «#? y* 1 Fig. 96. — Bacillus tetani. Spore stain. sede the vegetative ones. Very old cultures contain spore-bearing bacilli and spores only. The tetanus bacillus is easily stained by the usual anilin dyes, and reacts positively to Gram's stain. Flagella staining is successful only when very young cultures are employed. Distribution. — In nature, the tetanus bacillus has been found by Nicolaier and others to occur in the superficial layers of the soil. The 1 Vottaler, Zeit. f. Hyg., xxvii. 456 PATHOGENIC MICROORGANISMS earth of cultivated and manured fields seems to harbor this organism with especial frequency, probably because of its presence in the dejecta of some of the domestic animals. Biological Characteristics. — The bacillus of tetanus is generally de- scribed as an obligatory anaerobe. While it is unquestionably true that growth is ordinarily obtained only in the complete absence of oxygen, various observers, notably Ferran l and Belfanti,2 have successfully habituated the bacillus to aerobic conditions by the gradual increase of oxygen in cultures. Habituation to aerobic condi- tions has usually been accompanied by diminution or loss of pathogenicity and toxin-formation. Anaerobic conditions may likewise be dispensed with if tetanus bacilli be grown in symbiosis with some of the aerobic bacteria. The addition to culture media of suitable carbohydrates, and of fresh sterile liver tissue, has also been found to render it less exacting as to absolute anaerobiosis.3 xAnaerobically cultivated, Bacillus tetani grows readily upon meat-infusion broth, which it clouds within twenty-four to thirty-six hours. Anaerobic broth cultures may be simply made by covering the surface of the medium with a layer of albolin or any other oil, and removing the air by boiling. Upon meat-infusion gelatin at 20° to 22° C. the tetanus bacillus grows readily, growth becoming visible during the second or third day. There is slow nuidification of the gelatin. On agar, at 37.5° C, growth appears within forty- eight hours. Colonies on agar plates present a rather characteristic appearance, consisting of a compact center surrounded by a loose meshwork of fine fila- ments, not unlike the medusa-head appearance of subtilis colonies. In agar stabs, fine radiating processes growing out in all directions from the central stab tend to give the culture the appearance of a fluff of cotton. Milk is a favorable culture medium and is not coagulated. On potato, growth is delicate and hardly visible. 1 Ferran, Cent. f. Bakt., xxiv, No. 1. 2 Belfanti, Arch, per le sci. med., xvi. 3 Th. Smith, Brown, and Walker, Jour. Med. Res., N. S., ix, 1906. ! m • • • ■ . ...f , A Fig. 97. — Young Tetanus Culture in Glucose Agar. BACILLUS TETANI 457 The most favorable temperature for the growth of this bacillus is 37.5° C. Slight alkalinity or neutrality of the culture media is most ad- vantageous, though moderate acidity does not altogether inhibit growth. All the media named may be rendered more favorable still by the ad- dition of one or two per cent of glucose, maltose, or sodium formate.1 In media containing certain carbohydrates, tetanus bacilli produce acid. In gelatin and agar, moderate amounts of gas are produced, consisting chiefly of C02, but with the admixtures of other volatile substances which give rise to a characteristically unpleasant odor, not unlike that of putrefying organic matter. This odor is due largely to H2 S and methylmercaptan. The vegetative forms of the tetanus bacillus are not more resistant against heat or chemical agents than the vegetative forms of other microorganisms. Tetanus spores, however, will resist dry heat at 80° C. for about one hour, live steam for about five minutes; five per cent carbolic acid kills them in twelve to fifteen hours; one per cent of bichlo- rid of mercury in two or three hours. Direct sunlight diminishes their virulence and eventually destroys them.2 Protected from sunlight and other deleterious influences, tetanus spores may remain viable and virulent for many years. Henrijean 3 has reported her success in producing tetanus with bacilli from a splinter of wood infected eleven years before. Pathogenicity. — The comparative infrequency of tetanus infection is in marked contrast to the wide distribution of the bacilli in nature. Introduced into the animal body as spores, and free from toxin, they may often fail to incite disease, easily falling prey to phagocytosis and other protective agencies before the vegetative forms develop and toxin is formed. The protective importance of phagocyto- sis was demonstrated by Vaillard and Rouget,4 who introduced tetanus spores inclosed in paper sacs into the animal body. By the paper cap- Fig. 98.— Older Tetanus Culture in Glucose Agar. lKitasato, Zeit. f. Hyg., 1891. 2 v. Eisler und Pribram, in Levaditi, "Handbuch," etc., Jena, 1907. 3 Henrijean, Ann. de la soc. med. chir. de Liege, 1891. 4 Vaillard et Rouget, Ann. de l'inst. Pasteur, 1892. ±5S PATHOGENIC MICROORGANISMS sules the spores were protected from the leucocytes, not from the body fluids. Nevertheless, tetanus developed in the animals. The nature of the wound and the simultaneous presence of other microorganisms seem to be important factors in determining whether or not the tetanus bacilli shall be enabled to proliferate. Deep, lacerated wounds, in which there has been considerable tissue destruction, and in which chips of glass, wTood splinters, or grains of dirt have become embedded, are particularly favorable for the development of these germs. The injuries of compound fractures and of gunshot wounds are especially liable to supply these conditions, and the presence in such wounds of the common pus cocci, or of other more harmless parasites, may aid materially in furnishing an environment suitable for the growth of the tetanus bacilli. Apart from its occurrence following trauma, tetanus has been not infrequently ob- served after childbirth,1 and isolated cases have been reported in which it has followed diphtheria and ulcerative lesions of the throat.2 A definite period of incubation elapses between the time of infection with tetanus bacilli and the development of the first symptoms. In man this may last from five to seven days in acute cases, to from four to five weeks in the more chronic ones. Experimental inoculation of guinea-pigs is followed usually in from one to three days by rigidity of the muscles nearest the point of infection. This spastic condition rapidly extends to other parts and finally leads to death, which occurs within four or five days after infection. Autopsies upon human beings or animals dead of tetanus reveal few and insignificant lesions. The initial point of infection, if at all evident, is apt to be small and innocent in appearance. Further than a general and moderate congestion, the organs show no pathological changes. Bacilli are found sparsely even at the point of infection, and have been but rarely demonstrated in the blood or viscera. Nicolaier succeeded in producing tetanus with the organs of infected animals in but eleven out of fifty-two cases. More recently, Tizzoni 3 and Creite 4 have suc- ceeded in cultivating tetanus bacilli out of the spleen and heart's blood of infected human beings. Tetanus Toxin. — The pathogenicity .of the tetanus bacillus depends entirely upon the soluble toxin which it produces. This toxin is produced in suitable media by all strains of virulent tetanus bacilli, individual strains showing less variation in this respect than do the separate strains 1 Baginsky, Deut. med. Woch., 1893. 2 Foges, Wien. med. Woch., 1895. 3 Tizzoni, Ziegler's Beit., vii. 4 Creite, Cent. f. Bakt., xxxvii. BACILLUS TETANI 459 of diphtheria bacilli. While partial aerobiosis does not completely elimi- nate toxin formation, anaerobic conditions are by far more favorable for its development. The medium most frequently employed for the production of tetanus toxin is neutral or slightly alkaline beef-infusion bouillon containing five- tenths per cent Nad and one per cent pepton. Glucose, sodium formate, or tincture of litmus may be added, but while these substances increase the speed of growth of the bacilli they do not seem to enhance the de- gree of toxicity of the cultures. Glucose is said even to be unfavorable for strong toxin development. It is important, too, that the bouillon shall be freshly prepared.1 There does not seem to be any direct relationship between the amount of growth and the degree of toxicity of the cultures. Under anaerobic conditions in suitable bouillon and grown at 37.5° C, the maximum toxin content of the cultures is reached in from ten days to two weeks. After this time the toxin deteriorates rapidly. Tetanus toxin has been produced without resort to anaerobic methods by several observers, notably by Debrand,2 by cultivating the bacilli in bouillon in symbiosis with Bacillus subtilis. By this method, Debrand claims to have produced toxin which was fully as potent as that produced by anaerobic cultivation. The tetanus toxin, in solution in the bouillon cultures, may be sepa- rated from the bacteria by filtration through Berkefeld or Chamberland filters. Since the poison in such filtrates deteriorates very rapidly, much more rapidly even than diphtheria toxin, various methods have been devised to obtain the toxin in the solid state. The most useful of these is precipitation of the poison out of solution by oversaturation with ammonium sulphate.3 Very little of the toxin is lost by this method and, thoroughly dried and stocked in vacuum tubes, together with an- hydrous phosphoric acid, it may be preserved indefinitely without dete- rioration. The precipitate thus formed is easily soluble in water or salt solution, and therefore permits of the preparation of uniform solu- tions for purposes of standardization. Brieger and Boer 4 have also succeeded in precipitating the toxin out of broth solution with zinc chloride. Vaillard and Vincent 5 have procured it in the dry state by evaporation in vacuo. 1 Vaillard et Vincent, Ann. de l'inst. Pasteur, 1891. 2 Debrand, Ann. de l'inst. Pasteur, 1890, 1902. 3 Brieger und Cohn, Zeit. f. Hyg., xv. 4 Brieger und Boer, Zeit. f. Hyg., xxi. 5 Vaillard et Vincent, Ann. de l'inst. Pasteur, 1891. 460 PATHOGENIC MICROORGANISMS Brieger and Cohn,1 Brieger and Boer,2 and others have attempted to isolate tetanus poison, removing the proteids from the ammonium sul- phate precipitate by various chemical methods. The purest preparations obtained have been in the form of fine yellowish flakes, soluble in water, insoluble in alcohol and ether. Solutions of this substance have failed to give the usual proteid reactions. The toxin when in solution is extremely sensitive to heat. Kita- sato 3 states that exposure to 68° C. for five minutes destroys it com- pletely. Dry toxin is more resistant,4 often withstanding temperatures of 120° C. for more than fifteen minutes. Exposure to direct sunlight destro}^ the poison in fifteen to eighteen hours.0 Interesting experiments as to the action of eosin upon tetanus toxin have been carried out by various observers. Flexner and Noguchi6 found that five per cent eosin added to the toxin would destroy it within one hour. This action is ascribed to the photodynamic power of the eosin. The toxin exerts an extremely low osmotic pressure and is easily destroyed by electric currents. Tetanus toxin is one of the most powerful poisons known to us. Filtrates of broth cultures, in quantities of 0.000,005 c.c, will often prove fatal to mice of ten grams weight. Dry toxin obtained by ammo- nium sulphate precipitation7 is quantitatively even stronger, values of 0.000,001 grams as a lethal dose for a mouse of the given weight not being uncommon. Brieger and Cohn8 succeeded in producing a dry toxin capable of killing mice in doses of 0.000,000,05 gram. Different species of animals show great variation in their suscepti- bility to tetanus toxin. Human beings and horses are probably the most susceptible species in proportion to their body weight. The common domestic fowls are extremely resistant. Calculated for grams of body weight, the horse is twelve times as susceptible as the mouse, the guinea- pig six times as susceptible as the mouse. The hen, on the other hand, is 200,000 times more resistant than the mouse. 1 Brieger und Cohn, loc. cit. 2 Brieger und Boer, Zeit. f. Hyg., xxi. 3 Kitasato, Zeit. f. Hyg., x. 4 Morax et Marie, Ann. de l'inst. Pasteur, 1902. 5 Fermi und Pernossi, Cent. f. Bakt., xv. 6 Flexner and Noguchi, "Studies from Rockefeller Inst.," v., 1905. 7 Brieger und Cohn, loc. cit. 8 Brieger und Cohn., Zeit. f. Hyg., xv. BACILLUS TETAN1 461 After the inoculation of an animal with tetanus toxin there is always a definite period of incubation before the toxic spasms set in. This period may be shortened by increase of the dose, but never entirely eliminated.1 When the toxin is injected subcutaneously, spasms begin first in the muscles nearest the point of inoculation. Intravenous inoculation,2 on the other hand, usually results in general tetanus of all the muscles. The feeding of toxin does not produce disease, the poison being passed through the bowel unaltered. The harmful action of tetanus toxin is generally attributed to its affinity for the central nervous system. Wassermann and Takaki 3 showed that tetanus toxin was fully neutralized when mixed with brain substance. Other organs — liver and spleen, for instance — showed no such neutralizing power. The central origin of the tetanic contractions was made very evident by the work of Gumprecht,4 who succeeded in stop- ping the spasms in a given region by division of the supplying motor nerves. The manner in which the toxin reaches the central nervous system has been extensively investigated, chiefly by Meyer and Ransom, and Marie and Morax. Meyer and Ransom 5 from a series of careful experi- ments reached the conclusion that the toxin is conducted to the nerve centers along the paths of the motor nerves. Injected into the circu- lation,6 the toxin reaches simultaneously all the motor nerve endings, producing general tetanus. In this case too, therefore, the poison from the blood can not pass directly into the central nervous system, but must follow the route of nerve tracts. These observations have been of great practical value in that they pointed to the desirability of the injection of tetanus antitoxin directly into the nerves and the central nervous system in active cases. Tetanolysin. — Tetanus bouillon contains, besides the "tetano- spasmin " described above which produces the familiar symptoms of the disease, another substance discovered by Ehrlich 7 and named b}^ him " tetanolysin." Tetanolysin has the power of causing hemolysis of the red blood corpuscles of various animals, and is an entirely separate 1 Courmont et Doyen, Arch, de phys. , 1893. 2 Ransom, Deut. med. Woch., 1893. 3 Wassermann und Takaki, Berl. klin. Woch., 1898. 4 Gumprecht, Pfliiger's Arch., 1895. 5 Meyer und Ransom, Arch. f. exp. Pharm. u. Path., xlix. 6 Marie et Morax, Ann. de Finst. Pasteur, 1902. < Ehrlich, Berl. klin. Woch., 1898. 462 PATHOGENIC MICROORGANISMS substance from tetanospasmin. It may be removed from toxic broth by admixture of red blood cells, is more thermolabile than the tetano- spasmin, and gives rise to an antihemolysin when injected into animals. For the production, standardization, and use of tetanus antitoxin, see p. 220 et seq. CHAPTER XXXII BACILLUS OF SYMPTOMATIC ANTHRAX, BACILLUS OF MALIGNANT EDEMA, BACILLUS AEROGENES CAPSULATUS, BACILLUS BOTULINUS BACILLUS OF SYMPTOMATIC ANTHRAX (Bacillus anthracis symptomatici, Rauschbrand, Charbon symptomatique, Sarcophysematos bovis) Symptomatic anthrax is an infectious disease occurring chiefly among sheep, cattle, and goats. It is colloquially spoken of as " quarter- evil" or "blackleg." The disease has never been observed in man. It was formerly, and is often to-day, confused with true anthrax, largely because of a superficial similarity between the clinical symptoms of the two maladies. Bacteriologically, the two microorganisms are in entirely different classes. Geographically, symptomatic anthrax is of wide distribution and infection is usually through the agency of the soil in which the bacillus is present, probably in the form of spores which may retain viability and pathogenicity for several years. Morphology and Staining. — The bacillus of symptomatic anthrax is a bacillus with rounded ends, somewhat shorter and relatively thicker than the bacillus of malignant edema, being about four to six micra long, and five-tenths to six-tenths micra wide. It is usually seen singly and never forms long chains. The bacillus in its vegetative form is actively motile and possesses numerous flagella placed about its periphery. In artificial media it forms spores which are oval, broader than the rod itself, and placed near, though never actually at, the end of the bacillary body. This gives the bacillus a racket-shaped appearance. It is readily stained with the usual anilin dyes, but is decolorized by Gram's method of staining. Cultivation. — The bacillus is a strict anaerobe. It was obtained in pure culture first by Kitasato.1 Under anaerobic conditions it is easily 1 Kitasato, Woch. f. Hyg., 1889. 463 464 PATHOGENIC MICROORGANISMS cultivated upon the usual laboratory media, all of which are more favorable after the addition of glucose, glycerin, or nutrose. In all media there is active gas formation, which, owing to an admixture of butyric acid, is of a foul, sour odor. The bacillus is not very delicate in its requirements of a special reaction of media, growing equally well on those slightly acid or slightly alkaline. On gelatin plates, at 20° C, colonies appear in about twenty-four hours, usually round or oval, with a compact center about which fine radiating filaments form an opaque halo. The gelatin is fluidified. V •*- \ I Fig. 99. — Bacillus of Symptomatic Anthrax. (After Zettnow.) Surface colonies upon agar plates are circular and made up of a slightly granular compact center, from which a thinner peripheral zone emanates, containing microscopically a tangle of fine threads. In agar stabs, at 37.5° C, growth appears within eighteen hours, rapidly spreading from the line of stab as a diffuse, fine cloud. Gas formation, especially near the bottom of the tube, rapidly leads to the formation of bubbles and later to extensive splitting of the medium. In gelatin stab cultures growth is similar to that in agar stabs, though less rapid. Pathogenicity. — Symptomatic anthrax bacilli are pathogenic for cattle, sheep, and goats. By far the largest number of cases, possibly the only spontaneous ones, appear among cattle. Guinea-pigs are very susceptible to experimental inoculation. Horses are very little suscep- BACILLUS OF SYMPTOMATIC ANTHRAX 465 tible. Dogs, cats, rabbits, and birds are immune. Man also appears to be absolutely immune. Spontaneous infection occurs by the en- trance of infected soil into abrasions or wounds, usually of the lower extremities. Infection depends to some extent upon the relative de- gree of virulence of the bacillus — a variable factor in this species. Twelve to twenty-four hours after inoculation there appears at the point of entrance a soft, puffy swelling, which on palpation is found to emit an emphysematous crack- ling. The emphysema spreads rapidly, often reaching the abdomen and chest within a day. The course of the disease is extremely acute, the fever high, the general prostration extreme. Death may result within three or four days after inoculation. At autopsy the swollen area is found to be infiltrated with a thick exudate, blood-tinged and foamy. Subcutaneous tissue and muscles are edematous and crackle with gas. The internal organs show parenchymatous degeneration and hemorrhagic areas. The bacilli, immediately after death, are found but sparsely distributed in the blood and internal organs, but are demonstrable in enormous numbers in the edema surrounding the central focus. If carcasses are allowed to lie unburied for some time, the bacilli will attain a general distribution, and the entire body will be found bloated with gas, the organs filled with bubbles. Practically identical conditions are found after experimental inocula- tion. Toxins. — According to the investigations of Le- clainche and Vallee,1 the bacillus of symptomatic anthrax produces' a soluble toxin. It is not formed to any extent in ordinary broth, but is formed in considerable quantities in broth containing blood or albuminous ani- mal fluids. The best medium for obtaining toxin, according to the same authors, is the bouillon of Martin,2 made up of equal parts of veal infusion and a Fig. 100.— Ba- cillus of Symp- tomatic Anthrax. Culture in glucose agar. 30 1 Leclainche et Vallee, Ann. de Tinst. Pasteur, 1900. 2 Martin, Ann. de l'inst. Pasteur, 1898. 466 PATHOGENIC MICROORGANISMS pepton solution obtained from the macerated tissues of the stomachs of pigs. The toxin contained in filtrates of such cultures is quite resistant to heat, but rapidly deteriorates if free access of air is allowed. Immunity. — Active immunization against the bacillus of symptom- atic anthrax was first accomplished by Arloing * and his collaborators by the subcutaneous inoculation of cattle with tissue-extracts of in- fected animals. The work of these authors resulted in a practical method of immunization which is carried out as follows: Two vaccines are prepared. Vaccine I consists of the juice of in- fected meat, dried and heated to 100° C. for six hours. Vaccine II is a similar meat-juice heated to 90° C, for the same length of time. By the heating, the spores contained in the vaccines are attenuated to relatively different degrees. Vaccine I in quantities of 0.01 to 0.02 c.c. is emulsified in sterile salt solutions and injected near the end of the tail of the animal to be protected. A similar quantity of Vaccine II is injected in the same way fourteen days later. This method has been retained in principle, but largely modified in detail by various workers. Kitt 2 introduced the use of the dried and powdered whole meat instead of the meat juice, and made only one vaccine, heated to 94° C, for six hours. This method has been largely used in this country.3 Passive immunization with the serum 4 of actively immunized sheep and goats has been used in combination with the methods of active immunization. BACILLUS OF MALIGNANT EDEMA (Bacillus oedematis maligni, Vibrion septique) In 1877, Pasteur 5 described a bacillus which he had found in guinea- pigs and rabbits experimentally inoculated with putrefying animal tissues. This bacillus, which he named "Vibrion septique," he suc- ceeded in cultivating only under anaerobic conditions and in an impure state, and described as its pathognomonic characteristics the formation of an extensive edema in and about the point of inoculation. 1 Arloing, Cornevin, et Thomas, " Le Charbon Sympt.," etc., Paris, 1887. Ref. from Grassberger und Schattenfroh, Kraus und Levaditi, "Handbuch," etc., vol. i, pt. 2. 2 Kitt, Ref. from Grassberger und Schattenfroh, loc. cit. 3 Report of Bureau of Animal Ind., Wash., 1902. 4 Arloing, Leclainche, et Vallee, loc. cit. 5 Pasteur, Bull, de 1'acad. de med., 1877, p. 793. BACILLUS OF MALIGNANT EDEMA 467 Koch,1 who studied this infection in connection with his work upon anthrax in 1881, called attention to the fact that the bacillus described by Pasteur did not produce a true septicemia, and suggested the term " Bacillus of malignant edema," which is now in general use. Gaffky 2 found that, apart from its presence in putrid material, the bacillus occurred in the upper layers of garden soil and in dust. It has since been found to be widely distributed in nature and in the intestines of animals and of man. Its wide distribution is unques- tionably due to the great resistance of its spores. Morphology and Staining. — The bacillus of malignant edema is a Fig. 101. — Bacillus of Malignant Edema. (After Frankel and Pfeiffer.) long slender rod, not unlike the anthrax bacillus, but decidedly more slender. Its average measurements are 1 micron in thickness and 3 to 8 micra in length. It usually occurs as single rods, but frequently appears in long threads showing irregular subdivisions. Often no sub- divisions can be seen and the threads appear as long, homogeneous filaments. These threads are less frequently seen in preparations from solid media than in those from bouillon or edema fluid. The bacilli are motile and possess numerous laterally placed flagella. Their motil- ity is never very marked and is often entirely absent. The bacillus 1 Koch, Mitt. a. d. kais. Gesundheitsamt, i, 1881, p. 52 et seq. 2 Gaffky, Mitt. a. d. kais. Gesundheitsamt, 1881. 468 PATHOGENIC MICROORGANISMS produces spores at temperatures above 20° C, which are oval, irregularly placed either in the center or slightly nearer one or the other end, and cause a bulging of the bacillary body. It is readily stained by any of the usual anilin dyes. Stained by Gram's method it is decolorized. Cultivation. — Bacillus cedematis maligni is strictly anaerobic. Under anaerobic conditions it develops readily upon any of the usual artificial media. The bacillus is not very sensitive to the reaction of media and grows more luxuriantly in all media to which glucose has been added. In all media it forms, by the cleavage of proteids, putridly offensive gases. In gelatin at room temperature, colonies develop in about three days as small grayish spherical growths, which microscopically show an arrangement in radial filaments. The gelatin is fluidified. In gelatin stab cultures growth begins as a white column extending to within a centimeter of the top of the medium. Soon irregularly radiating processes develop laterally and gas bubbles appear, breaking up the medium. Stab cultures in agar show growth within twenty- four to thirty-six hours at 37.5° C, appearing at first as a white line, but soon showing a cloud-like lateral extension along the entire line of the stab. If sugar is present bubbles appear throughout the medium. In broth there is general clouding and a granular sediment; no pellicle is formed. Milk is slowly coag- ulated. On blood serum growth is very luxuriant. On potato, a medium used in the earliest studies of the bacillus by Gaffky, the bacillus grows readily. Isolation may be accomplished by any of the ordinary anaerobic plating methods. The bacillus can usually be obtained for subsequent isolation by injection of a susceptible animal with soil, especially that of gardens or manured fields. Pathogenicity. — The bacillus is pathogenic for mice, guinea-pigs, rabbits, horses, dogs, sheep, pigs, some birds, and man. Cattle were formerly regarded as immune, an opinion which has since been found to be erroneous. Fig. 102.— Ba- cillus of Ma- lignant Edema. Culture in glu- cose agar. BACILLUS AEROGENES CAPSULATUS 469 Subcutaneous inoculation of pure culture into a susceptible subject produces, within twenty-four to thirty-six hours, an acute edematous inflammation about the point of inoculation. The edema extends throughout the subcuticular and deeper layers, and consists of thin, slightly bloody fluid. Neighboring lymph nodes become swollen and hemorrhagic. In the mixed infections of accidental inoculation, but more rapidly in experimental inoculations with pure cultures, gas is formed and consequent subcutaneous emphysema. Together with this there are symptoms of general toxemia. In the smaller test animals this disease is usually fatal. At autopsy the bacilli are found in the edema fluid about the local lesion. At autopsies done soon after death, the organisms are not found in the blood or internal organs. Later they may be generally distributed throughout the body. In mice only may the bacilli enter the blood stream before death. The internal organs of animals dead of this infection usually show parenchymatous degen- eration and occasionally hemorrhages. Malignant edema is not a frequent disease. It has been occasionally observed in horses, in cattle, and in sheep. In man the infection usually appears after traumatism or secondarily after compound fractures or upon the site of suppurating wounds. Isolated cases have been de- scribed as arising after hypodermic injections. One case has been reported as arising in the uterus after instrumental abortion. Immunity. — Recovery from an infection with the bacillus of malig- nant edema produces immunity against subsequent infections.1 The bacillus in fluid media produces small amounts of a soluble toxin which in bacteria-free filtrates is capable of killing guinea-pigs. Relatively large quantities of filtrate must be employed. Roux and Chamberland,2 the first to work upon these toxins, were able, by means of them, to immunize guinea-pigs. Similar immunity could be produced by treat- ment with the toxic, filtered sera of animals dead of the disease.3 BACILLUS AEROGENES CAPSULATUS Bacillus aerogenes capsulatus was first observed by Welch and fully described by Welch and Nuttall 4 in 1892. It is identical with a bacillus 1 Arloing et Chauveau, Ann. de med. vet., 1884. 2 Roux et Chamberland, Ann. de Finst. Pasteur, 1887. 3 Sanfelice, Zeit. f. Hyg., xiv. A Welch and Nuttall, Bull. Johns Hopkins Hosp., iii, 1892, p. 81. 470 PATHOGENIC MICROORGANISMS later described by Frankel,1 and named by him Bacillus phlegmonis emphysematosa. Similar, probably identical, bacilli have been found and reported sub- sequently by other observers, ignorant of the work of Welch and Nuttall. Such are the B. perfringens (Veillon and Zuber), and B. emphysematis vaginae (Lindenthal, Wien. klin. Woch., 1897), and others. Welch 2 first obtained the bacillus from the intravascular blood of a case of ruptured aortic aneurysm, autopsied six hours after death, his attention being called to the blood particularly by the existence of air bubbles throughout the vessels. Apart from its occurrence in infected subjects, the bacillus finds wide distribution in nature, being found in soil, dust, and brackish water, and in the normal intestinal tracts of human beings and mammals.3 Morphology and Staining. — Bacillus aerogenes capsulatus appears usually as a straight rod, not unlike the anthrax bacillus, but more variable in length and somewhat thicker in proportion than the latter. Occasionally bacilli are seen which are slightly curved, but these are rare. The bacillus averages 3 to 6 micra in length, but may be three or four times longer than this. More rarely the bacillus may appear so short as to be almost coccoid. In artificial cultures it is usually thicker and shorter than it is in animal tissues. The bacilli are generally single, but are often seen in short chains. Their ends are usually slightly rounded but, especially when in chains, may be almost square. Chain- formation seems to occur chiefly in the blood, long chains never occurring in artificial media. This characteristic is regarded by Welch as a dis- tinguishing feature in differentiating this bacillus morphologically from anthrax. In their first publication, Welch and Nuttall did not describe spores as appearing in these bacilli. Dunham,4 however, in 1895, found spores in cultures grown upon blood serum. The spores seem to be formed only upon special media, rarely upon plain agar, never in the animal body. The spores are oval, and may be placed centrally or toward one end, one in each bacillus. The bacillus is non-motile, and does not possess flagella. It pos- i Frankel, Cent. f. Bakt., xiii, 1893, p. 13. 2 Welch, Bull. Johns Hopkins Hosp., xi, 1900, p. 185. 3 Although B. aerogenes capsulatus and B. phlegmonis emphysematosae are separately described in many books, notably Migula's " System d. Bakteriologie," the microorganisms have been shown beyond question to be identical and are acknowledged to be so by Frankel himself. 4 Dunham, Johns Hopkins Hosp. Bull., viii, 1897, p. 68. BACILLUS AEROGENES CAPSULATUS 471 sesses a capsule which, however, can not be constantly demonstrated. The capsules are best seen when preparations are made from animal fluids, but can often be demonstrated in those stained from artificial cultures. They are demonstrated best by one or the other of the ordinary capsule stains. The bacillus is stained easily by the usual anilin dyes. In tissue preparations, the bacilli regularly retain the gentian-violet when stained by Gram's method. In smears from artificial culture media, while most of the bacilli stain by Gram, many will be seen wholly or partially decolorized, owing probably to the rapid production of involution forms. Cultivation. — Bacillus aerogenescapsulatus is an obligatory anaerobe. The first cultivations by Welch and Nuttall were made in deep agar stabs. It grows well upon all the usual media, preferring a neutral or slightly alkaline reaction. All media are improved for the cultivation of this bacillus by the addition of glucose, lactose, or some other easily fermented carbohydrate. Upon agar or gelatin plates, growth appears at 37.5° C. within twenty-four hours, as a flat, grayish translucent round disk. The margins of colonies are slightly irregular and fringed. Gelatin is slowly liquefied by the large majority of cultures, but Welch states that occa- sionally liquefaction does not occur. In deep agar stabs or in agar slant cultures, especially in those con- taining a carbohydrate, there is a rapid formation of gas bubbles, a characteristic which is especially well developed and lends the cultures of this bacillus their chief diagnostic feature. In broth, growth is heavy and abundant. At first there is general clouding. Within forty-eight hours, however, a heavy, white, flocculent sediment is formed. Owing to the formation of gas, broth tubes if undisturbed usually show a light froth of bubbles on the surface. On potato, growth is scanty and the medium possesses no advantages either for cultivation or diagnosis. On coagulated blood serum, growth is heavy and rapid and this medium is especially adapted for spore formation. There is slight peptonization of the blood serum. In milk, there is rapid coagulation, rapid acidification, and gas formation. The carbohydrates, glucose, lactose, and saccharose, are fermented by this bacillus. Mannit is apparently not fermented. Welch and Nuttall state that the bacillus is capable of producing gas from proteid matter. The gas formed, according to Dunham,1 consists of 1 Dunham, loc. cit. 472 PATHOGENIC MICROORGANISMS 64 per cent of hydrogen, 28 per cent of C02, and 8 per cent of a mix- ture of gases, chiefly nitrogen. The gas from the infected animal body is ignitable and burns with a bluish hydrogen flame. Biological Considerations. — The bacillus, as stated, is anaerobic. Its anaerobic requirements, however, are less exacting than those of some other anaerobes, and in stab cultures it will often grow up to the surface of the stab. It grows best at 37.5° C, but will also develop at room temperature (20° to 22° C). Isolation. — The bacillus may, of course, be isolated by anaerobic plating methods. It is best isolated, however, from mixed cultures by animal inoculation. If, for instance, it is desired to obtain it from a mixed culture or from feces, a suspension of about 1 c.c. of the suspected material is made in 5 c.c. of sterile salt solution. This is thoroughly emulsified and filtered through a sterile paper. One to two c.c. of this suspension is then injected into the ear vein of a rabbit. After four or five minutes the rabbit is killed. It is then placed in the incubator for five to eight hours. At the end of this time, the animal is usually found tensely distended with gas. At autopsy, gas bubbles will be found distributed through- out the organs, most characteristically in the liver, where isolated bubbles are found covering the surface. From these bubbles cul- tures or smears may be taken for identification. Identification is easily made from its morphology, its capsule, lack of motility, and gas formation. Pathogenicity. — Bacillus aerogenes capsulatus is highly pathogenic for guinea-pigs, but very slightly for rabbits. Its virulence is subject to great variations, however, some strains showing little if any pathogen- icity even for guinea-pigs. In general, its pathogenicity for the ordi- nary laboratory animals may be regarded as slight. In man,1 the bacillus has been isolated from numerous cases of so-called "emphyse- matous gangrene " 2 (gangrene foudroyant). The infection usually occurs upon the extremities and is characterized by a rapidly necro- tizing inflammation, with which there occurs extensive subcutaneous emphysema. The infection usually follows traumatism,3 especially compound fractures, and is extremely grave. The bacillus has also been found in the uterus in puerperal infection,4 and in the fetus 1 Welch and Flexner, Jour. Exp. Med., 1, 1896. 2 Mann, Ann. of Surgery, xix, 1894. 3 Bloodgood, Progressive Med., 1899. i Dobbin, Johns Hopkins Hosp. Bull., viii, 1897. BACILLUS BOTULINUS 473 dead in utero. It has been found in the blood before death, by Gwyn,1 in a case of chorea. The bacillus has also been found in infectious processes of various other parts of the body, such as the gastrointes- tinal and biliary tracts, the lungs, the pleura, and the meninges. As stated above, this bacillus is frequently present in the normal intestinal contents. Its presence in abnormally high proportions, as indicated by a Gram stain of a smear of the feces, has been associated by a number of observers with various pathological conditions. Herter 2 has recently studied this subject and believes that the abnormal prolif- eration of the bacillus in the gastrointestinal tract has in some way (probably by toxin absorption) an etiological relationship to pernicious anemia. This assertion, however, can in no way be regarded as conclusively proven. * BACILLUS BOTULINUS Meat poisoning was formerly regarded as universally dependent upon putrefactive changes taking place in infected meat, resulting in the production of ptomains or other harmful products of bacterial putre- faction. It was not until 1888 that certain of these cases were definitely recognized as true bacterial infections, in which the preformed poison probably aided only in establishing the infection. Gartner, in that year, discovered the Bacillus enteritidis, a microorganism belonging to the group of the paracolon bacilli, and demonstrated its presence both in the infecting meat and in the intestinal tracts of patients. The char- acteristics of this type of meat poisoning have been discussed more particularly in the section describing the bacillus of Gartner and its allied forms. There is another type of meat poisoning, however, which is not only much more severe (ending fatally in almost 25 per cent of the cases) , but is characterized by a clinical picture more significant of a profound systemic toxemia than of a mere gastroenteric irritation. The etio- logical factor underlying this type of infection was first demonstrated by van Ermengem,3 in 1896, and named Bacillus botulinus. van Ermengem isolated the bacillus from a ham, the ingestion of which had caused disease in a large number of persons. Of the thirty-four individuals who had eaten of it, all were attacked, about ten of them 1 Gwyn, Johns Hopkins Hosp. Bull., x, 1899. 2 Herter, " Bacterial Infection of the Intestinal Tract," New York, Macmillan, 1907. 3 van Ermengem, Cent. f. Bakt., xix, 1896; Zeit. f. Hyg., xxvi, 1897. 474 PATHOGENIC MICROORGANISMS very severely, van Ermengem found the bacilli in large numbers lying between the muscle fibers in the ham, and was able to cultivate the same microorganism from the stomach and spleen of one of those who died of the infection. The results of van Ermengem have been confirmed by Romer,1 and others. Morphology and Staining. — Bacillus botulinus is a large, straight rod with rounded ends, 4 to 6 micra in length by 0.9 to 1.2 micra in thickness. The bacilli are either single or grouped in very short chains. Involu- tion forms are numerous on artificial media. The bacillus is slightly motile and possesses from four to eight undulated flagella, peripherally arranged. Spores are formed in suitable media, most regularly in glucose-gelatin of a distinctly alkaline titer. The spores are oval and usually situated near the end of the bacillus, rarely in its center. Spores are formed most abundantly when cultivation is carried out at 20° to 25° C, and are usually absent when higher temperatures are em- ployed. The bacillus is easily stained by the usual aqueous anilin dyes, and retains the anilin-gentian- violet when stained by Gram. It is necessary, however, in carrying out Gram's stain to decolorize carefully with alco- hol since overdecolorization is easily accomplished, leaving the result doubtful. Cultivation. — The bacillus is a strict anaerobe. In anaerobic en- vironment it is easily cultivated on the usual meat-infusion media. It grows most readily at temperatures about 25° C, less luxuriantly at temperatures of 35° C. and over. The bacillus is delicately susceptible to the reaction of media, growing only in those which are neutral or moderately alkaline. In deep stab cultures in one per cent glucose agar, growth is at first noticed as a thin, white column, not reaching to the surface of the medium. Soon the medium is cracked and split by the abundant formation of gas. On agar plates, the colonies are yellowish, opalescent, and round, and show a finely fringed periphery. On gelatin, at 20° to 25° C, growth is rapid and abundant, and differs little from that on agar, except that, besides the formation of gas, there is energetic fluiciification of the medium. On glucose-gelatin plates, van Ermengem describes the colonies as round, yellowish, transparent, and composed of coarse granules which, along the periphery 1 Romer, Cent. f. Bakt., xxvii, 1900. BACILLUS BOTULINUS 475 in the zone of fluidification, show constant motion. The appearance of the surface colonies on glucose-gelatin plates is regarded by the discov- erer as diagnostically characteristic. In glucose broth there is general clouding and large quantities of gas are formed. At 35° C. and over, the gas formation ceases after four or five days, the broth becoming clear with a yellowish-white flocculent sediment. At lower temperatures this does not occur. Milk is not coagulated and disaccharids and polysaccharids are not fermented. The gas formed in cultures consists chiefly of hydrogen and methane. All cultures have a sour odor, like butyric acid, but this is not so offensive as that of some of the other anaerobic organisms. The bacillus lives longest in gelatin cultures, but even upon these, transplantations should be done every four to six weeks, since the spores of this bacillus show less viability and resistance than do those of most spore-formers. Pathogenicity. — Botulism or allantiasis, as noticed in human beings, is, as far as we know, always due to ingestion of infected meat, usually of ham, canned meats, or sausages (botulus = sausage). Symptoms appear only after a definite period of incubation which varies from twenty-four to forty-eight hours. The first definite symptoms are chilliness, trembling, and giddiness. These manifestations are soon followed by headache, occasionally by vomiting. In contradistinction to the meat poisonings caused by other microorganisms, those due to Bacillus botulinus may show few or no symptoms directly referable to the intestinal tract. The chief diagnostic characteristics of the disease are a group of symptoms referable to toxic interference with the cranial nerves. Loss of accommodation, dilated pupils, ptosis, aphonia, and dysphagia may occur. Fever is usually absent. Consciousness is rarely lost. The characteristic symptoms may be produced in various animals by injection of living cultures or culture filtrates, i.e., toxins. The most susceptible animals are guinea-pigs. These may be killed by the injec- tion of minute quantities of bouillon cultures or of toxin. Preceding death, which occurs within twenty-four to thirty-six hours, there may be general motor paralysis, dyspnea, and hypersecretion of mucus from nose and mouth. Guinea-pigs may be infected per os as well as by hypodermic injections. Cats, mice, and monkeys are highly susceptible; rabbits are less so, but still favorable subjects for experimental studies. Birds, especially pigeons, are highly resistant, but react typically to large doses. Autopsies upon man or animals dead of botulism show 476 PATHOGENIC MICROORGANISMS general hyperemia of the organs with much parenchymatous degenera- tion and many minute hemorrhages. The bacilli have been found in the spleen after death/ but van Ermengem does not believe that they are generally distributed during the course of the disease. It is believed by most of those who have studied this disease that poisoning in the human subject is due to the toxins preformed in the infected meat by this bacillus. Experiments have shown that little or no poison is produced by the bacilli after gain- ing entrance to the human or animal body. The Toxin of B. botulinus. — Bacillus botulinus produces disease chiefly by means of a strong soluble toxin secreted by it, and absorbed by the infected subject. This toxin is active in animals and presumably in man, not only when injected subcutaneously, but also when intro- duced through the gastrointestinal canal. The poison has been par- ticularly studied by Brieger and his collaborators. It is obtained in filtrates of alkaline bouillon cultures. It has been precipitated out of the filtrate by Brieger and Kempner 2 by means of a three per cent zinc chlorid solution (2 volumes of 3 per cent ZnCl2). The toxin thus obtained was sufficiently powerful to kill a 250-gram guinea-pig in fifty hours. Specific action of the toxin for the nerve-cells of the spinal ganglia has been shown by Marinesco.3 A specific antitoxin has been produced by Kempner and Pollack.4 1 Stricht, Quoted from van Ermengem, in Kolle und Wassermann, 2 Brieger und Kempner, Deut. med. Woch., xxxiii, 1897. 3 Marinesco, Compt. rend, de Tacad. des sci., Nov., 1896. 4 Kempner und Pollack, Deut. med. Woch., xxxii, 1897. N CHAPTER XXXIII THE TUBERCLE BACILLUS In view of the clinical manifestations of tuberculosis, it is not sur- prising that the infectious nature of the disease has been suspected for many centuries. Transmission by means of tuberculous material was first successfully accomplished by Klencke, in 1843, and, more elabo- rately, by Villemin,1 in 1865. It was not until 1882, however, that Koch 2 succeeded in isolating and cultivating the tubercle bacillus. Baumgarten 3 had previously seen the bacillus in tissue sections, but his researches were limited to purely morphological observations. Koch, in addition to demonstrating the bacillus in tuberculous tissues from various sources, produced characteristic lesions in guinea-pigs and other animals by infecting them with pure cultures, and established beyond doubt the etiological relationship of the bacillus to the disease. Morphology. — Tubercle bacilli appear as slender rods, 2 to 4 micra in length, 0.2 to 0.5 micra in width. Their ends are usually rounded. The rods may be straight or slightly curved; their diameters may be uniform throughout; more often, however, they appear beaded and irregularly stained. The beaded appearance is due to different causes. Unstained spaces may occur along the body of the bacillus, especially in old cultures. These are generally regarded as vacuoles. The bodies of the bacilli, on the other hand, may bulge slightly here and there, often in three or four places, showing oval or rounded knobs which stain with great depth and are very resistant to decolorization. These thickenings were formerly regarded as spores, but in view of the fact that the bacilli are not more resistant against heat and disinfectants than other vegeta- tive forms, this interpretation is probably incorrect. The bacilli are said to possess a cell membrane which confers upon them their resistance against drying and entrance of stains. This membrane gives a cellulose reaction and is believed to contain most of the waxy substances which can be extracted from the cultures. 1 Villemin, Gaz. hebdom., 1865. 2 Koch, Berl. klin. Woch., 1882; Mitt. a. d. kais. Gesundheitsamt, 1884. 3 Baumgarten, Virchow's Arch., lxxxii. 477 478 PATHOGENIC MICROORGANISMS Various observers, notably Nocard and Roux,1 Mafucci,2 and Klein,3 have demonstrated branched forms of the tubercle bacillus. These ob- servations, variously extended and confirmed, make it probable that Bacillus tuberculosis is not a member of the family of schizomycetes Fig. 103. — Tubercle Bacilli in Sputum. but belongs rather to the higher bacteria, closely related to the actino- myces. Staining. — Tubercle bacilli do not stain easily with the ordinary anilin dyes; to these they are made permeable only by long exposure or by heating of the staining solution. Once stained, however, the dye is tenaciously retained in spite of treatment with alcohol and strong 1 Nocard et Roux, Ann. de l'inst. Pasteur, 1887. 2 Mafucci, Zeit. f. Hyg., ii. 3 Klein, Cent. f. Bakt., 1890. THE TUBERCLE BACILLUS 479 acids. For this reason, this bacillus, together with some other bacteria to be mentioned later, is spoken of as " acid-fast." The acid-fast nature of the bacillus seems to depend upon the fatty substances contained in it,1 and has furnished the basis for differential staining methods. All the staining methods devised for the recognition of the tubercle bacillus thus depend upon the use of an intensely penetrating staining solution, followed by vigorous decolorization which deprives all but the acid-fast group of their color. Counterstains of any of the weaker dyes may then be used to stain the decolorized elements. One of the first of the staining solutions to be of practical use was the anilin-water-gentian- violet solution of Ehrlich 2 (11 c.c. saturated alcoholic gentian-violet to 89 c.c. 5 per cent anilin water). This dye, although of sufficient penetrating power, has the disadvantage of deteriorating rapidly and has in practice been almost entirely displaced by ZiehPs 3 carbol-fuchsin solution. (Fuchsin 1 gm. in 10 c.c. alcohol absolute, added to 90 c.c. 5 per cent carbolic.) This staining solution is the one now in general use and is employed as follows: Thin smears, on slides or cover-slips, are covered with the dye and gently heated. In the case of cover-glasses, these may be floated, face downward, in staining dishes filled with the dye. The dye is allowed to act for about three minutes, steaming but not allowed to boil. At the end of this time the preparation is washed either with 5 per cent nitric acid, 5 to 20 per cent sulphuric acid, or 1 per cent hydro- chloric acid, until most of the red color has disappeared (a few seconds), and the preparation appears pale pink. This results in decolorization of all microorganisms with the exception of members of the acid-fast group. Thorough washing in 80 to 95 per cent alcohol is now employed to complete the decolorization. The preparation is then rinsed in water and counterstained with 1 per cent aqueous methylene-blue . Tubercle-bacillus staining has been further simplified by Gabbett,4 who combines decolorization and counterstaining. In this method preparations are stained with ZiehFs carbol-fuchsin as in the preceding; they are then rinsed in water and covered with a solution containing methylene-blue 1 gram, concentrated sulphuric acid 25 grams, and distilled water 100 c.c. This is allowed to act for from two to four » Bienstock, Fort. d. Med., 1886; Weyl, Deut. med. Woch., 1891. 2 Ehrlich, Deut. med. Woch., 1882; Weigert, Deut. med. Woch., 1885. 3 Ziehl, Deut. med. Woch., 1883; Neelsen, " Lehrb. d. allg. Path./' 1894. * Gabbett, Lancet, 1887. 480 PATHOGENIC MICROORGANISMS minutes, at the end of which time all elements in the preparation except the acid-fast bacilli will be decolorized and counterstained. While the acid-fast group of bacteria is composed of a number of organisms to be mentioned later, a few only of these offer difficulties of differentiation from the tubercle bacillus. Those to be considered practically are the bacillus of leprosy and that of smegma. The latter bacillus, because of its distribution, is not infrequently found to con- taminate feces, urine, or even sputum, and it is important to apply to suspected specimens one or the other of the stains devised for the differentiation of the smegma bacillus from Bacillus tuberculosis. The one most frequently employed is that of Pappenheim.1 The preparations are stained in hot carbol-fuchsin as before; the carbol-fuchsin is then poured off without washing and the preparation immersed in a solution made by saturating a 1 per cent alcoholic solution of rosolic acid with methylene-blue and adding 20 per cent of glycerin. In such prepara- tions tubercle bacilli remain red, smegma bacilli appear blue. Stained by Gram, tubercle bacilli retain the gentian- violet. Isolation and Cultivation. — Tubercle bacilli are not easily cultivated. Their slowness of growth precludes their isolation by the usual plating methods. The first isolations by Koch 2 were made upon coagulated blood serum from bits of tuberculous tissue smeared over its surface. Isolation from tuberculous material may be greatly aided by in- oculation into guinea-pigs. These animals will often withstand the acute infection which may be produced by the contaminating organisms and succumb at a later date (four to six weeks) to the tuberculous infection. The bacilli may then be obtained, after sterile dissection, by making cultivations from lymph nodes or other tubercu- lous foci which contain only tubercle bacilli. When isolation from sputum is attempted, whether directly or by means of animal inocula- tion, the sputum may be rendered comparatively free from contaminat- ing bacteria by a process of washing devised by Koch. The sputum is thoroughly rinsed in running water to free it from its outer covering of pharyngeal mucus. It is then washed in eight or ten changes of sterile water. The material selected for cultivation is taken from the center of the washed mass, if possible from the small flakes of caseous material often visible in such sputum. On blood serum at 37.5° C, colonies usually become visible at the end of eight to fourteen days. They appear at first as small, dry, gray- 1 Pappenheim, Berl. klin. Woch., 1898. 2 Koch, loc. cit. THE TUBERCLE BABILLUS 481 ish-white, scaly spots with corrugated surfaces. After three or four weeks' cultivation, these join together, covering the surface of the medium as a dry, whitish, wrinkled membrane. Coagulated dog serum is regarded by Theobald Smith * as one of the most favorable media for the growth of tubercle bacilli. Slants of agar, to which whole rabbit's blood has been added in quantities of from 1 to 2 c.c. to each tube, make an excellent medium for this bacillus, both for isolation and continuous cultivation. Cultivation methods were simpli- fied by the discovery by Roux and Nocard that growth can be obtained upon media to which glycerin has been added. Upon glycerin-agar (glycerin 3 to 6 per cent), at 37.5° C, colonies become visible at the end of from ten days to two weeks, at first as dry, white spots; later, as deli- cately corrugated membranes. Glycerin bouillon (made of beef or veal with pepton one per cent, gly- cerin six per cent, and rendered slightly alkaline) is an extremely favorable medium. It should be filled, in shallow la}^ers, into wide- mouthed flasks, since free access of oxygen is essential for growth. Trans- plants to this medium should be made by carefully floating flakes of the cul- ture upon the surface. In this medium the bacilli will spread out upon the surface, at first as a thin, opaque, floating membrane. This rapidly thickens into a white, wrinkled, or granular layer, spreading out in all directions, and covering the entire surface of the fluid in from four to six weeks. Later, portions of the membrane sink to the bottom. In old cultures, the membrane as- sumes a yellowish hue. These cultures emit a peculiar aromatic odor. Glycerin potato forms a favorable culture medium for the bacillus. Fig. 104. — Culture of Bacillus Tuberculosis in Flask of Glyc- erin Bouillon. 1 Th. Smith, Jour. Exp. Med., iii, 1898. 31 482 PATHOGENIC MICROORGANISMS Hesse * has devised a medium containing a proprietary preparation known as " Nahrstoff Heyden," upon which tubercle bacilli are said to proliferate more rapidly than other bacteria. His method has yielded excellent results in the hands of other observers, both in isolation and in rapid cultivation. It is prepared as follows: "Nahrstoff Heyden " 2 10 grams Sodium chlorid 5 " Glycerin 30 " Agar 10 " Normal sodium solution 5 c.c. Aq. dest 1,000 " A variety of other culture media have been devised, none of them, however, possessing any marked advantages over those given. Biological Considerations. — The tubercle bacillus is markedly depend- ent upon the free access of oxygen. The optimum temperature for its development is 37.5° C. Temperatures below 30° and above 42° C. inhibit its growth. In fluid media, the bacilli are killed by a tempera- ture of 60° in fifteen to twenty minutes, by one of 80° in five minutes, by one of 90° in one to two minutes. They will withstand dry heat at 100° C. for one hour. They are resistant to cold. The comparatively high powers of resistance of the bacillus are attributed to the protective qualities of the waxy cell membrane;5 The natural life of cultures, kept in favorable environment, is from two to eight months, varying to some extent with the nature of the culture medium. The viability of the bacilli in sputum is of great hygienic importance. In moist sputum they may remain alive and virulent for as long as six weeks, in dried sputum for more than two months.4 Five per cent carbolic acid kills the bacilli in a few minutes.5 If used for sputum disinfection, however, where the bacilli are protected by mucus, complete disinfection by this method requires five to six hours. Bichlorid of mercury is not very efficient for sputum disinfection be- cause of the formation of albuminate of mercury. 1 Hesse, Zeit. f. Hyg., xxxi. 2 " Nahrstoff Heyden " is prepared in Germany. It is a white powder similar to nutrose. 3 Th. Smith, Jour. Exper. Med., 1899; Grancher et Ledoux-Lebard, Arch, de m£d. exper., 1892; Galtier, Compt. rend, de l'acad. des sci., 1887. * Schell und Fischer, Mitt. a. d. kais. Gesundheitsamt, 1884. « De Toma, Ann. di med., 1886. THE TUBERCLE BACILLUS 483 For room disinfection, formaldehyde gas if thoroughly employed is efficient. Direct sunlight kills tubercle bacilli in a few hours. Pathogenicity. — The tubercle bacillus gives rise in man and suscep- tible animals to specific phenomena of inflammation which are so characteristic that a diagnosis of tuberculosis may usually be made on the basis of the histological examination of material, even without the finding of tubercle bacilli. The foci of inflammation known as tubercles have been systematically studied by Baumgarten 1 and many others and descriptions of them may be found in any text-book of pathological anatomy. In man, tuberculosis is by far the most common of diseases. Naegeli,2 in a large series of autopsies, found lesions of healed or active tuberculosis in an appalling percentage of cases. His figures are in- teresting in showing not only the frequency of the disease, but its rela- tion to age. Before one year of age, he finds it very rare. From the first to the fifth year it is rare, but usually fatal when occurring. From the fifth to the fourteenth year, one-third of his cases showed tuberculous lesions ; from the fourteenth to the eighteenth year, one-half of the cases. Between the ages of eighteen and thirty, almost all the cases examined showed some trace of tuberculous infection. Three-quarters of these were active, one-quarter healed. Two-fifths of all deaths occurring at these ages were due to tuberculosis. After the age of thirty, active lesions gradually diminished in number, healed lesions increased. In 1900, at a public hearing of the New York Tenement House Commission, Pryor 3 stated that the average yearly mortality from tuberculosis in New York amounted to 6,000, and that in Manhattan alone there were constantly 20,000 persons suffering from the disease. Cornet 4 estimates that in 1894 the deaths in Germany from all other infectious diseases amounted to 116,705, while those from tuberculosis alone amounted to 123,904. Similar statistics might be chosen at will from the health reports of any large city. While the disease is less common in rural districts than in large towns, the difference is not so striking as is generally supposed. In man, pulmonary infection is by far the commonest type. Be- sides this, however, tuberculous processes may be found in the skin, the 1 Baumgarten, Berl. klin. Woch., 1901. 2 Naegeli, Virchow's Arch., cix, 1900, p. 462. 3 Pryor, Med. News, lxxvii, 1900. * Cornet, " Die Tuberculose, " Wien, 1899, p. 1. 484 PATHOGENIC MICROORGANISMS bones, the joints, the organs of special sense, and the abdominal viscera and peritoneum. No part of the human body is exempt from the danger of infection. Infection in the human subject may take place by inhalation or through the skin or the digestive apparatus, v. Behring 1 has within recent years expressed the belief that a large percentage of all cases of tuberculosis originate in childhood from infection by way of the intes- tinal tract. He determined, as have others since his publication, that tubercle bacilli may penetrate the intestinal mucosa without causing lesions. Behring's contention has caused a great deal of discussion, and the question he has raised is intimately bound up with the problem of the virulence of bovine tubercle bacilli for human beings, as he assumes that the infection is due to the use of infected milk. In spite of many investigations 2 to settle the points in question, our present knowl- edge, however, is insufficient to permit a positive opinion upon the subject, except in so far as we may recognize the original sweeping- statements of Behring as extravagant. The problem is discussed at greater length in a later section of this chapter. Suffice it to say in this place that the opinion, at the present time, of such investi- gators as Calmette tends toward the belief that infection by inhala- tion may take place, but that infection by the path of the digestive tract is probably more common. Bacillus tuberculosis (typus humanus) is pathogenic for guinea- pigs, less markedly for rabbits, and still less so for dogs. It is slightly pathogenic for cattle, a question spoken of more extensively below. Chemical Analysis of Tubercle Bacilli.3 — Diligent efforts by many investigators to isolate the specific toxins which lend tubercle bacilli their pathogenic properties have led to careful chemical analysis of the organisms. About 85.9 per cent of the bacillus consists of water; 20 to 26 per cent of the residue can be extracted with ether and alcohol. This material consists of fatty acids and waxy substances (fatty acids in combination with the higher alcohols). The residue after alcohol- ether extraction is composed chiefly of proteids. These can be extracted with dilute alkaline solutions, and consist chiefly of nucleo-albumins. i v. Behring, Deut. med. Woch., 39, 1903. 2 Battel, Internat. Tube. Conferenz, Wien, 1907; Oehlecker, Arb. a..d. kais. Gesundheitsamt, H. 7, 1907. 3 Hammer schlag, Cent. f. klin. Med., 1891; Weyl, Deut. med. Woch., 1891; De Schweinitz and Dorset, Jour. Amer. Chem. Soc., 1895; Hammerschlag, loc. cit. THE TUBERCLE BACILLUS 485 A nuclein present in .this fraction shows extremely high toxicity and has,1 therefore, been suspected of being the pathogenic principle of the bacillus. After these extractions the remainder contains "cellulose/' supposed to represent the framework of the cell membrane, and an ash rich in calcium and magnesium. Toxins of the Tubercle Bacillus. — The Tuberculins. — Filtrates of bouillon cultures of Bacillus tuberculosis 2 will occasionally produce slight emaciation when injected into guinea-pigs, and when administered to tuberculous subjects in sufficient quantity will give rise to marked increase of temperature. It is likely, therefore, that the tubercle bacillus actually secretes a soluble toxin.3 The chief toxic principles, however, of Bacillus tuberculosis are probably endotoxins or bacterial proteins, bound during cell life to the body of the bacillus. Dead bacilli will produce sterile abscesses when injected into animals. Prudden and Hodenpyl,4 Straus and Gamaleia,5 and others,6 moreover, have shown that the injection of dead and care- fully washed cultures of this bacillus will produce lesions histologi- cally similar to those occurring after infection with the living germs, and will often lead to marasmus and other systemic symptoms of poisoning. The hope of actively immunizing with substances obtained from dead bacilli led Koch to employ various methods of extraction of cultures for the manufacture of tuberculin. " Old Tuberculin" \Koch) (" T. A. K") .—The first tuberculin made by Koch is produced in the following manner: Tubercle bacilli are grown in slightly alkaline 5 per cent glycerin-pepton-bouillon for six to eight weeks. At the end of this time, growth ceases and the corrugated pellicle of tubercle bacilli, which during growth has floated on the surface, begins, here and there, to sink to the bottom. The entire culture is then heated on a water-bath at about 80° C, until reduced to one-tenth of its original volume. It is then filtered either through sterile filter paper or through porcelain filters. The resulting filtrate is a rich brown, syrupy fluid, containing the elements of the original cul- 1 Behring, Berl. klin. Woch., 1899. 2 Straus and Gamaleia, Arch. med. exp., 1891. 3 Denys, " Le Bouillon Filtre," Louvain, 1905. 4 Prudden and Hodenpyl, N. Y. Med. Jour., June, 1891; Prudden, ibid., Dec. 5. 5 Straus and Gamaleia, loc. cit. 6 Mafucci, Cent. f. allg. Path., 1890. ^ Koch, Cent. f. Bakt., 1890; Deut. med. Woch., 1891. 4S6 PATHOGENIC MICROORGANISMS ture medium and a 50 per cent glycerin extract of the tubercle bacilli. While the glycerin is of sufficient concentration to preserve it indef- initely, 0.5 per cent phenol may be added as an additional precaution. Dilutions of this fluid are used for diagnostic and therapeutic purposes. "New Tuberculin"1 (Koch) (T A,. TO, TR).— Koch believed that the immunity resulting from treatment with the old tuberculin was purely an antitoxic immunity, devoid of all antibacterial action. The use of whole dead tubercle bacilli for immunization purposes, however, wTas impracticable; because, injected subcutaneously, they were not absorbed, and introduced intravenously they were deposited in the lungs and gave rise to lesions. Koch was led, therefore, to resort to more energetic extraction of the bacilli in the hope of procuring a substance which could be easily absorbed and would at the same time give rise, when injected, to antibodies more definitely bactericidal. By extract- ing tubercle bacilli with decinormal NaOH, for three days, filtering through paper and neutralizing, he obtained his TA (alkaline tubercu- lin). This preparation seemed to fulfil some of the hopes of its dis- coverer, but had the disadvantage of often producing abscesses at the points of injection. Koch then resorted to mechanical trituration of the bacilli. The method he subsequently followed for tuberculin pro- duction is now extensively used, and is carried out as follows: 2 Virulent cultures of tubercle bacilli are dried in vacuo and thoroughly ground in a mortar. Grinding is continued until stained preparations reveal no intact bacilli. (This is done by machinery in all large manu- factories.) One gram of the dry mass is shaken up in 100 c.c. of sterile distilled water. This mixture is then centrifugalized at high speed. The supernatant fluid, known as TO (Tuberculin-Oberschicht) , contains the water-soluble constituents of the bacillus, gives no precipitate on the addition of 50 per cent glycerin, and has the same physiological action as the old tuberculin. The residue, TR (Tuberculin- Ruckstand) , after pouring off TO, is again dried and ground up, and again shaken in water and centrifugalized. This process is repeated several times, and eventually, after three or four repetitions, all the TR goes into emulsion. The total volume of water used for these TR extractions should not exceed 100 c.c. All of the TR emulsions are then mixed to- gether. This TR gives a precipitate with 50 per cent of glycerin, and is supposed by Koch to contain substances important in producing an antibacterial immunity. For purposes of standardization, the amount 1 Koch, Deut. med. Woch., 14, 1897. 2 Ruppel, Lancet, March 28, 1908. THE TUBERCLE BACILLUS 487 of solid substance in 5 c.c. of the TR is determined by evaporation in vacuo and drying. To the rest is added a little glycerin and formalde- hyde and enough water to allow each cubic centimeter of the solution to contain 0.002 grams of solid material. Thus the culture and the medium remaining the same, fairly accurate standardization is possible. "New Tuber culin-Bacillary Emulsion." * — In 1901, Koch combined "TO" and "TR" by putting forth a preparation referred to as "Bazillenemulsion." This consists of an emulsion of pulverized bacilli 1 : 100 in distilled water. After several days of sedimentation to re- move the coarser particles, the supernatant fluid is poured off and fifty per cent volume of glycerin is added to it for purposes of preservation. This preparation contains 5 milligrams of solid substance in each cubic centimeter. Bouillon Filtre (Denys).2 — This preparation consists of the filtrate (through Chamberland filters) of 5 per cent glycerin-pepton-bouillon cultures of Bacillus tuberculosis. Phenol 0.25 per cent is added to insure sterility. The filtered bouillon corresponds to the unconcentrated old tuberculin of Koch, but, not having been heated, is supposed by Denys to contain important soluble and possibly thermolabile secretory products of the bacillus. Tuber culoplasmin (Buchner and Hahri).3 — Buchner and Hahn, by crushing tubercle bacilli by subjecting them to a pressure of 400 atmospheres, obtained a cell-juice in the form of an amber fluid, to which they attributed qualities closely analogous to those of TR. Other tuberculins are those of Beraneck,4 highly recommended clinically by Sahli,5 that of Klebs,6 and the tuberculin produced from bovine tubercle bacilli by Spengler.7 Diagnostic Use of Tuberculin. — Subcutaneous Use. — The preparation usually employed for diagnostic purposes is Koch's "Old tuberculin" (Alttuberculin) . This preparation is administered by hypodermic injec- tion of small quantities obtained by means of dilutions. The dilutions are best made with a 0.5 per cent aqueous carbolic acid solution. In practice a 1 per cent solution is made by pipetting 0.1 c.c. of tuberculin 1 Koch, Deut. med. Woch., 1901. 2 Denys, " Le Bouillon Filtre," Lou vain, 1905. 3 Buchner und Hahn, Munch, med. Woch., 1897; Hahn, ibid. 4 Beraneck, Compt. rend, de l'acad. des sci., 1903. s Sahli, Corrbl. d. Schw. Aerzte, 1906. e Klebs, Cent. f. Bakt., 1896; Deut. med. Woch., 1907. 7 Spengler, Deut. med. Woch., xxxi, 1904; xxxi and xxxiv, 1905. 488 PATHOGENIC MICROORGANISMS into 9.9 c.c. of the 0.5 per cent carbolic solution. A cubic centimeter of this then contains 0.01 c.c. of tuberculin. One c.c. of this solution added to 9 c.c. of 0.5 per cent carbolic acid gives a solution in which each cubic centimeter contains 0.001 c.c, or 1 milligram of tuberculin. The initial dosage in adults in Koch's * early work, and as used by Beck 2 on a large number of patients, was 1 milligram. This, according to present opinions, is too high, and most clinicians to-day prefer 0.1 to 0.2 of a milligram . If after three or four days no reaction has occurred, a second dose of 1 milligram is given. In the absence of reaction after three further days, a third dose of 5 mgm. may be given and, under similar conditions, a fourth of 10 mgm. This is the largest dose which should ever be given, and absence of a reaction to this dose may gener- ally be regarded as proof that the patient is free from tuberculosis. Doses larger than 10 mgm. may give reactions in perfectly healthy subjects. Increase in dosage should be carried out only when the preceding dose has been entirely without reaction. In all cases it should be remembered that absolute rules of dosage can not be made and the condition and physique of each patient must be separately judged. The reaction itself is recognized chiefly by the changes in tem- perature. In a positive reaction the patient's temperature will begin to increase within six to eight hours after injection, rising sharply within a few hours to 0.5 or 1.5° higher than the temperature before injection. It then sinks more gradually than it rose, the reaction usually being complete within thirty to thirty-six hours. With the temperature there may be nausea, a chill, rapid pulse, and general malaise. Locally visible tuberculous processes, such as lupus, lymph nodes, etc., may become more tender or swollen, and if the tuberculosis is pulmonary, there may be coughing and increased expectoration. The tempera- tures of persons subjected to the test should be taken regularly for three or four days before tuberculin is used. Ophthalmo-Tuberculin Reaction. — Wolff-Eisner 3 and, soon after him, Calmette 4 proposed a method of using tuberculin for diagnostic purposes by instillation into the conjunctival sac. In tuberculous patients this process is followed by a sharp conjunctival congestion last- ing from one to several days. 1 Koch, Deut. med. Woch.; 1890. 2 Beck, Deut. med. Woch., 1899. s Wolf-Eisner, Berl. med. Gesell., May 15, 1907. 4 Calmette, Acad, des sci., June 17, 1907. THE TUBERCLE BACILLUS 489 The preparation used for this purpose is produced in the following- way: "Old tuberculin" is treated with double the quantity of 95 per cent alcohol, and the precipitate allowed to settle and the alcohol then filtered off through paper. The sediment is washed with 70 per cent alcohol until the filtrate runs clear, then pressed between layers of filter paper to remove excess of moisture, scraped into a dish, dried in vacuo over H2S04, and broken up in a mortar under a hood. S Solutions of the powder are made in sterile normal salt solution, 1 per cent by weight, boiled and filtered. The solutions are used in strengths of 0.5 to 1 per cent, a drop of which is instilled into the con- junctival sac.1 Cutaneous Tuberculin Reaction. — Von Pirquet 2 has suggested the cutaneous use of tuberculin for diagnostic purposes. A 25 per cent solution of "old tuberculin " is made in the following way: Tuberculin 1 Salt solution 2 5 per cent carbolic acid in glycerin 1 After sterilization of the patient's forearm, two drops of this solution are placed upon the skin about 6 cm. apart. Within each of these drops scarification is done, and the skin between them is scarified as a con- trol. Within twenty-four to forty-eight hours, in tuberculous patients, erythema, small papules, and herpetiform vesicles will appear. The reaction is irregular and more reliable in children than in adults. Ac- cording to recent investigations, about 70 per cent of adults show a positive reaction and in such cases it is probable that an old healed tuberculosis may give rise to a positive test where absolutely no active process exists. Recently, v. Pirquet has modified his procedure by using instead of the 25 per cent solution given above, the pure, undiluted " old tubercu- lin." Moro 3 has modified this by simply making a 50 per cent ointment of tuberculin in lanolin and rubbing it into the skin without scarification. It is more simple and equally efficient to massage into the skin a drop of undiluted "old tuberculin." The Tuberculin Tests as Applied to Cattle. — In cattle, the symptoms 1 Method in use at Saranac and kindly communicated by Dr. Baldwin. 2 v. Pirquet, Berl. klin. Woch., xx, 1907; Med. Klinik, xl, 1907. » Moro, Munch, med. Woch., 1906, p. 216 490 PATHOGENIC MICROORGANISMS of tuberculosis are not easily detected by methods of physical diag- nosis until the disease has reached an advanced stage. In conse- quence, cows may be elements of danger without appearing in any way diseased to those who handle them. In consequence, routine examination of herds by the tuberculin test has become one of the necessary measures in public sanitation. According to Mohler,1 an accurate diagnosis may be established in at least 97 per cent of the cases. It is natural that a good deal of objection to the test is encoun- tered on the part of dairy farmers and cattle raisers, and recently it has been publicly claimed that the cattle are injured by the test. There is, however, no scientific basis for this belief, if the test is carried out care- fully and intelligently. As a matter of fact, the systematic use of the test would eventually be distinctly advantageous to the owners of the cattle themselves, since it has been shown that cows, even in the early stages of the disease, may expel tubercle bacilli, either during respira- tion or in the feces, and thus become a menace to healthy members of the herd. The tuberculin test on cattle should be made as follows: (The directions given below are taken directly from the circular sent out from the Bureau of Animal Industry at Washington.) 1. Begin to take the rectal temperature at 6 a.m., and take it every two hours thereafter until midnight. 2. Make the injection at midnight. 3. Begin to take the temperature next morning at 6 a.m., and con- tinue as on preceding day. To those who have large herds to examine, or are unable to give the time required by the above directions, the following shortened course is recommended: 1. Begin to take the temperature at 8 a.m., and continue every 2 hours until 10 p.m. (omitting at 8 p.m., if more convenient) ; or take the temperature at 8 a.m., 12 m., and 6 and 10 p.m. 2. Make the injection at 10 p.m. 3. Take the temperature next morning at 6 or 8 a.m., and every 2 hours thereafter until 6 or 8 p.m. Each adult animal should receive 2 c.c. of the tuberculin as it is sent from the laboratory. (The tuberculin sent out from the central labora- tory at Washington is already diluted; 2 c.c. represents 0.25 c.c. of the concentrated old tuberculin of Koch.) Yearlings and two-year-olds, » Mohler, Pub. H. and Mar. Hosp. Serv. Bull. 41, 1908. THE TUBERCLE BACILLUS 491 according to size, should receive from 1 to 1.5 cubic centimeters. Bulls and very large animals may receive three cubic centimeters. The injec- tion should be made beneath the skin of the neck or shoulders behind the scapula, after washing the area with a weak carbolic acid solution. There is usually no marked local swelling at the seat of the injection. There are now and then uneasiness, trembling, and the more fre- quent passage of softened dung. There may also be slight acceleration of the pulse and of the breathing. The febrile reaction in tuberculous cattle following the subcutaneous injection of tuberculin begins from six to ten hours after the injection, reaches the maximum nine to fifteen hours after the injection, and returns to normal eighteen to twenty-six hours after the injection. A rise of two or more degrees Fahrenheit above the maximum tem- perature observed on the previous day should be regarded as an indica- tion of tuberculosis. For any rise less than this a repetition of the injection after four or six weeks is highly desirable. It is hardly necessary to suggest that for the convenience of the one making the test the animals should not be turned out, but fed and watered in the stable. It is desirable to make note of the time of feed- ing and watering and of any temperature fall after watering. The tuberculin should not be used later than six weeks after the date on the bottle, nor if there is a decided clouding of the solution. Therapeutic Uses of Tuberculin. — Tuberculin was first used therapeu- tically, shortly after its discovery, by Koch.1 Hailed with the most optimistic enthusiasm, its possibilities were overestimated and hope- less cases were treated unskilfully, with unsuitable dosage. The conse- quence was that harm was done, the method was attacked by Virchow and others and the new therapy fell into almost complete neglect. At present, the use of tuberculin has again been revived, but with greater caution and with a thorough understanding of its limitations. The tendency has been toward smaller dosage and the limitation of the agent to early cases. No two institutions use tuberculin in exactly the same manner, and it is, therefore, impossible to do more than outline the general scheme of treatment. It must never be forgotten, however, that all forms of tuberculin treatment consist in an " active immuniza- tion " in which, for the time being, the toxemia of the patient is increased rather than neutralized. It is obvious, therefore, that only such cases are at all suitable for treatment in which the process is not a very acute 1 Koch, Deut. med. Woch., iii, 1891. 492 PATHOGENIC MICROORGANISMS one. The general principle of modern tuberculin- therapy seems to lie in choosing doses so small that no marked general reaction shall follow. The preparations most frequently employed are Koch's "Alttuber- culin," his "TR," his "Neu Tuberkulin-Bazillen Emulsion/' and the Bouillon nitre of Denys. Initial doses of Alttuberculin range from 0.1 to 0.01 of a milligram. In case of successful avoidance of a reaction, the injection may be repeated, gradually increasing, about twice a week. The occurrence of a reaction should be the signal for a longer interval and a slower advance in the size of the dose. The initial dose of " TR " is, as advised by Koch,1 about 0.002 mgm. This usually causes no reaction. The dose is doubled, at reason- able intervals, up to 1 mgm. After this, further increase is care- fully gauged by the clinical indications. The maximum dose is about 20 mgm. " Neu Tuberkulin-Bazillen Emulsion," 2 is begun with a dose of 0.001 mgm. Gradual increase as with the other preparations is then prac- ticed. The maximum dose is about 10 mgm. Bouillon nitre has been used chiefly by Denys 3 and with apparently excellent results. Denys is very emphatic in advising the absolute avoidance of any reaction. He begins with a millionth or even the tenth of a millionth of a cubic centimeter of the bouillon and increases with extreme caution. His dilutions are made with glycerin broth. Passive Immunization in Tuberculosis. — Numerous attempts have been made to immunize tuberculous subjects with the sera of actively immune animals. The most widely used method of producing such serum is that of Maragliano. Maragliano's Serum.4 — Maragliano believes that a toxalbumin is present in tubercle-bacillus cultures which is destroyed by the heating employed in the usual tuberculin production. He procures this sub- stance by filtration of unheated cultures and precipitation with alcohol (tossina prsecipitata) . He furthermore makes an aqueous extract of the bacillary bodies. With these two substances he immunizes horses. He draws blood from these after four to six months of treatment. The serum is extensively used in Italy. Its value is, at present, very doubtful. 1 Koch, Deut. med. Woch., xiv, 1897. 2 Bandelier und Roepke, " Lehrb. d. spezifisch. Tub. Ther.," Wurzburg, 1908; Koch, Deut. med. Woch., 1901. 3 Denys, "Le Bouillon filtre," Louvain, 1905. * Maragliano, Berl. klin. Woch., 1899; Soc. de biol., 1897. THE TUBERCLE BACILLUS 493 Marmorek's Serum.1 — Marmorek claims that the poisons produced by Bacillus tuberculosis depend largely upon the medium on which it is grown. He advanced the view in 1903 that the substances obtained in tuberculin were not the true toxins of the tubercle bacillus, that there was a marked difference between these and the poisons elaborated by a younger (primitive) phase of the bacillus as it occurs only within the animal body or on media composed of animal tissue. He consequently grows his cultures on a medium composed of a leucotoxic serum (pro- duced by inoculating calves with guinea-pig leucocytes) and liver tissue. Such cultures, he claims, contain no tuberculin. To the sera produced by immunization with these cultures he attributes high curative powers. Bacilli Closely Related to the Tubercle Bacillus. — The Bacillus of Bovine Tuberculosis. — Tuberculosis of cattle (Perlsucht) was studied by Koch 2 in connection with his early work on human tuberculosis. Koch did not fail to recognize differences between the reactions to in- fection in the bovine type of the disease and that of man. He attrib- uted these, however, to the nature of the infected subject rather than to any differences in the infecting agents. This point of view met with little authoritative contradiction, until Theobald Smith 3 in 1898, made a systematic comparative study of bacilli isolated from man and from cattle and pointed out differences between the two types. The opinion of Smith was fully accepted by Koch 4 in 1901. Since that time, the question, because of its great importance to prophylaxis, has been the subject of many investigations, most of them confirming Smith's original work. Morphologically, Smith 5 found that the bovine bacilli were usually shorter than those of the human type and grew less luxuriantly than these upon artificial media. He determined, furthermore, that, grown upon slightly acid glycerin bouillon, the bovine bacillus gradually reduces the acidity of the culture medium until the reaction reaches neutrality or even slight alkalinity. Fluctuations, after this, do not exceed 0.1 or 0.2 per cent on either side of neutrality. In the case of the human bacillus, on the other hand, there is but slight reduction of the acidity during the first weeks of growth; after this acidity increases and, though subject to fluctuations, never reaches neutrality. This behavior is probably due to action exerted upon the 1 Marmorek, Berl. klin. Woch., 1903, p. 1108; Med. Klinik, 1906. 2 Koch, Arb. a. d. kais. Gesundheitsamt, 11, 1882. 3 Th. Smith, Jour. Exp. Med., Ill, 1898. *Koch, Deut. med. Woch., 1901. 5 Th. Smith, Jour. Exp. Med., 1905. 494 PATHOGENIC MICROORGANISMS glycerin, since on ordinary bouillon no such differences between the two varieties can be noticed. These observations of Smith were confirmed by Ravenel,1 Vagedes,2 and others. The cultural differences between the two types have been studied with especial care by Wolbach and Ernst,3 and Kossel, Weber, and Heuss.4 All of these observers bear out Smith's contention that luxuriance and speed of growth are much more marked in the human than in the bovine variety. Marked differences, furthermore, have been shown to exist in the pathogenic qualities of these bacilli toward various animal species. Guinea-pigs inoculated with the bovine type 5 die more quickly and show more extensive lesions than those infected with human bacilli. The difference in the pathogenicity of the two organisms for rabbits is sufficiently striking to be of diagnostic value. The bovine bacilli usually kill a rabbit within two to five weeks; the human bacilli produce a mild and slow disease, lasting often for six months, and occasionally fail to kill the rabbits at all. The practical importance of distinguishing between the two types, of course, attaches to the question as to whether the bovine and the human disease are mutually intercommunicable. Extensive attempts to infect cattle with bacilli of the human type have been made,6 for the most part with very little or no success. Infections of human beings with bovine bacilli, however, have been reported and proved beyond reason- able doubt, by Smith,7 Ravenel,8 Kossel, Weber, and Heuss,9 and others. Most of these infections have been in children. It is likely, therefore, that while cattle are to a considerable degree immune against the bacillus of the human type, human beings do not enjoy the same safeguard in respect to the bovine bacillus. During adult life, the danger of such in- fection, however, is far less than it is during infancy and early youth. The Bacillus of Avian Tuberculosis. — A disease resembling in many features the tuberculosis of man is not uncommon among chickens, pigeons, and some other bird species. Koch was the first to discover in 1 Ravenel, Lancet, 1901; Univ. Penn. Med. Bull., 1902. 2 Vagedes, Zeit. f. Hyg., 1898. 3 Wolbach and Ernst, " Studies from the Rockefeller Inst.," 11, 1904. 4 Kossel, Weber, und Heuss, Arb. a. d. kais. Gesundheitsamt, 1904 and 1905. 5 Smith, loc. cit., and Medical News, 1902. « Beck, "Festsch. R. Koch," 1902; Smith, loc. cit. 7 Smith, Trans. Assn. Amer. Phys., 1903. " Ravenel, Univ. Penn. Med. Bull., 1902. 9 Kossel, Weber, und Heuss, loc. cit. THE TUBERCLE BACILLUS 495 the lesions of diseased fowl bacilli much resembling Bacillus tuberculosis. It was soon shown, however, by the studies of Nocard and Roux,1 Mafucci,2 and others, that the bacillus of the avian disease represented a definitely differentiable species. Morphologically, and in staining characteristics, the bacillus is almost identical with that of the human disease. In culture, however, growth is more rapid and takes place at a temperature of 41° to 45° C.3 (the normal temperature of birds), while the human type is unable to thrive at a temperature above 40°. Guinea-pigs, very susceptible to human tuberculosis, are very refractory to infection with the avian type; while, on the other hand, rabbits which are resistant to the human type, succumb rapidly to in- fection with avian tuberculosis.4 Prolonged cultivation and passage through the mammalian body is said to cause these bacilli to approach more or less closely to the mammalian type. Conversely, Nocard 5 succeeded in rendering mammalian tubercle bacilli pathogenic for fowl by keeping them in the peritoneal cavities of hens in celloidin sacs for six months. Recently Koch and Rabinovitsch 6 have isolated from the spleen of a young man dead of tuberculosis, a microorganism which, culturally, morphologically, and in its pathogenic action upon birds, seemed to belong to the avian type. Lowenstein 7 describes a similar organism cultivated from a human case which seems to be a transitional type. Observations of this order are, however, too few at the present time to be used as the basis of a definite opinion as to the relationship between the two varieties. Bacillus of Fish Tuberculosis. — This bacillus, isolated by Dubarre and Terre,8 resembles Bacillus tuberculosis in morphology and in a certain degree of acid-fastness. It grows at low temperatures, 15° to 30° C. It is non-pathogenic for animals, but kills frogs within a month. Except for the acid-fastness it has little in common with Bacillus tuberculosis. 1 Nocard et Roux, Ann. de l'inst. Pasteur, 1887. 2 Mafucci, Zeit. f. Hyg., xi. 3 Mafucci, loc. cit. 4 Straus et Gamaleia, Arch, de med. exper., 1891; Courmont et Dor, Arch, de med. exp., 1891. 5 Nocard, Ann. de l'inst. Pasteur, 1898. 6 Koch and Rabinovitsch, Virch. Arch., Beiheft to Bd. 190, 1907. 7 Lowenstein, quoted from Koch and Rabinovitsch, loc. cit. 8 Dubarre et Terre, Compt. rend, de la soc. de biol., 1897. 496 PATHOGEi'IC MICROORGANISMS Bacillus of Timothy. — Moeller isolated from timothy-grass and from the dust in haylofts bacilli, which appear like Bacillus tuberculosis and are acid-fast. They grow rapidly on agar, colonies soon showing a deep red or dark yellow color. Bacillus butyricus {Butter Bacillus). — Bacilli morphologically re- sembling Bacillus tuberculosis and possessing the acid-fast property, though to but a slight degree, have been isolated from milk and butter by Petri,1 Rabinovitsch,2 Korn,3 and others. These bacilli are easily differentiated from Bacillus tuberculosis culturally. They are slightly pathogenic for guinea-pigs, but not for man. Bacillus smegmatis and the bacillus of leprosy will be discussed in separate sections. The differentiation of these organisms by means of staining reactions has been discussed in the section on staining methods. 1 Petri, Arb. a. d. kais Gesundheitsamt, 1897. 2 Rabinovitsch, Zeit. f. Hyg., 1897. s Korn, Cent. f. Bakt., 1899. CHAPTER XXXIV THE SMEGMA BACILLUS AND THE BACILLUS OF LEPROSY BACILLUS SMEGMATIS In 1884, Lustgarten 1 announced that he had succeeded in demon- strating, in a number of syphilitic lesions, a characteristic bacillus, which he declared to be the etiological factor in the disease. The great importance of the subject of Lustgarten's communication caused nu- merous investigators to take up the study of the microorganisms found upon the genitals of normal and diseased individuals. As a result of these researches the presence of the Lustgarten bacilli upon the genitals of many syphilitics was confirmed; but at the same time bacilli, which in all essential particulars were identical with them, were found in the secretions about the genital organs and anus of many normal persons. The first to throw' doubt upon the etiological significance of Lustgarten's bacillus, and to describe in detail the microorganism now recognized as Bacillus smegmatis, were Alvarez and Tavel.2 Similar studies were made soon afterward by Klemperer,3 Bitter,4 and others. The smegma bacilli are now known to occur as harmless sapro- phytes in the preputial secretions of the male, about the external genital organs of the female, and within the folds of thighs and buttocks. They are usually found, in these situations, in clumps upon the mucous mem- brane, and occasionally in the superficial layers of the epithelium, intra- and extra-cellularly. Morphology. — The smegma bacilli are very similar to tubercle bacilli, but show greater variations in size and appearance than do the latter. In length the individuals may vary from two to seven micra. They are usually straight or slightly curved, but according to Alvarez and Tavel may show great polymorphism, including short comma-like forms, and occasional S-shaped spiral forms. 1 Lustgarten, Wien. med. Woch., 47, 1884. 2 Alvarez et Tavel, Arch. d. physiol. norm, et path., Oct., 1885. 3 Klemperer, Deut. med. Woch., xi, 1885. 4 Bitter t Virchow's Arch., ciii. 32 497 498 PATHOGENIC MICROORGANISMS They are not easily stained, and though less resistant in this respect than the tubercle bacillus, they yet belong distinctly to the group of acid-fast bacilli. Once stained by the stronger dyes, such as carbol- fuchsin or anilin-water-gentian-violet, they are tenacious of the dye, though less so than tubercle bacilli. The identification of the smegma bacillus by staining methods has become of great practical importance since Fraenkel,1 Miiller,2 and others have demonstrated the occasional presence of acid-fast bacilli, probably of the smegma group, in sputum, and in secretions from the tonsillar crypts and throat. The methods of differentiation which have been found most practical are those which depend upon differences in the retention of stain shown by these bacilli. While it may be stated as a general rule that the smegma bacilli are more easily decolor- ized than tubercle bacilli, it is nevertheless important that a con- trol, as suggested by Wood, be made with known tubercle bacilli whenever a slide of suspected smegma bacilli is examined. For the actual differentiation an excellent method is that of Pappenheim, described in detail in the section on Staining, page 106. This method depends upon the fact that prolonged treatment with alcohol and rosolic acid decolorizes the smegma bacilli but not the tubercle bacilli. Coles 3 has stated that smegma bacilli will resist Pappenheim's decolorizing agent for four hours at the most, while tubercle bacilli will retain the stain, in spite of such treatment, for as long as twenty- four hours. Although minor differences between the smegma bacillus and that of Lustgarten have been upheld by Doutrelepont 4 and others, never- theless, the etiological significance of Lustgarten's bacillus in syphilis has been finally discredited, and, if not identical with the smegma bacillus, it at least belongs to the same group. The smegma bacilli have no pathogenic significance. They are found upon human beings as harmless saprophytes, and all attempts to infect animals have so far been unsuccessful. They are cultivated with great difficulty, first cultivations from man being successful only upon the richer media containing human serum or hydrocele fluid. After prolonged cultivation upon artificial media they may be kept alive upon glucose agar or ascitic agar. Their growth is slow; 1 Fraenkel, Berl. klin. Woch., 1898. 2 Miiller, Deut. med. Woch., 1898. 3 Coles, Jour, of State Med., 1904. 4 Doutrelepont, quoted from Klemperer, loc. cit. BACILLUS LEPRAE AND LEPROSY 499 and the colonies, appearing within five or six days after inoculation, are yellowish white, corrugated, and not unlike tubercle-bacillus colonies. BACILLUS LEPRA! AND LEPROSY The bacillus of leprosy was first seen and correctly interpreted as the etiological factor in the disease in 1879, by G. Armauer Hansen,1 a Norwegian observer. Hansen found the bacilli in the tissues of the nodular lesions of patients, lying in small clumps, intra- and extra- cellularly, as well as in the serum oozing from the tissue during its removal. Hansen's observation was the fruit of over six years of careful study and as to his priority in making this great discovery, there can be no doubt. Almost simultaneously with his publication, however, Neisser 2 published similar results, obtained by him during a brief stay at Bergen, during the preceding summer. The bacilli described by these workers are now recognized as being unquestionably the cause of the various forms of the disease known as leprosy. Morphology and Staining. — The leprosy bacillus is a small rod measuring about 5 to 7/z in length and has a close morphological re- semblance to Bacillus tuberculosis, except in that it is less apt to display the beaded appearance and is slightly less slender than the latter. It is non-motile, possesses no flagella, and forms no spores. Like tubercle bacilli, furthermore, the leprosy bacilli belong to the class of so-called acid-fast bacteria, being stained with much difficulty; but when once stained they are tenacious of the color, offering con- siderable resistance to the decolorizing action of acids. It is necessary for differential diagnosis, however, to note that both the difficulty of staining and the resistance to decolorization are less marked in the case of this microorganism than in the case of Bacillus tuberculosis. It was this peculiar behavior to stains that caused the delay of several years in Hansen's publications, since he failed in obtaining good morphological specimens until the work of Koch upon bacterial staining had supplied him with proper methods. The bacillus is stained most easily with anilin-water-gecitian-violet or with carbol-fuchsin solution. Stained by Gram's method, it is not decolorized and appears a deep blue. Differ- ential staining by the Ziehl-Neelsen method shows the bacillus stained red unless decolorization by means of the acid and alcohol are prolonged 1 Hansen, Virch. Arch., 79, 1879. 2 Neisser, Breslauer arztl. Zeitschr., 20, 1879. 500 PATHOGENIC MICROORGANISMS for an unusual time. A differentiation from tubercle bacilli by virtue of greater ease of decolorization is of value only in the hands of those having much experience with these bacilli, and follows no regular laws of acid-strengths or time of application which can be generally applied by the inexperienced. In tissues, the bacilli are easily stained by the methods used for staining tubercle bacilli. The sections are left in the Ziehl carbol-fuchsin solution either from two to twelve hours at incu- bator temperature or for twenty-four hours at room temperature. Subsequent treatment is that employed in the case of tuberculous tissue sections (see p. 112). Cultivation. — Cultivation of the leprosy bacillus has not met with success. Hansen and others who have approached the problem with a thorough knowledge of the microorganism, combined Avith a com- petent bacteriological training, have failed in all their attempts. Numerous positive results reported by observers have always lacked adequate confirmation. Recently, Rost,1 of the British Army Medical Corps, has claimed success in cultivation of leprosy bacilli upon salt-free bouillon, his point of departure being the previous observation that salt-free media favored the growth of tubercle bacilli. His results have not been confirmed. In all cases, in fact, in which successful cultiva- tion has been claimed, no positive proof as to the leprous nature of the cultivated organisms has been brought. Pathogenicity. — Innumerable attempts to transmit leprosy to ani- mals by inoculation have been unsuccessful. Xicolle,2 however, has recently claimed successful experiments upon monkeys (macacus) in whom inoculation with tissue from infected human beings was followed, in sixty-two days, by the development of a small nodule at the site of inoculation, in which, upon excision, leprosy bacilli were found. In most cases, however, inoculation has given rise merely to a transient inflammatory reaction. Among human beings, leprosy has been a widely spread disease since the beginning of history, and much evidence is found in ancient lit- erature which testifies to a wide distribution of the disease long before the Christian era and throughout the Middle Ages. At the present day, leprosy is most common in the eastern countries, especially in India and China. In Europe the disease is found in Norway, in Russia, and in Iceland. In other European countries, while the disease occurs, it is not at all common. In the United States, there are, according to Osier, 1 Rost, Brit. Med. Jour., 1, 1905. 2 Nicolle, Sem. medicale, 10, 1905. BACILLUS LEPRAE AND LEPROSY 501 three important centers of leprosy situated in Louisiana, in California, and among the Norwegian settlers in Minnesota. The disease is also present in several provinces of Canada. In all countries in which segregation of lepers is rigidly practiced, the disease is diminishing. In Norway, according to Hansen, proper sanitary measures have reduced the number of lepers from 2,870 in 1856, to 577 in 1900. Clinically, the disease appears in two chief varieties, tubercular leprosy and the so-called anesthetic leprosy. In the former variety, hard nodular swellings appear, usually in the face but often on other parts of the body as well. These lead to frightful disfigurement and are accompanied by a falling-out of hair and a loss of sensation in the affected areas. In the anesthetic form, there is usually at first pain in definite areas of the extremities and the trunk, which is soon followed by the formation of flat or slightly raised pigmented areas, within which there is absolute anesthesia with, later, atrophy and often secondary necrosis in the atrophied parts. The disease is usually chronic in its course. The bacilli are found in large numbers in the cutaneous lesions. In the knobs of the nodular variety, they lie in clumps between the con- nective-tissue cells and within the large spheroidal cells which make up the nodules. They are found, also, in advanced cases, in the liver and in the spleen, lying within the cells, and, to a slighter extent, in the intercellular spaces. They have also been found within the kidneys, the endothelium of the blood-vessels, and in the testicles.1 In the blood, the bacilli have frequently been demonstrated, especially during the febrile attacks which occur during the disease. Westphal and Uhlen- hut 2 have found the bacilli within the central nervous system, and these observers, as well as others, have found them tying within the substance of the peripheral nerves, thus explaining the anesthesia. A fact of enormous importance to the question of transmission is the observation made by various observers, more especially by Sticker, that the bacilli are found with great regu- larity in considerable numbers in the nasal secretions of persons suffering from the disease. Sticker is inclined to regard the nose as the primary path of infection. Whether or not this be true can not, at present, be decided. As a source of infection, however, the nasal mucus and, secondarily, the saliva, are certainly the vehicles 1 Sticker, Munch, med. Woch., 39, 1897. 2 Westphal und Uhlenhut, Klin. Jahrb., 1901. 502 PATHOGENIC MICROORGANISMS by which large numbers of the bacilli leave the infected patient, and, therefore, tend to spread the disease. The contagiousness of leprosy is far less than is that of most other bacterial diseases. Physicians and others who come into direct contact with large numbers of leprous patients, observing at the same time the ordinary precautions of cleanliness, rarely contract the disease. On the other hand, intimate contact with lepers without such precautions is the only possible means of transmission. The demonstration of leprosy bacilli in dust, soil, etc., must always be looked upon with sus- picion, since, apart from actual human inoculation, there is no method of positively differentiating the bacilli from similar acid-fast organisms. Instances of transmission by contact are on record, not the least famous of which is the case of Father Damien, who contracted the disease while taking care of the lepers upon the island of Molokai. Hansen states that in his knowledge no case of leprosy can be found in which careful examination of the past history will not reveal direct contact with a previous case. Direct inoculation of the human being with material from a leprous patient has been successfully carried out by Arning,1 upon a Hawaiian criminal. In this case a piece of a leprous nodule was planted into the subcutaneous tissue of the left arm. One month after the inoculation, pain appeared in the arm and shoulder, and four and a half months later a typical leprosy nodule was formed. Four years after the inoculation, the patient was a typical leper. Although our inability to cultivate the leprosy bacillus, and the lack of success attending animal inoculation, have made it impossible to study more closely the toxic action of this microorganism, there is, neverthe- less, some evidence which points toward the production of a poisonous substance of some kind by the bacillus. Rost,2 who claims to have cultivated the bacillus, manufactured from his cultures, by the technique for the production of "old tuberculin," a substance which he called "leprolin," and which he employed therapeutically in the same manner in which tuberculin is employed in tuberculosis. As stated before, the results of Rost still lack confirmation. Of far greater importance, both in demonstrating the probability of the existence of a definite toxin as well as in indicating the close relationship between the leprosy bacillus and the Bacillus tuberculosis, are the investigations upon the action of tuberculin upon leprous patients. When tuberculin is adminis- tered to lepers, a febrile reaction occurs usually twenty-four or more 1 Arning, Vers. d. Naturfor. u. Aerzte, 1886. 2 Rost, loc. cit. BACILLUS LEPRAE AND LEPROSY 503 hours after the administration. The fever differs from that produced by the use of the same substance in tuberculous patients in that it is of late occurrence and lasts considerably longer. At the same time, there may be marked redness and tenderness of the nodules. In isolated cases, Babes * has noticed alarmingly high and prolonged fever together with systemic symptoms such as nausea, headache, and even uncon- sciousness, following the injection of tuberculin. The same writer claims to have extracted from the organs of lepers, which contained enormous numbers of bacilli, substances which showed an action similar to that of the tuberculin. 1 Babes, in Kolle und Wassermann, " Handbuch," etc., Erst. Erganz. Bd.; 1907. CHAPTER XXXV BACILLUS DIPHTHERIA, BACILLUS HOFFMANNI, AND BACILLUS XEROSIS BACILLUS DIPHTHERIA Since 1821, when Bretonneau of Tours published his observa- tions, diphtheria has been an accurately recognized clinical entity. Our knowledge of the disease in the sense of modern bacteriology, however, begins with the first description of Bacillus diphtheria by Klebs in 1883. Klebs 1 had observed in the pseudomembranes from diphtheritic throats, bacilli which in the light of more recent knowledge we can hardly fail to recognize as the true diphtheria organism. His work, however, was purely morphological and, therefore, inconclusive. One year after this announcement, Loefner 2 isolated and cultivated an organism which corresponded in its morphological characters to the one described by Klebs. He obtained it from thirteen clinically unques- tioned cases of diphtheria, and, by inoculating it upon the injured mucous surfaces of animals, succeeded in producing lesions which resembled closely the false membranes of the human disease. His failure to find the bacillus in all the cases he examined, his finding it, in one instance, in a normal throat, and his inability to explain to his own satisfaction some of the systemic manifestations of the infection which we now know to be due to the toxin, caused him to frame his conclusions in a tone of the utmost conservatism. The second and third publications of Loefner,3 however, and the inquiry into the nature of the toxins produced by the bacillus, published in 1888 by Roux and Yersin,4 eliminated all remaining doubt as to the etiological relationship existing between this organism and the disease. Innumerable observations, both clinical and bacteriological, by other workers, have, since that time, confirmed the early investigations, 1 Klebs, Verh. d. 2. Kongr. f. inn. Medizin, Wiesbaden, 1883. 2 Loeffler, Mittheil. a. d. kais. Gesundheitsamt, 1884. * Loeffler, Cent. f. Bakt., 1887 and 1890. * Roux and Yersin, Ann. de l'inst. Pasteur, 1888 and 1889. 504 BACILLUS DIPHTHERIA 505 and it is to-day a scientific necessity to find the bacillus of Klebs and Loefner in the lesion before a diagnosis of " diphtheria " can properly be made. Morphology and Staining. — While Bacillus diphtherias presents certain characteristic appearances which facilitate its recognition, it is, at the same time, subject to a number of morphological variations with Fig. 105. — Bacillus diphtheria. all of which it is important to be familiar. These variations are, to a limited extent, dependent upon the age of the culture and upon the constitution of the medium on which it has been grown. These factors, however, do not control the appearance of the organism with any degree of regularity, and any or all of its various forms may occur in one and the same culture. It is likely -that these different appear- ances represent stages in the growth and degeneration of the indi- vidual bacilli, but there does not seem to be any just reason for believing that, as several observers have stated, there is definite correla- tion between its microscopic form and its biological characteristics, such as virulence, toxicity, etc. 506 PATHOGENIC MICROORGANISMS The bacilli are slender, straight, or slightly curved rods. In length they vary from 1.2 micra to 6.4 micra, in breadth from 0.3 to 1.1. As seen most frequently when taken from the throat they are about 4 to 5 micra in length. They are rarely of uniform thickness throughout their length, showing club-shaped thickening at one or both ends. Occasionally they may be thickest at the center and taper toward the extremities. When thickened at one end only, a slender wedge-shape results. Such forms are usually straight, of smaller size than their neighbors, and are more often stained with great uniformity. These are spoken of by Beck1 as the "ground type," and assumed, for in- sufficient reasons, to be the young individuals. Branched forms have been described by some investigators. They are rare and probably to be regarded as abnormal or involution forms due to un- favorable environment. The organisms stain with the aqueous anilin dyes. A characteristic irregularity of staining which is of great aid in diagnosis is best obtained with Loeffler's " alkaline methylene-blue." (For preparation see section on Staining, p. 96.) Stained with this solution for five to ten minutes many of the bacilli appear traversed by unstained transverse bands which give fhem a striped or beaded appearance. The longer indi- viduals often have a strong resemblance to short chains of strepto- cocci. Others may appear unevenly granular. In cultures which are about eighteen hours old, many of the bacilli may show deeply stained oval bodies situated most frequently at the ends. These are the so-called "polar7' or "Babes-Ernst" bodies.2 Special stains have been devised for the demonstration of these appearances. One of these was originated by Neisser,3 who claims for it differential value in distinguishing these organisms from pseudodiphtheria and xerosis bacilli. His method requires two solutions: 1. Methylene blue (Grubler) 1 gram. Alcohol, 96 per cent 20 c.c. Glacial acetic acid 50 Water 950 " 2. Bismarck brown 2 grams. Water 1,000 c.c. 1 Beck, in Kolle und Wassermann, ii, p. 773. 2 Babes, Zeit. f. Hyg., Bd. v, 1889. 3 Neisser, Zeit. f. Hyg., xxiv, 1897. BACILLUS DIPHTHERIA 507 The cover-slip preparation, after having been fixed, is stained with so- lution No. 1 for one to three seconds. It is then washed in water and immersed for from three to five seconds in solution No. 2. With this stain the bodies of the bacilli appear brown, the polar granules blue. Another method which has been extensively used is that of Roux. The solutions required for this are : 1. Dahlia violet 1 gram. Alcohol, 90 per cent 10 c.c. Aq. dest ad 100 " 2. Methyl green 1 gram. Alcohol, 90 per cent 10 c.c. Aq. dest ad 100 The two solutions are mixed, one part of 1 being added to three parts of 2. Preparations are stained in this mixture for two minutes. The polar bodies appear a dark violet. Other methods for the staining of polar bodies have been recommended. There is very little advantage in the use of these double stains and most bacteriologists employ for routine work the simple stain with Loeffler's alkaline methylene blue. The significance of the polar bodies is not well understood. Their discoverer, Ernst, regarded them as bodies analogous to the spores of other organisms. The ease with which they are stained, however, and the low temperatures to which the bacteria succumb make this appear very unlikely. A more probable interpretation seems to be that of Escherich 1 who regards them as chromatic granules. Stained by Gram's method, the diphtheria bacilli retain the gentian- violet. Care must be used in carrying out this method and strict timing adhered to, since slight carelessness in this respect may lead to irregular results. In stained smears from the throat or from cultures a characteristic grouping of the bacilli has been observed. They lie usually in small clusters, four or five together, parallel to each other, or at sharp angles. Two organisms may often be seen attached to . W * " '*» *~ tv^ J< IV » <$ .'*..' : # * '<**• fall' ' ' ■ £ ♦ ' j* t *# .' G * fc# '«■ ' * w[ « • in. ' f '«*, , -y f}' . :< » 6*1 ' .. |K • *■ , 4/ . *" •»*.*<» • .#: g^A . 9 " d&ii - * • * ' % ' ; •• * '^ Ov » ■ V | v * ** * // / vv^. ♦, _ * ;'** *, V >• '*>' («£ "f ' ' ■ 4» " • #■ * # ■ m ' " * * 1* '■ V^ ••>.»'- ■' v ' H * " -'^^StM&i(S ' .** ^ "** ' Vr- ; ll\ *lT* » * * # . " . ..• im; - * * . - ->■«* • '' .&- *-^- '• ' ■ t ■ u * * * ~jr~C It . • -;f$ '> • *'. ■' i** •* ' ■ * '"'it ^c ^0 : :• ■ ■ '** '■' * ? * ' -' • .#.' ,g - ». *> &«* f .* . / * •»,** >» #*■ ♦ «f '%* ' * i •4* 4 '-•'r «. * * " fi ' ^ :: -c *•,'■' /^M* *!* ■'■■?* (■ ' ' «;>J& ''•■ '0 *' :• '> ^ '*"$T ' * ," . . • 1* * *' : ♦ - £ ■ * Fig. 111. — Glanders Bacilli in Tissue. (From a drawing furnished by Dr. James Ewing.) Finally, there is involvement of the lungs and death within four to six weeks. When the disease takes the chronic form the onset is more gradual. Concomitant with the nasal inflammation there is a formation of subcu- taneous swellings all over the body, some of which show a tendency to break down and ulcerate. Together with this the lymphatics all over the body become enlarged. The disease may last for several years, and occasionally may end in complete cure. In horses the chronic form of 524 PATHOGENIC MICROORGANISMS the disease is by far the more frequent. In man the disease is similar to that of the horse except that the point of origin is more frequently in some part of the skin rather than in the nasal mucosa, and the clinical symptoms differ accordingly. The onset is usually violent, with fever and systemic symptoms. At the point of infection a nodule appears, surrounded by lymphangitis and swelling. A general papular eruption may occur. The papules may become pustular, and the clinical features may thus simulate variola. This type of the disease usually ends fatally in eight to ten days. The chronic form of the disease in man is much like that in the horse, but is more frequently fatal. The histological appearance of the glanders nodules is usually one of diffuse leucocytic infiltration and the formation of young connective tissue which preponderates more and more as the disease becomes chronic. Virchow has classed these lesions with the granulomata. From the center of such nodules B. mallei may often be obtained in pure culture. The nodules may be generally distributed throughout the internal organs. The bacilli themselves are found, apart from the nodules, in the nasal secretions, and occasionally in the circulating blood.1 The bacteriological diagnosis of glanders may be made by isolating and identifying the bacilli from any of the above-mentioned sources. When superficial nodules can be opened for the purpose of diagnosis this may prove an easy task. The most diagnostic ally helpful medium in such cases is potato. In a majority of cases, however, isolation is ex- tremely difficult and resort must be had to animal inoculation. The most suitable animal for this purpose is the male guinea-pig. Intra- peritoneal inoculation of such animals with material containing glanders bacilli leads within two or three days to tumefaction and purulent inflammation of the testicles. Such an experiment, spoken of as the " Strauss test,"2 should always be reinforced by cultural examination of the testicular pus, the spleen, and the peritoneal exudate of the animals employed. Toxin of Bacillus mallei. — The toxin of B. mallei, or mallein, belongs to the class of endotoxins. The toxic products have been invariably obtained by extraction of dead bacilli.3 Mallein differs from many other bacterial poisons in being extremely resistant. It withstands » Wassilieff, Deut. med. Woch., 1883. 2 Strauss, Arch, de med. exp., 1889. 3 Kresling, Arch. d. sci. biol., 1892; Preuser, Berl. thieriirzt. Woch., 1894. BACILLUS MALLEI 525 temperatures of 120° C. and prolonged storage without noticeable loss of strength.1 In its physiological action upon healthy animals, mallein is not a powerful poison. It can be given in considerable doses without causing death. Mallein may be obtained by a variety of methods. Helman and Kalning, the discoverers of this toxin, used filtered aqueous and glycerin extracts of potato cultures. Roux 2 cultivates virulent gland- ers bacilli in flasks containing 250 c.c. each of 5 per cent glycerin bouillon. Growth is allowed to continue at 35° C. for one month. At the end of this time, the cultures are sterilized at 100° for thirty min- utes, and evaporated on a water bath to one-tenth their original volume. They are then filtered through paper. This concentrated poison is diluted ten times with 0.5 per cent carbolic acid before use. Concen- tration is done merely for purposes of conservation. The diagnostic dose of such mallein for a horse is 0.25 c.c. of the undiluted fluid. At the Washington Bureau of Animal Industry, mallein is prepared by growing the bacilli for five months at 37.5° C. in glycerin-bouillon. This is then boiled for one hour and allowed to stand in a cool place for one week. The supernatant fluid is then decanted and filtered through clay filters by means of a vacuum pump. The filtrate is evaporated to one-third its original volume on a water bath, and the evaporated volume resupplied by a 1 per cent carbolic acid solution containing about 10 per cent of glycerin. Diagnostic Use of Mallein. — The injection of a proper dose of mallein into a horse suffering from glanders is followed within six to eight hours by a sharp rise of temperature, often reaching 104° to 106° F. (40° C. +). The high temperature continues for several hours and then begins gradually to fall. The normal is not usually regained for several days. Locally, at the point of injection, there appears within a few hours a firm, hot, diffuse swelling, which gradually extends until it may cover areas of 20 to 30 centimeters in diameter. The swelling is in- tensely tender during the first twenty-four hours, and lasts for three to nine days. Together with this there are marked symptoms of general intoxication. In normal animals the rise of temperature following an injection is trifling, and the local reaction is much smaller and more transient. Injections are best made into the breast or the side of the neck. 1 Wladimiroff, in Kraus und Levaditi, " Handbuch," etc., 1908. 2 Roux et Nocard, Bull. d. 1. soc. centr. vet., 1892. 526 PATHOGENIC MICROORGANISMS The directions given by the United States Government for using mallein for the diagnosis of glanders in horses are as follows : "Make the test, if possible, with a healthy horse, as well as with one or more affected or supposed to be affected with glanders. Take the temperature of all these animals at least three times a day for one or more days before making the injections. "The injection is most conveniently made at 6 or 7 o'clock in the morning, and the maximum temperature will then usually be reached by or before 10 p.m. of the same day. "Use for each horse one cubic centimeter of the mallein solution as sent out, and make the injection beneath the skin of the middle of one side of the neck, where the local swelling can be readily detected. "Carefully sterilize the syringe after injecting each horse by naming the needle over an alcohol lamp or, better, use separate syringes for healthy and suspected animals. If the same syringe is used, inject the healthy animals first, and flame the needle of the syringe after each injection. "Take the temperature every two hours for at least eighteen hours after the injection. Sterilize the thermometer in a 5 per cent solu- tion of carbolic acid, or a 0.2 per cent solution of corrosive sublimate, after taking the temperature of each animal. "The temperature, as a rule, will begin to rise from four to eight hours after the injection, and reach its maximum from ten to sixteen hours after injection. On the day succeeding the injection take the tempera- ture at least three times. " In addition to the febrile reaction, note the size, appearance, and duration of any local swelling at the point of injection. Note the general condition and symptoms of the animal, both before, during, and after the test. " Keep the solution in the sealed bottle and in a cool place, and do not use it when it is clouded or if it is more than six weeks old ■ when it leaves the laboratory of the Bureau it is sterile.' ' If the result of first injection is doubtful, the horse should be isolated and retested in from one to three months, when the slight immunity conferred by the first injection will have disappeared. The second injection into healthy horses usually shows no reaction whatever. Mallein may cause reactions in the presence of other diseases than glanders, such as bronchitis, periostitis, and other inflammatory lesions and is not so specifically valuable as tuberculin for diagnosis. BACILLUS MALLEI 527 Immunity. — Recovery from a glanders infection does not confer immunity against a second inoculation.1 Artificial active immunization has been variously attempted by treatment with attenuated cultures, with dead bacilli, and with mallein, but without convincing results. The serum of subjects suffering from glanders contains specific agglutinins.2 These are of great importance diagnostically if the tests are made with dilutions of, at least, 1 in 500, since normal horse serum may agglutinate B. mallei in dilutions lower than this. 1 Finger, Ziegler's Beitrage, vi, 1899. 2 Galtier, Jour, de med. vet., 1901. CHAPTER XXXVII » BACILLUS INFLUENZAE AND CLOSELY RELATED BACTERIA There is no other epidemic disease which spreads over such enormous territories, and with such speed, as influenza. Epidemics have been numerous and reports of the disease, unquestionably recognizable, are extant even from the most remote times. The last serious epidemic occurred in the years 1S89 to 1890, when the disease, spreading from the East, traveled through Russia and, pandemic ally, attacked all of Europe, then reached America, and eventually, having traveled east- ward as well as westward from its point of origin, became prevalent in China, Japan, Australia, and Africa. Hundreds of thousands were attacked and the mortality of this epidemic was high. Its enormous scope and the rapidity of its spread were facilitated probably by the activity of modern international commerce. The character of the disease pointed so definitely to a bacterial etiology that numerous attempts to isolate a specific microorganism were, of course, made. Pfeiffer 1 finally, in 1892, described the bacillus which is at present definitely recognized as the etiological factor of influenza. Morphology and Staining. — The bacillus of influenza (Pfeiffer bacillus) is an extremely small organism, about 0.5 micron long by 0.2 to 0.3 micron in width. They are somewhat irregular in length, but show rounded ends. They rarely form chains. They are non-motile, and do not form spores. Influenza bacilli stain less easily than do most other bacteria with the usual anilin dyes, and are best demonstrated with 10 per cent aqueous fuchsin (5 to 10 minutes), or with Loeffler's methylene-blue (5 minutes). They are Gram-negative, giving up the anilin-gentian- violet stain upon decolorization. Occasionally slight polar staining may be noticed. Grouping, especially in thin smears of bronchial secretion, is characteristic, in that the bacilli very rarely form threads 1 Pfeiffer, Deut. med. Woch., ii, 1892; Zeit. f. Hyg., xiii, 1892; Pfeiffer und Beck, Deut. med. Woch., xxi. 1893. 528 BACILLUS INFLUENZA 529 or chains, usually lying together in thick, irregular clusters without definite parallelism. Isolation and Cultivation. — Isolation of the influenza bacillus is not easy. Pfeiffer * succeeded in growing the bacillus upon serum-agar plates upon which he had smeared pus from the bronchial secretions of patients. Failure of growth in attempted subcultures made upon agar Fig. 112. — Bacillus influenzae. Smear from pure culture on blood agar. and gelatin, however, soon taught him that the success of his first culti- vations depended upon the ingredients of the pus carried over from the sputum. Further experimentation then showed that it was the blood, and more particularly the hemoglobin, in the pus which had made growth possible in the first cultures. Pfeiffer made his further cultivations 34 Pfeiffer, loc. cit. 530 PATHOGENIC MICROORGANISMS upon agar, the surface of which had been smeared with a few drops of blood taken sterile from the finger. Hemoglobin separated from the red blood cells was found to be quite as efficient as whole blood. This method of Pfeiffer is still the one most frequently employed for isolation and cultivation. AYhole blood taken from the finger may be either smeared over the surface of slants or plates, or mixed with the melted meat-infusion agar. In isolating from sputum, only that secretion should be used which is coughed up from the bronchi and is uncont animated by microorganisms from the mouth. It may be washed in sterile water or bouillon before transplantation, to remove the mouth flora adhe- -•^ k» '. i Fig. 113. — Bacillus influenzae. Smear from sputum. (After Heim.) rent to the outer surface of the little clumps of pus. The blood of pigeons or that of rabbits may be substituted for human blood. The former seems to be the more favorable of the two and even more so than human blood. Pigeons may be easily bled for this purpose from the large veins under the wing. Huber * has succeeded in cultivating in- fluenza bacilli upon media containing a soluble hemoglobin derivative known as hematogen. This substance, however, offers some difficulties to sterilization and is not so favorable as whole blood. The absence of oxyhemoglobin from the hematogen, however, is theoretically im- portant in that it shows that hemoglobin is suitable for the growth 1 Huber, Zeit, f. Hyg., xv, 1893. BACILLUS INFLUENZA 531 of this bacillus because of its nutrient qualities and not by virtue of its oxygen-carrying properties. Although the presence of hemo- globin seems to be a necessity for the successful cultivation of the bacillus, the quantity present need not be very large. Ghon and Preyss 1 showed that an amount too small to be demonstrated spectroscopically sufficed for its growth. Other substances which, added to neutral or slightly alkaline agar, have been used for the cultivation of influenza bacilli are the yolk of eggs 2 (not confirmed) and spermatic fluid.3 None of these, however, is as useful as the blood media. Symbiosis with staphylococci,4 too, Fig. 114. — Colonies of Influenza Bacillus on Blood Agar. (After Heim.) has been found to create an environment favorable for their develop- ment. Influenza bacilli do not grow at room temperature. Upon suitable media at 37.5° C. colonies appear at the end of eighteen to twenty-four hours, as minute, colorless, transparent droplets, not unlike spots of moisture. These never become confluent. The limits of growth are reached in two or three days. To keep the cultures alive, tubes should 1 Ghon und Preyss, Cent. f. Bakt., xxxv, 1904. 2 Nastjukoff, Cent. f. Bakt., Ref., xix, 1896. 3 Cantani, Cent. f. Bakt., xxii, 1897. * Grassberger , Zeit. f. Hyg., xxv, 1897. 532 PATHOGENIC MICROORGANISMS be stored at room temperature and transplantations done at intervals not longer than four or five days. Biology. — The bacillus is aerobic, growing in broth-blood mixtures only upon the surface, hardly at all in agar stab cultures, and not at all under completely anaerobic conditions. As it does not form spores, it is exceedingly sensitive to heat, desicca- tion, and disinfectants.1 Heating to 60° C. kills the bacilli in a few min- utes. In dried sputum they die within one or two hours. They are easily killed even by the weaker antiseptics. Upon culture media the bacilli, if not transplanted, die within a week or less, the time depend- ing to some extent upon the medium used. Pathogenicity. — The relationship between the clinical disease known as influenza or grippe and the PfeifYer bacillus has been definitely estab- lished by numerous investigators.2 During epidemics, the bacilli are found with much regularity in the nasal passages and bronchial secre- tions of tkose afflicted with the malady. The organs most frequently attacked in man are the upper respiratory passages and lungs. Here the disease most frequently takes the form of a broncho- or lobular pneumonia, and sections of the lung tissue of those who have died of the infection show innumerable bacilli upon and within the mucosa of the bronchioles. [Thin sections are stained for one-half to one hour in dilute carbol fuchsin and are then dehydrated in slightly acid alcohol (alcohol absolute J i, glacial acetic acid gtt. i-ij).] Clinically, influenzal broncho-pneumonias are not essentially dif- ferent from those due to other microorganisms, and it must always be left to the bacteriological examination to make the positive diagnosis. The pulmonary influenzal infection is not infrequently followed by abscess or gangrene of the lung, or may occasionally develop into a chronic interstitial process. Apart from the lungs, the bacilli have been found in the middle ear,3 in the meninges,4 and in the brain and spinal cord. Demonstration of the bacilli in the circulating blood has never been satisfactorily accomplished, although the general charac- ters of the symptoms of the infection would suggest the existence of a septicemia. The short incubation period5 of the disease has been in- 1 Kruse, in Fliigge, "Die Mikroorg.," Leipzig, 1896. 2 Weichselbaum, Wien. klin. Woch., 32, 1892; Baumler, Munch, med. Woch., 1894; Huber, loc. cit. 3 Kossell, CharitS-Annalen, 1893. « Pfuhl, Berl. klin. Woch., xxxix, 1892. 6 Quoted from Tedesco, Cent. f. Bakt., xliii, 1907- BACILLUS INFLUENZAE 533 voluntarily determined by Kretz, who became violently ill twenty-four hours after accidentally breaking an agar plate of a pure culture which he was photographing. The bacilli are said to remain in the bronchial secretions of con- valescents or even of normal individuals for many years. They are found for long periods in the lungs of those suffering from tuberculosis. To such sources, probably, are attributable the sporadic cases develop- ing constantly in crowded communities. It is not improbable, further- more, that occasional reactivation of the influenzal infection may often aggravate the condition of phthisical patients. It is a peculiar fact that cases of influenza observed apart from the large epidemics are rarely due to an unmixed Pfeiffer bacillus infection, but that these sporadic cases are usually due to a mixed infection, including with this bacillus, pneu- mococci, streptococci, and other secondary invaders.1 This may, in part, account for the frequently atypical courses of such attacks. Experimental infection of animals has revealed susceptibility only in monkeys. Pfeiffer and Beck 2 succeeded in producing influenza-like symptoms in monkeys by rubbing a pure culture of the bacillus upon the unbroken nasal mucosa. Severe symptoms have been produced in rabbits by intravenous inoculation, but the bacilli do not seem to proliferate in these animals, the reaction produced probably beiag purely toxic. Dead cultures (killed with chloroform) may produce severe transient toxic symptoms in rabbits.3 The immunity produced by an attack of influenza, if present at all, is of very short duration. Bacteria Closely Related to Influenza Bacillus. — Pseudo-influenza Bacillus: In the broncho-pneumonic processes of children, Pfeiffer4 found small, non-motile, Gram-negative bacilli, which he was forced to separate from true influenza bacilli because of their slightly greater size, and their tendency to form threads and involution forms. These micro- organisms are strictly aerobic and grow, like true influenza bacilla, only upon blood media. They are differentiated entirely by their morphology upon twenty-four-hour-old blood-agar cultures. Wollstein,5 who has made a careful study of influenza-like bacilli, both culturally and by agglutination tests, has come to the conclusion that these bacilli are so similar to the true influenza organisms that the term pseudo-influenza should be discarded. Strains of similar bacilli isolated from cases of 1 Tedesco, loc. cit. 2 Pfeiffer und Beck, Deut. med. Woch., xxi, 1893. 3 Pfeiffer, loc. cit. < Pfeiffer, Zeit. f. Hyg., xiii, 1892. 5 Wollstein, Jour. Exp. Med., viii, 1906. 534 PATHOGENIC MICROORGANISMS pertussis, while differing from the others in some of their characteristics, could not properly be maintained as distinct species. Koch-Weeks Bacillus. — Koch,1 in 1883, Weeks 2 and Kartulis, in 1887, described a small Gram-negative bacillus found in connection with a form of acute conjunctival inflammation, which occurs epidemically. The bacillus is morphologically similar to B. influenza?, but is generally longer than this and more slender. The bacilli grow only at incubator temperature, but, unlike influenza bacilli, can be cultivated upon media of serum or ascitic fluid, without hemoglobin. In fact, growth upon serum-agar is more active than upon hemoglobin media.3 Bacillus of Pleuro-Pneumonia of Rabbits. — This is a small Fig. 115. — Koch- Weeks Bacillus. Gram-negative bacillus, described by Beck, not unlike that of influenza. These microorganisms are slightly larger than the Pfeiffer bacilli and grow upon ordinary media even without animal sera or hemoglobin. Bacillus murisepticus and Bacillus RHusioPATHiiE. — While mor- phologically similar to the microorganisms of this group, these bacilli are culturally easily separated because of their luxuriant growth on simple media. (The last two microorganisms are more closely related to the groups of the bacilli of hemorrhagic septicemia. See page 543.) 1 Koch, Arb. a. d. kais. Gesundheitsamt, iii; Cent. f. Bakt., 1, 1887. 2 Weeks, N. Y. Eye and Ear Infirmary Rep., 1895; Arch. f. Augenheilk., 1887. 3 Kamen, Cent. f. Bakt., xxv, 1899; Weichselbaum and Mutter, Arch. f. Ophthalm., xlvii, 1899; Knapp, Studies from Dept. of Path., Coll. of P. and S., 1903. CHAPTER XXXVIII BORDET-GENGOU BACILLUS, MORAX-AXENFELD BACILLUS, ZUR NEDDEN'S BACILLUS, DUCREY BACILLUS BORDET-GENGOU BACILLUS (" Microbe de la Coqueluche," Pertussis bacillus, Bacillus of whooping- cough.) In 1900 Bordet and Gengou x observed in the sputum of a child suffering from pertussis a small ovoid bacillus which, though similar to the influenza bacillus, showed a number of morphological characteristics which led them to regard it as a distinct species. As they were at first unable to cultivate this organism, their discovery remained ques- tionable until 1906, when cultivation succeeded and the biology of the microorganism was more fully elucidated. Morphology. — The morphology of this organism is described by them as follows: The organism in the sputum, early in the disease, is scattered in enormous numbers indiscriminately among the pus cells, and at times within the cells. It is extremely small and ovoid, and frequently is so short that it resembles a micrococcus. Often its poles stain more deeply than the center. In general, the form of the or- ganisms is constant, though occasionally slightly larger individuals are encountered. They are usually grouped separately, though occa- sionally in pairs, end to end. Compared with the influenza bacillus in morphology, the bacillus of pertussis is more regularly ovoid and somewhat larger. It has, furthermore, less tendency to pleomorphism and involution. Staining. — The Bordet-Gengou bacillus may be stained with alkaline methylene-blue, dilute carbol-fuchsin, or aqueous fuchsin solutions. Bordet and Gengou recommended as a staining-solution carbolated toluidin-blue made up as follows: Toluidin-blue 5 gms. Alcohol . 100 c.c. Water 500 c.c. 1 Bordet et Gengou, Ann. de l'inst. Pasteur, 1906. 535 536 PATHOGENIC MICROORGANISMS Allow to dissolve and add 500 c.c. of 5 per cent carbolic acid in water. Let this stand one or two days and filter. Stained by the method of Gram, the bacillus of Bordet and Gengou is decolorized. Cultivation. — Early attempts at cultivation made by the discover- ers upon ordinary ascitic agar or blood agar were unsuccessful. They Fig. 116. — Bordet-Gengou Bacillus. finally obtained successful cultures from sputum by the use of the following medium: One hundred grams of sliced potato are put into 200 c.c. of 4 per cent glycerin in water. This is steamed in an autoclave and a glycerin extract of potato obtained. To 50 c.c. of this extract 150 c.c. of 6-per- cent salt solution and 5 grams of agar are added. The mixture is melted in the autoclave and the fluid filled into test tubes, 2 to 3 c.c. each, and sterilized. To each tube, after sterilization, is added an equal volume of sterile defibrinated rabbit blood or preferably human blood, the sub- stances are mixed, and the tubes slanted. On such a medium, inoculated with sputum, taken preferably BORDET-GENGOU BACILLUS 537 during the paroxysms of the first day of the disease, colonies appear, which are barely visible after twenty-four hours, 1 plainly visible after forty-eight hours. They are small, grayish, and rather thick. After the first generation the organisms grow with markedly greater luxu- riance and speed. On the potato-blood medium, after several gen- erations of artificial cultivation, they form a grayish glistening layer which, after a few days, becomes heavy and thick, almost resembling the growth of typhoid bacilli. In these later generations, also, they develop readily upon plain blood agar or ascitic agar and in ascitic broth or broth to which blood has been added. In the fluid media they form a viscid sediment, but no pellicle. Culturally, the bacillus varies from B. influenzae in growing less readily on hemoglobin media than the latter, on first cultivation from the sputum. Later it grows much more heavily on such media and shows less dependence upon the presence of hemoglobin than does B. influenzae. It also grows rather more slowly than the influenza bacillus. It is strictly aerobic and in fluid cultures is best grown in wide flasks with shallow layers of the medium. The Bordet-Gengou bacillus grows moderately at temperatures about 37.5° C, but does not cease to grow at temperatures as low as 5° to 10° C. On blood agar and in ascitic broth it may remain alive for as long as two months (Wollstein). Pathogenicity. — As regards the pathogenicity and etiological spe- cificity of this organism for whooping-cough, no positive statement can as yet be made. The fact that it has been found in many cases in almost pure cultures during the early paroxysms, renders it likely that the organism is the specific cause of the disease. However, in early cases true influenza bacilli have often been found, and these latter seem to remain in the sputum of such patients for a longer period and in larger numbers than the bacillus of Bordet and Gengou. Endotoxins have been obtained from the cultures of the bacilli by Bordet and Gengou by the method of Besredka.2 The growth from slant cultures is washed up in a little salt solution, dried in vacuo, and ground in a mortar with a small, measured quantity of salt. Finally, enough distilled water is added to bring the salt into a solution of 0.75 per cent and the mixture is centrifugalized and decanted. One to two c.c. of such an extract will usually kill a rabbit within twenty-four hours after intravenous inocula- 1 Wollstein, Jour. Exp. Med., xi, 1909. 2 Bordet, Bull, de la Soc. Roy. de Brux., 1907. 538 PATHOGENIC MICROORGANISMS tion. Subcutaneous inoculation produces non-suppurating necrosis and ulceration without marked constitutional symptoms. Inoculation of monkeys with the bacilli themselves by the respira- tory path has failed to produce the disease. Specific agglutinins may be obtained in immunized animals which prove absolutely the distinctness of this organism from Bacillus in- fluenzae.1 In the serum of afflicted children the agglutination is too irregular to be of value. Specific complement fixation with the serum of patients is reported by Bordet and Gengou, but failed in the hands of Wollstein. MORAX-AXENFELD BACILLUS In 1896 Morax 2 described a diplo-bacillus, which he associated etiologically with a type of chronic conjunctivitis to which he applied the name " conjonctivite subaigue." Soon after this, a similar micro- organism was found in cases corresponding to those of Morax by Axen- feld.3 The condition which these microorganisms characteristically produce is a catarrhal conjunctivitis which usually attacks both eyes. The inflammation is especially noticeable in the angles of the eye, most severe at or about the caruncle. There is rarely much swelling of the conjunctiva and hardly ever ulceration. The condition runs a subacute or chronic course. Its diagnosis is easily made by smear preparations of the pus which is formed with especial abundance during the night. Morphology. — In smear preparations from the pus, the microorgan- isms appear as short, thick bacilli, usually in the form of two placed end to end, but not infrequently singly or in short chains. Their ends are distinctly rounded, their centers slightly bulging, giving the bacillus an ovoid form. They are usually about two micra in length. They are easily stained by the usual anilin dyes, and, stained by the method of Gram, are completely decolorized. Cultivation. — The Morax-Axenfeld bacillus can be cultivated only upon alkaline media containing blood or blood serum. It grows poorly, or not at all, at room temperature. Upon Loeffler's blood serum, colonies appear after twenty-four to thirty-six hours as small indentations which indicate a liquefaction of the medium. Axenfeld states that eventually the entire medium may 1 Wollstein, loc. cit. 2 Morax, Ann. de Finst. Pasteur, 1896. 3 Axenfeld, Cent. f. Bakt., xxi, 1897. ZUR NEDDEN'S BACILLUS 539 become liquefied. Upon serum agar delicate grayish drop-like colonies are formed which are not unlike those of the gonococcus. In ascitic bouillon general clouding occurs within twenty-four hours. Pathogenicity. — Attempts to produce lesions in the lower animals with this bacillus have been universally unsuccessful. Subacute con- FlG. 117. MORAX-AXENFELD DlPLO-BACILLUS. junctivitis, however, has been produced in human beings by inocula- tions with pure cultures. ZUR NEDDEN'S BACILLUS In ulcerative conditions of the cornea, Zur Nedden has frequently found a bacillus to which he attributes etiological importance. The bacillus which he has described is small, usually less than one micron in length, often slightly curved, and generally found singly. It may be found in the diplo form but does not form chains. It is stained by the usual dyes, often staining poorly at the ends. Stained by Gram's method it is decolorized. The bacillus is non-motile. Cultivation. — It is easily cultivated upon the ordinary laboratory media. Upon agar it forms, within twenty-four hours, transparent, slightly fluorescent colonies which are round, raised, rather coarsely granular, and show a tendency to confluence. Gelatin is not liquefied. 540 PATHOGENIC MICROORGANISMS Milk is coagulated. Upon potato, there is a thick yellowish growth. Upon dextrose media, there is acid formation, but no gas. The bacillus forms no indol in pepton solutions. Pathogenicity. — Corneal ulcers have been produced by inoculation of guinea-pigs. BACILLUS OF DUCREY The soft chancre, or chancroid, is an acute inflammatory, destructive lesion which occurs usually upon the genitals or the skin surrounding the genitals. The infection is conveyed from one individual to an- other by direct contact. It may, however, under conditions of surgical manipulation, be transmitted indirectly by means of dressings, towels, or instruments. The lesion begins usually as a small pustule which rapidly ruptures, leaving an irregular ulcer with undermined edges and a necrotic floor which spreads rapidly. It differs clinically from the true or syphilitic chancre in the lack of induration and in its violent inflammatory nature. Usually it leads to lymphatic swellings in the groin which, later, give rise to abscesses, commonly spoken of as " buboes." In the discharges from such lesions, Ducrey,1 in 1889, was able to demonstrate minute bacilli to which he attributed an etiological rela- tionship to the disease, both because of the regularity of their presence in the lesions and the successful transference of the disease by means of pus containing the microorganisms. Morphology and Staining. — The Ducrey bacillus is an extremely small bacillus, measuring from one to two micra in length and about half a micron in thickness. It has a tendency to appear in short chains and in parallel rows, but many of the microorganisms may be seen irregularly grouped. It is not motile, possesses no nagella, and does not form spores. Stained by the ordinary anilin dyes, it has a tendency to take the color irregularly and to appear more deeply stained at the poles. By the Gram method, it is decolorized. In tissue sections, it may be demon- strated by Loefner's methylene-blue method, and in such preparations has been found within the granulation tissues forming the floor of the ulcers. In pus, the bacilli are often found within leucocytes. 1 Ducrey, Monatschr. f. prakt. Dermat., 9, 1889. BACILLUS OF DUCREY 541 Cultivation and Isolation. — Early attempts at cultivation of this bacillus were universally unsuccessful in spite of painstaking experi- ments with media prepared of human skin and blood serum. In 1900, Besancon, Griffon, and Le Sourd * finally succeeded in obtaining growths upon a medium containing agar to which human blood had been added. They were equally successful when dog's or rabbit's blood was substi- tuted for that of man. Since the work by these authors, the cultiva- tion by similar methods has been carried out by a number of investiga- tors. Coagulated blood, which has been kept for several days in sterile tubes, has been found to constitute a favorable medium. Freshly clotted blood can not be employed, probably because of the bacteri- cidal action of the serum. Serum-agar has occasionally been used with success but does not give results as satisfactory as those obtained by the use of the whole blood. The best method of obtaining pure cultures upon such media con- sists in puncturing an unruptured bubo with a sterile hypodermic needle and transferring the pus in considerable quantity directly to the agar. If possible, the inoculation of the media should be made immediately before the pus has had a chance to cool off or to be exposed to light. When buboes are not available, the primary lesion may be thoroughly cleansed with sterile water or salt solution, and material scraped from the bottom of the ulcer or from beneath its overhanging edges with a stiff platinum loop. This material is then smeared over the surface of a number of blood-agar plates. Upon such plates, isolated colonies appear, usually after forty-eight hours. They are small, transparent, and gray, and have a rather firm, finely granular consistency. The colonies rarely grow larger than pin- head size, and have no tendency to coalesce. At room temperature, the cultures die out rapidly. Kept in the incubator, however, they may remain alive and virulent for a week or more. On the simpler media, glucose-agar, broth, or gelatin, cultivation is never successful. On moist blood-agar and in the condensation water of such tubes, the bacilli have a tendency to grow out in long chains. Upon media which are very dry, they appear singly or in short chains. Pathogenicity. — Besancon, Griffon and Le Sourd, and others, have succeeded in producing lesions m man by inoculation with pure cultures. Inoculation of the lower animals has, so far, been entirely without result. 1 Besangon, Griffon, and Le Sourd, Presse med., 1900. 542 PATHOGENIC MIRCOORGAXISMS MICROCOCCUS MELITENSIS (MALTA FEVER) (Bacillus melitensis) Malta fever is a disease occurring along the Mediterranean coast and its islands. It has been recently found to occur also in South Amer- ica and in the West Indies. The disease is not very unlike typhoid fever, though more irregular and with a lower mortality. It is accompanied by joint pains, sweating, constipation, and occasionally orchitis. The spleen is almost always enlarged. The microorganism causing the disease was isolated in 1887 by Bruce,1 a British army surgeon. Morphology. — Micrococcus melitensis is a minute bacterium ap- pearing coccoid in smears from agar cultures, in broth cultures assum- ing the form of a short, slightly wedge-shaped bacillus resembling B. in- fluenz2e. Babes2 regards it as unquestionably a bacillus. Earlier observers wrongly believed it to be a coccus. It appears in irregularly parallel groups, and occasionally forms short chains. It is easily stained with the ordinary dyes, and is decolorized by Gram's method. Cultivation. — It can usually be cultivated from the spleens of those who have succumbed to the disease. It grows on nutrient agar at 37.5° C, forming small, pearly white colonies at the end of two or three days. It grows easily on all of the ordinary laboratory media. Animal experiments have proved it pathogenic only for monkeys, in whom it produces a disease similar to that occurring in man.3 Both in patients and in injected animals, infection with this bacte- rium produces specific agglutinins which are of great practical aid in diagnosis.4 1 Bruce, Practitioner. 1887. 2 Babes, Kolle und Wassermann. iii, p. 443. 3 Hughes, Ann. de l'inst. Pasteur. 1893. * Wright and Lamb, Jour. Path, and Bact., v, 1899. CHAPTER XXXIX THE BACILLI OF THE HEMORRHAGIC SEPTICEMIA GROUP AND BACILLUS PESTIS. In many of the lower animals there occur violently acute bacterial infections characterized by general septicemia, usually with petechial hemorrhages throughout the organs and serous membranes and severe intestinal inflammations. These diseases, spoken of as the "hemor- rhagic septicemias," are caused by a group of closely allied bacilli, first classified together by Hueppe ' in 1886. Some confusion has existed as to the forms which should be considered within Hueppe's group of "hemorrhagic septicemia," a number of bacteriologists including in this class bacilli such as Loeffler's Bacillus typhi murium, and Salmon and Smith's hog-cholera bacillus, microorganisms which, because of their motility and cultural characteristics, belong more properly to the "Gartner," " enteritidis," or "paratyphoid" group, intermediate be- tween colon and typhoid. The organisms properly belonging to this group are short bacilli, more plump than are those of the colon type, and showing a marked ten- dency to stain more deeply at the poles than at the center. They are non-motile, possess no flagella, and do not form spores. They grow readily upon simple media, but show a very marked preference for oxygen, growing but slightly below the surface of media. By some observers they are characterized as "obligatory aerobes," but this is undoubtedly a mistake. While showing considerable variations in form and differences in minor cultural characteristics, the species characteristics of polar stain- ing, decolorization by Gram, immobility, lack of gelatin liquefaction, and great pathogenicity for animals, stamp alike all members of the group. Its chief recognized representatives are the bacillus of chicken cholera, the bacillus of swine-plague (Deutsche Schweineseuche) , 1 Hueppe, Berl. klin. Woch., 1886. 543 544 PATHOGENIC MICROORGANISMS and the Bacillus pleurosepticus which causes an acute disease in cattle and often in wild game. Because of certain cultural and pathogenic characteristics, it seems best to consider the bacillus of bubonic plague with this group. BACILLUS OF CHICKEN CHOLERA (Bacillus avisepticus) The bacillus of chicken cholera was first carefully studied by Pas- teur ' in 1880. It is a short, non-motile bacillus, measuring from 0.5 to 1 micron in length. Stained with the ordinary anilin dyes, it displays marked polar staining qualities, which often give it the appearance of being a diplococcus. It is decolorized by Gram's method. It does not form spores, but may occasionally form vacuolated degeneration forms, not unlike those described for Bacillus pestis. The bacillus is easily cultivated from the blood and organs of infected animals, it grows well upon the simplest media at temperatures vary- ing from 25° to 40° C. In broth, it produces uniform clouding with later a formation of a pellicle. Upon agar it forms, within twenty-four to forty-eight hours, minute colonies, white or yellowish in color, which are at first transparent, later opaque. Upon gelatin, it grows without liquefaction. Upon milk, the growth is slow and does not produce co- agulation. According to Kruse,2 indol is formed from pepton bouillon. Acid, but no gas, is formed in sugar broth. Among barnyard fowl, this disease is widely prevalent, attacking chickens, ducks, geese, and a large variety of smaller birds. The infection is extremely acute, ending fatally within a few days. It is accompanied by diarrhea, often with bloody stools, great exhaustion, and, toward the end, a drowsiness bordering on coma. Autopsy upon the animals re- veals hemorrhagic inflammation of the intestinal mucosa, enlargement of the liver and spleen, and often bronchopneumonia. The specific bacilli may be found in the blood, in the organs, in exu- dates, if these are present, and in large numbers in the dejecta. Infection takes place probably through the food and water contaminated by the discharges of diseased birds.3 Subcutaneous inoculation or feeding of such animals with pure cultures, even in minute doses, gives rise to a quickly developing septicemia which is uniformly fatal. The bacillus is extremely patho- 1 Pasteur, Comptes rend, de l'acad. des sci., 1880. 2 Kruse, in Fliigge's "Die Mikroorganismen." 3 Salmon, Rep. of the Com. of Agriculture, 1880, 1881, and 1882. BACTLLI OF HEMORRHAGIC SEPTICEMIA GROUP 545 genie for rabbits, less so for hogs, sheep, and horses, if infection is prac- ticed by subcutaneous inoculation. Infection by ingestion does not seem to cause disease in these animals. Historically, the bacillus of chicken cholera is extremely interesting, since it was with this microorganism that Pasteur x carried out some of his fundamental researches upon immunity, and succeeded in immu- nizing chickens with attenuated cultures. The first attenuation ex- periment made by Pasteur consisted in allowing the bacilli to remain in a broth culture for a prolonged period without transplantation. With minute doses of such a culture (vaccin I) he inoculated chickens, fol- lowing this, after ten days, with a small dose of a fully virulent culture. Although enormously important in principle, the practical results from this method, as applied to chicken cholera, have not been satisfactory. It was with this bacillus, furthermore, that Pasteur was first able to demonstrate the existence of a free toxin which could be separated from the bacteria by filtration. BACILLUS OF SWINE PLAGUE (Bacillus suisepticus, Schweineseuche) This microorganism is almost identical in form and cultural charac- teristics with the bacillus of chicken cholera. It is non-motile, forms no spores, is Gram-negative, and does not liquefy gelatin. The bacillus causes an epidemic disease among hogs, which is characterized almost regularly by a bronchopneumonia followed by general septicemia. There is often a sero-sanguineous pleural exudate, a swelling of bronchial lymph glands and of liver and spleen. The gastrointestinal tract is rarely affected. The bacilli at autopsy may be found in the lungs, in the exudates, in the liver and spleen, and in the blood. The disease is rarely acute, but, in young pigs, almost uniformly fatal. It is probable that spontaneous infection usually occurs by inhala- tion. Experimental inoculation is successful in pigs, both when given subcutaneously and when administered by the inhalation method. Mice, guinea-pigs, and rabbits are also susceptible, dying within three or four days after subcutaneous inoculation of small doses. Active and passive immunization of animals against Bacillus suisep- ticus has been attempted by various observers. Active immunization, if carried out with care, may be successfully done in the laboratory. 1 Pasteur, loc. cit. 35 546 PATHOGENIC MICROORGANISMS Passive immunization of animals with the serum of actively immunized horses has been practiced by Kitt and Mayr,1 Schreiber,2 and Wasser- mann and Ostertag. The last-named observers, working with a poly- valent serum produced with a number of different strains of the bacillus, have obtained results of considerable practical value. The researches of Kitt and Mayr have revealed a fact pointing to the interrelationship of the bacilli of the "hemorrhagic septicemia" group. They were able to show that the serum of horses immunized with chicken cholera bacilli was able to protect, somewhat, against Bacillus suisepticus. Infection with the bacillus of swine plague, in hogs, is often ac- companied by an infection with the hog-cholera bacillus (Schweinepest) . The latter, as we have seen, is a microorganism belonging to the enteri- tidis group, intermediate between Bacillus coli and Bacillus typhosus, and differing from suisepticus in being actively motile, possessing flagella, not showing the polar staining, having a more slender morphology, and producing gas upon dextrose broth. A confusion between the two bacilli frequently occurs because of their nomenclature. Bacteriologic- ally and pathogenically, they are quite distinct. Bacillus suisepticus produces an acute septicemia, accompanied by bronchopneumonia and usually not affecting the gastro-intestinal canal. The bacillus of hog cholera produces an infection localized in the intestinal canal. BACILLUS PESTIS (Bacillus of Bubonic Plague) The history of epidemic diseases has no more terrifying chapter than that of plague.3 Sweeping, time and again, over large areas of the civilized world, its scope and mortality were often so great that all forms of human activity were temporarily paralyzed. In the reign of Justinian almost fifty per cent of the entire population of the Roman Empire perished from the disease. The "Black Death" which swept over Europe during the fourteenth century killed about twenty-five million people. Smaller epidemics, appearing in numerous parts of the world during the sixteenth, seventeenth, and eighteenth centuries, have claimed innumerable victims. In 1893, plague appeared in Hong Kong. During the epidemic which followed, Bacillus pestis, now recognized as the etiological factor of the disease, was discovered by 1 Kitt und Mayr, Monatsh. f. prakt. Thierheilk., 8, 1897. 2 Schreiber, Berl. tierarztl. Woch., 10, 1899. 3 Hirsch, " Handb. d. histor.-geogr. Path.," 1881. BACILLUS PESTIS 547 Kitasato l and by Yersin,2 independently of each other. By both ob- servers the bacillus could invariably be found in the pus from the buboes of afflicted persons. It could be demonstrated in enormous numbers in the cadavers of victims. The constancy of the occurrence of the bacillus in patients, shown in the innumerable researches of many bacteriologists, would alone be sufficient evidence of its etiological relationship to the disease. This evidence is strengthened, moreover, by accidental infections which occurred in Vienna in 1898, with labora- tory cultures. Morphology and Staining. — Bacillus pestis is a short, thick bacillus with well-rounded ends. Its length is barely two or two and a half times Fig. 118. — Bacillus pestis. (After Mallory and Wright.) its breadth (1.5 to 1.75 micra by 0.5 to 0.7 micron). The bacilli appear singly, in pairs, or, more rarely, in short chains of three or more. They show distinct polar staining. In size and shape these bacilli are sub- ject to a greater degree of variation than are most other microorganisms. In old lesions or in old cultures the bacilli show involution forms which may appear either as swollen coccoid forms or as longer, club-shaped, diphtheroid bacilli. Degenerating individuals appear often as swollen, oval vacuoles. All these involution forms, by their very irregularity, are of diagnostic importance. They appear more numerous in artificial cultures than in human lesions. According to Albrecht and Ghon,3 the plague bacillus may, by 1 Kitasato, Lancet, 1894. 2 Yersin, Ann. de l'inst. Pasteur, 1894. 3 Albrecht und Ghon, Wien, 1898, 548 PATHOGENIC MICROORGANISMS special methods, be shown to possess a gelatinous capsule. It does not possess flagella and does not form spores. The plague bacillus is easily stained with all the usual anilin dyes. Diluted aqueous fuchsin and methylene-blue are most frequently employed. With these stains the characteristically deeper staining of the polar portions of the bacillus is usually easy to demonstrate. Special polar stains have been devised by various observers. Most of these depend upon avoidance of the usual heat fixation of the prepara- tions, which, in some way, seems to interfere with good polar staining. Fixation of the dried smears with absolute alcohol is, therefore, prefer- able. The bacillus is decolorized by Gram's method. Fig. 119. — Bacillus pestis, Involution Forms. (After Zettnow.) Isolation and Cultivation. — The bacillus is easily isolated in pure culture from the specific lesions of plague patients, during life or at autopsy. It grows readily and luxuriantly upon the meat-infusion media. The optimum temperature for its cultivation is about 30° C. Below 20° C. and above 38° C, growth is sparse and delayed, though it is not entirely inhibited until exposed to temperatures below 12° C, or above 40° C. The most favorable reaction of culture media is neu- trality or moderate alkalinity, though slight acidity does not prevent development. On agar, growth appears within twenty-four hours as minute colonies with a compact small center surrounded by a broad, irregularly indented, granular margin. BACILLUS PESTIS 549 On gelatin, similar colonies appear after two or three days at 20° to 22° C. The gelatin is not liquefied. In bouillon, the plague bacilli grow slowly. They usually sink to the bottom or adhere to the walls of the tube as a granular deposit and may occasionally form a delicate pellicle. Chain-formation is not un- common. In broth cultures, moreover, a peculiar stalactite-like growth is often seen, when the culture fluid is covered with a layer of oil. Delicate threads of growth hang down from the surface of the medium into its depths like stalactites. Milk is not coagulated. In litmus-milk there is slight acid forma- tion. On potato and on blood serum the growth shows nothing char- acteristic or of differential value. On pepton media no indol is formed. Biological Considerations. — Bacillus pestis is aerobic. Absence of free oxygen is said to prevent its growth, at least under certain condi- tions of artificial cultivation. It is non-motile. Outside of the animal body the bacilli may retain viability for months and even years if preserved in the dark and in a moist environment. In cadavers they may live for weeks and months if protected from dryness. In pus or sputum from patients they may live eight to fourteen days. These facts are of great hygienic importance. Complete drying in the air kills the bacilli within two or three days.1 Thoroughly dried by artificial means they die within four or five hours. Dry heat at 100° C. kills the bacillus in one hour.2 Live steam or boil- ing water is effectual in a few minutes. The bacilli possess great resist- ance against cold, surviving a temperature of 0° C. for as many as forty days. Direct sunlight destroys them within four or five hours. The common disinfectants are effectual in the following strengths : carbolic acid, one per cent kills them in two hours, five per cent in ten minutes; bichlorid of mercury 1 : 1,000 is effectual in ten minutes. Pathogenicity. — In man, plague is acquired 3 by entrance of the bacil- lus either through the skin or by the respiratory tract. The period of incubation is about three to seven days. Two distinct clinical types of the disease occur, depending upon the mode of infection. When cutaneous infection has occurred the disease is first localized in the lymph nodes nearest the point of inoculation. If the respiratory tract 1 Kitasato, Lancet, 1894. 2 Abel, Cent. f. Bakt., xxi, 1897. 3 Gottschlich, Zeit. f. Hyg., xxxv, 1900. 550 PATHOGENIC MICROORGANISMS has been the portal of entrance the disease primarily takes the form of a pneumonia. Infection may take place through the most minute lesions, hardly visible to the naked eye. Even the unbroken skin may admit the microorganisms if these are rubbed in with sufficient energy. From the primary lymphatic swellings, the bacilli enter the blood and produce secondary foci. The general symptoms are largely referable to endo- toxins. The pneumonic form of plague usually begins with symptoms not unlike a typical pneumonia and is usually fatal within four or five or even fewer days. This form of the disease is especially menacing as a means of dissemination, because of the enormous numbers of plague bacilli in the sputum. One of the chief characteristics of the general systemic plague infec- tion is the very marked cardiac depression. The bacteriological diagnosis during life may be made by finding the bacilli in the sputum or in aspiration fluid from a bubo. The micro- organisms are identified morphologically, culturally, by animal experi- ment, and by agglutination reaction. Blood cultures from plague pa- tients often yield positive results, especially when the blood is well diluted in neutral broth to prevent any inhibiting action of the anti- bodies in the serum. At autopsy, in man, the bacilli are found in the primary lesions, in the blood, and in the spleen, the liver, and the lymphatics. There may be hemorrhages into the serous cavities. When pneumonia exists, it usually takes the form of a bronchopneumonia with extensive swelling of the bronchial lymph nodes. In cases in which the disease is prolonged, there are often tubercle- like foci in the spleen, liver, and lungs. Histologically these foci show central necrosis surrounded by the usual inflammatory cell reactions. In more chronic cases connective-tissue encapsulation may appear. Bacillus pestis is extremely pathogenic for rats, mice, guinea-pigs, rabbits, and monkeys. The most susceptible of these animals are rats and guinea-pigs, in whom mere rubbing of plague bacilli into the un- broken skin will often produce the disease. This method of experimen- tal infection of guinea-pigs is of great service in isolating the plague bacillus from material contaminated with other microorganisms. For the same purpose, infection of rats subcutaneously at the root of the tail may be employed. Such inoculation in rats is invariably fatal. The virulence of separate strains of plague bacilli is subject to great BACILLUS PESTIS 551 variations. Animal passage usually increases it, but prolonged growth upon artificial media often results in gradual reduction of the virulence. In rats, spontaneous infection with plague is common and plays an important role in the spread of the disease. Rats become infected from the cadavers of plague victims or by gnawing the dead bodies of other rats dead of the disease. The pneumonic type of the disease is common in these animals and has been produced in them by inhalation experi- ments. During every well-observed plague epidemic, marked mortality among the domestic rats has been noticed. Plague is transmitted either by direct contact or inhalation, or in- directly by clothing, linen, and other objects worn or handled. The role in the transmission of the disease played by rats is probably of great importance. The animals vomit, defecate, and die in cellars, storerooms, etc., and thus set free vast numbers of plague bacilli for indirect accidental transmission to human beings. The actual mode in which this transmission takes place is by no means certain. The fact that in countries where plague is prevalent many of the natives go insufficiently shod or barefooted, may account for many infections. Simond x lays great stress upon transmission to man by means of fleas, the Indian rat-flea often being parasitic upon man. HLs conclu- sions are probably too far-reaching, though the possibility of such in- fection can not be denied.2 Toxin Formation. — The systemic symptoms of plague are largely due to the absorption of poisonous products of the bacteria. Albrecht and Ghon,3 Wernicke,4 and others were unable to obtain any toxic action with broth-culture filtrates and concluded that the poisons of B. pestis were chiefly endotoxins, firmly attached to the bacterial body. Kossel and Overbeck,5 however, on the basis of a careful investigation, came to the conclusion that, in addition to the endotoxin, there is formed in older broth cultures a definite and important true, soluble toxin. Immunization. — A single attack of plague usually protects human beings from reinfection. A second attack in the same individual is extremely rare. Immunization in animals produces specific agglutinat- ing and bacteriolytic substances which are of great importance in the bacteriological diagnosis of the bacillus. The agglutinating action of the 1 Simond, Ann. de Pinst. Pasteur, 1893. 2 Nuttall, Cent. f. Bakt., xxii, 1897; Nuttall, Hyg. Rund., ix, 1899. 3 Albrecht und Ghon, loc. cit. * Wernicke, Cent. f. Bakt., Ref., xxiv, 1898. 5 Kossel und Overbeck, Arb. a. d. Gesundh., xviii, 1901. 552 PATHOGENIC MICROORGANISMS serum of patients is clinically important in the diagnosis of the disease even in dilutions of one in ten, since undiluted normal human serum has no agglutinating effect upon plague bacilli. Active immunization of animals * is accomplished by inoculation of the whole dead bacteria. Haffkine has attempted active immunization in human beings by subcutaneous treatment with sterilized broth cultures of B. pestis. Gaffky 2 and his collaborators recommend, for similar purposes, forty-eight-hour agar cultures of a bacillus of standard viru- lence, emulsified in bouillon and sterilized at 65° C. 1 Yersin, Calmette et Roux, Ann. de l'inst. Pasteur, 1895. 2 Gaffky, Pfeiffer, Sticker und Dieudonne, Arb. a. d. kais. Gesundheitsamt, xvi, 1899. CHAPTER XL BACILLUS ANTHRACIS AND ANTHRAX (Milzbrand, Charbon) Anthrax is primarily a disease of the herbivora, attacking especially cattle and sheep. Infection not infrequently occurs in horses, hogs, and goats. In other domestic animals it is exceptional. Man is susceptible to the disease and contracts it either directly from the living animals or from the hides, wool, or other parts of the cadaver used in the industries. The history of the disease dates back to the most ancient periods and anthrax has, at all times, been a severe scourge upon cattle- and sheep- raising communities. Of all infections attacking the domestic animals no other has claimed so many victims as anthrax. In Russia, where the disease is most common, 72,000 horses are said to have succumbed in one year (1864). * In Austro-Hungary, Germany, France, and the Eastern countries, each year thousands of animals and numerous human beings perish of anthrax. In England and America the disease is relatively infrequent. No quarter of the globe, however, is entirely free from it. Especial historical interest attaches to the anthrax bacillus in that it was the first microorganism proved definitely to bear a specific etio- logical relationship to an infectious disease. The discovery of the an- thrax bacillus, therefore, laid, as it were, the cornerstone of modern bacteriology. The bacillus was first observed in the blood of infected animals by Pollender in 1849, and, independently, by Brauell in 1857. Davaine,2 however, in 1863, was the first one to produce experimental infection in animals with blood containing the bacilli and to suggest a direct etiological relationship between the two. Final and absolute proof of the justice of Davaine's contentions, however, was not brought until the further development of bacteriological technique, by Koch,3 had made it possible for this last observer to isolate the bacillus upon 1 Quoted from Sobernheim, Kolle und Wassermann., vol. ii. 2 Davaine, Comptes rend, de Pacad. des sci., lvii, 1863. 3 Koch, Cohn's " Beitr. z. Biol. d. Pflanzen," ii, 1876. 553 554 PATHOGENIC MICROORGANISMS artificial media and to reproduce the disease experimentally by inocu- lation with pure cultures. Morphology and Staining. — The anthrax bacillus is a straight rod, 5 to 10 micra in length, 1 to 3 micra in width. It is non-motile. In preparations made from the blood of an infected animal, the bacilli are usually single or in pairs. Grown on artificial media they form tangles of long threads. Their ends are cut off squarely, in sharp con- Fig. 120. — Bacillus anthracis. From pure culture on agar. trast to the rounded ends of many other bacilli. The corners are often sharp and the ends of bacilli in contact in a chain often touch each other only at these points, leaving in consequence an oval chink between the ends of the organisms. The appearance of a chain of anthrax bacilli, therefore, has been not inaptly compared to a rod of bamboo. On artificial media the anthrax bacillus forms spores. Oxygen is necessary for the formation of these spores and they are consequently not found BACILLUS ANTHRACIS AND ANTHRAX 555 in the blood of infected subjects. The spores are located in the middle of the bacilli and are distinctly oval. They are difficult to stain, but may be demonstrated by any of the usual spore-staining procedures, such as Holler's or Novy's methods. The bacilli themselves are easily stained by the usual anilin dyes, and gentian-violet or fuchsin in aque- ous solution may be conveniently employed. They are not decolorized by Gram's method. In preparations from animal tissues or blood, stained by special pro- Fig. 121. — Bacillus anthracis. In section of kidney of animal dead of anthrax. cedures, the anthrax bacillus may occasionally be seen to possess a cap- sule. The capsule is never seen in preparations from the ordinary artificial media. Some observers have demonstrated them in cultures grown in fluid blood serum. In chains of anthrax bacilli, the capsule when present seems to envelop the entire chain and not the individual bacteria separately. Isolation. — Isolation of the anthrax bacillus from infected material 556 PATHOGENIC MICROORGANISMS is comparatively simple, both because of the ease of its cultivation and because of the sharply characteristic features of its morphological and cultural appearance. Cultivation. — The anthrax bacillus is an aerobic, facultatively anaero- bic bacillus. While it may develop slowly and sparsely under anaerobic conditions, free oxygen is required to permit its luxuriant and charac- teristic growth. The optimum temperature for its cultivation ranges about 37.5° C. It is not, however, delicately susceptible to moderate variations of tern- Fig. 122. — Bacillus anthracis. In smear of spleen of animal dead of anthrax. perature and growth does not cease until temperatures as low as 12° C. or as high as 45° C. are reached. By continuous cultivation at some of the temperatures near either the higher or the lower of these limits, the bacillus may become well adapted to the new environment and attain luxuriant growth.1 The anthrax bacillus may be cultivated on all of the usual artificial media, growing upon the meat-extract as well as upon the meat-infusion media. Dieudonne, Arb. a. d. kais. Gesundheitsamt, 1894. BACILLUS ANTHRACIS AND ANTHRAX 557 It may be cultivated also upon hay infusion, various other vegetable media, sugar solutions, and urine. While moderate acidity of the medium does not prevent the growth of this bacillus, the most favorable reaction for media is neutrality or slight alkalinity. Cn gelatin plates, colonies develop within twenty-four to forty-eight hours as opaque, white disks, pin-head in size, irregularly round and rather flat. As the colonies increase in size their outlines become less regular and under the microscope they are seen to be made up of a hair-like tangle of threads spreading in thin wavy layers from a more compact central knot. The microscopic appearance of these colonies has been aptly described as resembling a Medusa head. Fragments of a Fig. 123. — Anthrax Colony on Gelatin. (After Giinther.) colony examined on a slide with a higher power show the individual threads to be made up of parallel chains of bacilli. After a day or two of further growth, the gelatin about the colonies becomes fluidified. In gelatin stab cultures, growth appears at first as a thin white line along the course of the puncture. From this, growth proceeds in thin spicules or filaments diverging from the stab, more abundantly near the top than near the bottom of the stab, owing to more active growth in well oxygenated environment. The resulting picture is that of a small inverted "Christmas tree." Fluidification begins at the top, at first a shallow depression filled with an opaque mixture of bacilli and fluid. 558 PATHOGENIC MICROORGANISMS Later the bacilli sink to the bottom of the flat depression, leaving a clear supernatant fluid of peptonized gelatin. In broth, growth takes place rapidly, but does not lead to an even, general clouding. There is usually an initial pellicle formation at the top where the oxygen supply is greatest. Simultaneously with this a slimy mass appears at the bottom of the tube, owing to the sinking of Fig. 124. — Anthrax Colony on Agar. bacilli to the bottom. Apart from isolated flakes and threads the inter- vening broth is clear. Shaken up, the tube shows a tough, stringy mass, not unlike a small cotton fluff, and general clouding is produced only by vigorous mixing. Upon agar plates, growth at 37.5° C. is vigorous and colonies appear BACILLUS ANTHRACIS AND ANTHRAX 559 within twelve to twenty-four hours. They are irregular in outline, slightly wrinkled, and show under the microscope the characteristic tangled-thread appearance seen on gelatin, except that they are more compact than upon the former medium. The colonies are slightly glisten- ing and tough in consistency. On agar slants, the colonies usually become confluent, the entire surface soon being covered by a grayish, tough pellicle which, if fished, has a tendency to come away in thin strips or strands. On potato, growth is rapid, white, and rather dry. Sporulation upon potato is rapid and marked, and the medium is favorable for the study of this phase of development. Milk is slowly acidified and slowly coagulated. This action is chiefly upon the casein; very few, if any, changes being produced either in the sugars or in the fats of the milk. The acids formed are, according to Iwanow,1 chiefly formic, acetic, and caproic acids. Biological Considerations. — The anthrax bacillus is aerobic and facul- tatively anaerobic. It is non-motile and possesses no flagella. In the animal body it occasionally forms capsules. In artificial cultures in the presence of oxygen, it sooner or later invariably forms spores. The spores appear after the culture has reached its maximum of develop- ment. Sporulation never occurs in the animal body, probably because of the absence of sufficient free oxygen. Spores are formed most exten- sively 2 at temperatures ranging from 20° C. to 30° C. Spore formation ceases below 18° C. and above 42° C. For different strains these figures may vary slightly, as has been shown from the results of various observers. Spores appear most rapidly and regularly upon agar and potato media. The spore — one in each bacillus — appears as a small, highly refractile spot in the center of the individual bacterium. As this enlarges, the body of the bacillus around it gradually undergoes granular degenera- tion and loses its staining capacity.3 If anthrax bacilli are cultivated for prolonged periods upon media containing hydrochloric or rosolic acid or weak solutions of carbolic acid,4 cultures may be obtained which do not sporulate and which seem permanently to have lost this power, without losing their virulence to the same degree. Similar results may be obtained by continuous cul- 1 Iwanow, Ann. de Tinst. Pasteur, 1892. 2 Koch, loc. cit. 3 Behring, Zeit. f. Hyg., vi and vii, 1889; Deut. med. Woch., 1889. 4 Chamberland et Roux, Comptes rend, de l'acad, des sci.7 xcvi, 1882, 560 PATHOGENIC MICROORGANISMS tivation at temperatures above 42° C. By this procedure, however, virulence, too, is considerably diminished. Resistance. — Because of its property of spore formation, the anthrax bacillus is extremely resistant toward chemical and physical environ- ment. The vegetative forms themselves are not more resistant than most other non-sporulating bacteria, being destroyed by a temperature of 54° C. in ten minutes. Anthrax spores may be kept in a dry state for many years without losing their viability.1 While different strains of anthrax spores show some variation in their powers of resistance, all races show an extremely high resistance to heat. Dry heat at 140° C. kills them only after three hours.2 Live steam at 100° kills them in five to ten minutes. Boiling in water destroys them in about ten min- utes. Low temperatures have but little effect upon them. Ravenel 3 found that, frozen by liquid air, they were still viable after three hours. The variability shown by different strains of spores in their resistance to heat is even more marked in their behavior toward chemicals.4 Some strains will retain their viability after exposure to five-per-cent carbolic acid for forty days,5 while others are destroyed by the same solution in two days. Corrosive sublimate, 1 : 2,000, kills most strains of anthrax in forty minutes. Direct sunlight destroys anthrax spores within six to twelve hours.6 Pathogenicity. — The anthrax bacillus is pathogenic for cattle, sheep, guinea-pigs, rabbits, rats, and mice. The degrees of susceptibil- ity of these animals differ greatly, variations in this respect existing even among different members of the same species. Thus, the long-haired Algerian sheep show a high resistance, while the European variety are highly susceptible; and, similarly, the gray rat is much more resistant than the white rat. Dogs, hogs, cats, birds, and the cold-blooded ani- mals are relatively insusceptible. For man the bacillus is definitely pathogenic, though less so than for some of the animals mentioned above. While separate races of anthrax bacilli may vary much in their de- gree of virulence, a single individual strain remains fairly constant in this respect if preserved, dried upon threads or kept in sealed tubes, in 1 Surmont et Arnould, Ann. de l'inst. Pasteur, 1894. 2 Koch und Wolffhugel, Mitt. a. d. kais. Gesundheitsamt, 1881. 3 Ravenel, Medical News, vii, 1899. * Frankel, Zeit. f. Hyg., vi, 1889. 5 Koch, loc. cit. 6 Momont, Ann. de l'inst. Pasteur, 1892. BACILLUS ANTHRACIS AND ANTHRAX 561 a cold, dark place. Virulence may be reduced * by various attenuating laboratory procedures which are of importance in that they have made possible prophylactic immunization. Heating the bacilli to 55° C. for ten minutes considerably reduces their virulence. Similar results are obtained by prolonged cultivation at temperatures of 42° to 43° C, or by the addition of weak disinfectants to the culture fluids.2 Once reduced, the new grade of virulence remains fairly constant. Increase of virulence may be artificially produced by passage through animals. Experimental infections in susceptible animals are most easily accom- plished by subcutaneous inoculations. The inoculation is followed, at first, by no morbid symptoms, and some animals may appear perfectly well and comfortable until within a few hours or even moments before death, when they suddenly become visibly very ill, rapidly go into collapse, and die. The length of the disease depends to some extent, of course, upon the resistance of the infected subject, being in guinea- pigs and mice from twenty-four to forty -eight hours. The quantity of infectious material introduced, on the other hand, has little bearing upon the final outcome, a few bacilli, or even a single bacillus, often sufficing to bring about a fatal infection. Although the bacilli are not demonstrable in the blood until just before death, they nevertheless invade the blood and lymph streams immediately after inoculation, and are conveyed by these to all the organs. This has been demonstrated clearly by experiments where inoculations into the tail or ear were im- mediately followed by amputation of the inoculated parts without pre- vention of the fatal general infection. The bacilli are probably not at first able to multiply in the blood. At the place of inoculation and probably in the organs they proliferate, until the resistance of the in- fected subject is entirely overcome. At this stage of the disease, no longer held at bay by any antagonistic qualities of the blood, they enter the circulation and multiply within it. Autopsy upon such animals reveals an edematous hemorrhagic infiltration at the point of inocu- lation. The spleen is enlarged and congested. The kidneys are con- gested, and there may be hemorrhagic spots upon the serous mem- branes. The bacilli are found in large numbers in the blood and in the capillaries of all the organs. The mode of action of Bacillus anthracis is as yet an unsettled point. It is probable that death is brought about to a large extent by purely 1 Toussaint, Comptes rend, de l'acad. des sci., xci, 1880; Pasteur, Chamberland et Roux, Comptes rend, de l'acad. des sci., xcii, 1881. 2 Chamberland et Roux, ibid., xcvir 1882. 36 562 PATHOGENIC MICROORGANISMS mechanical means, such as capillary obstruction. Neither a true secretory toxin nor an endotoxin has been demonstrated for the anthrax bacillus. The decidedly toxemic clinical picture of the disease, however, in some animals and in man, precludes our definitely concluding that such poisons do not exist. It is a matter of fact, however, that neither culture filtrates nor dead bacilli have any noticeable toxic effect upon test animals, and exert no appreciable immunizing action. Spontaneous infection of animals takes place largely by way of the alimentary canal, the bacilli being taken in with the food. The bacteria are swallowed as spores, and therefore resist the acid gastric juice. In the intestines they develop into the vegetative forms, increase, and gradually invade the system. The large majority of cattle infections are of this type. Direct subcutaneous infection may also occur sponta- neously when small punctures and abrasions about the mouth are made by the sharp spicules of the hay, straw, or other varieties of fodder. When infection upon a visible part occurs, there is formed a diffuse, tense local swelling, not unlike a large carbuncle. The center of this may be marked by a black, necrotic slough, or may contain a pustular de- pression. Infection by inhalation is probably rare among animals. Trans- mission among animals is usually by the agency of the excreta or un- burned carcasses of infected animals. The bacilli escaping from the body are deposited upon the earth together with animal and vegetable matter, which forms a suitable medium for sporulation. The spores may then remain in the immediate vicinity, or may be scattered by rain and wind over considerable areas. The danger from buried car- casses, at first suspected by Pasteur, is probably very slight, owing to the fact that the bacilli can not sporulate in the anaerobic environment to which the burying-process subjects them. The disease, in infected cattle and sheep, is usually acute, killing within one or two days. The mortality is extremely high, fluctuating about eighty per cent. In man the disease is usually acquired by cutaneous inoculation. It may also occur by inhalation and through the alimentary tract. Cutaneous inoculation occurs usually through small abrasions or scratches upon the skin in men who habitually handle live-stock, and in butchers, or tanners of hides. Infection occurs most frequently upon the hands and forearms. The primary lesion, often spoken of as " malig- nant pustule," appears within twelve to twenty -four hours after inocula- tion, and resembles, at first, an ordinary small furuncle. Soon, however, its center will show a vesicle filled with sero-sanguineous, later sero- BACILLUS ANTHRACIS AND ANTHRAX 563 purulent fluid. This may change into a black central necrosis sur- rounded by an angry red edematous areola. Occasionally local gangrene and general systemic infection may lead to death within five or six days. More frequently, however, especially if prompt excision is practiced, the patient recovers. The early diagnosis of the condition is best made bacteriologically by finding the bacilli in the local discharge. The pulmonary infection, known as "wool-sorter's disease," occurs in persons who handle raw wool, hides, or horse hair, by the inhala- tion or by the swallowing of spores. The disease is fortunately rare in this country. The spores, once inhaled, develop into the vegetative forms l and these travel along the lymphatics into the lungs and pleura. The disease manifests itself as a violent, irregular pneumonia, which, in the majority of cases, leads to death. The bacilli in these cases can often be found in the sputum before death. Infection through the alimentary canal may occasionally, though rarely, occur in man, the source of infection being usually ingestion of the uncooked meat of infected animals. This form of infection is rare, because in many cases the bacilli have not sporulated in the animal and the ingested vegetative forms are injured or destroyed by the acid gastric juice. When viable spores enter the gut, however, infection may take place, the initial lesion being localized usually in the small intes- tine. The clinical picture that follows is one of violent enteritis with bloody stools and great prostration. Death is the rule. The diagnosis is made by the discovery of the bacilli in the feces. General hygienic prophylaxis against anthrax consists chiefly in the destruction of infected animals, in the burying of cadavers, and in the disinfection of stables, etc. The practical impossibility of destroying the anthrax spores in infected pastures, etc., makes it necessary to re- sort to prophylactic immunization of cattle and sheep. Immunity against Anthrax. — Minute quantities of virulent anthrax cultures usually suffice to produce death in susceptible animals. Dead cultures are inefficient in calling forth any immunity in treated subjects. It is necessary, therefore, for the production of active immunity to resort to attenuated cultures. The safest way to accomplish such at- tenuation is the one originated by Pasteur,2 consisting in prolonged cultivation of the bacillus at 42° to 43° C. in broth. Non-spore-forming races are thus evolved. The longer the bacilli are grown at the above temperature the greater 1 Eppinger, Wien. med. Woch., 1888. 2 Pasteur, loc. cit. 564 PATHOGENIC MICROORGANISMS is the reduction in their virulence. Koch, Gaffky, and Loeffler,1 utilizing the variations in susceptibilities of different species of animals, devised a method by means of which the relative attenuation of a given culture may be estimated and standardized. Rabbits are less susceptible than guinea- pigs, and virulent anthrax cultures, grown for two or three days under the stated conditions, lose their power to kill rabbits, but are less virulent for guinea-pigs. After ten to twenty days of further cultivation at 42° C. the virulence for the guinea-pig disappears, but the culture is potent against the still more susceptible mouse. Even the virulence for mice may be entirely eliminated by further cultivation at this temperature. The method of active immunization first practiced by Pasteur, and still used extensively, is carried out as follows: Two anthrax cultures of varying degrees of attenuation are used as vaccins. The premier vaccin is a culture w^hich has lost its virulence for guinea-pigs and rabbits, and is potent only against mice. The deuxieme vaccin is a cul- ture which is still definitely virulent for mice and guinea-pigs, but not potent for rabbits. Forty-eight-hour broth cultures of these strains, grown at 37.5° C, form the vaccin actually employed. Vaccin I is subcutaneously injected into cattle in doses of 0.25 c.c, sheep receiving about half this quantity. After twelve days have elapsed similar quan- tities of Vaccin II are injected. Pasteur's method has given excellent results and confers an im- munity which lasts about a year. Chauveau 2 has modified Pasteur's method by growing the bacilli in bouillon at 38° to 39° C, at a pressure of eight atmospheres. Cul- tures are then made of races attenuated in this way, upon chicken bouillon and allowed to develop for thirty days. Single injections of 0.1 c.c. each of such cultures are said to protect cattle. Active immunization of small laboratory animals is very difficult, but can be accomplished by careful treatment with extremely attenu- ated cultures. Passive immunization by means of the serum of actively immune animals was first successfully accomplished by Sclavo.3 The subject of passive immunization has been especially investigated and practically applied by Sobernheim.4 The serum used is produced by actively immunizing sheep. It is necessary to carry immunization to an 1 Koch, Gaffky, und Loeffler, Mitt. a. d. kais. Gesundheitsamt, 1884. 2 Chauveau, Comptes rend, de Pacad. des sci., 1884. a Sclavo, Cent. f. Bakt,, xviii, 1895. * Sobernheim, Zeit. f. Hyg., xxv, 1897; xxxi, 1899. BACILLUS ANTHRACIS AND ANTHRAX 565 extremely high degree in order to obtain any appreciable protective power in the serum. This is accomplished by preliminary treatment with Pasteur's or other attenuated vaccines, followed by gradually increasing doses of fully virulent cultures. Treatment continued at intervals of two weeks, for two or three months, usually produces an effective serum. Horses and cattle may also be used for the process, but they are believed by Sobernheim to give less active sera than sheep. Bleeding is done about three weeks after the last injection. The sera are stable and easily preserved. Injections of 20 to 25 c.c. of such a serum have been found to protect animals effectually from anthrax and to confer an immunity lasting often as long as two months. Animals already infected are said to be saved by treatment with 25 to 100 c.c. of the serum. Neither specific bactericidal nor bacteriolytic properties have, so far, been demonstrated in these immune sera. In fact, these properties are distinctly more pronounced against Bacillus anthracis in the normal sera of rats and dogs. Agglutinins have not been satisfactorily demon- strated in sera, partly because of the great technical difficulties en- countered in the active chain-formation of the bacillus in fluid media. An increase of opsonic power of such serum over normal serum has not been satisfactorily demonstrated. Bacteria Closely Resembling Bacillus anthracis. — In most laboratory collections there are strains of true anthrax bacilli so attenuated that they are practically non-pathogenic. These do not differ from the virulent strains in any morphological or cultural characteristics. Besides such strains there are numerous non-virulent bacteria culturally not identical with Bacillus anthracis, but resembling it very closely. B. anthracoides (Hueppe and Wood 1). — A Gram-positive bacillus, morphologically different from B. anthracis in that the ends are more rounded. Culturally, somewhat more rapid in growth and more rapid in gelatin nuidification. Non-pathogenic. Otherwise indistinguishable from B. anthracis. B. radicosus (Wurzel Bacillus) . — Cultivated from water — city water supplies. Morphologically somewhat larger than Bacillus anthracis, and the individual bacilli more irregular in size. Very rapid nuidification of gelatin and growth most active at room temperature. Non-pathogenic. B. subtilis (Hay Bacillus). — Although not very closely related to the anthrax group, this bacillus is somewhat similar and conveniently 1 Hueppe und Wood, Berl. klin. Woch., xvi, 1889. 566 PATHOGENIC MICROORGANISMS described in this connection. It is of importance to workers with patho- genic bacteria, because of the frequency with which it is found as a saprophyte or secondary invader in chronic suppurative lesions. Morphology. — Straight rod, 2 to 8 micra long, 0.7 micron wide. Spores formed usually slightly nearer one pole than the other. Grows in long chains and only in such chains are spores found. It does not decolor- ize by Gram's method. Is actively motile in young cultures in which Fig. 125. — Bacillus Subtilis. (Hay Bacillus.) the bacilli are single or in pairs. In older cultures chains are formed and the bacilli become motionless. Gelatin is liquefied. On gelatin and agar the bacilli grow as a dry corrugated pellicle. Microscopically, the colonies are made up of interlacing threads, being irregularly round with fringed edges. There is a tendency to confluence. The bacillus is found in brackish water, infusions of vegetable matter, etc., and is practically non -pathogenic, occurring only occasionally as a saprophyte in old sinuses and infected wounds. CHAPTER XLI BACILLUS PYOCYANEUS It is a matter of common surgical experience that many suppurating wounds, especially sinuses of long standing, discharge pus which is of a bright green color. The fact that this peculiar type of purulent inflam- mation is due to a specific chromogenic microorganism was first demon- strated by Gessard l in 1882. The bacillus which was described by Ges- sard has since become the subject of much careful research and has been shown to hold a not unimportant place among pathogenic bacteria.2 Morphology and Staining. — Bacillus pyocyaneus is a short rod, usu- ally straight, occasionally slightly curved, measuring, according to Flugge, about 1 to 2 micra in length by about 0.3 of a micron in thickness. The bacilli are thus small and slender, but are subject to considerable variation from the measurements given, even in one and the same cul- ture. While ordinarily single, the bacilli may be arranged end to end in short chains of two and three. Longer chains may exceptionally be formed upon media which are especially unfavorable for its growth, such as very acid media or those containing antiseptics. Spores are not found. The bacilli are actively motile and possess each a single flagellum placed at one end. Bacillus pyocyaneus is stained easily with all the usual dyes, but is decolorized by Gram's method. Irregular staining of the bacillary body is common, but is always an indication of degeneration, and not a normal characteristic, as, for instance, in the diphtheria group. Cultivation. — The pyocyaneus bacillus is aerobic and facultatively anaerobic. It can be adapted to absolutely anaerobic environments, but does not produce its characteristic pigment without the free access of oxygen. The bacillus grows readily upon the usual laboratory media and is not very sensitive to reaction, growing equally well upon moder- ately alkaline or acid media. Development takes place at temperatures as low as 18° to 20° C, more rapidly and luxuriantly at 37.5° C. 1 Gessard, These de Paris, 1882. 2 Charrin, " La maladie pyocyanique, " Paris, 1889. 567 568 • PATHOGENIC MICROORGANISMS On agar slants, growth is abundant and confluent, the surface of the agar being covered by a moist, grayish or yellowish, glistening, even layer. The pigment which begins to become visible after about eighteen hours soon penetrates the agar itself and becomes diffused throughout it, giving the medium a bright green fluorescent appearance, which grows darker as the age of the culture increases. In gelatin stabs, growth takes place much more rapidly upon the surface than in the depths. A rapid liquefaction of the gelatin takes place, causing a saucer-shaped depression. As this deepens, pigment begins to form in the upper layers, often visible as a greenish pellicle. In gelatin plates, the colonies have a characteristic appearance. They are round and are composed of a central dense zone, and a peripheral, loosely granular zone, which extends outward into the peripheral fluidi- fied area in a fringe of fine filaments. When first appearing, they are grayish yellow, later assuming the characteristic greenish hue. In broth, growth is rapid and chiefly at the surface, forming a thick pellicle. Below this, there is moderate clouding. The pigment is formed chiefly at the top. In old cultures there is a heavy flocculent precipitate. In fluid media containing albuminous material, strong alkalinity is produced. On potato, growth develops readily and a deep brownish pigment ap- pears, which is not unlike that produced by B. mallei upon the same medium. Milk is coagulated by precipitation of casein and assumes a yellowish- green hue. In older cultures the casein may again be digested and liquefied The pigment of Bacillus pyocyaneus has been the subject of much investigation. It was shown by Charrm 1 and others that this pigment had no relation to the pathogenic properties of the bacillus. It is found in cultures as a colorless leukobase which assumes a green color on the addition of oxygen. Conversely, the typical green "pyocyanin," as the pigment is called, may be decolorized by reducing substances. This explains the fact that it is not found in cultures sealed from the air. Pyo- cyanin may be extracted from cultures with chloroform and crystallized out of such solution in the form of blue stellate crystals. These, on chemical analysis, have been found to belong to the group of aromatic compounds, with a formula, according to Ledderhose,2 of C14H14N20. Besides pyocyanin, Bacillus pyocyaneus produces another pig- 1 Charrin, loc. cit. 2 Ledderhose, quoted from Boland Cent. f. Bakt., xxv, 1889. BACILLUS PYOCYANEUS 569 ment which is fluorescent and insoluble in chloroform, but soluble in water.1 This pigment is common to other fluorescent bacteria, and not peculiar to Bacillus pyocyaneus. The reddish-brown color seen in old cultures2 and supposed by some writers to be a third pigment, is probably a derivative from pyocyanin by chemical change. Chloroform extraction of pyocyanin from cultures may serve oc- casionally to distinguish the pyocyaneus bacilli from other similar fluorescent bacteria. Ernst has claimed that there are two types of B. pyocyaneus, an «-type which produces only the fluorescent, water- soluble pigment, and a /5-type which produces both this and pyocyanin.3 Pathogenicity. — Bacillus pyocyaneus is one of the less virulent patho- genic bacteria. It is widely distributed in nature and may be found frequently as a harmless parasite upon the skin or in the upper respira- tory tracts of animals and men. It has, however, occasionally been found in connection with suppurative lesions of various parts of the body, often as a mere secondary invader in the wake of another incitant, or even as the primary cause of the inflammation. In most cases where true pyocyaneus infection has taken place, the subject is usually one whose general condition and resistance are abnormally low.4 Thus pyocyaneus may be the cause of chronic otitis media in ill-nour- ished children. It has been cultivated out of the stools of children suf- fering from diarrhea, and has been found at autopsy generally distributed throughout the organs of children dead of gastro-enteritis.5 It has been cultivated from the spleen at autopsy from a case of general sepsis following mastoid operation. The bacillus has been found, further- more, during life in pericardial exudate and in pus from liver abscesses.6 Brill and Libman,7 as well as Finkelstein,8 have cultivated B. pyocyaneus from the blood of patients suffering from general sepsis. Wassermann 9 showed the bacillus to have been the etiological factor in an epidemic of umbilical infections in new-born children. Similar exam- ples of B. pyocyaneus infection in human beings might be enumerated in large numbers, and there is no good reason to doubt that, under given 1 Boland, loc. cit. *Gessard, Ann. de l'inst. Pasteur, 1890, 1891, and 1892. 3 Ernst, Zeit. f. Hyg., ii, 1887. * Rohner, Cent. f. Bakt., xi, 1892. 5 Neumann, Jahrb. f. Kinderheilk., 1890. 6 Kraunhals, Zeit. f. Chir., xxxvii, 1893. 7 Brill and Libman, Amer. Jour. Med. Sci., 1899. s Finkelstein, Cent. f. Bakt., 1899. 9 Wassermann, Virchow's Arch., clxv, 1901. 570 PATHOGENIC MICROORGANISMS conditions, fatal infections may occur. Such cases, however, are still to be regarded as depending more upon the low resistance of the individual attacked than upon the great pathogenicity of B. pyocyaneus. Many domestic animals are susceptible to experimental pyocyaneus infection, chief among these being rabbits, goats, mice, and guinea- pigs. Guinea-pigs are killed by this bacillus with especial ease. Intra- peritoneal inoculation with a loopful of a culture of average virulence usually leads to the death of a young guinea-pig within three or four days. Toxins and Immunization. — Emmerich and Low have shown that filtrates of old broth cultures of B. pyocyaneus contain a ferment-like substance which possesses the power to destroy some other bacteria, apparently by lysis. They have called this substance " pyocyanase " and claim that, with it, they have succeeded in protecting animals from anthrax infection. During recent years pyocyanase has been employed locally for the removal of diphtheria bacilli from the throats of convales- cent cases. Broth-culture filtrates evaporated to one-tenth their volume in vacuo are used for this purpose. Pyocyanase is exceedingly thermostable, resisting boiling for several hours, and is probably not identical with any of the other toxins or peptonizing ferments produced by B. pyocyaneus. The toxins proper of B. pyocyaneus have been the subject of much investigation, chiefly by Wassermann.1 Wassermann found that filtrates of old cultures were far more poisonous for guinea-pigs than extracts made of dead bacteria. He concludes from this and other observations that B. pyocyaneus produces both an endotoxin and a soluble secreted toxin. The toxin is comparatively thermostable, resisting 100° C. for a short time. Animals actively immunized with living cultures of B. pyo- cyaneus give rise in their blood serum to bacteriolytic antibodies only. Immunized with filtrates from old cultures, on the other hand, their serum will contain both bacteriolytic and antitoxic substances. The true toxin of B. pyocyaneus never approaches in strength that of diph- theria or of tetanus. Active immunization of animals must be done carefully if it is desired to produce an immune serum, since repeated injections cause great emaciation and general loss of strength. Specific agglutinins have been found in immune sera by Wassermann 2 and others. Eisenberg 3 claims that such agglutinins are active also against some of the fluorescent intestinal bacteria. 1 Wassermann, Zeit. f. Hyg., xxii, 1896. 2 Wassermann, Zeit. f. Hyg., 1902. 3 Eisenberg, Cent. f. Bakt., 1903. BACILLUS PYOCYANEUS 571 Bulloch and Hunter l have recently been able to show that old broth cultures of B. pyocyaneus contain a substance capable of hemolyzing the red blood corpuscles of dogs, rabbits, and sheep. This " pyocyanolysin " seems intimately attached to the bacterial body. Prolonged heating of cultures does not destroy it. Heating of hemolytic filtrates, however, destroys it in a short time. The filtration of young cultures yields very little pyocyanolysin in the filtrate. In old cultures, however, a considerable amount passes into the filtrate. Whether or not the hemolytic power is due to a specific bacterial product or is dependent upon changes in the culture fluid, such as alkalinization, etc., can not yet be regarded as certain. Gheorghiewski 2 claims to have found a leucocyte-destroying ferment in pyocyaneus cultures. 1 Bulloch und Hunter, Cent. f. Bakt., xxviii, 1900. 2 Gheorghiewski, Ann. de Tinst. Pasteur, xiii, 1899. CHAPTER XLII ASIATIC CHOLERA AND THE CHOLERA ORGANISM (Spirillum cholera asiaticce, Comma Bacillus) The organism of Asiatic cholera was unknown until 1883. In this year, Koch,1 at the head of a commission established by the German government to study the disease in Egypt and India, discovered the "comma bacillus " in the defecations of patients, and satisfactorily de- termined its etiological significance. Koch's investigations were carried out on a large number of cases and many investigations have since then corroborated his results. The numerous morphologically similar spirilla which were later found in normal individuals and in connection with other conditions, have been shown by accurate bacteriological methods to be closely related, but not identical. Apart from the evidence of the constant association of the cholera vibrio with the disease, the etiological relationship has been clearly demonstrated by several accurately recorded accidental infections oc- curring in bacteriological workers, and by the famous experiment of Pettenkofer and Emmerich, who purposely drank water containing cholera bacilli. Both observers became seriously ill with typical clini- cal symptoms of cholera, and one of them narrowly escaped death. Morphology and Staining. — The vibrio or spirillum of cholera is a small curved rod, varying from one to two micra in length. The degree of curvature may vary from the slightly bent, comma-like form to a more or less distinct spiral with one or two turns. The spirals do not lie in the same plane, being arranged in corkscrew fashion in three dimensions. The spirillum is actively motile and owes its motility to a single polar flagellum, best demonstrated by Van Ermengem's flagella stain. Spores are not found. In young cultures the comma shapes predominate, in older growths the longer forms are more nu- merous. Strains which have been cultivated artificially for prolonged 1 Koch, Dent. med. Woch., 1883 and 1884. 572 ASIATIC CHOLERA AND THE CHOLERA ORGANISM 573 periods without passage through the animal body have a tendency to lose the curve, assuming a more bacillus-like appearance. The spirilla are stained with all the usual aqueous anilin dyes. The}r are decolor- ized by Gram's method. In histological section they are less easily stained, but may be demonstrated by staining with alkaline methylene blue. Cultivation. — The cholera spirillum grows easily upon all the usual culture media, thriving upon meat-extract as well as upon meat-infusion Fig. 126. — Cholera Spirillum. (After Frankel and Pfeiffer.) media. Moderate alkalinity of the media is preferable, though slight acidity does not prevent growth. In gelatin plates growth appears at room temperature within twenty- four hours as small, strongly refracting yellowish-gray, pin-head colonies. As growth increases the gelatin is fluidified. Under magnification these colonies appear coarsely granular with margins irregular because of the liquefaction. Liquefaction, too, causes a rapid development in such colonies of separate concentric zones of varying refractive power. Old strains, artificially cultivated for long periods, lose much of their liquefying power. In gelatin stab cultures fluidification begins at the surface, rapidly giving rise to the familiar funnel-shaped excavation. Upon agar plates, within eighteen to twenty-four hours, grayish, opalescent colonies appear, which are easily differentiated by their 574 PATHOGENIC MICROORGANISMS transparency from the other bacterfa apt to appear in feces. Agar plates, therefore, are important in the isolation of these organisms. Coagulated blood serum is fluidified by the cholera vibrio. On potato, growth is profuse and appears as a brownish coarse layer. In milk, growth is rapid and without coagulation. In broth, general clouding and the formation of a pellicle result. The rapidity and luxuri- ance of growth of the cholera spirillum upon alkaline pepton solutions render such solutions peculiarly useful as enriching media in isolating this microorganism from the stools of patients. In pepton solution, Fig. 127. — Colonies of Spirillum choler.e on Gelatin Plate, Thirty Hours Old. (After Frankel and Pfeiffer.) too, the cholera spirillum gives rise to abundant indol, demonstrated in the so-called " cholera-red " reaction. This reaction has a distinct diagnostic value, but is by no means specific.1 In the case of the cholera vibrio the mere addition of strong sulphuric acid suffices to bring out the color reaction. This is due to the fact that, unlike some other indol- producing bacteria, the cholera organism is able to reduce the nitrates present in the medium to nitrites, thus itself furnishing the nitrite necessary for the color reaction. The medium which is most suitable for this test is that proposed by Dunham,2 consisting of a solution of one per cent of pure pepton and five-tenths per cent NaCl in water. 1 See indol reaction, p. 167. 2 Dunham, Zeit. f. Hyg., ii, 1887. ASIATIC CHOLERA AND THE CHOLERA ORGANISM 575 Isolation. — Isolation of the cholera vibrio from the feces, while easy in many cases, is occasionally attended with some difficulty owing to the large number of other bacteria present. The most satisfactory method of procedure is to inoculate a set of gelatin plates, another of agar plates, and a number of Dunham's pepton- broth tubes, with small quantities of the suspicious material. When the spirilla are numerous they can frequently be fished directly from sus- picious colonies in the plates and isolated for further identification. When less numerous, they can usually be found in relatively increased numbers after eight or ten hours at 37.5° C. in the topmost layers of the Dunham broth, which is an almost selectively favorable medium for these organisms. They collect at the surface where free oxygen is readily obtained. From the pepton broth, plate dilutions can then be 12 3 Fig. 128. — Cholera Spirillum. Stab Cultures in Gelatin: 1. Two days old. 2. Three days old. 3. Six days old. (After Frankel and Pfeiffer.) prepared and colonies fished.1 Once isolated, the spirilla are identified by their morphology, by the appearance of their colonies, by their manner of growth upon gelatin stabs, by the cholera-red reaction, and, finally, by agglutinative and bacteriolytic tests in immune sera. Owing to the existence of other spirilla morphologically and cultu- rally similar, the serum reactions are the only absolutely positive dif- ferential criteria. For isolation of the bacteria from water, it is, of course, necessary to use comparatively large quantities. Flugge 2 and Bitter advise the distribution of about a liter of water in ten or twelve Erlenmeyer flasks. To each of these they add 10 c.c. of sterile pepton-salt solution (pepton ten per cent, NaCl five per cent) . After eighteen hours at 37.5° C. the 1 Abel und Claussen, Cent. f. Bakt., xvii, 1895, 2 Flugge, Zeit. f. Hyg., xiv, 1893. 576 PATHOGENIC MICROORGANISMS surface growths in these flasks are examined both microscopically and culturally as before. Biological Considerations. — The cholera spirillum is aerobic and facultatively anaerobic. It does not form spores. The optimum tem- perature for its growth is about 37.5° C. It grows easily, however, at a temperature of 22° C. and does not cease to grow at temperatures as high as 40°. Frozen in ice, these bacteria may live for about three or four days. Boiling destroys them immediately. A temperature of 60° C. kills them in an hour. In impure water, in moist linen, and in food stuffs, they may live for many days. Associated with sapro- phytes in feces and other putrefying material, and wherever active acid formation is taking place, they are destroyed within several days. Complete drying kills them in a short time. The common disin- fectants destroy them in weak solutions and after short exposures (carbolic acid, five-tenths per cent in one-half hour; bichlorid of mercury, 1 : 100,000 in ten minutes; mineral acids, 1 : 5,000 or 10,000 in a few minutes).1 Pathogenicity. — Cholera is essentially a disease of man. Endemic in India and other Eastern countries, it has from time to time epidemically invaded large territories of Europe and Asia, not infrequently assuming pandemic proportions and sweeping over almost the entire earth.2 Five separate cholera epidemics of appalling magnitude occurred during the nineteenth century alone; several of these, spreading from India to Asia Minor, Egypt, Russia, and the countries of Central Europe, reached even to North and South America. The last great epidemic began about 1883, traveled gradually westward, and in 1892 reached Germany where it appeared with especial virulence in Hamburg, and thence, fol- lowing the highways of ocean commerce, entered America and Africa. During this epidemic in Russia alone 800,000 people fell victims to the disease. In man the disease is contracted by ingestion of cholera organisms with water, food, or any contaminated material. The disease is essen- tially an intestinal one. The bacteria, very sensitive to an acid reaction, may often, if in small numbers, be checked by the normal gastric secre- tions. Having once passed into the intestine, however, they proliferate rapidly, often completely outgrowing the normal intestinal flora. Fatal cases, at autopsy, show extreme congestion of the intestinal walls. 1 Forster, Hyg. Rundschau, 1893. 2 Hirsch, " Handb. d. histor.-geoj •geogr. Path.," 1881. ASIATIC CHOLERA AND THE CHOLERA ORGANISM 577 Occasionally ecchymosis and localized necrosis of the mucosa may be present and swelling of the solitary lymph-follicles and Peyer's patches. Microscopically the cholera spirilla may be seen to have penetrated the mucosa and to lie within its deepest layers close to the submucosa. The most marked changes usually take place in the lower half of the small intestine. The intestines are filled with the characteristically fluid, slightly bloody, or "rice-water" stools, from which often pure cultures of the cholera vibrio can be grown. The microorganisms can be cultivated only from the intestines and their contents, and the parenchymatous degenera- tions taking place in other organs must be interpreted as being purely of toxic origin. In animals, cholera never appears as a spontaneous disease. Nikati and Rietsch 1 have succeeded in producing a fatal disease in guinea-pigs by opening the peritoneum and injecting cholera spirilla directly into the duodenum. Koch 2 succeeded in producing a fatal cholera-like disease in animals by introducing infected water into the stomach through a catheter after neutralization of the gastric juice with sodium carbonate. At the same time, he administered opium to prevent active peristalsis. A method of infection more closely analogous to the infec- tion in man was followed by Metchnikoff,3 who successfully produced fatal disease in young suckling rabbits by contaminating the maternal teat. Subcutaneous inoculation of moderate quantities of cholera spirilla into rabbits and guinea-pigs rarely produces more than a temporary illness. Intraperitoneal inoculation, if in proper quantities, generally leads to death. It will be remembered that when working with intra- peritoneal cholera inoculations the phenomenon of bacteriolysis was discovered by Pfeiffer.4 Different strains of cholera spirilla vary greatly in their virulence. The virulence of most of them, however, can be enhanced by repeated passages through animals. Most of our domestic animals enjoy consid- erable resistance against cholera infection, though under experimental conditions successful inoculations upon dogs, cats, and mice have been reported. Doves are entirely insusceptible.5 1 Nikati und Rietsch, Deut. med. Woch., 1884. 2 Koch, Deut. med. Woch., 1885. 3 Metchnikoff, Ann. d. l'inst. Pasteur, 1894 and 1896. 4 Pfeiffer, loc. cit. 5 Pfeiffer und Nocht, Zeit. f. Hyg., vii, 1889. 37 578 PATHOGENIC MICROORGANISMS Hygienic Considerations. — The cholera spirillum leaves the body of the infected subject with the defecations only. Infection takes place, so far as we know, only by way of the mouth. From these two facts it follows that the chief source of danger for a community lies in infection of its water supply. As a matter of fact the bacteria have been fre- quently found in the wells, lakes, rivers, and harbors of afflicted terri- tories and in several cases it has been possible to define the limits of an epidemic almost precisely by the distribution of the contaminated water supply. A classic example of this is that of the Hamburg epi- demic, during which Altona, a town as close to Hamburg as Brooklyn is to New York, with unrestricted interurban traffic but with separate water supply, was almost spared, while Hamburg itself was undergoing one of the most virulent epidemics of its histoiy. It has been statistically noted, moreover, chiefly by Koch, that cholera in its spread not infre- quently follows the water courses. Apart from infection through the water supply, cholera may be transmitted directly or indirectly by con- tact with contaminated linen, bedclothes, etc., the organism being con- veyed to the mouth by the fingers, or by infected food. Epidemics due to this mode of infection alone, however, are apt to be more narrowly localized and more sporadic in their manifestations. It is probable that this mode of infection is of great importance in countries where the disease is endemic, but its significance in producing epidemics is limited owing to the fortunately low resistance of the spirillum to desiccation. The sudden appearance of cholera in a place far distant from the seat of a prevalent epidemic may be explained by the occasional presence of cholera spirilla in the dejecta of convalescents as late as two or three weeks after ap- parent recovery from the disease and consequent release from quaran- tine. Cholera Toxin. — The absence of the cholera spirilla from the in- ternal organs of fatal cases, in spite of the severe general symptoms of the disease, points distinctly to the existence of a strong poison pro- duced in the intestine by the microorganisms and absorbed by the patient. It was in this sense, indeed, that Koch first interpreted the clinical picture of cholera. Numerous investigations into the nature of these toxins have been made, the earlier ones defective in that definite identification of the cultures used for experimentation was not carried out. Pfeiffer,1 in 1892, was able to show that filtrates of young bouillon 1 Pfeiffer, Zeit. f. Hyg., xi, 1892. ASIATIC CHOLERA AND THE CHOLERA ORGANISM 579 cultures of cholera spirilla were but slightly toxic, whereas the dead bodies of carefully killed agar cultures were fatal to guinea-pigs even in small quantities. In consequence, he regarded the cholera poison as consisting chiefly of an endotoxin.1 The opinion as to the endotoxic nature of the cholera poison is not, however, shared by all workers. Metchnikoff, Roux, and Salimbeni,2 in 1896, succeeded in producing death in guinea-pigs by introduction into their peritoneal cavities of cholera cultures inclosed in celloidin sacs. Brau and Denier,3 and, more recently, Kraus,4 claim that they have succeeded not only in demonstrating a soluble toxin in alkaline broth cultures of cholera spirilla, but in producing true antitoxins by immunization with such cultures. It appears, therefore, that the poisonous action of the cholera organisms may depend both upon the formation of true secretory toxins and upon endotoxins. Which of these is paramount in the produc- tion of the disease can not be at present definitely stated. In favor of the great importance of the endotoxic elements is the failure, thus far, to obtain successful therapeutic results with supposedly antitoxic sera. Cholera Immunization. — One attack of cholera confers protection against subsequent infection. Active immunization of animals may be accomplished by inoculation of dead cultures, or of small doses of living bacteria. In the serum of immunized animals specific bacterio- lytic and agglutinating substances are found. The discovery of bacte- riolytic immune bodies, in fact, was made by means of cholera spirilla. Both the bacteriolysins and the agglutinins, because of their specificity, are of great importance in making a bacteriological diagnosis of true cholera organisms. Protective inoculation of man has been variously attempted by Ferran5 and others. Experiments on a large scale were done, more re- cently, by Haffkine,6 who succeeded in producing an apparently dis- tinct prophylactic immunization by the subcutaneous inoculation of dead cholera cultures. Similar immunization with bacterial filtrates has been attempted by Bertarelli.7 1 Pfeiffer und Wassermann, Zeit. f. Hyg., xiv, 1893. 2 Metchnikoff, Roux et Salimbeni, Ann. de l'inst. Pasteur, 1896. 3 Brau et Denier, Comptes rend, de l'acad. des sci., 1906. * R. Kraus, Cent. f. Bakt., 1906. 5 Ferran, Comptes rend, de l'acad. des sciences, 1885. 6 Haffkine, Bull, med., 1892. i Bertarelli, Deut. med. Woch., 33, 1904. 580 PATHOGENIC MICROORGANISMS CHOLERA-LIKE SPIRILLA The biological group of the vibriones, to which the cholera spirillum belongs, is a large one, numbering probably over a hundred separate species. Most of these are of bacteriological importance chiefly because of the difficulties which they add to the task of differentiation, for while some of them simply bear a morphological resemblance to the true cholera vibrio, others can be distinguished only by their serum reac- tions and pathogenicity for various animals. Additional difficulty, too, is contributed by the fact that within the group of true cholera organisms occasional variations in agglutinability and bacteriolytic reactions may exist. Certain strains, too, the six El Tor cultures isolated by Gottschlich, while in every respect similar to true cholera spirilla, are considered as a separate sub-species by Kraus,1 because of their ability to produce hemolytic substances, a function lacking in other cholera strains. Spirillum Metchnikovi. — This spirillum was discovered by Gamaleia 2 in the feces and blood of domestic fowl, in which it had caused an in- testinal disease. Morphologically and in staining reactions it is identical with Spirillum cholera? asiaticse. It possesses a single polar flagellum, and is actively motile. Culturally it is identical with Vibrio cholerae except for slightly more luxuriant growth and more rapid fluidification of gelatin. It gives the cholera-red reaction in pepton media. It is differentiated from the cholera vibrio by its power to produce a rapidly fatal septicemia in pigeons after subcutaneous inoculation of minute quantities.3 It is much more pathogenic for guinea-pigs than the cholera vibrio. It is not subject to lysis or agglutinated by cholera immune sera. Spirillum Massaua. — This organism was isolated at Massaua by Pasquale 4 in 1891 from the feces of a clinically doubtful case of cholera. Culturally and morphologically it is much like the true cholera vibrio, but in pathogenicity is closer to Spirillum Metchnikovi, in that small quantities produce septicemia in birds. It possesses four flagella. It does not give a specific serum reaction with cholera immune serum. 1 Kraus, Kraus und Levaditi, "Handbuch," vol. i, p. 186. 2 Gamaleia, Ann. de l'inst. Pasteur, 1883. 3 Pfeiffer und Nocht, Zeit. f. Hyg., vii, 1889. 4 Pasquale, Giorn. med. de r. eserc. ed. R. Marina, Roma, 1891 ASIATIC CHOLERA AND THE CHOLERA ORGANISM 581 . Spirillum of Finkler-Prior.1 — Isolated by Finkler and Prior from the feces of a case of cholera nostras. Morphologically it is like the true cholera spirillum, though slightly larger and less uniformly curved. Culturally it is much like the cholera vibrio, but grows more rapidly and thickly upon the usual media. It does not give the cholera-red reaction, nor does it give specific serum reactions with cholera im- mune serum. Spirillum Deneke.2 — A vibrio isolated by Deneke from butter. Much like that of Finkler-Prior. It does not give the cholera-red reaction. . . 1 Finkler und Prior, Erganz. Hefte, Cent. f. allg. ges. Phys., 1884. 2 Deneke, Deut. med. Woch., iii, 1885. CHAPTER XLIII DISEASES CAUSED BY SPIROCHETES The microorganisms known as spirochetes are slender, undulating, corkscrew-like threads which show definite variations both structurally and culturally from the bacteria as a class. Most important among them are the spirochete of relapsing fever, Spirochete pallida of syphilis, the spirillum of Vincent, Spirochete refringens, Spirillum gallinarum, a microorganism whicu causes disease in chickens, Spirochete anserina, which causes a similar condition in geese, and several species which have been found as parasites, both in animals and in man, without having definite etiological connection with disease. The classification of these various species in one group is rather more a matter of convenience than one of scientific accuracy, since our knowl- edge of them is not far advanced, and our inability to cultivate almost all of them has not permitted their detailed biological study. Formerly many of these organisms were regarded as bacteria belonging to the gen- eral group of the spirilla. Recently Schaudinn,1 the discoverer of the syphilis spirochete, has claimed, upon the basis of a careful morphological study, that many of these forms are actually protozoa. He based this claim upon the observation that stained preparations often showed undu- lating membranes extending along the long axis of the microorganisms and that definite nuclear structures were demonstrable. This observer also claimed that many of the spiral forms reproduce by cleavage along the longitudinal axis. Other observers have not agreed with this view, Laveran,2 Novy and Knapp,3 and others asserting that their own obser- vations indicate a close relationship of these microorganisms to the true bacteria. Whatever the final conclusion may be, the question is more or less an academic one, in that our ideas as to the exact line of division between the unicellular animals and the unicellular plants is not by any means founded upon a sound basis. In common with the bacteria, most 1 Schaudinn, Arb. a. d. kais. Gesundheitsamt, 1904. 2 Laveran, Comptes rend, de l'acad. des sci., 1902 and 1903. 3 Novy and Knapp, Jour, of Infec. Dis., 3, 1906. 582 KQQ DISEASES CAUSED BY SPIROCHETES 58 of these microorganisms have the power of multiplication by transverse fission. They possess flagella and, in the case of some of them at least, definite immune bodies can be demonstrated in the serum of infected subjects similar to those produced by bacteria during infection. The undulating membranes and the definite differentiation between nucleus and cytoplasm claimed for them by some observers have not been uni- formly confirmed, and their similarity to the trypanosomes has not therefore been established. On the other hand, none of these micro- organisms has so far been successfully cultivated upon artificial media, with the exception of the spirilla which occur in Vincent's angina. For some of the diseases caused by this class of parasites, moreover, trans- mission by an intermediate host, in which the spirilla undergo multipli- cation, has been definitely shown, a fact which corresponds with the conditions observed in many protozoan infections. Upon a careful re- view of these various data, it seems to be fully justified, on the basis of our present knowledge, to group these microorganisms, as Kolle and Hetsch * have done, in a class intermediate between bacteria and protozoa. The terms spirochete and spirillum have been indiscriminately used. In the original classification of Migula the difference between the two groups was based upon the rigidity of the cell body in the case of the spirilla and the sinuous or flexible nature of the cell in the case of the spirochsetae. Although the term spirillum is still colloquially used for some members of this group, merely because of past usage, it would be better to speak of all the microorganisms here grouped together by the term "spirochsetse." SYPHILIS AND SPIROCHETE PALLIDA The peculiar manifestations of syphilis, its mode of transmission, and the fact that its primary lesion was always located at the point of contact with a preceding case, have always stamped it as unques- tionably infectious in nature. Until very recently the microorgan- ism which gives rise to syphilis was unknown 4 Many bacteriologists had attacked the problem and many microorganisms for which defi- nite etiological importance was claimed had been described. Most of these announcements, however, aroused little more than a sensational interest and received no satisfactory confirmation. A bacillus described 1 Kolle und Hetsch, "Die experimentelle Bakt.," Berlin, 1906. 584 PATHOGENIC MICROORGANISMS by Lustgarten * in 1884 seemed, for a time, to have solved the mystery. The Lustgarten bacillus was an acid-fast organism very simi- lar to Bacillus tuberculosis, and found by its discoverer in a large num- ber of syphilitic lesions. The observation, at first, aroused much interest and received some confirmation. Later extensive investigations, how- Fig. 129. — Spirochete pallida. Smear preparation from chancre stained by the india-ink method. ever, failed to uphold the etiological relationship of this bacillus to the disease and practically identified it with the smegma bacillus, so often a saprophyte upon the mucous membranes of the normal genitals. In 1905, Schaudinn,2 a German zoologist, working in collaboration 1 Lustgarten, Wien. med. Woch., xxxiv, 1884. 2 Schaudinn und Hoffmann, Arb. a. d. kais. Gesundheitsamt, 22, 1905. DISEASES CAUSED BY SPIROCHETES 585 with Hoffmann, investigated a number of primary syphilitic indurations and secondarily enlarged lymph nodes, and in both lesions discovered a spirochete similar to, but easily distinguished from, the spirochete already known. He failed to find similar microorganisms in uninfected human beings. The microorganism described by him as "Spirochete pallida" is an extremely delicate undulating filament measuring from four to ten micra in length, with an average of seven micra, and varying in thickness from an immeasurable delicacy to about 0.5 of a micron. It is thus distinctly smaller and more delicate than the spirochete of relasping fever. Ex- amined in fresh preparations it is distinctly motile, its movements con- sisting in a rotation about the long axis, gliding movements backward and forward, and, occasionally, a bending of the whole body. Its con- volutions, as counted by Schaudinn, vary from 3 tb 12 and differ from those observed in many other spirochete by being extremely steep, or, in other words, by forming acute, rather than obtuse, angles. The ends of the microorganism are delicately tapering and come to a point. In his first investigations, Schaudinn was unable to discover flagella and believed that he saw a marginal undulating membrane similar to that noticed in the trypanosomes. Later observations by this observer, as well as by others, revealed a delicate flagellum at each end, but left the existence of an undulating membrane in doubt. Uncertain, in his later investigations, whether the microorganisms described by him could scientifically be classified with the spirochete proper, Schaudinn sug- gested the name of "Treponema pallidum." In the same preparations in which Spirochete pallida was first seen, other spirochete were present, which were easily distinguished from the former by their coarser contours, their flatter and fewer undula- tions, their more highly refractile cell bodies, and, in stained prepara- tions, their deeper color. These microorganisms were not found regularly, and were interpreted merely as fortuitous and unimportant companions. To them Schaudinn gave the name of "Spirochete re- fringens" The epoch-making discovery of Schaudinn and Hoffmann was soon confirmed by many observers, and the etiological relationship of Spiro- chete pallida to syphilis may now be regarded as an accepted fact. Although our inability to cultivate the microorganism has made it impossible to- carry out Koch's postulates, nevertheless indirect evi- dence of such a convincing nature has accumulated that no reasonable doubt as to its causative importance can be retained. The spirochete 586 PATHOGENIC MICROORGANISMS have been found constantly present in the primary and secondary lesions of all carefully investigated cases, and, so far, have invariably been absent in subjects not afflicted with syphilis. Schaudinn himself, not long after his original communication, was able to report seventy cases of primary and secondary syphilis in which these microorganisms were found. Spitzer * found them constantly present in a large number of similar cases. Sobernheim and Tomas- czewski 2 found the spirochetes in fifty cases of primary and secondary syphilis, but failed to find them in eight tertiary cases. Mulzer,3 who found the microorganisms invariably in twenty cases of clinical syphilis, failed to find them in fifty-six carefully investigated non-syphilitic sub- jects. The voluminous confirmatory literature which has accumulated upon the subject can not here be reviewed. The presence of these spirochete in the blood at certain stages of the disease has been demon- strated by Bandi and Simonelli 4 who found them in the blood taken from the roseola spots, and by Levaditi and Petresco 5 who found them in the fluid of blisters produced upon the skin. In tertiary lesions the spirochete have been found less regularly than in the primary and secondary lesions, but positive evidence of their presence has been brought by Tomasczewski,6 Ewing,7 and others who succeeded in demonstrating them in gummata. In congenital syphilis, many observers have found Spirochete pallida in the lungs, liver, spleen, pancreas, and kidneys, and, in isolated cases, in the heart muscle. The organisms were always present in large numbers and practically in pure culture. These results more than any others seem to furnish positive proof of the etiological relationship be- tween the spirochete and the disease. Demonstration of Spirochete pallida. — In the living state the spirochetes have been observed in the hanging drop or under a cover- slip rimmed with vaseline. It is extremely important, in preparing such specimens from primary lesions or from lymph glands, to obtain the material from the deeper tissues, and thus as uncontaminated as possible by the secondary infecting agents present upon the surface 1 Spitzer, Wien. klin. Woch., 1905. 2 Sobernheim und Tomasczewski, Munch, med. Woch., 1905. s Mulzer, Berl. klin. Woch., 1905, and Archiv f. Dermat. u. Syph., 79, 1906. * Bandi und Simonelli, Cent, f . Bakt., 40, 1905. 5 Levaditi and Petresco, Presse med., 1905. 6 Tomasczewski, Munch, med. Woch., 1906. 7 Ewing, Proc. N. Y. Path. Soc, N. S., 5, 1905. DISEASES CAUSED BY SPIROCHETES 587 of an ulcer, and also as free from blood as possible. An ordinary micro- scope and condenser may be used, provided that the light is cut down considerably by means of the iris diaphragm. This method is, how- ever, difficult and uncertain. It is better to employ a special device, known as a " condenser for dark-field illumination " (Dunkel-Kammer- Beleuchtung) . This apparatus is screwed into the place of the Abbe condenser. The preparation is made upon a slide and covered with a cover-slip as usual. A drop of oil is then placed upon the upper sur- face of the dark chamber and the slide laid upon it so that an even layer of oil, without air-bubbles, intervenes between the top of the dark chamber and the bottom of the slide. The preparation is then best examined with a high-power dry lens. An arc light furnishes the most favorable illumination. In such preparations the highly refractive cell- bodies stand out against the black background, and the motility of the organisms may be observed.1 Examination in Smears. — The Spirochete pallida can not be stained with the weaker anilin dyes, and even more powerful dyes, such as carbol-fuchsin and gentian- violet, give but a pale and unsatisfactory preparation. The staining method most commonly used is the one originally recommended by Schaudinn and Hoffmann. This depends upon the use of Giemsa's azur-eosin stain employed in various modi- fications. The most satisfactory method of applying this solution is as follows: Make smears upon slides or cover-slips, if possible from the depth of the lesions, as free as possible from blood. Fix in methyl alcohol for ten to twenty minutes and dry. Cover the preparation with a solution freshly prepared as follows : Distilled water 10 c.c. Potassium carbonate 1 : 1,000 5-10 gtt. Add to this: Giemsa's solution (Fur Romanowski Fdrbung) 10-12 gtt. This staining fluid is left on for one to four hours, preferably in a moist chamber. Wash in running water. Blot. By this method Spirochete pallida is stained characteristically with a violet or reddish tinge. 1For a critical summary of the various methods of dark-field illumination, the reader is referred to an article by Siedentopf, Zeit. f. wiss. Mikrosc, xxv., 1908. 588 PATHOGENIC MICROORGANISMS A rapid and convenient method for staining such smears consists in the use of azur I and eosin in aqueous solutions as recommended by Wood (see section on Staining, page 109). The smears are fixed in methyl alcohol as before and are then flooded with the azur I solution. The eosin solution is then dropped on the preparation until an iridescent pellicle begins to form. Satisfactory preparations may be obtained by this method after ten or fifteen minutes of staining. Goldhorn 1 has prepared a stain which gives excellent results and is extremely rapid. He describes the preparation of his staining fluid as follows : One gram of lithium carbonate is dissolved in 200 c.c. of water. To this are added 2 grams of methylene-blue and the mixture is care- fully heated, filtered, and divided into two equal parts. To one of these parts is added 5 per cent acetic acid until acid to litmus. The two parts are then mixed, and a weak solution of eosin is added until a pale blue color is obtained. The fluid is then allowed to stand for a day and the precipitate which is formed is filtered off and al- lowed to dry without heat. One gram of this precipitate is dissolved in 100 c.c. of methyl alcohol. This stain is applied for five minutes or longer after methyl-alcohol fixation. Excellent results are usually obtained with this stain, but variations due to the difficulty of manu- facturing it make it less reliable than the two methods previously mentioned. Recently a rapid and extremely simple and reliable method for the demonstration of Spirochete pallida in smears, by the use of India ink, has been described. Smears are prepared in the following way: A drop of the fluid squeezed out of the syphilitic lesion, as free as possible from blood cells, is mixed, on a slide, with a drop of India ink (best variety is " Chin chin " Giinther- Wagner Liquid Pearl ink) , and the mixture smeared with the edge of another slide as in making blood smears. When the smear dries, which takes about a minute, it may be immediately examined with an oil-immersion lens. The organisms are seen unstained on a black back- ground. (See Fig. 129, p. 584.) Demonstration of Spirochetes in Tissues. — Ordinary his- tological staining methods do not reveal the spirochetes in tissue sections. It is customary, therefore, to employ some modification of Cajal's silver impregnation. The technique most commonly employed » Goldhorn, Proc. N. Y. Path. Soc, N. S„ 5, 1905. DISEASES CAUSED BY SPIROCHETES 589 is that known as Levaditi7 s method,1 which is carried out as fol- lows: The fresh tissue is cut into small pieces which should not be thicker than 2 to 4 millimeters. Fix in ten-per-cent formalin (four per cent formaldehyde) for twenty-four hours. Wash in water. Dehydrate in 96-per-cent alcohol twenty-four hours. Wash in water. Place in a 3-per-cent silver-nitrate solution at incubator temperature (37.5° C.) and in the dark for three to five days. Wash in water for a short time. Place in the following solution (freshly prepared) : Pyrogallic acid ., 2-4 grams. Formalin 5 c.c. Distilled water 100 " Leave in this for twenty-four to forty-eight hours at room tem- perature. Wash in water. Dehydrate in graded alcohols. Embed in paraffin and cut thin sections. The sections may be examined without further staining, or, if de- sired, may be weakly counterstained with Giemsa's solution or hema- toxylin. A modification of this method which has been much recommended is that of Levaditi and Manouelian.2 The directions given by these authors are as follows: Fix in formalin as in previous method. Dehydrate in 96-per-cent alcohol twelve to twenty-four hours. Wash in distilled water. Place in a 1-per-cent silver-nitrate solution to which 10 per cent of pyridin has been added just before use. Leave in this solution for two to three hours at room temperature and from four to six hours at 50° O. approximately. Wash rapidly in 10-per-cent pyridin. Place in a solution containing 4 per cent of pyrogallic acid to which 10 per cent of C. P. acetone, and 15 per cent (per volume) of pyridin 1 Levaditi, Comptes rend, de la soc. de biol., 59, 1905. 2 Levaditi and Manouelian, Comptes rend, de la soc. de biol., 60, 1906. 590 PATHOGENIC MICROORGANISMS have been added just before use. Leave in this solution two to three hours. Wash in water, dehydrate in graded alcohols, and embed in paraffin by the usual technique. Examined after treatment by either of these methods, the spiro- chetal appear as black, untransparent bodies lying chiefly extracellu- larly. They are characteristically massed about the blood-vessels of the V *■ mA Fig. 130. — Spirochete pallida. Spleen, congenital syphilis. (Levaditi method.) organs and only exceptionally seem to penetrate into the interior of the parenchyma cells. Attempts at cultivating Spirochete pallida have not so far met with success. Recently Schereschewsky x has reported that he has succeeded in obtaining multiplication of the organisms on artificial media as follows: Sterile horse serum in centrifuge tubes was coagu- lated at 60° C. until it assumed a jelly-like consistency. It was then placed in the incubator at 37.5° C. for three days before being used. The cultures were planted by snipping off a small piece of tissue from a syphilitic lesion, dropping it into such a tube, and causing it to sink to the bottom by means of centrifugalization. The tube was then tightly 1 Schereschewsky, Deut. med. Woch., N. S., xix and xxix, 1909. DISEASES CAUSED BY SPIROCHETES 591 stoppered with a cork. In such anaerobic serum cultures Schereschew- sky claims to have grown the organisms for several generations, though not in pure culture. Animal Pathogenicity. — Until very recently, all experimental inocu- lation of animals was unsuccessful. During the year 1903 Metchnikoff Fig. 131. — Spirochete pallida. Liver, congenital syphilis. (Levaditi method.) and Roux 1 finally succeeded in transmitting the disease to monkeys. The monkey first used by these observers was a female chimpanzee. At the point of inoculation, the clitoris, there appeared, twenty-six days after inoculation, a typical indurated chancre, which was soon followed by swelling of the inguinal glands. Fifty-six days after the inoculation 1 Metchnikoff and Roux, Ann. de l'inst. Pasteur, 1903, 1904, and 1905. 592 PATHOGENIC MICROORGANISMS there appeared a typical secondary eruption, together with swelling of the spleen and of the lymph nodes. Similar successful experiments were made soon after this by Lassar.1 Soon after the experiments of Metch- nikoff and Roux, successful inoculations upon lower monkeys (maca- cus) were carried out by Nicolle.2 Since that time, it has been found by various observers that almost all species of monkeys are susceptible. Simple subcutaneous injection is not sufficient to produce a lesion. The technique, which has given the most satisfactory results, consists in the cutaneous implantation of small quantities of syphilitic tissue obtained by excision or curetting of primary and secondary lesions. A small pocket is made under the mucous membrane of the genitals or of the eyebrows and the tissue placed in this under aseptic precautions. The inoculation may be made directly from the human being, but can also be successfully carried out from monkey to monkey for many generations. Attempts at transmission from tertiary lesions have so far been unsuccessful. The spirochsetaB can be demonstrated both in the primary lesions of the inoculated animal and in the secondarily enlarged glands. The successful inoculation of rabbits with syphilis has been recently performed by Bertarelli.3 He obtained ulcerative lesions by inoculation upon the cornea and into the anterior chamber of the eye and was able to prove the syphilitic nature of these lesions by finding the spirochsetae within the tissue. In these animals as well as in the lower monkeys, the disease usually remains localized. Immunization in Syphilis. — It is a well-known fact observed by clinicians that one attack of syphilis usually protects the infected in- dividual at least from the development of another chancrous lesion. That this immunity develops quite rapidly was shown by Metchnikoff and Roux, who found that reinfection of a monkey was possible if attempted within two weeks of the first inoculation, but was unsuccess- ful if delayed beyond this period. On the basis of this knowledge as to the actual development of an immunity, Metchnikoff,4 Finger and Landsteiner,5 and others have made extensive attempts to devise some method of active immunization. Working along the line of Pasteur's original attenuation of virus, these observers attempted to attenuate the syphilitic virus by repeated pas- i Lassar, Berl. klin. Woch., xl, 1903. 2 Nicolle, Ann. de l'inst.. Pasteur, 1903. 3 Bertarelli, Cent. f. Bakt., xli, 1906. 4 Metchnikoff, Arch. gen. de med., 1905. 5 Finger und Landsteiner, Sitzungsber. d. Wien. Akad. d. Wiss., 1905. DISEASES CAUSED BY SPIROCHETES 593 sage through monkeys. These experiments were entirely without suc- cess, the last-mentioned observers finding absolutely no attenuation after twelve generations of monkey inoculation. Attempts at passive immunization have been entirely without success. The experiments of Wassermann l and others, made upon the serum of syphilitic patients, have shown apparently that the blood of infected subjects contains an immune body comparable to the anti- bodies found in the serum of animals and man infected with various bacteria. The antibodies were demonstrated by them by a modification of the Bordet-Gengou method of complement fixation, now spoken of as the Wassermann test. The technique and critical review of this test have been discussed in another place. (See page 262.) The fact that the syphiltic virus does not pass through a filter has been demonstrated by Klingmuller and Baermann,2 who inoculated themselves with filtrates from syphilitic material. THE SPIROCH-ffiT-ffi OF RELAPSING FEVER » The microorganisms causing relapsing fever were first observed in 1873, by Obermeier,3 who demonstrated them in the blood of patients suffering from this distinct type of fever. Since his time extensive studies by many other observers have proven beyond question the etiological connection between the disease and the organisms. Morphology and Staining. — The spirochete of Obermeier is a delicate spiral thread measuring from 7 to 9 micra in length (Novy) , and about 1 micron in thickness. While this is its average size, it may, according to some observers, be considerably longer than this, its undulations varying from 4 to 10 or more in number. Compared with the red blood cells among which they are seen, the microorganisms may vary from one-half to 9 or 10 times the diameter of a corpuscle. In fresh prepara- tions of the blood, very active corkscrew-like motility and definite lateral oscillation are observed. In stained preparations no definite cellular structure can be made out, the cell body appearing homogeneous, except in degenerated individuals, in which irregular granulation or beading has been observed. Flagella have been described by various observers. 1 Wassermann, Neisser, Bruck, und Schucht, Zeit. f. Hyg., lv, 1906. 2 Klingmuller und Baermann, Deut. med. Woch., 1904. 3 Obermeier, Cent. f. d. med. Wiss.? 11, 1873. 38 594 PATHOGENIC MICROORGANISMS Novy and Knapp x believe that the organisms possess only one terminal flagellum. Zettnow,2 on the other hand, claims to have demonstrated lateral flagella by special methods of staining. Norris, Pappenheimer, and Flournoy,3 in smears stained by polychrome methods, have described long, filamentous tapering ends which they interpreted as bipolar, / m Fig. 132. — Spirochete of Relapsing Fever. (After Norris, Pappenheimer, and Flournoy.) terminal flagella, never observing more than one at each end. Spores are not found. Cultivation. — Innumerable attempts to induce these microorganisms to multiply upon artificial media have been made. Novy and Knapp succeeded in keeping the microorganisms alive and virulent in the original blood for as long as forty days, and call attention to the fact that the length of time for which they may be kept alive depends to a 1 Novy and Knapp, Jour, of Infec. Dis., 3, 1906. 2 Zettnow, Deut. med. Woch., 32, 1906. 3 Norris, Pappenheimer, and Flournoy, Jour, of Inf. Dis., 3, 1906. DISEASES CAUSED BY SPIROCHETES 595 great extent upon the stage of fever at which the blood is removed from the patient. They do not, however, believe that extensive multiplica- tion, or, in other words, actual cultivation, had taken place in their experiments. Norris, Pappenheimer, and Flournoy, on the other hand, have obtained positive evidence of multiplication of the spirochaetae in fluid media. They obtained their cultures by inoculating a few drops of spirochetal rat blood into 3 to 5 c.c. of citrated human or rat blood. Smears made from these tubes, after preservation for twenty-four hours at room temperature, showed the microorganisms in greater number than in the original infected blood. A similar multiplication could be / Fig. 133. — Spirochete of Relapsing Fever. Citrated normal rat blood. (After Norris, Pappenheimer, and Flournoy.) observed in transfers made from these " first-generation " tubes to other tubes of citrated blood. Attempts at cultivation for a third generation, however, failed. Pathogenicity. — Inoculation with blood containing these spirochaetae produces disease in monkeys, rats, and mice. Attempts to transmit the disease experimentally to dogs, rabbits, and guinea-pigs have so far been unsuccessful. The subcutaneous inoculation of monkeys is followed after from two to four days by a rise of temperature which occurs abruptly as is the case in the disease in man and which may last several days. During this time, the spirochaetae can be found in the blood of the animals just as it is found in that of infected human beings. 596 PATHOGENIC MICROORGANISMS The temperature subsides after a day or more, when it again rapidly returns to normal. As a rule, the paroxysms are not repeated. Occa- sionally, however, two or three attacks may supervene before immunity is established. In rats, an incubation time of from two to five days occurs. At the end of this time the spirochsetse may be found in large numbers in the blood, and the animals show symptoms of a severe Fig. 134. — Spirochete of Relapsing Fever. (From preparation furnished by Dr. G. N. Calkins.) systemic infection. The attack lasts from four to five days, at the end of which time the microorganisms again disappear. Occasionally even in these animals relapses have been observed. Gross pathological changes are not found, with the exception of an enlargement of the spleen. DISEASES CAUSED BY SPIROCHETES 597 In man the disease caused by the spirocharte of Obermeier, commonly known as relapsing fever, is common in India, Africa, and most of the warmer countries. It has, from time to time, been observed epidemically in Europe, especially in Russia, and a few epidemics have occurred in the Southern United States. The disease comes on abruptly, beginning usually with a chill accompanied by a sharp rise of temperature and gen- eralized pains. Together with the rise of temperature, which often ex- ceeds 104°, there are great prostration and occasionally delirium. Early in the disease the spleen becomes palpable and jaundice may appear. The spirochete are easily detected in the blood during the persistence of the fever, which lasts usually from three to ten days. At the end of this time the temperature usually drops as suddenly as it rose, and the general symptoms rapidly disappear. After a free interval of from one to three weeks a relapse may occur, which is usually less severe and of shorter duration than the original attack. Two, three, or even four attacks may occur, but the disease is not very often fatal. When patients do succumb, however, the autopsy findings are not particularly characteristic. Apart from the marked enlargement of the spleen, which histologically shows the changes indicating simple hyperplasia, and a slight enlargement of the liver, no lesions are found. The diagnosis is easily made during the febrile stage by examination of a small quantity of blood under a cover-slip or in the hanging-drop preparation. Several types of relapsing fever have been described. In Africa the disease has long been prevalent in many regions and the investigations of Ross and Milne,1 Koch,2 Dutton and Todd,3 and others have brought to light that many conditions occurring among the natives, formerly regarded as malarial, are caused by a species of spirochete. Whether or not the microorganisms observed in the African disease are exactly identical with the spirochete observed by Obermeier is yet a question about which several opinions are held. Dutton and Todd believe that the same microorganism is responsible for both diseases. Koch, on the other hand, believes that the slightly smaller size of the African spiro- chete and the milder course of the clinical symptoms indicate a defi- nite difference between the two. Animal experiments made with the African organism, furthermore, usually show a much more severe in- fection than do similar inoculations with the European variety. The 1 Ross and Milne, Brit. Med. Jour., 1904. 2 Koch, Deut. med. Woch., xxxi, 1905. 3 Dutton and Todd, Lancet, 1905, and Jour, of Trop. Med., 1905. 598 PATHOGENIC MICROORGANISMS spirochete found in the African disease is usually spoken of at present as "Spirochete Duttoni." Novy and Knapp,1 after extensive studies with microorganisms from various sources, have come to the conclusion that, although closely related, definite species differences exist between the two types mentioned above, and that these again are definitely dis- Fig. 135. — Spirochete of Dutton, African Tick Fever. tion furnished by Dr. G. N. Calkins.) (From prepara- tinguishable from similar organisms described by Turnbull 2 as occurring in a similar disease observed in India. The mode of transmission of this disease is not clear for all types. Button and Todd, however, were able to show satisfactorily that, in the case of the African disease at least, transmission occurs through the intermediation of a species of tick. The conditions under which such intermediation occurs have been carefully studied by Koch.3 The tick (Ornithodorus moubata) infects itself when sucking blood from an infected human being. The spirochete may remain alive and 1 Novy and Knapp, loc. cit. 2 Turnbull, Indian Med. Gaz., 1905. 3 Koch, Berl. med. Woch., 1906. DISEASES CAUSED BY SPIROCHETES 599 demonstrable within the body of the tick for as long as three days. Koch has shown, furthermore, that they may be found also within the eggs laid by an infected female tick. He succeeded in producing experi- mental infection in monkeys by subjecting the animals to the bites of the infected insects. For the European variety of the disease no such intermediate host has as yet been demonstrated. Immunity. — It has long been a well-known fact that recovery from an attack of relapsing fever usually results in a more or less definite immunity. The blood of human beings, monkeys, and rats which have recovered from an attack of this disease show definite and specific bactericidal and agglutinating substances, and Novy and Knapp have demonstrated that the blood serum of such animals may be used to confer passive immunity upon others. VINCENT'S ANGINA The condition known as Vincent's angina consists of an inflamma- tory lesion in the mouth, pharynx, or throat, situated most frequently upon the tonsils. The disease usually begins as an acute stomatitis, pharyngitis, or tonsillitis, which soon leads to the formation of a pseudo- membrane, which, at this stage, has a great deal of resemblance to that caused by the diphtheria bacillus. At later stages of the disease there may be distinct ulceration, the ulcers having a well-defined margin and " punched-out " appearance, so that clinically they have often been erroneously diagnosed as syphilis. Apart from the localized pain, the disease is usually mild, but occasionally moderate fever and systemic disturbances have been observed. Unlike diphtheria and syphilis, this peculiar form of angina usually yields, without difficulty, to local treat- ment. The nature of lesions of this peculiar kind was not clear until Plaut,1 Vincent,2 and others reported uniform bacteriological findings in cases of this description. These observers have been able to demonstrate in smears from the lesions a spindle-shaped or fusiform bacillus, together with which there is usually found a spirillum not unlike the spirillum of relapsing fever. The two microorganisms are almost always found together in this form of disease and were re- 1 Plaut, Deut. med. Woch., xlix, 1894. 2 Vincent, Ann. de l'inst. Pasteur, 1896, and Bull, et mem. de la soc. med. des hop. de P., 1898. 600 PATHOGENIC MICROORGANISMS garded by the first observers as representing two distinct forms dwelling in symbiosis. More recently Tunnicliff,1 on the basis of experimental work, has claimed identity for the two forms, believing that they repre- sent different developmental stages of the same organism. The fusiform bacilli described by Vincent, Plaut, Babes, and others, are from 3 to 10 micra in length, and have a thickness at the center varying from 0.5 to 0.8 micron. From the center they taper gradually - 1 9 *s • s > ■ i Fig. 136. — Smear from the throat of a Case of Vincent's Angina. Giemsa Stain. toward the ends, ending in blunt or sharp points. The length of these bacilli may vary greatly within one and the same smear preparation. They are usually straight, sometimes slightly curved. They do not stain very easily with the weaker anilin dyes, but are readily stained by Loefner's methylene-blue, carbol-fuchsin, or better, by Giemsa's stain. Stained by Gram, they are usually decolorized, though in this respect the writers have found them to vary. Stained preparations show a charac- 1 Tunnicliff, Jour, of Infec. Dis.; 3; 1906. DISEASES CAUSED BY SPIROCHETES 601 teristic inequality in the intensity of the stain, the bacilli being more deeply stained near the end, and showing a banded or striped alternation of stained and unstained areas in the central body. Their staining qualities in this respect are not unlike those of the diphtheria bacillus, and according to Babes * the dark areas are to be interpreted as meta- chromatic granules. The bacilli are not motile. The spirilla found in Vincent's angina are usually somewhat longer than the fusiform bacilli, and are made up of a variable number of un- dulations, shallow and irregular in their curvatures, unlike the more Fig. 137. — Throat Smear, Vincent's Angina. Fusiform bacilli and spirilla. regularly steep waves of Spirochete pallida. They are stained with even more difficulty than are the bacilli and usually appear less distinct in the preparations. The stain, however, is taken without irregu- larity, showing none of the apparent metachromatism observed in the bacilli. By the earlier observers cultivation of these microorganisms was attempted without success. Recently, however, it has been shown that cultivation could be carried out under anaerobic conditions. Tunni- 1 Babes, in Kolle und Wassermann, 1. Erganzungsband , 1907. 602 PATHOGENIC MICROORGANISMS cliff l has cultivated the organisms anaerobically upon slants of ascitic agar at 37.5° C. This observer found that in such cultures, before the fifth day, bacilli only could be found, that after this time, however, spirilla gradually appeared and finally constituted the majority of the organisms in the culture. It appeared to TunniclifT from this study that the spirilla might be developed out of the fusiform microorgan- isms representing the adult form. The microorganisms of Vincent's angina, when occurring in the throat, are rarely present alone, being usually accompanied by other microorganisms, such as staphylococci, streptococci, and not infre- quently diphtheria bacilli. When occurring together with diphtheria, they are said, by some German observers, to aggravate the latter condition considerably. This frequent association with other micro- organisms renders it impossible to decide conclusively that the fusi- form bacilli and spirilla are the primary etiological factors in these inflammations. It has been frequently suggested that they may be present as secondary invaders upon the soil prepared for them by other microorganisms. Animal inoculation with these microorganisms has led to little result. Fusiform Bacilli other than those in Vincent's Angina. — Fusiform bacilli morphologically indistinguishable from those found in the angina of Vincent may frequently be found in smears taken from the gums, from carious teeth, and occasionally among the microorganisms in the pus from old sinuses. Several varieties of these bacilli have been de- scribed in connection with definite pathological conditions. Babes,2 in 1893, observed spindle-shaped bacilli not unlike those described above but somewhat shorter, in histological sections prepared from tissues from the gums of individuals suffering from scurvy. He found similar bacilli in rabbits intravenously inoculated with material from the patients and was able to cultivate the bacilli for several genera- tions. His descriptions, however, of the microorganisms as found in the secondary cultures vary considerably from those of the original findings in the gums of the patients. His results are not convincing. In noma, a gangrenous disease of the gums and cheeks, occurring occasionally in individuals who have been severely run down by acute infectious diseases or great hardship, Weaver and TunniclifT have found spirilla and fusiform bacilli in large numbers. The organisms were pres- ent not only in smears from the surface, but were also found by histo- 1 Tunnicliff, Jour, of Infec. Dis., 3, 1906. 2 Babes, Deut. med. Woch., xliii, 1893. DISEASES CAUSED BY SPIROCHETES 603 logical methods, in large numbers, lying in the tissues beyond the area of necrosis. Here again it is not entirely certain whether these micro- organisms were the primary etiological factors or whether they are to be regarded merely as secondary invaders of a necrotic focus. SPIROCHETE PERTENUIS In a disease known as "Frambcesia tropica/' or popularly "Yaws," occurring in tropical and subtropical countries and much resembling syphilis, Castellani,1 in 1905, was able to demonstrate a species of spirochsete which has a close morphological resemblance to Spirochsete pallida. The microorganism was found in a large percentage of the cases examined both in the cutaneous papules and in ulcerations. Confirm- atory investigations on a larger series of cases were later carried out by Vom dem Borne.2 The microorganism is from 7 to 20 micra in length with numerous undulations and pointed ends. Examined in fresh preparations, it has an active motility similar to that of Spirochsete pallida. In smears it is easily stained by means of the Giemsa method. Both the clinical similarity between yaws and syphilis, as well as the similarity between the microorganisms causing the diseases, has opened the question as to the identity of the two microorganisms. According to most clinical observers, however, yaws, which is a disease characterized chiefly by a generalized papular eruption, is unquestion- ably distinct, clinically, from lues, and experiments of Neisser, Baermann, and Halberstadter,3 as well as of Castellani4 himself, have tended to show that there is a distinct difference between the immunity produced by attacks of the two diseases. The disease is transmissible to monkeys, as is syphilis, but it has been satisfactorily shown that monkeys inocu- lated with syphilitic material, while no longer susceptible to infection with Spirochete pallida, may still be successfully inoculated with Spirochete pertenuis. SPIROCHETE GALLINARUM An acute infectious disease occurring among chickens, chiefly in South America, has been shown by Marchoux and Salimbeni 5 to . be 1 Castellani, Brit. Med. Jour., 1905, and Deut. med. Woch., 1906. 2 Vom dem Borne, Jour. Trop. Med., 10, 1907. 3 Neisser, Baermann, und Halberstadter, Munch, med. Woch., xxviii, 1906. 4 Castellani, Jour, of Hyg., 7, 1907. 5 Marchoux and Salimbeni, Ann. de l'inst. Pasteur, 1903. 604 PATHOGENIC MICROORGANISMS caused by a spirochete which has much morphological similarity to the spirochete of Obermeier. The disease comes on rather suddenly with fever, diarrhea, and great exhaustion, and often ends fatally. The spirochete is easily demon- strated in the circulating blood of the animals by staining blood-smears with Giemsa's stain or with dilute carbol-fuchsin. Fig. 138. — Spirochete gallinarum. (From preparation furnished by Dr. G. N. Calkins. ) Artificial cultivation of the microorganism has not yet been ac- complished. Experimental transmission from animal to animal is easily carried out by the subcutaneous injection of blood. Other birds, such as geese, ducks, and pigeons, are susceptible; mammals have, so far, not been successfully inoculated. According to the investigations of DISEASES CAUSED BY SPIROCHETES 605 Levaditi and Manouelian,1 the spirochete are found not only in the blood but thickly distributed throughout the various organs. Under natural conditions, infection of chickens seems to depend upon a species of tick which acts as an intermediate host and causes infection by its bite. The spirochete, according to Marchoux and Sal- imbeni, may be found in the intestinal canal of the ticks for as long as five months after their infection from a diseased fowl. In the blood of animals which have survived an infection, agglutin- ating substances appear and active immunization of animals may be carried out by the injection of infected blood in which the spirochete have been killed, either by moderate heat or by preservation at room temperature. The serum of immune animals, furthermore, has a pro- tective action upon other birds. It is not impossible that the Spirochete gallinarum may be identical with the Spirochcete anserina previously discovered by Sacharoff.2 This last-named microorganism causes a disease in geese, observed espe- cially in Russia and Northern Africa, which both clinically and in its pathological lesions corresponds closely to the disease above described as occurring in chickens. The spirochete is found during the febrile period of the disease in the circulating blood, is morphologically indis- tinguishable from the spirochsete of chickens, and can not be cultivated artificially. The similarity is further strengthened by the fact that Spirochsete anserina is pathogenic for other birds, but not for animals of other genera. 1 Levaditi and Manouelian, Ann. de l'inst. Pasteur, 1906. 2 Sacharoff, Ann. de l'inst. Pasteur, 1891. CHAPTER XLIV THE HIGHER BACTERIA {Chlamydobacteriacece, Trichomycetes) Standing midway between the true bacteria and the more complex molds or Hyphomycetes, there are a number of pathogenic micro- organisms which offer great difficulties to classification. In the classifi- cation of Migula most of these forms have been placed in a rather heterogeneous group, the Chlamydobacteriacese. By other authors, notably Lachner-Sandoval,1 Berestnew,2 and by Petruschky,3 the close relationship of these forms to the higher hyphomycetes has been em- phasized and they have been grouped as a subdivision of the true molds under the family name of Trichomycetes. Petruschky 4 proposes the following clear schematization, which, even though possibly defective from a purely botanical point of view, is at least serviceable for the purposes of the bacteriologist. Hyphomycetes True molds Trichomycetes Leptothrix Cladothrix Streptothrix Actinomyces Leptothrix is used to designate those forms which appear as simple threads without branching. Cladothrix is a thread-like form in which false branching may be recognized. By false branching is meant an appearance resulting from the fragmentation of threads. The terminal cell breaks away from the main stem, is set at an angle by the elongation of the thread itself, and, 1 Lachner-Sandoval, "Ueber Strahlenpilze." Diss. Strassburg, 1898. 2 Berestnew, Ref. Cent. f. Bakt., xxiv, 1898. 3 Petruschky, in Kolle und Wassermann, "Handbuch," etc. * Petruschky, loc. cit. 606 THE HIGHER BACTERIA 607 as both continue dividing, the simulation of true branching is pro- duced. Streptothrix denotes forms with numerous true branches and spores which usually appear in chains. Actinomyces is of more complicated structure, characterized by the formation of club-shaped ends and the stellate arrangement of its threads. LEPTOTHRIX Members of the leptothrix group have been observed in connection with inflammations of the mouth and pharynx by Frankel,1 Michelson,2 Epstein,3 and others. In many of these cases the organism was identi- fied by morphology chiefly, pure cultures not having been obtained. The disease in none of these cases was accompanied by severe systemic symptoms and it is likely that when found in human beings the organ- isms may be regarded simply as comparatively harmless saprophytes appearing in connection with some other specific inflammation. Cultivation of the Leptothrices is not easy and has been successful only in the hands of Vignal 4 and Arustamoff.5 CLADOTHRIX Owing to much confusion in the differentiation of these forms from the streptothrices, it is not possible to determine whether cases of true cladothrix infection have been observed. It is likely that most cases ascribed to microorganisms of this class have really been due to strep- tothrix infection. The deciding criterion is, of course, the formation of branches and these seem to have been observed in most of the cases described. A closer differentiation, in the future, between true and false branching can alone determine whether or not cases of cladothrix infection proper may occur. STREPTOTHRIX Reports of cases of streptothrix infection of various parts of the body, in both animals and man, are abundant in the literature. The 1 Frankel, Eulenburg's " Realencycl. d. gesam. Heilkunde," 1882. 2 Michelson, Bed. klin. Woch., ix, 1889. 3 Epstein, Prag. med. Woch., 1900. . j * Vignal, AmC3fe=p&?&, viii, 1886. J^'veAv. cLt o\uj$ .^tfjwJ ■ VT QOJdfiJ. 5 Arustamoff, Quoted from Petruschky, loc. cit. ' 608 PATHOGENIC MICROORGANISMS earliest observations were made upon microorganisms isolated from the human conjunctiva. Nocard * in 1888 described a member of this group as the etiological factor in a disease " farcies du bceuf " occurring among cattle in Guadeloupe. Eppinger 2 found streptothrices in the pus of a cerebral abscess. Petruschky,3 Berestneff,4 Flexner,5 Norris and Fig 139. — Cladothrix, Showing False Branching. Larkin,6 and a number of other observers have found these microor- ganisms in cases of pulmonary disease, simulating tuberculosis. Sup- purations of bone and of the skin and the intestinal canal have been reported. The infection, therefore, is not very rare, but the diverse experiences of workers who have attempted to cultivate these micro- 1 Nocard, Ann. de l'inst. Pasteur, ii, 1888. 2 Eppinger, Wien. klin. Woch., 1890. 3 Petruschky, Verhandl. d. Kongr. f. innere Mediz., 1898. a Berestneff, Zeit. f. Hyg., xxix, 1898. s Flexner, Jour. Exp. Med., iii, 1896. e Norris and Larkin, Proc. of N. Y. Path. Soc, March, 1899. THE HIGHER BACTERIA 609 organisms seem to indicate that not all of the incitants described be- longed to one and the same variety, but that probably a number of different types may exist. Morphology. — Morphologically the streptothrices show considerable variation. In material from infectious lesions they have most often appeared as rods and filaments with well-marked branching. Occasion- ally the filaments are long and interwined, and branches have shown bulbous or club-shaped ends. In Norris and Larkin's case, the young cultures in the first generations seem to have consisted chiefly of rod- shaped forms not unlike bacilli of the diphtheria group, showing marked metachromatism when stained with Loeffler's methylene-blue. They are Fig. 140. — Streptothrix, Showing True Branching. easily stained with this dye or with aqueous fuchsin. In tissue sections they may be demonstrated by the Gram-Weigert method. Cultivation. — Direct cultivation upon agar and gelatin plates has occasionally been successful. At the end of four or five days grayish- white, glistening, flat colonies may appear which attain a diameter of several millimeters within two weeks. The colonies later may take on a yellowish hue and begin to liquefy the gelatin. In bouillon flocculent precipitates and surface pellicles of interwined threads may form, with- out clouding of the medium. Norris and Larkin1 found much difficulty in cultivating, but finally succeeded by making smears of the infectious material upon fresh, sterile kidney- tissue of rabbits. The micro- Norris and Larkin, loc. cit. 39 610 PATHOGENIC MICROORGANISMS organisms grew abundantly upon this, but failed to grow on any of the other tissues. After growth of several generations upon this medium, cultures were finally obtained upon agar plates and upon broth. Inoculation of cultures into rabbits and guinea-pigs have given rise to subcutaneous abscesses, bronchopneumonia, and suppuration, accord- ing to the mode of infection. ACTINOMYCES Among the diseases caused by the Trichomycetes or higher bacteria, the most important is actinomycosis. Occurring chiefly in some of the domestic animals, notably in cattle, the disease is observed in man with sufficient frequency to make it of great clinical importance. In cattle the specific microorganism which gives ries to the disease was first observed by Bollinger 1 in 1877. In the following year Israel 2 dis- covered a similar microorganism in human cases. The parasites appear in the pus from discharging lesions as small granular bodies, plainly visible to the naked eye and somewhat resem- bling sulphur granules, of a grayish or of a pale yellow color. In size they measure usually a fraction of a millimeter. Ordinarily they are soft and easily crushed under a cover-slip, but occasionally, especially in old lesions, they may be quite hard, owing to calcification. Microscopically they are most easily recognized in fresh preparations prepared by crushing the granules upon the slide under a cover-slip and examining them without staining. They may be rendered more clearly visible by the addition of a drop or two of 20 per cent potassium hydrate. When the granules are calcareous, the addition of a drop of concentrated acetic acid will facilitate examination. Fresh preparations may be examined after staining with Gram's stain. Observed under the micro- scope, the granules appear as rosette-like masses, the centers of which are quite opaque and dense, appearing to be made up of a closely meshed network of filaments. Around the margins there are found radially arranged striations which in many cases end in characteristically club- shaped bodies. Inside of the central network there are often seen coccoid or spore-like bodies which have been variously interpreted as spores, as degeneration products, and as separate cocci fortuitously found in symbiosis with the actinomyces. Individually considered, 1 Bollinger, Deutsch. Zeit. f. Thiermed., iii, 1877. 2 Israel, Virch. Arch., 74, 1878, and 78, 1879. THE HIGHER BACTERIA 611 the central filaments have approximately the thickness of an anthrax bacillus and are, according to Babes,1 composed of a sheath within which the protoplasm contains numerous and different sized granules. About the periphery of the granules the free ends of the filaments become gradually thickened to form the so-called actinomycosis " clubs." These clubs, according to most observers, must be regarded as hyaline thickenings of the sheaths of the threads and are believed to represent a form of degeneration and not, as some of the earlier observers believed, organs of reproduction. They are homogeneous, and in the smaller and presumably younger granules are extremely fragile and soluble in water. In older lesions, especially in those of cattle, the clubs are more re- sistant and less easily destroyed. They appear only in the parasites taken from active lesions in animals or man, or, as Wright 2 has found, from cultures to which animal serum or whole blood has been added. In cultures from media to which no animal fluids have been added, such as glucose agar or gelatin, no clubs are found. In preparations stained by Gram's method the clubs give up the gentian-violet and take counter-stains, such as eosin. The coccus-like bodies found occasionally lying between the filaments of the central mass, most observers now agree, do not represent any- thing comparable to the spores of the true hyphomycetes. In many cases they are unquestionably contaminating cocci; in others again they may represent the results of degeneration of the threads. In tissue sections, the microorganisms may be demonstrated by Gram's method of staining or by a special method devised by Mallory.3 This is as follows for paraffin sections : 1. Stain in saturated aqueous eosin 10 minutes. 2. Wash in water. 3. Anilin gentian- violet, 5 minutes. 4. Wash with normal salt solution. 5. Gram's iodin solution 1 minute. 6. Wash in water and blot. 7. Cover with anilin oil until section is clear. 8. Xylol, several changes. 9. Mount in balsam. Cultivation. — The isolation of actinomyces from lesions may be easy or difficult according to whether the pus is free from contamination i Babes, Virch. Arch., 105, 1886. 2 J. H. Wright, Jour. Med. Res., viii, 1905. a Mallory, Method No. 1, Mallory and Wright, " Path. Technique," Phila., 1908. 612 PATHOGENIC MICROORGANISMS or whether it contains large numbers of other bacteria. In the latter case it may be almost impossible to obtain cultures. The descriptions of methods of isolation and of cultural characteristics given by various writers have shown considerable differences. The most extensive cultural work has been done by Bostroem,1 Wolff and Israel, and by J. H. Wright. Bostroem has described his cultures as aerobic, but Wolff and Israel 2 and Wright 3 agree in finding that the microorganisms iso- Fig. 141. — Actinomyces Granule Crushed Beneath a Cover-glass. Un- stained. Low power. Shows radial striations. (After Wright and Brown.) lated by them from actinomycotic lesions grow but sparsely under aerobic conditions and favor an environment which is entirely free from oxygen, or at least contains it only in small quantities. The method for isolation recommended by Wright is, briefly, as follows: Pus is obtained, if possible, from a closed lesion and washed in sterile water or broth. The granules are then crushed between two sterile slides and examined for 1 Bostroem, Beitr. z. path. Anat. u. z. allg. Path., ix, 1890. 2 Wolff und Israel, Virch. Arch., 126, 1891. *J. H. Wright, Jour. Med. Res., viii, 1905. THE HIGHER BACTERIA 613 the presence of filaments. If these are present in reasonable abundance, the material is distributed in tubes of glucose agar, which are then allowed to solidify. If these first cultivations show a large number of contaminations, Wright recommends the preservation of other washed granules in test tubes for several weeks, in the hope that contaminating microorganisms may thus be killed by drying before the actinomyces lose their viability. If cultivation is successful colonies will ap- pear, after two to four days at 37.5° C, as minute white specks, which, in Wright's cultures, ap- peared most abundantly within a zone situated 5 to 10 millimeters below the surface of the medium. Above and below this zone they are less numerous, indicating that a small amount of oxygen furnishes the best cultural environment. Upon the surface of agar slants, growth, if it takes place at all, is not luxuriant. In alkaline meat-infusion broth growth takes place in the form of heavy, flocculent masses which appear at the bottom of the tubes. Surface growth and clouding do not take place. Milk and potato have been used as culture media but are not particularly favorable. Pathogenicity. — As stated above, actinomy- cosis occurs spontaneously most frequently among cattle and human beings. It may also occur in sheep, dogs, cats, and horses. Its loca- tions of predilection are the various parts adjacent to the mouth and pharynx. It occurs also, however, in the lungs, in the intestinal canal, and upon the ekin. When occurring in its most frequent location, the lower jaw, the disease presents, at first, a hard nodular swell- ing which later becomes soft because of central necrosis. It often involves the bone, causing a rarefying osteitis. As the swellings break down, sinuses are formed from which the granular pus is discharged. The neighboring lymph nodes show painless, hard swell- ings. Histologically, about the filamentous knobs or granules, there is a formation of epithelioid cells and a small round-cell infiltration. In older cases there may be an encapsulation in connective tissue and a WattsrSBA ijKy^JEV^- Bw '■ .-/' !'->'^ :--\ . ■ . rjL' u • Aiarf * i Fig. 142. — Actino- myces Granule Crushed Beneath a Cover-glass. Un- stained. The prepara- tion shows the margin of the granule and the "clubs." (After Wright and Brown.) 614 PATHOGENIC MICROORGANISMS calcification of the necrotic masses, leading to spontaneous cure. As a rule, this process is extremely chronic. Infection in the lungs or in the intra-abdominal organs is, of course, far more serious. When death occurs acutely, it is often due to secondary infection. The disease is acquired probably by the agency of hay, straw, and grain. Berestnew ! has succeeded in isolating actinomyces from straw and hay which he covered with sterile water in a potato jar and placed in the incubator. After a few days small white specks looking like chalk powder appeared upon the stalks, which, upon further cultivation, he was able to identify as the organism in question. Animal inoculation, carried out extensively both with pus and with pure cultures by several observers, has yielded little result. Progressive - -.- T >s I t ' V ' 9 J>««rf4i t * Fig. 143. — Branching Filaments of Actinomyces. (After Wright and Brown.) actinomycotic lesions were never obtained, although occasionally small knobs containing colonies surrounded by epithelioid cells and connective tissue were observed, showing that the invading microorganisms were able to survive and grow for a short time, but were not sufficiently virulent to give rise to an extensive disease process. Transmission from animal to animal, or from animal to man directly, has not been satis- factorily proven. Whether or not there are various forms of actinomyces must as yet be regarded as an open question. The investigations of Wolff and Israel, however, together with those of W'right, who alone observed thirteen different strains, seem to indicate that most, if not all, of the cases clinically observed are due to one and the same microorganism. 1 Berestnew, Ref. Cent. f. Bakt., 24, 1898. THE HIGHER BACTERIA 615 MYCETOMA (MADURA FOOT) The disease known by this name is not unlike actinomycosis. It is more or less strictly limited to warmer climates and was first recog- nized as a clinical entity, in India, by Carter.1 Clinically it consists of a chronic productive inflammation most frequently attacking the foot, less often the hand, very infrequently other parts of the body. Nodular swellings occur, which break down in their centers, leading to the formation of abscesses, later of sinuses. Often the bones are in- volved and a progressive rarefying osteitis results. From the sinuses a purulent fluid exudes, in which are found characteristic granular bodies. These may be hard, brittle, and black, resembling grains of gunpowder, or may be grayish- white or yellow and soft and grumous. According to the appearance of these granules, two varieties of the disease are dis- tinguished, the "melanoid" and the "ochroid." Many observers believe that the yellow orochroid variety is, in fact, actinomycosis. The black variety, which is certainly a distinct disease, is caused by a member of the hyphomycetes group. The parasite has been carefully studied by Wright,2 from whose description the following points are taken : The small, brittle granules observed under the microscope show a dark, almost opaque center along the edges of which, filaments, or hyphaB, may be seen in a thickly matted mass. By crushing the granules under a cover-slip in a drop of sodium hypochlorite or of strong sodium hydrate, the black amorphous pigment is dissolved and the structural elements of the fungus may be observed. They seem to be composed of a dense meshwork of mycelial threads which are thick and often swollen, and show many branches. Transverse partitions are placed at short distances and the individual filaments may be very long. Spores were not observed by Wright. In a series of over fifty cultiva- tions on artificial media from the original lesion, Wright obtained growth in a large percentage. In broth, he obtained at first a rapid growth of long hyphse which eventually formed a structure which he compares in appearance to a powder-puff. On agar, growth appeared within less than a week and spread over the surface of the medium as a thick meshwork of spreading hyphae 1 Carter on Mycetoma, etc., London, 1874. 2 Wright, Jour, of Exper. Med., 3, 1898. 616 PATHOGENIC MICROORGANISMS of a grayish color. In old cultures black granules appeared among the mycelial meshes. On 'potato, he observed a dense velvety membrane, centrally of a pale brown, white at the periphery. Small brown droplets appeared on the growth in old cultures. Animal inoculation with this microorganism has so far been un- successful. CHAPTER XLV THE YEASTS (Blastomycetes, Saccharomycetes) The yeasts or blastomycetes form a distinctive family among uni- cellular microorganisms, characterized essentially by their method of multiplication by budding. By this, they are sharply separated from the bacteria. Their differentiation from the higher fungi, the hypho- mycetes, however, is less definitely established, since the chief character- istic of this latter class, the formation of hyphae and mycelial threads, has occasionally been described for some of the forms otherwise identified with the yeasts. It is probable that a gradual transition between the two families exists, represented by a number of connecting forms, some- times spoken of as oidia. For the practical purposes of the bacteriolo- gist, the yeast family is sufficiently distinct, both morphologically and biologically, to make a separate classification extremely useful. The yeast cell, as a rule, is oval, but among the wild yeasts, or "torulse," spherical forms are common. In size, great variations occur, but in general the yeasts are much larger than bacteria, measuring usually from 10 to 20 micra in length with a width of about one-half or two-thirds of the long diameter. They possess a well-defined, doubly-contoured cell-membrane, composed chiefly of cellulose, and their body protoplasm, unlike that of the bacteria, shows definite structure. Within a mass of finely granul ar cytoplasm, a number of highly refractive globules and vacuoles may be observed. Some of the globules have been interpreted as fat-droplets. Other granules, revealed by special staining methods, are interpreted as nuclear material. When budding takes place, the mother cell sends out a small, globular evagination of the cell membrane into which maternal proto- plasm flows. This bud gradually enlarges until it has attained approxi- mately the same size as the original cell. Until that time, free inter- communication between the protoplasm of mother and daughter cell exists. Finally, by gradual narrowing of the isthmus connecting the two, the daughter cell becomes complete and free. By some observers 617 618 PATHOGENIC MICROORGANISMS definite karyokinetic changes in the nuclear structures have been de- scribed as accompanying the budding process. This observation, how- ever, has not been generally confirmed. Under conditions of special stress, such as unfavorable environment or lack of nutrition, most yeasts possess the power of forming spores. These, called " ascospores," are formed within the yeast cell itself, each spore forming a separate membrane of its own, but all of them lying well protected within the original cell-membrane. The number of ascospores formed is constant for each species, and rarely exceeds four. The yeasts have been studied most extensively in connection with fermentation and are industrially of great importance in the production t * - « Fig. 144. — Yeast Cells. Young culture unstained. (After Zettnow.) of wine and beer. Although Schwann, as early as 1837, recognized the fact that many fermentations could not occur without the presence of yeast, it was not until considerably later that the study of yeast fermen- tation was put upon a scientific basis. The typical fermentative action consists in the transformation of sugar into ethyl alcohol according to the following formula: C6 Hl2 Ott = 2 C2 H5 OH + 2 C02 The enzyme by which this fermentation is produced is known as "zy- mase," and is, according to Buchner, in most cases, an endo-enzyme which may be procured from the cell by expression in a hydraulic press. In addition to this, however, the various yeasts also produce THE YEASTS 619 other ferments by means of which they may split higher carbohydrates, such as saccharose, maltose, and even starch, and prepare them for action of the zymase. The manner in which this is accomplished, and the by-products which are formed during the process, vary among different species, and it is for this reason that the employment of pure cultures is of such great importance in the wine and beer industries where differences in flavor and other qualities may be directly dependent upon the particular species of yeast employed for the fermentation. It is due to the work chiefly of Pasteur ? and of Hansen 2 that the beer and wine industries have been carried on along exact and scientific lines. As the incitants of disease in man, the yeasts have been much studied since 1894, when Busse 3 reported a case of fatal, generalized yeast in- fection, beginning from a tibial bone abscess. The microorganism which was found in this case he named " Saccharomyces hominis." In morpho- logical and biological characters it appeared to be a typical yeast, grew readily upon most artificial media, and produced active fermentation in sugars. Mycelia were not observed. When inoculated into animals, this yeast proved pathogenic for mice and rats. A peculiarity of Busse's culture, observed since then in the case of many pathogenic yeasts, was the formation of gelatinous capsules, of varying thicknesses, about the individual cells, developing with particular luxuriance in the animal lesions. In 1896, Gilchrist 4 described a peculiar skin disease, which he spoke of as pseudo-lupus vulgaris, in the lesions of which he demonstrated a large number of round, doubly-contoured bodies which, though differing somewhat from Busse's saccharomyces, were unquestionably members of, or closely related to, the family of blastomycetes. In the same year, Curtis,5 in France, isolated a similar form from a myxoma of the leg. Ophuls 6 has described a number of fatal cases occurring in California, which at first were wrongly interpreted as protozoan in origin, but later were determined by him to be caused by a species of blastomycetes. In a case observed by Zinsser 7 simi- lar microorganisms were isolated from an abscess of the back, which 1 Pasteur, " Etudes sur la biere," Paris, 1876. 2 Hansen, " Prac. Studies in Fermentation," London, 1896. 3 Busse, Cent. f. Bakt., I, xvi, 1894, and Virch. Arch., 140, 1895. 4 Gilchrist, Bull. Johns Hopkins Hosp., vii, 1896. 5 Curtis, Ann. de Pinst. Pasteur, 10, 1896. • Ophuls, Jour. Exp. Med., 6, 1901. 7 Zinsser, Proc. N. Y. Path. Soc, vii, 1907. 620 PATHOGENIC MICROORGANISMS in almost all respects corresponded to Gilchrist's cultures. Animal inoculation in rabbits and guinea-pigs proved positive in this case and the organism seemed to show selective action for the lungs and spleen. In the lungs of the animals, especially, lesions were found with surpris- ing regularity even when the inoculation was made intraperitoneally. Cases of blastomycotic infection in man have been reported in large numbers and appear to be less rare than they were formerly believed to be. The clinical course of the disease is by no means uniform. A well- defined clinical picture seems to characterize the cases of blastomycotic dermatitis first described by Gilchrist. The eruption is very chronic and begins usually as a small pimple or papule with moderate induration of the skin. Scabs and pustules then form, which discharge yellowish- white pus. As the lesion slowly spreads, the older areas show a tendency to spontaneous healing. In Gilchrist's 1 case, it took four years for the lesion to spread two inches. When not purely cutaneous, blastomycotic infection takes the form of chronic abscess formation occurring in various parts of the body. In the latter, metastatic lesions in the lungs have been occasionally observed, and in one case cited by Ophuls,2 the lung seemed to have been the primary focus. The fact that blastomycetes have frequently been found in tumor tissue has led several Italian observers 3 to assume an etiological relationship between these microorganisms and malignant growths. Absolutely no satisfactory evidence in favor of such a belief has been advanced, however, and the yeasts in these conditions must be regarded as purely fortuitous findings. In animals, diseases caused by members of the yeast family have been reported by various observers. The most important communica- tion of this kind is by Tokishige,4 who found these microorganisms in a nod alar skin disease occurring among horses in Japan. Sanfelice 5 has isolated similar microorganisms from the lymph glands of a horse which was apparently suffering from a primary carcinoma of the liver. The same author has described a member of this group which he obtained from a cheesy consolidation occurring in the lung of a hog. Demonstration of the organisms offers little difficulty either in fresh preparations of the pus under a cover-slip, or in smears stained with 1 Rixford and Gilchrist, Johns Hopkins Hosp. Rep., i, 1896. 2 Ophuls, loc. cit. s Sanfelice, Cent. f. Bakt., I, xxxi, 1902. * Tokishige, Cent. f. Bakt., I, xix, 1896. 6 Sanfelice, Cent. f. Bakt. I, xviii, 1895, and Zeitschr. f. Hyg., xxi, 1895. THE YEASTS G21 thionin, methylene-blue, or the polychrome stains. In fresh prepara- tions the addition of a little NaOH in weak solution facilitates the search. When found, the organisms are easily recognized by their size, their highly refractive doubly-contoured cell-membrane, their vacuolated protoplasm, and, most important, by the discovery of budding forms. In tissue sections they are recognizable by the ordinary hematoxylin- eosin technique, but may be better demonstrated by the Loeffler methy- lene-blue method in use for bacterial tissue-staining. Excellent prep- arations are obtained by staining frozen sections with thionin, a method well adapted for the demonstration of capsules. The cultivation of the blastomycetes is comparatively easy after they have once been obtained in pure culture. Their isolation, however, usually is difficult when they occur in material contaminated with bac- teria. Growing more slowly than the bacteria, cultures taken from such contaminated material usually show very few yeast colonies. No special methods of facilitating the procedure have been devised, but success will often attend painstaking and oft-repeated plating of the mixed cultures in high dilution. The most favorable medium for this process is glucose agar. When once obtained in pure culture, the blastomycetes can readily be kept alive for indefinite periods by transplantation re- peated every two or three months. On agar or glucose agar, colonies appear after about three or four days as minute, glistening, white hemispherical spots which are not unlike colonies of staphylococcus albus. Planted in agar stab cultures, the microorganisms indicate their preference for a well-oxygenated environment by growing but slightly along the course of the stab, but by heaping up in a thick, creamy layer upon the surface of the medium. This layer in old cultures may be a quarter of an inch high. At first white, the growth, after three or four weeks, may turn distinctly yellow or even brown. In broth, the microorganisms form a stringy, gelatinous, and uneven cloud. On Loeffler' s blood-serum media, and upon gelatin, growth is easily obtained. The gelatin is not liquefied. Sugar media are fermented by few of the pathogenic blastomycetes, a fact which places them rather in distinct contrast with the fermenting yeasts used in the industries. Great and fundamental differences seem to exist between the patho- genic species described by various observers, and attempts at system- atizing the various members of the group, such as that by Rickets,1 1 Rickets, Jour. Med. Res., 6, 1902. 622 PATHOGENIC MICROORGANISMS have met with almost insurmountable obstacles. Some of the forms described, like that of Busse, have fermented sugars and have not formed mycelia, while organisms like that of Gilchrist did not cause fermentation in carbohydrate media, but, by their formation of my- celia under certain conditions, have indicated their close relationship or possibly their identity with the o'idia, transitional forms between the yeasts and the hyphomycetes. It is thus, in the light of our pres- ent knowledge of these microorganisms, quite impossible to establish within this group a distinct classification that is at all reliable. In considering the possible origin of blastomycotic infection in animals and man, it is, of course, important that we should have some knowledge as to the pathogenic properties of the yeast met with and handled in daily life. Rabinowitsch,1 with this in view, has investigated the pathogenic properties of fifty different species of yeasts obtained from fruit, manure, dough, and other sources, and among them found only seven varieties that had any pathogenicity for rabbits, mice, or guinea-pigs. In most of her successful inoculations, however, the disease produced in the animals had but very little resemblance to the blastomycotic conditions observed in man. 1 Rabinowitsch, Zeit. f. Hyg., xxi, 1895. CHAPTER XLVI HYPHOMYCETES (Eumycetes, Molds) The hyphomycetes or molds interest the bacteriologist chiefly because of the frequency with which they appear as contaminants during bacterial cultivation, and because they play the role of incitants in a few common diseases of the skin and mucous membranes. Morphologically they are entirely distinct from and much more complex than the bacteria. To the yeasts they are more closely related, the gap between the two classes being bridged by certain forms often spoken of as " oiidia " which, though usually appearing in the budding yeast-form, may occasionally grow out in mycelial threads. The characteristic feature of the hyphomycetes as a class is the formation of long, interlacing filaments or threads, known as mycelia. From these, branches come off which are spoken of as "hyphse." Each mycelial thread possesses a well-marked, doubly-contoured sheath or cell-wall and a finely granular protoplasmic cell-body, which, in some of the forms, is multinucleated. Two main classes of hyphomycetes are distinguished, the phycomy- cetes, and the so-called higher forms, or mycomycetes. The former class is characterized by the fact that no partitions exist within the mycelial threads or hyphse, the entire meshwork of a single microorganism con- sisting of one multinucleated cell. This group, furthermore, possesses the power of reproduction by both a sexual and an asexual process. The second class, or the mycomycetes, possess a mycelial meshwork which is divided into numerous partitions, and reproduces usually by the asexual process only. The process of reproduction, upon the basis of which the separation of groups within this class is determined, is best illustrated by citing a common example of each of the main divisions. As an example of the phycomycetes, the division most commonly met with upon contaminated gelatin plates, or upon exposed and moist organic matter of any description, is the one spoken of as the muco- 623 624 PATHOGENIC MICROORGANISMS rince. Most forms belonging to this division appear, grossly, as a light, cotton-like fluff, spreading in a thin fur over the surface of the culture medium. Examined with the low power of a microscope, there may be seen a complicated network of fine mycelial threads, which show no septa and from which delicate hyphal branches arise. In the forma- tion of the asexual spore organs near the tip of each hypha a septum appears. The tip of the hypha then gradually enlarges and forms a spherical capsule which is known as the sporangium. The unswollen Fig. 145. — Mucor mucedo. Single-celled mycelium with three hyphae and one developed sporangium. (After Kny, from Tavel.) portion of the hypha which projects into the sporangium is spoken of as the columella. Within the sporangium, a large number of small, round spores are formed. When these are ripe, the wall of the spor- angium bursts and the spores escape. Upon suitable media, then, new mycelia develop from these spores. The sexual reproduction, which oc- curs in this group, takes place in the following way: From two hyphae, placed in close apposition, lateral branches grow toward each other. These are spoken of as gametophores. The tips of the gametophores soon come in contact and, for a time, their protoplasm freely inter- communicates. Septa are then formed which cut off from the original hyphai a central cell, the zygospore. This zygospore gradually enlarges HYPHOMYCETES 625 and, under favorable conditions, sends out a branch which terminates in a non-sexual sporangium. Among the higher molds, or mycomycetes, various methods of sporulation occur, but sexual reproduction does not usually take place. One of the most commonly found genera is that of Penicillium. In this form the mycelial threads are partitioned off, by many transverse septa, into a number of separate cells. From these, hyphse, also sep- tate, are given off. From the ends of these hyphse, germinating branches arise which are known as conidiophores . These conidiophores divide into two or three septate branches, the sterigmata. From the m- Fig. 146. — Mucor mucedo. 1. Sporangium, c. columella, m. sporangium capsule, sp. spores. 2. Columella, after bursting of sporangium. 3. Poorly de- veloped sporangia. 4. Germinating spore. 5. Emptying of sporangium. (After Brefeld.) ends of these, other sterigmata may be given off and from the tip of each of these a single chain of spores or conidia are constricted off. The result is an appearance not unlike a hand in which the wrist represents the conidiophore; the metacarpal bones, the sterigmata; and the fingers, the long streptococcus-like chains of conidia. Similar to and even more common than the penicillia are the varieties known as Aspergillus. These forms, like the preceding, form delicate mycelial meshworks from which branches or conidiophores about 3-10 mm. in length, arise. These develop club-shaped expansions at their free ends and from these club-shaped expansions radially arranged sterig- 40 626 PATHOGENIC MICROORGANISMS mata arise. On the ends of these sterigmata spores or conidia develop similar to those developed by penicillium. Other forms of sporulation occur within this group. Thus, tubular spore capsules may be formed within the end segments of the hyphse, known as ascospores. In other cases, within a mycelial thread, a swelling may take place into which protoplasm flows from the neighbor- ing cells, at both ends. In this way, an oval spore case is developed Fig. 147. — Mucor mucedo. Formation of zygospore. 1. Two branches coa- lescing. 2 and 3. Process of conjugation. 4. Ripe zygospore. 5. Germination of zygospore. 6 and 7. Mucor erectus. Azygo sporulation. No two branches meet, but form spores without conjugation. 8 and 9. Mucor tenuis. Azygo sporulation. The spores grow out from side branches without sexual union. (1-5 after Brefeld; 6-9 after Bainier, from Tavel.) within the course of the mycelial thread. This is known as a chlamy- dospore. The segments on each side of the chlamydospore die out and the spore capsule is liberated from the mycelium. The classification of the various divisions of the hyphomycetes is a problem requiring much study and great botanical insight, and can hardly be discussed in a general work on bacteriology. Upon artificial media, the members of this group are not at all fastidious, growing easily upon organic matter of all kinds, provided HYPHOMYCETES 627 moisture is present. In laboratories they are frequently found as con- taminants, and in order to procure them for purposes of study it is only necessary to expose agar or gelatin plates in a dusty, dark corner. In households they are frequently found growing in store-rooms upon stale bread, shoes, leather trunks, and on remnants of food. Most of them prefer an acid environment and are dependent upon a free supply of oxygen. At room temperature they grow more readily than at the usual incubator temperature. DISEASES CAUSED BY HYPHOMYCETES Pityriasis versicolor (Microsporon furfur). — Pityriasis is a disease of the skin observed chiefly among persons living under conditions of uncleanliness, or among those who combine these conditions with a Fig. 148. — Mucor ramosus. Ripe sporangia on columellae. (After Lindt.) tendency to profuse perspiration. It begins as a small, light brown or yellowish patch upon the skin of the abdomen, breast, or back, is flat and barely raised from the cutaneous surface. It spreads and coalesces with similar spots until the entire area resembles strongly the figures of a map with irregular continents and islands. The disease does not penetrate into the skin itself, but consists, as Plaut has pointed out, of a simple saprophytism of the inciting agent upon the skin. The condition is caused by a member of the group of Hyphomycetes, discovered in 1846 by Eichstedt, and later named Microsporon furfur. 628 PATHOGENIC MICROORGANISMS The microorganism consists of a dense meshwork of mycelial threads, from which septate hyphse arise in large numbers. From the ends of these, spores arise in rows, after the manner depicted for penicillium (Fig. 149). The hyphse, according to Unna,1 show a characteristic bending at right angles, due to a slight flattening of their diameters. Characteristic of the microsporon proper, in preparations made from Fig. 149. — Penicillium glaucum. A. Showing septate mycelia. B. End of a hypha — branching into two conidiophores, from which are given off the sterigmata. From the ends of these are developed the round conidia. (After Zopf.) cutaneous scrapings, are the fragments of bent hyphse and the large numbers of free spores. Cultivation of Microsporon furfur has been successfully carried out by many observers.2 Growth is particularly characteristic upon potato, white or yellowish-white colonies appearing within four or five days and rapidly spreading over the entire surface of the potato. « Unna, " Histopathol. of Skin," transl., N. Y., Macmillan, 1896. 2 Kotjar, Ref. Baumgarten's Jahresbericht, 1892. HYPHOMYCETES 629 Thrush (Soor or Muguet ; Oidium albicans) . — Thrush is a localized disease of the mouth occurring most frequently in children suffering from malnutrition or it occurs, under conditions of uncleanliness, upon an area of catarrhal inflammation of the mucous membrane. The microorganism which causes the condition was first described by Langenbeck in 1839, and has, since then, been studied by many observers. It was successfully cultivated by Grawitz l in 1886 and recognized by him as belonging to the hyphomycetes. The most careful cultural studies which have been made upon the Oidium albicans are those of Linossier and Roux.2 c Fig. 150. — Aspergillus glaucus. m. Mycelial threads, s. Sterigmata. r. Ascospore. p. Germinating conidium. A. Ascus. (After de Bary.) Morphologically, the oidium consists of budding cells, resembling those of yeast, which, under certain conditions, can produce mycelia and hyphse from which spores are developed. Two main varieties are described, that which produces large spores and liquefies gelatin in culture, and that which gives rise to smaller spores and fails to liquefy gelatin. In many cases only the yeast-like budding cells can be found ; these, however, when subjected to unfavorable cultural conditions, may be induced to send out hyphae and form spores. Like yeasts, but to a lesser degree, the Oidium albicans causes fermentation in sugars. It 1 Grawitz, Virch. Arch., 1886. 2 Linossier and Roux, Comptes rend, de l'acad. des sci.; 1889. 630 PATHOGENIC MICROORGANISMS develops best under slightly acid conditions and in the presence of free oxygen, upon gelatin and agar. Favus (Achorion Schoenleinii). — Favus is a disease attacking chiefly the hairy portions of the body of man and some domestic animals. In man, it is found most frequently in undernourished children upon the scalp. It is a disease of extremely chronic course and is very resistant to treatment. Beginning as a small erythematous spot, it soon develops into small sulphur-yellow cupped crusts, which are placed usually about the base of a hair. These may spread and coalesce. The small inden- tated crust is spoken of as a scutulum. When such a scutulum is re- moved and examined under a microscope, teased out in a few drops of Fig. 151. — Thrush. Oidium albicans, stained. (After Zettnow.) strong sodium hydrate solution (20 per cent) , the incitant of the disease may be easily recognized and studied. In such a preparation the cen- ter of the scutulum is found to be made up chiefly of small doubly- contoured spores which are irregularly oval or round, and may be ar- ranged in chains, lying scattered among an extremely dense meshwork of fine mycelial threads. Toward the periphery of the scutulum, the spores are less numerous and the looser arrangement of the meshwork permits us to distinguish distinct filaments of mycelia which give off hyphse, the ends of which are often swollen into small knobs. The interior of the scutulum usually contains a pure culture of the fungus. HYPHOMYCETES 631 Many varieties of achorion have been described, but Plaut 1 believes that, at the present time, it is not possible to separate these from one another, owing to the fact that selective cultivation has succeeded in altering many of the characteristics displayed by many of the strains. The same observer recommends the following method for obtaining pure cultures of this microorganism. As much of the material as can be conveniently obtained is gently rubbed up in a sterile mortar with fine sand or infusorial earth. The triturated material is then inoculated into fluid agar and plates are poured. Ordinary streaked plates upon agar may also be used with success with material directly from the centers of scutula. Fig. 152. — Thrush. Oidium albicans, unstained. (After Zettnow.) The achorion grows best upon acid agar at a temperature of 37.5° G. Growth appears within from forty-eight hours to three days as yellowish disks, which occasionally may be slightly furred with aerial hypha3. Ringworm (Trichophyton tonsurans). — Ringworm, Tinea circinata, or Herpes tonsurans, is a contagious disease of the skin and hair, occur- ring most often in children and appearing upon both the haired portions of the body, as well as upon free skin. It is characterized by the forma- tion of circular scaly patches, within which the hairs fall out. The disease is caused by several species of the trichophyton, a genus of hyphomycetes. These microorganisms were first recognized as inci- 1 Plaut, in Kolle und Wassermann's " Handbuch," I. 632 PATHOGENIC MICROORGANISMS tants of tlie disease by Gruby 1 in 1841, and were most thoroughly studied later, by Sabouraud.2 The fungi consist of finely interlaced narrow septate mycelia, within which characteristic swellings appear. From these swellings, chlamydospores develop. Hyphae, both aerial and deep, grow out of the mycelial threads, on the ends of which ascopores may develop. In the diseased skin, the fungi grow chiefly within the hair sheath, causing an area of secondary inflammation about the base of the hair. The infection probably begins first in the epidermis sur- rounding the hairs, from which it then spreads into the hair bulb and thence grows up into the substance of the hair in which mycelial threads Fig. 153. — Achorion Schoenleinii. Section of favus crust. Stained by Gram. (After Fraenkel and Pfeiffer.) and spores develop in large numbers. The demonstration of the micro- organisms from a case can easily be accomplished by epilating an affected hair, making sure that the hair bulb has been removed. This is then immersed under a cover-slip in a drop of sodium hydrate or potassium hydrate solution and examined under the microscope. In this way enormous numbers of short mycelial threads and spores may be seen to lie within the bulb. Many varieties of these fungi have been described from different cases. Their interrelationship is not entirely 1 Gruby, Comptes rend, de l'acad. des sci., 13, 1841. 2 Sabouraud, Ann. de dermat. et de syph., 3, 1892, and 4, 1893. HYPHOMYCETES 633 clear. In general, a division is made between those which develop large spores and a more common small-spored variety. Cultivation is comparatively simple and is best carried out upon acid glucose agar or gelatin. Upon such media, within five or six days, mycelial threads, which are septate and form chlamydospores, may be observed. Pigment, reddish or brown, is occasionally noted. Gelatin is liquefied. The disease may be produced with such cultures upon guinea-pigs. In man, the disease is usually acquired by infection from patient to patient. Other Diseases in which Hyphomycetes have been Found. — A number of cases have been described in which members of this group have been found at autopsy in the lungs of persons dying of bronchopneu- monia.1 In most of these cases, the fungus found belonged to the aspergillus group and was regarded as a merely secondary invader. A few cases, however, have been reported in which the fungus was re- garded as the primary cause of the disease. A single case is on record, in which an intestinal infection with a member of the genus mucor resulted in a generalized infection with pulmonary and secondary cerebral abscesses. In birds, a disease of the lungs has long been known to be due to various species of aspergillus. In many domestic animals, diseases of the skin and hair occur which are caused by micro- organisms similar to, or identical with, those occurring in man. i Sticker, Nothnagel, " Spez. Path. u. Ther.," 14, 1900. SECTION IV DISEASES OF UNKNOWN ETIOLOGY CHAPTER XLYII RABIES (Hydrophobia, Rage, Lyssa, Hundswuth) Rabies is primarily a disease of animals, infectious for practically all the mammalia, but most prevalent among carnivora, dogs, cats, and wolves. It is said also to occur spontaneously among skunks of the southwestern United States, and is readily inoculable upon guinea-pigs, rabbits, mice, rats, and certain birds, chickens and geese being especially susceptible. Man is subject to the disease. Infection usually occurs as a consequence of the saliva of rabid animals gaining entrance to wounds from bites or scratches. The disease is prevalent to an alarming extent in all civilized countries except England, where the careful supervision of dogs, enforcement of muzzling laws, and rigid legislation regarding the importation of dogs, have caused a practical eradication of the disease in that country. A fair estimate of the prevalence of the disease may be obtained from the statistics of animals dying or killed because of rabies in different countries. In Germany, according to Kolle and Hetsch, during the fifteen years ending in 1901, there were 9,069 dogs, 1,664 cattle, 191 sheep, 110 horses, 175 hogs, 79 cats, 16 goats, 1 mule, and 1 fox affected with rabies. In the eastern United States the dis- ease is not uncommon. The statistics of the New York Department of Health, for a period of six months ending December 31, 1907, show 74 cases of rabies among dogs in the city of New York and vicinity. Among human beings the disease is no longer common in civilized countries, since early preventive treatment is successfully applied in almost all infected subjects. Experimental infection in susceptible animals is best carried out by injections of a salt-solution emulsion of the brain or spinal cord of an 634 RABIES 635 afflicted animal, subdurally, through a trephined opening in the skull, but may also be accomplished by injection into the peripheral nerves, the spinal canal, or the anterior chamber of the eye. Intravenous and intramuscular injections are also successful, though less regularly so. The time of incubation after inoculation varies with the nature of the virus used, the location of the injection, and the quantity injected. In accidental infections of man and animals the incubation is shortest and the disease most severe when the wounds are about the head, neck, and upper extremities and are deeply lacerated. This is explained by the fact that the poison is conveyed to the central nervous system chiefly by the path of the nerve trunks. This has been experimentally shown by di Vestea and Zagari 1 who inoculated animals by injection into peripheral nerves, and showed that the nerve tissue near the point of inoculation becomes infectious more quickly than the parts higher up; thus the lumbar cord of an animal inoculated in the sciatic nerve is in- fectious several days before virus can be demonstrated in the medulla. In man, infected with " street virus/' that is, with the virus of a dog or other animal not experimentally inoculated, the incubation period varies from about forty to sixty days. Isolated cases have been reported in which this period was prolonged for several months beyond this. The virulence of rabic virus may be markedly increased or diminished by a number of methods. By repeated passage of the virus through rabbits, Pasteur 2 was able to increase its virulence to a more or less constant maximum. Such virus which had been brought to the highest obtainable virulence, he designated as "virus fixe." Inocu- lation of rabbits, dogs, guinea-pigs, rats, and mice with such virus usually results in symptoms within six to eight days. The same animals inoculated with street virus may remain apparently healthy for two to three weeks. In dogs and guinea-pigs inoculation usually results first in a stage of increased excitability, restlessness, and sometimes viciousness. This is followed by depression, torpor, loss of appetite, inability to swallow, and finally paralysis. In rabbits the disease usually takes the form of what is known as "dumb rabies," the animals gradually growing more somnolent and weak, with tremors and gradual paralysis beginning in the hind legs. In experimentally infected birds the disease is slow in appearing and 1 di Vestea and Zagari, Ann. de l'inst. Pasteur, iii, 2 Pasteur's work on rabies. Compt. rend, de Tacad. des sciences, 1881, 1882, 1884, 1885, 1886, and Ann. de l'inst. Pasteur, 1887 and 1888. 636 DISEASES OF UNKNOWN ETIOLOGY may show a course of gradually increasing weakness and progressive paralysis extending over a period of two weeks after the appearance of the first symptoms. In man, the disease begins usually with headache and nervous de- pression. This is followed by difficulty in swallowing and spasms of the respiratory muscles. These symptoms occur intermittently, the free intervals being marked by attacks of terror and nervous depression. Occasionally there are maniacal attacks in which the patient raves and completely loses self-control. Finally, paralysis sets in, ending event- ually in death. Pathological examination of the tissues of rabid animals and human beings reveals macroscopically nothing but ecchymoses in some of the mucous and serous membranes. Microscopically, however, many abnormal changes have been observed and were formerly utilized in histological diagnosis of the condition. Babes * has described a disap- pearance of the chromatic elements in the nerve cells of the spinal cord. This observation has been confirmed by others,2 but is no longer regarded as pathognomonic of rabies. The same observer has described a marked leucocytic infiltration which occurs about the blood-vessels of the brain and about the ganglia of the sympathetic system. These changes are not found in animals infected with virus fixe and are present only in animals and human beings inoculated with street virus. The causative agent of rabies is not known. A number of observers have reported the cultivation of bacteria from the central nervous system of infected subjects; none of these claims, however, has found confirmation. In 1903, Negri 3 of Pavia described peculiar structures which he observed in the cells of the central nervous system of rabid dogs. While present in all parts of the brain, these " Negri bodies " are most regularly present and numerous in the larger cells of the hippocampus major and in the Purkinje cells of the cerebellum. The presence of these structures in rabid animals and man has been confirmed by a large number of workers in various parts of the world and the specific association of these bodies with the disease is now beyond doubt. In consequence, the determination of " Negri bodies " in the brains of suspected animals has become an extremely important method of diagnosis — more rapid and accurate than the methods previously known. 1 Babes, Virch. Arch., 110, and Ann. de l'inst. Pasteur, 6> 1892. 2 Van Gehuchten, Bull, de l'acad. de med. et biol., 1900. 3 Negri, Zeit. f. Hyg., xliii and xliv. «A RABIES 637 The demonstration of Negri bodies in tissues is carried out as follows : A small piece of tissue is taken from the cerebellum or from the center of the hippocampus major (cornu ammonis), and is fixed for twelve hours in Zenker's fluid. It is then washed thoroughly in water and dehydrated as usual in graded alcohols, embedded in paraffin, and sectioned. The sections are best stained by the method of Mann, as follows : The sections, attached to slides in the usual way, are immersed in the follow- ing solution for from twelve to twenty-four hours. Methylene-blue (Gruebler 00), 1 per cent 35 c.c. Eosin (Gruebler BA), 1 per cent 35 c.c. Distilled water 100 c.c. They are then differentiated in: Absolute alcohol 30 c.c. Sodium hydrate, 1 per cent in absolute alcohol 5 c.c. In this solution blue is given off and the sections become red. After about five minutes the sections are removed from this solution, are washed in absolute alco- hol, and are placed in water where they again become faintly bluish. It is of advantage to immerse them, now, in water slightly acidified with acetic acid. Following this they are dehydrated with absolute alcohol and cleared in xylol, as usual. In preparations made in this way, the nerve cells are stained a pale blue, and in their cytoplasm, lying either close to the nucleus or near the root of the axis-cylinder process, are seen small oval bodies stained a deep pink. The bodies are variable in size, measuring from 1 to 27 micra in diameter. They are round or oval, show a more deeply stained periph- eral zone which has been interpreted as a cell membrane, and, in their interior, often show small vacuole-like bodies. There may be more than one, often as many as three or four, in a single cell. The rapid demonstration of Negri bodies in smears of brain tissue has recently been advocated by many observers and has been extensively used for diagnosis. It is carried out, according to Van Gieson,1 in the following way: A small pin-head-sized piece of brain tissue from the regions indicated above, is placed on one end of a slide under a cover- glass and the cover is gently squeezed with the finger until the tissue is flattened out into a thin layer. The glass cover is then gently shifted across the slide until the brain tissue is smeared along the entire surface. These smears may be fixed in methyl alcohol and stained by the Giemsa 1 Van Gieson, Proe of N. Y. Pathol. Soc, N. S., iv, 1906. 638 DISEASES OF UNKNOWN ETIOLOGY method, as described in the chapter on Spirochete pallida (see page 587). Stained in this way, the Negri bodies are stained light blue, in con- trast to the darker and more violet cell-bodies. The smears may also be stained by a method originated by Van Gieson, which gives an excellent contrast stain and reveals more clearly the inner structure of the Negri bodies. Van Gieson's stain is prepared as follows: Distilled water 10 c.c. Saturated alcoholic solution of rosanilin violet 2 drops. Saturated aqueous solution of methylene-blue diluted one-half with water 2 drops. This method has been modified by Williams and Lowden,1 who add to 10 c.c. of distilled water 3 drops of saturated alcoholic basic fuchsin and 2 c.c. of Loeffler's methylene-blue. The slides are fixed in methyl alcohol, washed in water, and covered with the freshly prepared stain. The slide is held over the flame until the solution steams and is then rinsed in water and dried. The Negri bodies assume a brilliant hue and contain in their interior darkly stained, irregular particles which have been interpreted as chromatin bodies. As to the nature of the Negri bodies opinions are still divided. Their constant presence in rabic brain tissue is unquestioned and their diagnostic significance well established. Cultivation experiments, however, have been uniformly unsuccessful. A number of observers, Negri himself, Calkins,2 Williams and Lowden,3 and others, believe these bodies to be protozoa. The last-named authors base this opinion upon the definite morphology of the bodies, and their staining properties, which in many respects are similar to those of protozoa. These observers also claim that the mor- phology of the bodies shows a number of regular cyclic changes which are found accompanying different stages of the disease; these changes correspond, according to these workers, to similar cycles occurring among known protozoa of the suborders of the class "Sporozoa." Much has been said for and against this view, but, at the present time, the evi- dence collected does not warrant the positive assertion that the Negri bodies are parasitic in nature. Many pathologists still look upon them as specific degenerations of the nerve-cells similar to the changes observed by Babes. 1 Williams and Lowden, Jour. Inf. Dis., 3, 1906. 2 Calkins, Discussion, Proc. N. Y, Pathol. Soc, N. S., vol. vi, 1906. 3 Williams and Lowden, loc. cit. . RABIES 639 The Specific Therapy of Rabies. — The treatment which is now pro- phylactically applied to patients infected with or suspected of infection with rabies has been but little altered either in principle or in technical detail since it was first worked out by Pasteur. In principle it con- sists of an active immunization with virus, attenuated by drying, admin- istered during the long incubation period in doses of progressively increasing virulence. By the repeated passage of street virus through rabbits, Pasteur obtained a virus of maximum and approximately constant virulence which he designated as virus fixe. By a series of painstaking experi- ments he then ascertained that such virus fixe could be gradually at- tenuated by drying over caustic potash at a temperature of about 25° C, the degree of attenuation varying directly with the time of drying. Thus, while fresh virus five regularly caused death in rabbits after six to seven days, the incubation time following the inoculation of dried virus grew longer and longer as the time of drying was increased, until finally virus dried for eight days was no longer regularly infectious and that dried for twelve to fourteen days had completely lost its virulence. The method of active immunization, which Pasteur used, consisted in injecting, subcutaneously, virus of progressively increasing viru- lence, beginning with that derived from cords dried for thirteen days and gradually advancing to a strong virus. Thus the patient was im- munized to a potent virus several weeks before the incubation time of his own infection had elapsed. Pasteur successfully proved the efficacy of his method upon dogs and finally upon human beings, the first human case being that of a nine-year-old child, — Joseph Meister. Technique of Rabies Therapy.- — The technique developed by Pasteur is still, in the main, followed by those who treat rabies to-day. I. As a preliminary, it is necessary to prepare or obtain virus fixe. This may generally be procured from an established laboratory or may be prepared independently by passing street virus through a series of young rabbits (weighing from 700 to 1,000 gms.). According to Hogyes,1 the passage of the virus through twenty-one to thirty rabbits, in this way, will reduce its incubation time to seven or eight days. Babes claims to obtain a virus fixe more rapidly by passing the virus alter- nately through rabbits and guinea-pigs. For purposes of inoculation, virus is prepared by emulsifying in 1 Hogyes, Quoted from Kraus and Levaditi, "Handb.," etc., I. 640 DISEASES OF UNKNOWN ETIOLOGY sterile salt solution, pieces of the medulla or cerebellum of animals dead of a previous inoculation. The brain tissue which is not emulsified may be preserved under sterile glycerin in a dark and cool place for fur- ther use. II. Rabbits are inoculated with virus fixe by intracranial injection. A small incision is made in the shaved scalp in the median line, and the skin is retracted. With a small trephine or a round chisel, an opening- is made in the skull in the angle between the coronary and sagittal Fig. 154. — Method of Drying Spinal Cord Attenuation. of Rabbit for Purposes of sutures. Through this opening about 0.2 to 0.3 c.c. of the virus fixe is in- jected, either directly into the brain substance or simply under the dura. As soon as a rabbit so inoculated has died it is autopsied. The animal before dissection should be washed in a disinfectant solution — lysol or carbolic acid. The skin is then removed and the animal fastened to a dissecting board, lying on its ventral surface. The spinal canal is then laid open with a pair of curved scissors and the spinal cord carefully removed. This is accomplished by cutting across the cord in the lumbar region, and lifting this with a forceps while the nerve roots are divided from below upward. The cord is suspended by a sterile thread within a large bottle into the bottom of which pieces of potassium hydrate have been placed. The bottle is then set away in a dark room or closet, the temperature of RABIES 641 which is regulated so as to vary little above 25° C. Bacteriological controls as to the sterility of the cord should also be made. After a suitable period of drying, pieces of the cord are prepared for injection. This is done in various ways at different laboratories. No attempt at exact dosage is made. At the New York Depart- ment of Health 1 cm. of the cord is emulsified in 3 c.c. of sterile eight-tenths per cent salt solution, the dose for injection being usu- ally 2.5 c.c. Marx * emulsifies 1 cm. of the cord in 5 c.c. of sterile bouillon or salt solution, using 1 to 3 c.c. of this for injection according to the age of the cord. For shipment an addition of 20 per cent of glycerin and 0.5 per cent of carbolic acid is made. The scheme of treatment is also subject to variations according to the individual customs of various laboratories. The following scheme is the routine of the Pasteur Institute in Paris, as quoted in Kraus and Levaditi, "Handbuch fur Immunitatsforschung," Vol. I, p. 713. Day of Treatment. Mild Cases. Medium Cases. Severe Cases. Days of Drying. Dose. Days of Drying. Dose. Days of Drying. Dose. 1 14+13 3 c.c. 14+13 3 c.c. A.M.14+13P.M.12 + 11 3 c.c. 2 12+11 3 c.c. 12+11 3 c.c. A.M. 10 + 9 P.M. 8+ 7 3 c.c. 3 10 + 9 3 c.c. 10 + 9 3 c.c. A.M. 7 P.M. 6 2 c.c. 4 8 + 7 3 c.c. 8 + 7 3 c.c. 5 2 c.c. 5 6 + 6 3 c.c. 6 + 6 3 c.c. 5 2 c.c. 6 5 1 c.c. 5 2 c.c. 4 2 c.c. 7 5 1 c.c. 5 2 c.c. 3 1 c.c. 8 4 1 c.c. 4 2 c.c. 4 2 c.c. 9 3 1 c.c. 3 1 c.c. 3 1 c.c. 10 5 2 c.c. 5 2 c.c. 5 2 c.c. 11 5 2 c.c. 5 2 c.c. 5 2 c.c. 12 4 2 c.c. 4 2 c.c. 4 2 c.c. 13 4 2 c.c. 4 2 c.c. 4 2 c.c. 14 3 2 c.c. 3 2 c.c. 3 2 c.c. 15 3 2 c.c. 3 2 c.c. 3 2 c.c. 16 5 2 c.c. 5 2 c.c. 17 4 2 c.c. 4 2 c.c. 18 3 2 c.c. 3 2 c.c. 19 5 2 c.c. 20 4 2 c.c. 21 3 2 c.c. 1 Marx, Deut. med. Woch., 1899, 1900. 41 642 DISEASES OF UNKNOWN ETIOLOGY The treatment at the New York Department of Health is as follows :l Mild Cases. Medium Cases. Severe Cases. Day of Treatment. Days of Drying. Dose. Days of Drying. Dose. Days of Drying. Dose. 1 14+13 4 c.c. 10 4 c.c. A.M. 10 + 9 P.M. 10 + 9 4 c.c. 2 12+11 4c.c. 9 4 c.c. a.m. 8 + 7 p.m. 8 + 7 4 c.c. 3 10 + 9 4 c.c. 9 4 c.c. 6 4 c.c. 4 8 + 7 4 c.c. 8 + 7 4 c.c. 4 4 c.c. 5 6 2 c.c. 6 2 c.c. 3 2 c.c. 6 5 2 c.c. 5 2 c.c. 4 2 c.c. 7 4 2 c.c. 4 2 c.c. 3 2 c.c. 8 3 2 c.c. 3 2 c.c. 2 2 c.c. 9 5 2 c.c. 2 2 c.c. 4 2 c.c. 10 4 2 c.c. 5 2 c.c. 1 2 c.c. 11 3 2 c.c. 4 2 c.c. 4 2 c.c. 12 5 2 c.c. 3 2 c.c. 3 2 c.c. 13 4 2 c.c. 2 2 c.c. 2 2 c.c. 14 3 2 c.c. 4 2 c.c. 4 2 c.c. 15 5 2 c.c. 3 2 c.c. 1 2 c.c. 16 4 2 c.c. 2 2 c.c. 4 2 c.c. 17 4 2 c.c. 3 2 c.c. 18 3 2 c.c. 2 2 c.c. 19 2 2 c.c. 4 2 c.c. 20 3 2 c.c. 21 2 2 c.c. 22 4 2 c.c. 23 3 2 c.c. 24 2 2 c.c. 25 4 2 c.c. 2G 3 2 c.c. The severity or mildness of cases is estimated from the depth and degree of laceration of the wounds, also from their location — bites about the face and upper extremities being the most dangerous. During the course of such treatment patients may show troublesome erythema about the point of injection and occasionally backache and muscular pains. Treatment need not be omittted unless these symp- toms become excessive. The efficiency of the Pasteur treatment in rabies is no longer prob- lematical. According to Hogyes, 50,000 people have been treated within ten years, with an average mortality of 1 per cent. Personal communication from Dr. Poor, of the New York Department of Health. RABIES 643 Although the method described above is the one which is extensively used in all established institutes for the treatment of rabies, other methods have been elaborated and used to a slight extent. One of the most important of these is the " dilution method" of Hogyes. This method is carried out as follows: A definite quantity of the spinal cord of a rabbit dead of virus fixe is emulsified in 100 c.c. of normal salt solution. Dilutions of this emulsion are made and the patient is injected at first with a dilution of 1 : 1,000, subsequent injections being made of gradually increasing concentration until a concentration of 1 : 100 is reached. This method, so far as it has been used, has been satisfactory, but it has not yet found extensive application. Attempts to treat active rabies with the sera of immunized animals have so far been unsuccessful. CHAPTER XLVIII SMALLPOX Smallpox or variola is one of the most virulent of infectious diseases. Throughout history it has been a severe scourge of mankind, prevailing in China and other Eastern countries many centuries before Christ and sweeping through medieval Europe, especially at the time of the Crusades, in a series of severe epidemics. All races of men are suscep- tible and no age from childhood to senility is exempt. In modern times the disease is endemic in most uncivilized countries, especially those of the East, and occurs sporadically in all parts of the globe. Owing to rigid enforcement of vaccination and of quarantine laws, however, the disease has been practically eradicated from civilized countries. The etiological factor which causes smallpox is still unknown. Numerous researches aimed at the discovery of cultivatable microorgan- isms in the lesions or blood of infected patients have met with uniform failure. Streptococci, though often found in the smallpox vesicles and pustules, and often undoubtedly contributing materially to the fatal outcome of the disease, may be regarded as purely secondary in signifi- cance. Communications which have claimed the discovery of a protozoan incitant of the disease have, on the other hand, been numerous and, in some cases, have seemed plausible. Yet absolute proof has always been lacking. The literature on this question is extensive and some of the earlier contributions, such as those of Grunhagen, * of Van der Loeff,2 and of Pfeiffer,3 possess historical interest only. The work which, of re- cent years, has attracted the most serious attention to this subject is that published by Guarnieri4 in 1892. This observer found, in the deeper cells of the epithelium covering the pustules, both of smallpox lesions and of vaccination lesions, small bodies which were easily stained by hematoxylin, safranin, or carmin. Similar bodies could be observed in 1 Grunhagen, Arch. f. Dermat. u. Syph., 1892. 2 Van der Loeff, Monat. f. prakt. Dermat., iv. *L. Pfeiffer, Zeit. f. Hyg., xxiii. * Guarnieri, Arch, perle sc. med., xxvi, 1892; Cent. f. Bakt., I, xvi, 1894. 644 SMALLPOX 645 the cells of corneal lesions experimentally produced in rabbits. Guar- nieri claimed that he distinguished both cytoplasm and nucleus in these bodies and described both binary division and reproduction by sporu- lation as in the sporozoa. He named the supposed protozoan "Cy- toryctes variola?." At about the same time Monti 1 described similar bodies in the cells of the Malpighian layer of the skin covering smallpox lesions and, a few years later, Clarke 2 confirmed the researches of Guarnieri. Subsequently, many researches were carried out on the same subject in this country, the most notable being those of Council- man,3 Magrath and BrinckerhofT, and of Calkins.4 The former authors came to the distinct conclusion that the bodies seen by Guarnieri were parasites, and the latter author even described a distinct life-cycle for these parasites comparable to that of some protozoa. These researches, however, are by no means absolutely convincing, and Ewing,5 while admitting that the vaccine bodies are probably specific for variola, calls attention to the fact that specific cell-degen- erations or inclusions are found in diphtheria, measles, glanders, rabies, and other infectious processes, which can not be regarded as in any way related to these diseases etiologically, and suggests the probability of a similar interpretation for the vaccine bodies. Much has been said on both sides of the question since that time, and the problem can not be regarded as settled. The burden of proof, of course, rests upon those who claim the discovery of a specific microorganism, and absolute proof will probably be lacking until our experimental methods are such as will permit of other than purely morphological demonstration. Whatever the actual causative agent may be, it is certain that the disease is transmitted with extreme ease — actual contact, direct or in- direct, with a patient being unnecessary for its transmission. For this reason the disease is often spoken of as being "air borne." While we have no certain knowledge of the portal of entry through which the virus invades the human body, many considerations have made it seem plau- sible that this may take place through the mucosa of the upper respira- tory tract. Our knowledge of the means of defense against the malady is for- tunately more advanced than is that of its etiology. It has been known 1 Monti, Cent. f. Bakt., I, xvi. 2 Clarke, Brit. Med. Jour., 2, 1894. 3 Councilman, Magrath, and Brinckerhoff, Jour. Med. Res., xi, 1904. * Calkins, Jour. Med. Res., xi, 1904. 5 Ewing, Jour. Med. Res., xiii, 1905. 646 DISEASES OF UNKNOWN ETIOLOGY for centuries that one attack of smallpox protects against subsequent attacks. This knowledge was made use of by the physicians of ancient China and India, who, during mild epidemics, exposed healthy children to infection, hoping that mild attacks would result which would confer immunity. While dangerous in the extreme, such "variolation/' never- theless, was not without some benefit and was even introduced into Europe in the eighteenth century by Lady Mary Wortley Montagu. Such practices, however, were made unnecessary by the classical investigations of Jenner * published in 1798. Jenner, as a student, had been impressed with the fact that country-people who had been infected with a disease known as cowpox, were usually immune against smallpox. His studies and observations came to a practical issue when, in 1796, he inoculated a boy, James Phipps, with pus from a cowpox lesion on the hand of an infected dairy -maid. Two months later the same boy was inoculated with material from a smallpox pustule without subsequent disease. With this experiment the principles of vaccination as in use at the present time were founded. The question as to the identity of cowpox and smallpox has been the basis of a long controversy. Many observers claimed from the be- ginning that the two diseases, though closely related to each other, were essentially different. Others, on the contrary, and this seems to be the prevailing opinion among scientists at the present day, maintain that cowpox or vaccinia, as it is called when inoculated into a human being, represents merely an altered and attenuated variety of variola. This latter view is based on the following considerations, which we take from Haccius as quoted by Paul.2 1. Variola is invariably transmissible to cattle, when proper methods of inoculation are employed. 2. Variola carried through several animals, in the above way, be- comes altered in character, approaching in nature typical vaccinia or cowpox. 3. Such virus, reinoculated into man, gives rise to purely local lesions which are mild and unlike smallpox. 4. Inoculation with such virus protects both man and animals against subsequent inoculation with cowpox, and, in the case of man, against smallpox as well. It has been claimed, moreover, that cowpox originally Was trans- tenner, "Inquiry into the Causes and Effects of the Variola-Vaccinae/' London, 1798. 2 Paul, "Vaccination"; Kraus and Levaditi, "Handbuch," etc., I. SMALLPOX 647 mitted to cattle by human beings affected with smallpox. This seems likely both because of the comparative rarity of the former disease and because of its spontaneous occurrence almost invariably upon the teats of cows, although both males and females are equally susceptible to experimental inoculation. The relationship of variola to chicken-pox or varicella has been more easily determined. Chicken-pox does not protect against smallpox nor is this the case vice versa. The two diseases are unquestionably quite distinct. The Production of Vaccine. — During the early days of vaccination, it was customary to inoculate human beings with the matter obtained from the pustules of those previously vaccinated. While this method was perfectly satisfactory for the immediate purposes in view, practical difficulties and the occasional accidental transmission of syphilis have rendered this practice undesirable. In consequence, at all institutes at which vaccine is produced for use upon man, the virus is obtained from animals. Horses and mules, both extremely susceptible to vac- cine, have been employed, and goats have, at times, been chosen because of their insusceptibility to tuberculosis. Rabbits have also been used more recently by Calmette and Guerin.1 The animals almost exclusively employed at the present day, how- ever, are calves, preferably at ages of from six months to two years. Very young suckling calves are unsuitable because of the great speed of development and small size of the lesions produced. The animals should be healthy and at some institutes (Dresden) are subjected, before use, to the tuberculin test; although, according to Paul,2 this produces a hypersusceptibility to the vaccine, and can be omitted without danger when careful supervision is observed. Some observers prefer to use light- colored animals rather than dark-skinned or black ones, both for reasons of greater ease of cleanliness and because the former are supposed to be more susceptible than the latter. This contention is denied by others. The sex of the animals seems to be immaterial. During the period of use, the calves are fed, according to age, with either an exclusive milk diet, or they are given, in addition, fresh hay. The greatest cleanliness in regard to the bedding and stalls must be observed and separate stables should be available for the animals under treatment and those under observation before treatment. These stables, if possible, should be so built that they can be easily scoured and flushed 1 Calmette and Guerin, Ann. de l'inst. Pasteur, 1902. 2 Paul, loc. cit. 648 DISEASES OF UNKNOWN ETIOLOGY with water, and stalls should be disinfected after occupation. If possible, stables should be artificially heated and a comfortable temperature maintained. Halters and fastenings should be so arranged that the animals can not lick the scarified surfaces. Careful veterinary control before vaccination and during the period of treatment must be observed in order to eliminate animals with systemic disease or other complica- tions. The calves may be vaccinated with material taken from previously vaccinated animals. They may, on the other hand, be inoculated with "seed virus" obtained from the vesicles of human vaccinia. This method of using humanized virus for the inoculation of calves for vaccine production is preferred by many workers and is spoken of as " retro vac c inat ion . ' f Actual vaccination of the animals is done as follows: Calves which have been kept under observation for at least a week are thoroughly washed and cleaned and the abdomen is clipped and shaved over an area extending from the ensiform cartilage to the pubic region, including the entire width of the belly and the inner folds of the thighs. It is best to shave the animal a day of two before vaccination so as to avoid fresh scratches and excoriations. Just before actual operation the animal is strapped to a specially constructed operating table in such a way as to allow free access to the shaved area. This area is now thor- oughly washed with soap and water followed by alcohol, or, in some institutes, by a weak solution of lysol. If the latter is used, the field of operation must again be thoroughly rinsed with sterile water. About a hundred small scarifications are made in this area, preferably by crossed scratches, covering for each scarification an area of about 3-4 square centimeters. Into these areas the virus is rubbed, using for each small area a quantity about sufficient to vaccinate three children. Two to three centimeter spaces are left between the lesions. The lesions are then allowed to dry and may be covered with sterile gauze or, as in Vienna,1 with a paste made up of beeswax, gum arabic, zinc oxid, water, and glycerin. In some institutes the lesions are left entirely uncovered. Ordinarily within about twenty-four hours after vaccination a narrow pink areola appears about the scratches. Within forty-eight hours the scratches themselves become slightly raised and papular, and within four or six days typical vaccinia vesicles have usually developed. 1 Paul, loc. cit. SMALLPOX 649 To obtain the vaccine from such lesions, the entire operative field is carefully washed with warm water and soap, followed by sterile water. In some cases two per cent lysol is employed, but must again be thor- oughly removed by subsequent washing with sterile water. Crusts, if present, are then carefully picked off and the entire contents of the vesi- icle, sticky serum, and pulpy exudate removed by the single sweep of a spoon-curette. The curetted masses are caught in sterile beakers or tubes and to them is added four times their weight of a mixture of glyce- rin fifty parts, water forty-nine parts, and carbolic acid one part.1 Ger- man workers prefer a mixture of glycerin eighty parts, and water twenty parts, omitting the use of carbolic acid. The glycerinated pulp is allowed to stand for three or four weeks in order to allow bacteria, which are invariably present, to die out. After preservation for such a length of time, moreover, thorough emulsification is obtained more easily than when this is attempted immediately after curettage. At the end of three or four weeks, the glycerinated pulp is thoroughly triturated, either with mortar and pestle or by means of specially constructed trit- urating devices. Pulp so prepared should remain active for at least three months if properly preserved in sealed tubes in a dark and cool place. From the serum oozing from the bases of the lesions, after curettage, bone or ivory slips may be charged for vaccination with dry virus. The glycerinated pulp is put up in small capillary tubes, sealed at both ends, and distributed in this form. Park states that a calf should yield about 10 grams of pulp (which when made up should suffice to vac- cinate one thousand five hundred persons) , and, in addition, about two hundred charged bone slips. The virus may be tested for its efficiency by a variety of methods. Calmette and Guerin 2 inoculate rabbits upon the inner surfaces of the ears and estimate the potency of the virus from the speed of develop- ment and extensiveness of the resulting lesions. Guerin 3 has estimated the potency of virus quantitatively by a method depending upon the inoculation of rabbits with a series of dilutions. Beginning with a mix- ture containing equal weights of glycerin and vaccine pulp, dilutions are made with sterile water ranging from 1 in 10 to 1 in 100. Rabbits are shaved over the skin of the back and 1 c.c. of each of these dilu- tions is rubbed into the shaved areas. Fully potent virus should cause 1 Huddleston, quoted in Park, "Pathogenic Bacteria," N. Y., 1908. 2 Calmette and Guerin, Ann. de l'inst. Pasteur, 1902. 3 Guerin, Ann. de l'inst. Pasteur, 1905. 650 DISEASES OF UNKNOWN ETIOLOGY closely approximated vesicles in a dilution of 1 in 500, and numerous isolated vesicles in a dilution as high as 1 in 1,000. Quantitative estimations of the bacteria in the glycerinated virus should be made by the plating method and the vaccine used only when after several weeks of preservation the numbers of the bacteria have been greatly diminished. In glycerinated pulp the bacteria will often disappear entirely in the course of a month. The vaccine should also be tested for the possible presence of tetanus bacilli, by the inoculation of white mice.1 Vaccination of human beings is performed by slightly scarifying the skin of the arm or leg with a sharp sterile needle or lancet and rubbing into the lesion potent vaccine virus. The virus was formerly dried upon wood, bone, or ivory slips and moistened with sterile water before the operation. At the present day the glycerinated pulp is almost univer- sally employed. That vaccination is of incalculable benefit to the human race is no longer a question of opinion, and opposition to the practice is explicable only on the basis of ignorance. Statistical compilations upon this point are very numerous. It may suffice to select from the voluminous literature a single example, taken from Jurgensen, which embodies the statistics of death from smallpox in Sweden, during the periods immedi- ately preceding and following the introduction of vaccination. In that country the first vaccination was done in 1801. By 1810 the practice was generally in use but not enforced. In 1816 it was legally enforced. The years from 1774 to 1855 can thus be divided into three periods. 1. Prevaccinal period, 1774-1801 (25 years). Deaths smallpox per million inhabitants 2,050 2. Transitional period, 1801-1810 (9 years) 680 3. Vaccination enforced, 1810-1855 (35 years) 169 Prevaccinal period death rate 20.00 per mille. Vaccinal period death rate 0.17 per mille. In considering the benefit of vaccination it must not be forgotten that revaccination is quite as important as the first vaccination, which confers immunity only for from seven to ten years. A child should there- fore be vaccinated soon after birth or at least before the eighth month, and the process should be repeated every seven years thereafter. 1 Paul, loc. cit. CHAPTER XLIX ACUTE ANTERIOR POLIOMYELITIS The disease known as acute anterior poliomyelitis has long been recognized as an acute infectious condition, both because of the charac- teristics of its clinical manifestations and of its epidemic occurrence. For these reasons it was classified with acute infectious diseases by Marie and by Strumpell long before any experimental evidence of in- fection was obtainable. Its contagiousness, while not a proven fact, seemed very likely from the evidence of its mode of spreading and has been removed from the sphere of mere conjecture by the careful study of a Swedish epidemic, comprising one thousand cases, made by Wickman.1 While acute anterior poliomyelitis is almost exclusively a disease of childhood, it is assumed by clinicians that it is etiologically closely re- lated to, possibly identical with, certain diseases of the adult, character- ized by bulbar paralysis and acute encephalitis. Into this category, also, some observers place the condition known as " Landry's paralysis.'' The basis for the identification of these conditions with poliomyelitis lies chiefly in the similarity of the pathological lesions and upon the fact that the last-named diseases occur most often during the course of poliomyelitis epidemics. In consequence of the emphatically expressed opinion as to the infec- tious nature of acute poliomyelitis, the efforts to isolate specific micro- organisms from cases have been many, and numerous microorganisms have been described as the causative agents of this disease. The out- come of all these investigations has been purely negative and the infec- tious agent of acute poliomyelitis still remains undiscovered.2 An important advance in the study of this disease was made in 1908 when Landsteiner and Popper3 succeeded in transmitting it to two monkeys (Cyanocephalus hamadryas and Macacus rhesus) . The trans- i Wickman, quoted from Landsteiner and Popper, Zeit. f. Immunitatsforch., ii, 1909. 2 For literature, see Landsteiner and Popper, loc. cit. 3 Loc. cit. 651 652 DISEASES OF UNKNOWN ETIOLOGY mission was accomplished by intraperitoneal injections of a saline emul- sion of the spinal cord of a child that had died during the fourth day of an attack of infantile paralysis — during the stage of acute fever. The first monkey injected became severely ill six days after the injection and died on the eighth day. The second animal became paralyzed seventeen days after the injection and was killed two days later. Cultural experi- ments with the substance injected were negative, as were also inocula- tion experiments carried out upon guinea-pigs, rabbits, and mice. The histological lesions produced in the inoculated monkeys were similar to those occurring in children afflicted with the disease. An attempt to transmit the disease to another monkey with spinal- cord substance of the animal that was killed resulted negatively. Soon after the successful experiments of Landsteiner and Popper, a similar result was recorded by Knoepfelmacher.1 An attempt to trans- mit the disease from monkey to monkey was again negative. Similar positive inoculation results were published, a little later than this, by Flexner and Lewis 2 in November, 1909, and by Strauss and Huntoon 3 in January, 1910. Flexner and Lewis, in their work, moreover, succeeded in trans- mitting the disease through several inoculation-generations of monkeys, proving thereby that successful inoculation did not depend merely upon the transfer of an unorganized toxic body, but was due to a true infection. The same workers 4 have ascertained that inoculation may be successfully applied not only by the intraperitoneal route but intra- cerebrally, subcutaneously, intravenously, and by the path of the larger nerves. They also proved that not only the brain and cord of afflicted animals contains the virus, but that this may be found, during the early days of the disease at least, in the spinal fluid, the blood, the nasopharyngeal mucosa, and lymph nodes near the site of inoculation. Landsteiner and Levaditi,5 meanwhile, experimenting with the virus independently, succeeded in transferring the disease from one animal to others, demonstrated that the virus could pass through the pores of a Berkefeld filter, and showed that the virus was present in the salivary glands — a fact which may prove of great importance in possibly estab- 1 Knoepfelmacher, Mediz. Klinik, v, 1909. 2 Flexner and Lewis, Jour. Am. Med. Assn., 53, 1909. 3 Strauss and Huntoon, N. Y. Med. Jour., Jan., 1910. * Flexner and Lewis, Jour. Exp. Med., 12, 1909. 5 Landsteiner and Levaditi, Comptes rend, de la soc. de biol., Nov., 1909, and Dec., 1909. ACUTE ANTERIOR POLIOMYELITIS 653 lishing a clew to the mode of contagion among human beings. The same authors, as well as Flexner and Lewis, were able to show that the virus was preservable under glycerin for as long as ten days and retained its virulence for from seven to eleven days when dried. According to Flexner and Lewis the virus remains active, when frozen, for as long as forty days, but is extremely sensitive to heat, being destroyed by a temperature of from 45° to 50° C. maintained for thirty minutes. Experiments aimed at the isolation or even morphological detection of a parasite in the virulent material have been entirely without success. We are, likewise, ignorant of any phenomena which throw light upon possible immunity to the disease. Repeated attacks of the disease in the same human being have not been noted; but this, as Flexner and Lewis point out, may be due to the fact that the epidemics are rare, and individuals once afflicted have passed beyond the susceptible age by the time of the second epidemic. As a matter of fact, however, these workers have not succeeded in reinfecting monkeys that had recovered, and incline to the belief that one attack protects against subsequent infections. Up to the present time monkeys only have responded to experimental inoculation; numerous attempts made upon a variety of other animals have been without success. CHAPTER L YELLOW FEVER Yellow fever is an acute infectious disease which prevails endemi- cally in the tropical countries of the Western Hemisphere, but occurs also along the western coast of Africa and has exceptionally appeared, in epidemic invasions, in the north temperate United States and Europe. Guiteras, as quoted by Osier, classifies the distribution of the disease into three areas of infection. 1. The area in which the disease is never absent, including tropical South American ports and Havana. 2. The area of periodic epidemics, including sea-ports of the tropical Atlantic in America and Africa. 3. The area of accidental epidemics, extending from parallel 45° north latitude to 35° south latitude. In the United States severe epi- demics have frequently occurred in Louisiana, Mississippi, and Alabama, and occasional but severe epidemics have occurred in Philadelphia and Baltimore. The disease occurs spontaneously only in man, and experimental inoculation of lower animals has been successful only in the chimpanzee in a single case reported by Thomas.1 In man afflicted with the malady the clinical picture is one of a rapidly developing fever with severe gastrointestinal symptoms, vomiting of blood, albuminuria, and often active delirium. The mortality is usually high, often reaching eighty per cent or more in the severe epidemics. Etiology and Method of Transmission. — The actual infective agent which causes yellow fever is, as yet, unknown. Numerous researches have been aimed at the elucidation of the problem, and microorganisms, for which etiological significance was claimed, have been isolated from the dejecta, the vomitus, and the secretions of afflicted patients. None of these claims has been supported by convincing proof and none of them has found subsequent confirmation. A few of these claims only have historical importance because of the 1 Thomas, Brit. Med. Jour., 1, 1907. 654 YELLOW FEVER 655 widespread interest they aroused among bacteriologists. Cornil and Babes,1 in 1883, described chained cocci to which they attributed etio- logical significance, but their contentions have remained entirely un- confirmed. Sternberg,2 in 1897, described a colon-like organism, " bacil- lus X," for which he made very conservative claims, which he himself, later, withdrew. The most active discussion was aroused by the announcement of Sanarelli,3 in 1897, that he had discovered, in the blood and tissues of patients dead of yellow fever, a Gram-negative bacillus, which he be- lieved to be the etiological agent of the disease. He based his contention upon the facts that he had isolated the organism from seven cases of yellow fever, had produced symptoms similar to the disease of the human being by the inoculation of pure cultures into dogs, and had obtained agglutination of the bacillus in the serum of convalescent patients. Later he inoculated five human beings subcutaneously with sterilized cultures of this " Bacillus icteroides," and obtained symptoms which he believed simulated closely those of yellow fever. The claims of Sanarelli at first found much apparent confirmation, but later work by Durham and Myers,4 Otto,5 Agramonte,6 and others has definitely refuted his original claims, and there is to-day no scientific basis for the assumption that the Bacillus icteroides has any etiological relationship to the disease. Protozoan incitants, also, have been described by Klebs,7 Schuller,8 Thayer,9 and others, without bringing conviction or even justifying extensive investigation of their claims. While thus the causative agent of yellow fever remains undiscovered, some of its biological properties are known. Reed, Carroll, Agramonte, and Lazear10 were able to show that the infecting agent is present in the blood serum of patients during the first three days of the disease and that it could pass through the pores of Berkefeld filters. Such filtered serum remained infectious for human beings — a fact which de- monstrates that the incitant is extremely small and possibly ultra- 1 Cornil and Babes, Comptes rend, de Tacad. des sci., 1883. 2 Sternberg, Cent. f. Bakt., I, xxii, 1897. 3 Sanarelli, Ann. de 1' inst. Pasteur, 1897, and Cent, f . Bakt. , I, xxii, xxvii, and xxix. 4 Durham and Myers, Thompson Yates Laboratory Reports 3 1902. 6 Otto, Vierteljahrsch. f. gericht. Medizin, etc., 27, 1904. 6 Agramonte, N. Y. Med. News, 1900. » Klebs, Jour. Am. Med. Assn., April, 1898. ■ Schuller, Berl. klin. Woch., 7, 1906. 9 Thayer, Med. Record, 1907. 10 Reed, Carroll, Agramonte, and Lazear, Phila. Med. Jour., 1900. 656 DISEASES OF UNKNOWN ETIOLOGY microscopic. BlOod serum, filtered or unfiltered, becomes non-infectious when heated to 56° C. for ten minutes. Mode of Transmission. — Until comparatively recent years the mode of transmission of yellow fever was not understood and many erroneous theories were prevalent. It was supposed that yellow fever was conta- gious, and transmitted from person to person by direct or indirect con- tact with those afflicted or by fomites. The first to make the definite assertion that yellow fever was transmitted by the agency of mosquitoes was Carlos Finlay. Finlay,1 as early as 1881, advanced the theory that mosquitoes were responsible for the transmission of this disease and, fur- thermore, recognized " Stegomyia f asciata " or " Stegomyia calopus " as the guilty species. Finlay 's opinion, although later proved to be correct, was at first based only upon such circumstantial evidence as the corre- spondence of the yellow-fever zones with the distribution of this species of mosquito and the great prevalence of mosquitoes at times during which epidemics occurred. His theory was, therefore, received with much skepticism and was neglected by scientists until its revival in 1900, when the problem was extensively investigated by a commission of American army surgeons. Reed, Carroll, Agramonte, and Lazear were the members of this commission. The courage, self-sacrifice, and scientific accuracy which characterized the work of these men have made the chapter of yellow fever one of the most brilliant in the annals of American scientific achievement. Their work was much facilitated by the experience of Gorgas 2 and others, who had demonstrated the absolute failure of ordinary sanitary regulations to limit the spread of yellow fever. They began their researches by investigating carefully the validity of Sanarelli's claims as to the etiological significance of his "Bacillus icteroides." The results of this work yielded absolutely no basis for confirmation. They then proceeded to investigate the possibility of an intermediate host. In August, 1900, the commission began its work on this subject by allowing mosquitoes,3 chiefly those of the stegomyia species, to suck 1 Finlay, Ann. Roy. Acad. d. Havana, 1881. 2 Gorgas, Jour, of Trop. Med., 1903. 3 Reed, Carroll, Agramonte, and Lazear, Phila. Med. Jour., Oct., 1900; also Am. Pub. Health Assn. Rep., 1903; Agramonte, N. Y. Med. News, 1900; Reed, Jour, of Hyg., 1902; Reed, Carroll, and Agramonte, Am. Medicine, July, 1901. Boston Med. YELLOW FEVER 657 blood from patients, later causing the same insects to feed upon normal susceptible individuals. The first nine experiments were negative. The tenth, of which Carroll was the subject, was successful. Four days after being bitten by the infected insect Carroll became severely ill with an attack of yellow fever, by which his life was endangered, and from the effects of which he died several years later. On the 13th of September, Lazear, while working in the yellow-fever wards, noticed that a stegomyia had settled upon his hand, and deliber- (a) (6) Fig. 155. — Stegomyia Fasciata. (a) Female, (b) Male. (After Carroll.) ately allowed the insect to drink its fill. Five days later he became ill with yellow fever and died after a violent and short illness. With these experiences as a working basis, the commission now decided upon a more systematic and thoroughly controlled plan of experimentation. In November of the same year, 1900, an experiment station, "Camp Lazear," was established in the neighborhood of Havana, about a mile from the town of Quemados. The camp was surrounded by the strictest quarantine. Volunteers from the army of occupation were called for, and twelve individuals were selected for the camp, three immunes and nine non-immunes. Two of the latter were physicians. The immunes and Surg. Jour., 14, 1901; Carroll, Jour. Am. Med. Assn., 40, 1903; Carrol, Fever" in Mense, " Handbuch der Tropen-Krankheiten," ii. 42 Yellow 658 DISEASES OF UNKNOWN ETIOLOGY and the members of the commission only were allowed to go in and out. All non-immunes who left the camp were prohibited from re-entering and their places taken by other non-immune volunteers. During December, five of the non-immune inmates were successfully inoculated with yel- low fever by means of infected mosquitoes. During January and Febru- ary five further successful experiments were made. Clinical observa- tions were made by experienced native physicians, Carlos Finlay among them, and the patients, as soon as they were unquestionably ill with yellow fever, were removed to a yellow-fever hospital. This was done to prevent the possibility of the disease spreading within the camp it- self. The mosquitoes used for the experiments were all cultivated from the larva and kept at a temperature of about 26.5° C. A further important experiment was now made A small house was erected and fitted with absolutely mosquito-proof windows and doors. The interior was divided by wire mosquito netting into two spaces. With- in one of these spaces fifteen infected mosquitoes were liberated. Seven of these had fed upon yellow-fever patients four days previously; four, eight days previously; three, twelve days previously; and one, twenty- four hours previously. A non-immune person then entered this room and remained there about thirty minutes, allowing himself to be bitten by seven mosquitoes. Twice after this the same person entered the room, remaining in it altogether sixty -four minutes and being bitten fif- teen times. After four days this individual came down with yellow fever. In the other room two non-immunes slept for thirteen nights with- out any evil results whatever. It now remained to show that mosquitoes were the sole means of transmission and to exclude the possibility of infection by contact with excreta, vomitus, or fomites. For this purpose another mosquito-proof house was constructed. By artificial heating its temperature was kept above 32.2° C. and the air was kept moist by the evaporation of water. Clothing and bedding, vessels, and eating utensils, soiled with vomitus, blood, and feces of yellow-fever patients were placed in this house and three non-immune persons inhabited it for twenty days. During this time they were strictly quarantined and protected from mosquitoes. Each evening, before going to bed, they unpacked and thoroughly shooL clothing and bedding of yellow-fever patients, and hung and scattered these materials about their beds. They slept, moreover, in contact with linen and blankets soiled by patients. None of these persons contracted yellow fever. The same experiment was twice re- peated by other non-immunes, in both cases with like negative results. YELLOW FEVER 659 All of the non-immunes taking part in these experiments were American soldiers. Four of them were later shown to be susceptible to yellow fever by the agencies of mosquito infection or blood-injection. The results obtained by the investigations of this commission may be summarized, therefore, as follows: Yellow fever is acquired spontaneously only by the bite of the Stegomyia fasciata. It is necessary that the infecting insect shall have sucked the blood of a yellow-fever patient during the first four or five days of the disease, and that an interval of at least twelve days shall have elapsed between the sucking of blood and the reinfection of an- other human being. Sucking of the blood of patients advanced beyond the fifth day of the disease does not seem to render the mosquito infec- tious, and at least twelve days are apparently required to allow the para- site to develop within the infected mosquito to a stage at which rein- fection of the human being is possible. The results of the American Commission were soon confirmed by Guiteras * and by Marchoux, Salimbeni, and Simond.2 These latter ob- servers, moreover, confirmed the fact that infection could be experi- mentally produced by injections of blood or blood serum taken from patients during the first three days of the disease. They showed that blood taken after the fourth day was no longer infectious: that 0.1 c.c. of serum sufficed for infection and finally that no infection could take place through excoriations upon the skin. They furthermore confirmed the observation of Carroll that the virus of the disease could pass through the coarser Berkefeld and Chamberland filters, — passing through a Chamberland candle " F " but held back by the finer variety known as " B." The fundamental factors of yellow-fever transmission thus discovered, we are in possession of logical means of defense. The most important feature of such preventive measures must naturally center upon the extermination of the transmitting species of mosquito. Stegomyia fasciata or calopus is a member of the group of " Culi- cidse." It is more delicately built than most of the other members of the group culicidae, is of a dark gray color, and has peculiar thorax- markings which serve to distinguish it from other species. The more detailed points of differentiation upon which an exact zoological recog- nition depends are too technical to be entered into at this place. Briefly described, they consist of lyre-like markings of the back, 1 Guiteras, Rev. d. med. trop., Jan., 1901, and Am. Med., 11, 1901. 2 Marchoux, Salimbeni, and Simond, Ann. de l'inst. Pasteur, 1903. 660 DISEASES OF UNKNOWN ETIOLOGY unspotted wings, white stripes and spots on the abdomen, and band- like white markings about the metatarsi and tarsi of the third pair of legs. The peculiar power of transmitting yellow fever possessed by this species is explained by Marchoux and Simond ' by the fact that Stegomyia fasciata is unique among culicidse in that the female lives for prolonged periods after sucking blood. Among other species — Culex fatigans, Culex confirmatus, and most others — the female lays its eggs within from two to eight days after feeding on blood and rarely lives longer than the twelfth day — the time necessary for the develop- ment of the yellow-fever parasite. The limitation of yellow fever to tropical countries 2 is explained by the fact that stegomyia develops only in places where high tempera- tures prevail. The optimum temperature for this species lies between 26° and 32° C. At 17° C. it no longer feeds, and becomes practically paralyzed at 15° C. In order to thrive, the species requires a temperature never going below 22° C. at night and rising regularly above 25° C. during the day. The females only are dangerous as sources of infection. The insect, like Anopheles, has the peculiarity of feeding chiefly at night. Experiments done by Reed, Carroll, Agramonte, and Lazear,3 to ascertain whether the power of infecting was hereditarily transmissible from the mosquito to following generations, were negative. A positive result, however, has been reported by Marchoux and Simond.4 This question must still await more extensive research. Immunity. — Natural immunity against yellow fever was formerly assumed to exist in the negro race. More recent investigations have not borne out this assumption. The negro soldiers of the American army in Guba were afflicted equally with the white troops. The rela- tive immunity of dark-skinned races, however, is explained possibly by the fact that the stegomyia prefers to attack light-colored surfaces. A single attack seems to protect against subsequent infection throughout life. Artificial immunization has, so far, been unsuccessful. Relative immunity was produced, however, by Marchoux, Salimbeni, and Simond, by injections of the serum of convalescents, serum heated to 55° C, and of defibrinated blood preserved for eight days in vessels sealed with vaseline. 1 Marchoux and Simond, Ann. de Finst. Pasteur, 1906. 2 Otto, in Kolle und Wassermann, "Handbuch," etc., 11, Erganzungsband. 8 Loc. cit. * Marchoux and Simond, Comptes rend, de la soc. de biol., 59, 3905. CHAPTER LI MEASLES, SCARLET FEVER, AND FOOT-AND-MOUTH DISEASE MEASLES The causative agent of measles is unknown to the present day, and it would be a thankless task to review the literature of the many attempts to isolate microorganisms from this disease, none of which has resulted in throwing any light on the etiology. Attempts to produce the disease experimentally have frequently been made, the earliest recorded being those of Home of Edinburgh, published in 1759. l Home took blood from the arms of patients afflicted with measles, and caught it upon cotton, and inoculated normal in- dividuals by placing this blood-stained cotton on to wounds made in the arm. Home claimed that in this way he produced measles of a modified and milder type in fifteen individuals. Home's results, how- ever, while at first accepted, were assailed by many subsequent writers and it is by no means certain that the disease produced by him was really measles. A number of other observers after Home attempted experimental inoculation of this disease, and positive results were reported by Stewart of Rhode Island (1799), Speranza of Mantua (1822), Katowa of Hungary (1842), and McGirr of Chicago (1850). The experiments of all these early writers, however, are unsatisfac- tory, owing to the necessarily unreliable technique of their methods. In 1905, Hektoen 2 succeeded in experimentally producing the dis- ease in two medical students by subcutaneous injection of blood taken from measles patients at the height of the disease (fourth day). The experiments were carefully carried out and the symptoms in the sub- jects were unquestionable. They demonstrated beyond doubt that the virus of the disease is present in the blood. Attempts at cultivation carried out with the same blood were entirely negative. It was also shown by Hektoen's experiments that the virus of measles may be kept alive for at least twenty-four hours when mixed with ascitic broth. 1 Home, " Medical Facts and Experiments," Edinburgh, 1759. 2 Hektoen, Jour. Inf. Dis., ii, 1905. 661 662 DISEASES OF UNKNOWN ETIOLOGY SCARLET FEVER (Scarlatina) The etiology of scarlet fever, like that of measles, is still obscure. Streptococci have been found with striking regularity in the throats of scarlet-fever patients, and a large number of investigations have seemed to furnish evidence for the etiological relationship of these microorgan- isms with the disease. According to von Lingelsheim, Crooke as early as 1885 demonstrated the presence of streptococci in the cadavers of scarlet-fever victims. Baginsky and Sommerfeld l in 1900 examined a number of scarlatina cases with reference especially to streptococcus infection, and reported the presence of streptococci in the heart's blood of eight patients who had died after a very acute and short illness. They expressed the belief that the acuteness of the illness and the rapidity of death in these cases precluded the possibility of the streptococci being merely secondary invaders. A large number of other observers have expressed similar opinions, but we can not, as yet, justly conclude that streptococci are actually the etiological agents in this disease. Class 2 in 1899 described a diplococcus which he cultivated from a large number of scarlatina patients and with which he was able to pro- duce exanthemata and acute fever in pigs. Subsequent investigations seem to show that Class was really working with a streptococcus. Moser,3 working in Escherich's clinic, has recently reported the very favorable influence upon the course of scarlet fever of polyvalent streptococcus antisera. This is not really very strong evidence in favor of the streptococcus etiology of the disease, since there is, of course, no doubt that streptococcus infection complicates the disease, and it is to be expected that antistreptococcus serum should, therefore, benefit the patient's condition by combating this complication. Mallory 4 in 1904 published observations on four scarlatina cases on which he bases the belief that scarlatina is caused by protozoa. In the skin, between the epithelial cells, he found small bodies which were easily stained with methylene-blue and which because of their arrange- 1 Baginsky and Sommerfeld, Berl. klin. Woch., 1900. 2 Class, Phila. Med. Jour., iii, 1899. 3 Moser, quoted by Escherich, Wien. klin. Woch., xxiii, 1903. 4 Mallory, Jour. Med. Research, x, 1904. FOOT-AND-MOUTH DISEASE 663 ment and form he interpreted as parasites not very unlike the Plasmo- dium of malaria. Subsequent investigations of Field * and others have failed to substantiate Mallory's conclusions. FOOT-AND-MOUTH DISEASE This malady occurs chiefly in cattle, sheep, and goats, more rarely in other domestic animals. It is characterized by the appearance of a vesicular eruption localized upon the mucosa of the mouth and upon the delicate skin between the hoofs. In the females similar eruptions may appear upon the udders. With the onset of the eruption there may be increased temperature, refusal of food, and general depression. Usually the disease is mild; the vesicles go on to the formation of small ulcers and pustules, and gradually heal with a disappearance of systemic symptoms. Occasionally, however, the disease is complicated by ca- tarrhal gastroenteritis or an inflammation of the respiratory tract, and death may ensue. The disease is unquestionably transmitted from animal to animal by means of virus contained in the vesicular contents. It is also held that infection may take place through the agency of milk. It has been claimed, moreover, though on the basis of insufficient proof, that infec- tion may take place through the air, without actual contact, direct or indirect, with lesions. On rare occasions the disease may be transmitted to man. Such infection, when it does take place, occurs usually among the milkers and attendants in dairies, and is transmitted by direct contact. The course of the disease in man is usually very mild. Mohler states that the disease may be transmitted to man through the consumption of milk from infected animals. He 2 adds, however, that in the United States the disease has been practically eradicated. The causative agent of foot-and-mouth disease is unknown. A num- ber of organisms have been cultivated from the vesicles and mucous membranes of afflicted animals, but none of these could be shown to have etiological significance. Loeffler and Frosch,3 moreover, have demonstrated that the virus contained in the vesicles may pass through the pores of a Berkefeld filter. It must be assumed that the causative 1 Field, Jour. Exper. Med., vii, 1905. 2 Mohler, Bull. No. 41, U. S. Pub. Health and Mar. Hosp. Serv., Wash., 1908. 3 Loeffler und Frosch, Cent. f. Bakt., 1, 1908. 664 DISEASES OF UNKNOWN ETIOLOGY agent of this disease is too small to be within the range of vision of our microscopes. The virus of the disease is easily destroyed by heating to 60° C. and by complete desiccation. It has been observed that one attack of foot-and-mouth disease pro- tects against subsequent attacks. This immunity in most cases lasts for years, though rare cases of recurrence within a single year have been reported. On the basis of such naturally acquired immunity, Loeffler has actively immunized horses and cattle with graded doses of virus obtained from vesicles and with the sera of such animals has produced passive immunity in normal subjects. SECTION V BACTERIA IN AIR, SOIL, WATER, AND MILK CHAPTER LII BACTERIA IN THE AIR AND SOIL BACTERIA IN THE AIR Bacteria reach the air largely from the earth's surface, borne aloft by currents of air sweeping over dry places. Their presence in air, therefore, is largely dependent upon atmospheric conditions; humid- ity and a lack of wind decreasing their numbers, dryness and high winds increasing them. Multiplication of bacteria during transit through the air probably does not take place. Apart from these considerations the presence of bacteria in air also depends upon purely local conditions prevailing in different places. They are most plentiful in densely populated areas and within buildings, such as theaters, meeting halls, and other places where large numbers of people congregate. On mountain tops, in deserts, over oceans, and in other uninhabited regions, the air is comparatively free from bacteria. A classical illustration of this fact is found in the experiments which Pasteur carried out in his refutation of the doctrine of spontaneous generation. Tyndall also, in working upon the same subject, demon- strated this fact. From the surface of the ground and other places where bacteria have been deposited, they reach the air only after complete drying. It is a fact of much importance, both in bacterio- logical work and in surgery, that bacteria do not rise from a moist surface. From dry surfaces they may rise, but only when the air is agitated either by wind or by air-currents produced in other ways. In closed rooms, therefore, even when bacteria are plentiful and the walls and floors are perfectly dry, there is little danger of the inhalation of bacteria unless the air is agitated in some way. The most favorable conditions for the occurrence of many bacteria in air are the existence of a prolonged drought followed by a dry wind. Under such condi- 665 666 BACTERIA IN AIR, SOIL,. WATER, AND MILK tions, even the dark places and unlighted corners of streets and habita- tions are thoroughly dried out, and bacteria are taken up and carried about together with particles of dust. At such times the dangers from inhalation are much multiplied. By experiments made in balloons, it has been found that bacteria are plentiful below altitudes of about fifteen hundred feet and may be present, though much reduced in numbers, as high up as a mile above the earth's surface. The species of bacteria found in the air are, of course, subject to great variation, depending upon locality. Molds and spore-forming bacteria, being more regularly resistant to the effects of sunlight and. diying than bacteria possessing only vegetative forms, are naturally more generally distributed. Out of air thus laden with bacteria, they may again settle when the wind subsides and the air becomes quiescent. The process of settling, however, is extremely slow, since the weight of a bacterium is probably less than a billionth of a gram, and it may be held in suspension in air for considerable periods. Rains, snow, or even the condensation of moisture from a humid atmosphere, hastens this process considerably, and large quantities of bacteria may settle out from air, in a com- paratively short time, in ice chests, in operating rooms, or in other places in which much condensation of water vapor takes place. The importance of the air as a means of conveying disease is still a problem upon which much elucidation is needed. The importance of this manner of conveyance in smallpox, in measles, in scarlet fever, and in other exanthemata, can not be denied. As regards the dis- eases of known bacterial origin, conveyance by air is of importance in the case of tuberculosis, where infection by inhalation may take place, and in the case of anthrax, where inhaled anthrax spores may give rise to the pulmonary form of the disease. The importance of air conveyance for any great distance in pneumonia, in influenza, in diph- theria, and in meningitis is by no means clear and requires much fur- ther study. The expulsion of bacteria from the lungs and naso-pharynx does not take place during simple expiration, since an air-current pass- ing over a moist surface is not sufficient to dislodge microorganisms. Expulsion of bacteria in these conditions must take place together with small particles of moisture carried out in sneezing, coughing, or any forced expiration. The bacteria thus discharged are then subject to the process of drying and often are exposed to direct sunlight for a con- siderable period before they are again taken up in the air. The methods of estimating the bacterial contents of the air are not BACTERIA IN THE AIR AND SOIL 667 entirely satisfactory. The simple exposure of uncovered gelatin or agar plates for a definite length of time, and subsequent estimation of the colonies upon the plates, yield a result which is variable according to the air-currents and the degree of moisture in the atmosphere, and furnish no volume standard for comparative results. The methods which are in use at the present time depend upon the suction of a definite quantity of air by means of a vacuum-pump through some substance which will catch the bacteria. One of the first devices used for this pur- pose was that of Hesse, who sucked air through a piece of glass tubing, about 70 cm. long and about 3.5 cm. in diameter, the inner surface of which had been coated with gelatin in the manner of an Esmarch roll tube. This method is not efficient, since a large number of the bacteria may pass entirely through the tube without settling upon the gelatin. One of the most satisfactory methods at present in use is that in which definite volumes of air are sucked through a sand-filter. Within a small glass tube, a layer of sterilized quartz sand, about 4 cm. in depth, is placed. The sand is kept from being dislodged by a small wire screen. After the air has been sucked through the filter the sand is washed in a definite volume of sterile water or salt solution, and measured fractions of this are planted in agar or gelatin in Petri plates. The colonies which develop are counted. Thus, if two liters of air have been sucked through the filter, and the sand has been washed in 10 c.c. of salt solution, and 1 c.c. of this is planted, with the result of fifteen colonies, then the two liters of air have contained one hundred and fifty bacteria. BACTERIA IN SOIL Besides the normal bacterial inhabitants of the soil, bacteria reach the soil from the air, in contaminated waters, in the dejecta, excreta, and dead bodies of animals and human beings, and in the substance of decaying plants. It is self-evident, therefore, that the distribution of bacteria in soil depends largely upon the density of population and the use of the soil for agricultural or other purposes. Thus, bacteria are most plentiful in the neighborhood of cess-pools or in manured fields and gar- dens. Such conditions, however, may be regarded as abnormal. Even in uncultivated fields there is a constant bacterial flora in the soil which is of great importance in its participation in the nitrogen cycle, a phase of the bacteriology of soil which has been discussed in detail in another 668 BACTERIA IN AIR, SOIL, WATER, AND MILK section. (See page 40.) There are, thus, regular and normal inhabitants of the soil which fulfil a definite function and may be found wherever plant life nourishes. In addition to these, innumerable varieties of sapro- phytes and pathogenic germs may be present, which vary in species and in number with local conditions. Numerous investigations into the actual numerical contents of the soil have been made. Houston * found an average of 1,500,000 bacteria per gram in garden soil, and about 100,000 bacteria per gram in the arid soil of uncultivated regions. Fraenkel,2 in studying the horizontal distribution of bacteria in the earth, has found that they are most numerous near the surface, a gradual diminution occurring down to a depth of about two yards. Beyond this, the soil may be often practically sterile. Pathogenic bacteria may at times be found in the surface layers, and these are often of the spore-bearing varieties. Most important among them from the medical standpoint are the bacillus of tetanus, of malignant edema, and the Welch bacillus. If a guinea-pig is inoculated subcutaneously with an emulsion of garden soil, death will result almost invariably with enormous bloating and swelling of the body due to gas production. This is due to the fact that the spore-bearing, gas-producing anaerobic bacilli are commonly present and are actively pathogenic for these animals. The frequent occurrence of tetanus in persons sustain- ing wounds of the bare feet and hands in fields and excavations, is a matter of common knowledge. Anthrax, also, may be easily conveyed by soil in localities where animals are suffering from this infection. It is not probable that pathogenic germs which are not spore-bearers sur- vive in the soil for any great length of time. Unless the soil is specially prepared by the presence of defecations or other other organic material, the nutrition at their disposal is not at all suitable for their needs, since rapid decomposition of organic materials by saprophytes is always going on in the upper layers. Furthermore, in the deeper layers the con- ditions of temperature and possibly oxygen supply are not at all favorable for the growth of most pathogenic bacteria. Within a short distance from the surface the temperature of the soil usually sinks below 14° or 15° C. An interesting series of experiments by Fraenkel 3 have demonstrated this point. This investigator buried freshly inoculated agar and gelatin cultures of cholera spirilla and of typhoid and anthrax bacilli at differ- ent levels, and examined them for growth after two weeks had elapsed. 1 Houston, Report Med. Officer, Local Govern. Bd., London, 1897. 2 Fraenkel, Zeit. f. Hyg., ii, 1887. s Fraenkel, Zeit. f. Hyg., xi, 1887. BACTERIA IN THE AIR AND SOIL 669 The anthrax bacilli hardly ever showed growth at a depth below about two yards, and cholera and typhoid developed colonies at these depths only during the summer months. Under natural conditions it must be remembered that, at these levels, suitable nutritive material is not found. A consideration of practical importance in this connection is the possibility of infection by means of buried cadavers. An elaborate series of experiments has been carried out upon this subject in Germany, with results which demonstrate that the danger from the burial of persons dead of infectious diseases was formerly much exaggerated. Experi- ments * usually failed to reveal the presence of cholera and typhoid bacilli within two to three weeks after burial, and tubercle bacilli were never found after three months had elapsed. It was only in the case of sporulating microorganisms, such as the anthrax bacillus, that the living incitants could be found for as long as two years after burial. The dangers of infection of human beings through the agency of soil, therefore, are chiefly those arising from the spore-bearing bacteria which are able to remain alive in spite of the unfavorable cultural conditions. It has been found by some observers,2 however, that, under special con- ditions, non-sporulating bacteria, more especially the typhoid bacillus, may remain alive in soil for several months. Although these bacteria, as well as those of cholera, diphtheria, etc., can not proliferate under the conditions found in the soil, the fact that they can remain viable for such prolonged periods in the upper layers suggests the possibility of danger from the use of unwashed vegetables, such as lettuce or radishes or other soil and sewage contaminated food products. The examination of soil for colon bacilli, while demonstrating the presence or absence of manure or sewage contamination, has no practical value, since colon bacilli are found in the dejecta of animals. Examination of specimens of soil for their numerical bacterial contents is extremely unsatisfactory because the bacteria there found can hardly ever all be cultivated together under one and the same cultural environment. A large number are anaerobic, others again thrive at low temperatures, while again another class may require un- usually high temperatures. When such examinations are made, how- ever, specimens of the soil from the surface layer may be taken in a sterile platinum spoon. When taken from the lower levels, a drill, 1 Petri, Arb. a. d. kais. Gesundheitsamt, vii. 2 Firth and Horrocks, Brit. Med. Jour., Sept., 1902. 670 BACTERIA IN AIR, SOIL, WATER, AND MILK such as that devised byFraenkel, maybe used. This consists of an iron rod the lower end of which is pointed. Just above the point a movable collar is fitted. This collar has a slit-like opening. The rod beneath the collar has a deep longitudinal groove corresponding to the slit in the collar. A flange on the collar permits opening and closing of the groove while the instrument is below the ground. The drill is forced into the earth to the desired depth, the groove is opened and earth is forced into the chamber by twisting the rod. In the same manner the groove may be closed. The soil obtained in this way is taken out of the chamber and a definite quantity, say one gram, is dissolved and washed thor- oughly in a measured volume of sterile water or sterile salt solution. Fractions of this are then mixed with the culture medium, plated, and cultivated aerobically or anaerobically as desired. CHAPTER LIII BACTERIA IN WATER All natural waters contain a more or less abundant bacterial flora. This fact, combined with our knowledge that the incitants of several epidemic diseases and a number of minor ailments of a diarrheal char- acter are water borne, gives the bacteriological investigation of water a place of great importance in hygiene. In nature, there are very few sources of water supply which do not contain bacteria of one or another description. While pathogenic bacteria are usually not present except in those waters which are directly contaminated from human sources, a thorough understanding of the quantitative and qualitative bacterial contents of all natural waters is necessary in order that we may in- telligently gather comparative data as to the fitness of any given water for human consumption. The gross appearance of water is rarely, if ever, an indication of its danger. The turbid waters of running streams in sparsely populated agricultural districts may be safe, while perfectly clear well waters subjected to the dangers of contamination from neighboring sinks or cess-pools may contain large numbers of pathogenic germs. The diseases which are known to be more directly connected with water supply are typhoid fever and cholera. Typhoid germs discharged from the bowel or from the urine of typhoid patients or convalescents may be carried by the sewage or from the neighboring soil into a river or lake and lead to infection of the population deriving its drinking water from this source. There are a great many investigations on record in which severe typhoid epidemics have been traced to such sources. In the case of cholera, where the germs are discharged from the bowels in enormous numbers, conveyance of the disease by water is even more apparent, and the discoverers of the cholera germ themselves, in their early work in Egypt and India, were able to isolate the bacteria from contaminated water supplies. In regard to the less clearly understood diarrheal diseases, dysen- tery, cholera infantum, etc., the direct relation to water supply has not 671 672 BACTERIA IN AIR, SOIL, WATER, AND MILK been so definitely proven, and can be deduced only from the diminu- tion of such infections after the substitution of pure water for the pre- viously used impure supply. It is thus seen that water bacteriology is one of the most important branches of the science of hygiene, and has led, and is constantly leading, to enormous diminution of the death rate in all communities where an intelligent study of the conditions has been made. The bacterial purity of natural waters, although dependent upon special and local conditions in relation to possible contamination, differs widely, according to the source from which such waters are derived. Rain water and snow water are usually contaminated with bacteria by the dust which they gather on their way to the ground, and are especially rich in bacteria when taken during the first few hours of a rain or snow storm when the air is still dusty and filled with floating particles. During the later hours of prolonged storms, rain water and snow water may be comparatively sterile. Miquel,1 who made exten- sive experiments in France on the bacterial contents of rain water, found that in country districts, where the air is less dusty, rain water contained an average of about 4.3 bacteria to the cubic centimeter. The bacterial counts of snow water are usually somewhat higher than those of rain. The waters of streams, ponds, and lakes are usually spoken of as surface waters, and these of all natural supplies contain the largest num- ber of bacteria. In each case, of course, the quantitative and quali- tative bacterial flora of such waters is intimately dependent upon the conditions of the surrounding country, the density of the population, and the relation of these waters to sewage. It is also, and to no less important degree, dependent upon weather conditions, the influence of light and temperature, and the food supply contained within the waters in the form of decayed vegetation. In all such surface waters there is constantly going on a process of self-purification. The chief factor in this process is sedimentation. In stagnant ponds and lakes with but sluggish currents there is a constant sedimentation of the heavier particles, which gradually but steadily leads to a diminution of the number of bacteria in the upper layers of the water. In rivers where sedimentation is to a certain extent prevented by rapidity of current, the effectiveness of such sedimentation is, of course, entirely dependent upon the speed of the current. 1 Miquel, Revue d'hyg., viii, 1886. BACTERIA IN WATER 673 The influence of light in purifying surface waters is important chiefly in ponds, lakes, and sheets of water which expose a large surface to the sunlight, and where the surroundings are such that the sun has free access throughout the day. According to the researches of Buchner/ the bac- tericidal effects of light penetrate through water to a depth of three feet. The effects of temperature in purifying surface waters under natural conditions are probably not great. There is, however, a general tendency toward diminution of the bacterial flora as the temperature of such waters becomes lower. The presence of protozoa in natural waters as purifying agents has recently been emphasized by Huntemuller,2 who claims that these organ- isms by phagocytosis greatly diminish the number of bacteria in any given body of water. It is self-evident that the number of bacteria in any of these waters is never constant, since all factors which tend to a diminution or increase in volume, such as drying up of tributary streams or the occurrence of heavy rains, would lead to differences of dilution which would materially change numerical bacterial estimations. The influence of rains, furthermore, may be a twofold one. On the one hand, heavy rain-falls, by washing a large amount of dirt into the rivers and lakes from the surrounding land, have a tendency to increase the bacterial flora. This influence would be especially marked in all bodies of water which are surrounded by cultivated land where manured fields and grazing-meadows supply a plentiful source of bacteria. On the other hand, in regions where arid, uninhabited lands surround any given river or lake, the rain would carry with it very little living con- tamination and would act chiefly as a diluent and diminish the actual proportion of bacteria in the water. Another and extremely important source of water supply is that spoken of technically as " ground water." The " ground waters " include the shallow wells employed in the country districts, springs, and deep or artesian wells. The shallow wells that form the water supply for a large proportion of farms in the eastern United States are usually very rich in bacteria and are by no means to be regarded as safe sources, ex- cept in cases where great care is observed as to cleanliness of the sur- roundings. In such wells the filtration of the water entering the well may be subject to great variation according to the geological con- ditions of the surrounding ground. The proximity of barns and sinks may lead to dangerous contamination of such waters. 1 Buchner, Arch. f. Hyg., xvii, 1895. 2 Huntemuller , Arch. f. Hyg., liv, 1905. 43 674 BACTERIA IN AIR, SOIL, WATER, AND MILK Examinations by various bacteriologists have shown that such wells frequently contain as many as five hundred bacteria to the cubic centi- meter. Perennial spring waters are usually pure. Examinations by the Mas- sachusetts State Board of Health 1 in 1901 showed an average of about forty bacteria per cubic centimeter. As sources of water supply for general consumption, however, springs can hardly be very important because of the insignificant quantities usually derived from them. Of much greater practical importance are deep artesian wells, which, under ordinary conditions, are largely free from bacterial contamination. Quantitative Estimations of Bacteria. — The quantitative estima- tion of bacteria in water is of necessity inexact, because of the difficulty of always securing fair average samples from any large body of water, and because of the large variations in cultural requirements of the flora present in them. All these methods depend upon colony enumera- tion in plates of agar or gelatin, preferably of both. For the sake of gaining some basis of comparison for results which, at best, can never be entirely accurate, an attempt has been made by the American Public Health Association 2 to standardize the methods of analysis. Water for analysis should always be collected in clean, sterile bottles, preferably holding more than 100 c.c. If water is to be taken from a running faucet or a well supplied with piping, it is important that it should be allowed to run for some time before the sample is taken, in order that any change in bacterial content occurring inside of the pipes may be excluded. It is obvious that in water pipes through which the flow is not constant, bacteria may find favorable conditions for growth and such a sample would not represent fairly the supply to be tested. When the water is taken from a pond, lake, or cess-pool, the bottle may be lowered into the water by means of a weight, or may be plunged in with the hand, great care being exercised not to permit contamina- tion from the fingers to occur. A number of devices for collecting water have been originated, a very excellent one for small samples, by Stern- berg,3 consisting of a small glass bulb with a capillary neck which is sealed while the bulb is hot. This is attached to a rod, and a wire noose is fastened to the neck of the bulb. When immersed in water the neck » Mass. State Bd. of Health, 33d Annual Report for 1901. 2 Fuller, Trans. Amer. Public Health Assn., xxvii, 1902. Report of Com. on Standard Methods of Water Analysis. Jour. Inf. Dis., Suppl. 1, 1905. 3 Sternberg, "Manual of Bact." BACTERIA IN WATER 675 may be broken off by means of the wire, and water will be forced into the bulb to satisfy the vacuum. After the water has been collected it is important to plate it before the bacteria in it have a chance to increase. The changes taking place during transportation, even when packing in ice has been resorted to, have been found by Jordan and Irons l to be considerable. It is impera- tive, therefore, that plating of the water, if possible, shall not be delayed for longer than one or two hours after collection. Before plating, the bottle containing the sample should be shaken at least twenty-five times in order to distribute the bacterial contents evenly. The quantities to be plated will depend to a certain extent upon the probability of there being a large or small number of bacteria. If less than two hundred are suspected, 1 c.c. of the water should be taken out of the bottle with a sterile pipette and placed in a sterile Petri dish. To this is added 10 c.c. of gelatin at a temperature of about 30°C, or of agar at a temperature of not over 40° C. The water is thoroughly mixed with the medium by repeated tilting of the plate, and finally al- lowed to harden in the regular way. If unusual pollution or other data lead to a suspicion that the bacterial count is apt to be extremely high, it is advisable to dilute the sample of water with sterile water before plating. The reason for mixing water and medium in the Petri dish, rather than in the tube, as was formerly done, is the fact that in the pouring of the mixture from the tube a certain amount of residuum is left which naturally leads to a diminution in the actual number of colonies developing in the plate. The gelatin is incubated at 20° C, the agar at 37.5° C. The gelatin and agar which are used should be made according to the standard methods recommended by the American Public Health Associa- tion. The gelatin should be made of meat infusion and not of meat ex- tract, and should contain one per cent of Witte's pepton and ten per cent of the best so-called French brand of gelatin. It should not soften when kept at a temperature of 25° C. The agar medium should also be made of meat infusion and of the highest grade of commercial thread agar. For general purposes the standard reaction of media should be one per cent acidity, but for long-continued work on water from the same source the optimum reaction should be ascertained and adhered to, and differences from the standard reaction should be mentioned in the report. 1 Jordan and Irons, Reports of the Amer. Pub. Health Assn., xxv, 1889. 67G BACTERIA IN AIR, SOIL, WATER, AND MILK Incubation of the gelatin should be continued for forty-eight hours in an atmosphere saturated with moisture. When agar is used, incubation for twenty-four hours is usually sufficient, and it is advantageous to employ Petri dishes supplied with porous earthenware covers.1 Simple inversion of the Petri dishes when placed in the incubator obviates the necessity of using the porous covers. In counting, the ordinary counting plates divided into 1 cm. squares may be used, but, whenever possible, all the colonies in a plate should be counted. The value of the quantitative estimation of bacteria in water is only a comparative one, and no arbitrary standards can be established for the purity of water on this basis. In general it may be said that water containing one hundred bacteria to the cubic centimeter or less is apt to be from a deep source and comparatively pure; that water containing five hundred bacteria to the cubic centimeter or over is open to suspicion, and that any water containing over one thousand to the cubic centimeter is probably from a polluted source. At the same time it is quite impossible to draw any direct conclusions from numerical colony counts, and all such results must be carefully weighed in the balance with qualitative analyses and chemical tests, and knowl- edge of environmental conditions. Qualitative Bacterial Analyses of Water. — Of far greater importance than quantitative analysis is the isolation of bacteria either distinctly pathogenic, such as the cholera spirillum and the typhoid bacillus, or of other species probably emanating from contaminating sources, such as aB. coli. Unfortunately there are no reliable methods by which typhoid and cholera germs can be isolated from water with any degree of regularity or certainty. Although frequently the isolation of such organisms is possible, a negative result in these cases is by no means eliminative of their presence. The isolation of typhoid bacilli from water is very difficult, chiefly because of the great dilution which contaminations undergo upon enter- ing any large body of water. The difficulty of isolating typhoid bacilli, even from the stools of infected patients, makes it clear that such diffi- culties are enhanced when a considerable dilution of the excreta takes place. Furthermore, water is by no means a favorable medium for the typhoid bacillus. Russell and Fuller 2 have shown that typhoid bacilli J Hill, Jour. Med. Res., xiii, 1904. 2 Russell and Fuller, Jour. Inf. Dis., Suppl. 2, 1908. BACTERIA IN WATER 677 may die in water within five days, and it is unquestionable that the rate of increase of these bacteria is by no means equal to that of many other microorganisms for which polluted water at the temperature en- countered in streams and lakes forms a much more favorable medium. It is thus clear that even in infected waters the number of typhoid bacilli encountered can never be very great.1 A large number of methods for the isolation of the typhoid bacillus from water have been devised. Most of the media used are identical with those employed for the isolation of these bacteria from the stools. These media have been discussed in the chapter dealing with the typhoid bacillus. Drigalski 2 has reported a method for which he claims considerable success in isolating typhoid bacilli from water, which depends upon the motility of the organisms. One- to two-liter samples of water are taken and allowed to stand at room temperature in high jars for one or two days. Small quantities are then removed from the surface and planted on Wurtz's lactose litmus agar. The method depends upon the probabil- ity of the settling out of non-motile organisms and the possibility, there- fore, of getting motile organisms only in the plates. Parietti 3 and others have attempted to eliminate other organisms by adding phenol and hydrochloric acid to neutral broth, in the hope that the high acidity and the antiseptic qualities of the phenol will destroy more delicate organisms than the typhoid bacillus. Inasmuch as B. coli, however, withstands these reagents as well as B. typhosus, or even better, these methods have not met with great success. A method which has proved useful in the hands of Adami and Chapin4 is one which depends upon the phenomenon of agglutination. These authors collect water in two-liter specimens and to each two liters add 20 c.c. of one per cent glucose broth. These samples are incubated at 37.5° C. for twenty-four hours, and at the end of this time quantities of 10 c.c. are withdrawn and placed in test tubes. To each of these tubes potent typhoid serum, preferably diluted 1 : 60, is added, and whenever agglutination occurs the flocculi are washed and plated on various media for identification. Vallet and others have attempted to precipitate typhoid bacilli out of water by chemical means. The purpose of these methods has been 1 Laws and Anderson, Rep. of Med. Officer, London County Council, 1894. 2 Drigalski, Arb. a. d. kais. Gesundheitsamt, xxiv, 1906. 3 Parietti, Rev. d'igiene e san. pub., 1890. * Adami and Chapin, Jour. Med. Res., xl, 1904. 678 BACTERIA IN AIR, SOIL, WATER, AND MILK to entangle the bacteria in an inert precipitate, and thus concentrate the bacteria in water for purposes of cultivation. Vallet's method is as follows: To two liters of water add 20 c.c. of a 7.75 per cent solution of sodium hyposulphite and 20 c.c. of a 10 per cent solution of lead nitrate. When the precipitate has settled, the clear supernatant fluid is decanted and the precipitate dissolved in a saturated sodium hypo- sulphite solution. This clear solution is then plated. Willson x has modified this method by adding to the water 0.5 gms. of alum to each liter. A precipitate is formed which may either be allowed to settle or may be brought down by centrifugalization. The supernatant fluid is removed and the precipitate plated. In actual work many of the methods which are aimed purely at B. coli may lead to success in the isolation of B. typhosus, because of the similarity of the two organisms in their reaction to definite media. Thus, the method of Jackson,2 who employs one per cent of lactose in pure ox-bile for the isolation of B. coli, has occasionally led to the simul- taneous isolation of B. typhosus. The isolation of the vibrio of cholera is less difficult than that of B. typhosus, primarily because of the much greater numbers of these microorganisms discharged into sewage. The number of cholera spirilla in the excreta of cholera patients is enormously higher than is that of B. typhosus in the stools of typhoid-fever patients. It is not infre- quent, therefore, that the source of a cholera infection may be directly traced to the water supply. Koch,3 the discoverer of the cholera vibrio, has indicated a method which has frequently found successful applica- tion. To 100 c.c. of the infected water are added one per cent of pepton and one per cent of salt. This mixture is then incubated at 37.5° C. and after ten, fifteen, and twenty hours, specimens from the upper layers are examined microscopically and are plated upon gelatin. Upon the weak pepton solution cholera spirilla increase very rapidly at in- cubator temperatures and then when plated in gelatin the detection of characteristic colonies is comparatively easy. Because of the great difficulties outlined above in isolating specific pathogenic germs from polluted waters, bacteriologists have attempted to form an approximate estimate of pollution by the detection of other microorganisms which form the predominating flora of sewage. 1 Willson, Jour, of Hyg., v, 1905. 2 Jackson, Journ. of Inf. Dis., Suppl. 2, 1907. » Koch, Zeit. f. Hyg., xiv, 1893. BACTERIA IN WATER 679 Chief among these is B. coli. The isolation and numerical estimation of B. coli in polluted water has been for a long time one of the criteria of pollution. This so-called colon test, however, should always be ap- proached with conservatism and should never be carried out qualita- tively only. Careful quantitative estimation should be made in every case. B. coli in water is by no means always the result of human con- tamination, since this bacillus is found in great abundance in the intestines of domestic animals. According to Poujol, B. coli does not even always point to fecal contamination, since this author was able to find the bacillus in the water of a number of wells where no possible contamination of any sort could be traced. Prescott 1 explains this, as well as similar cases, by the fact that organisms of the colon group may occasionally be parasitic upon plants. The opinions of hygienists are widely at variance as to the value of the colon test. While the discovery of isolated bacilli of the colon group may therefore be of little value, it is nevertheless safe to follow the opinion of Houston,2 who states that the discovery of B. coli in considerable numbers invariably points to sewage pollution, and that the absolute absence of B. coli is, as a rule, reliable evidence of purity. For the purpose of isolating B. coli from water, a large number of methods have been devised. In examining sewage or other pol- luted waters in which the number of colon bacilli is comparatively large, the direct use of lactose litmus agar plates yields excellent results. Varying quantities of water may be added to this medium and the development of red colonies at incubator temperatures usually indicates the presence of bacilli of this species. These colonies may be fished and further identified. Copeland 3 has proposed the addition of two-tenths per cent of phenol to this medium in order to inhibit other bacteria. In water less grossly polluted, some method of enrichment must be employed in order to increase the number of bacteria so that they may be found in plates. For this purpose glucose bouillon in fermentation tubes, according to the method of Theobald Smith, may be employed.4 In this medium, at a temperature of 37.5° C.,the colon bacilli grow with 1 Prescott, Science, xv, 1903. 2 Houston, Rep. Medical Officer, Local Gov. Board, London, 1900. 3 Copeland, Jour. Boston Soc. Med. Sciences, 1901. 4 Prescott, Science, xvi, 1902. 680 BACTERIA IN AIR, SOIL, WATER AND MILK great speed and transplants to plating media may be taken after eight or more hours' incubation. A medium which has found successful application is that proposed by Jackson.1 This medium, as stated above, consists of undiluted ox- bile, to which has been added one per cent of lactose. For quantitative estimation of colon bacilli in water, Theobald Smith 2 has proposed the use of dextrose bouillon in fermentation tubes, to which are added varying quantities of water, ranging from 0.001 to 1 c.c. The appearance of gas in any of these tubes would indicate the presence of B. coli, and the number can be approximately computed from the smallest quantity of water by which gas formation has been produced. According to Irons,3 the presence of B. coli in such fermentation tubes may be determined without actual isolation and cultivation of the organism, by estimating the comparative amount of carbon dioxide in the gas. Whenever carbon dioxide forms approximately thirty-three per cent of the gas present, Irons concludes that B. coli is present. Jackson 4 believes when lactose ox-bile is used that twenty-five per cent of gas within seventy -two hours may be regarded as positive for B. coli. 1 Jackson, Jour. Inf. Dis., Suppl. 2, 1907. 2 Th. Smith, 13th Ann. Rep. N. Y. S. Board of Health. 3 Irons, Trans. Amer. Pub. Health Assn., xxvi, 1900. * Jackson, "Biol. Studies of Pupils of W. T. Sedgwick," Boston, 1906. CHAPTER LIV BACTERIA IN MILK AND MILK PRODUCTS, BACTERIA IN THE INDUSTRIES BACTERIA IN MILK The universal use of cows' milk as a food, especially for the nourish- ment of infants, has necessitated its close study by bacteriologists and hygienists. It furnishes an excellent culture medium for bacteria and is, therefore, pre-eminently fitted to convey the germs of infectious dis- ease. The many changes which take place in milk, furthermore, and which add or detract from its nutritive value, are due largely to bacterial growth and have been elucidated by bacteriological methods. Within the udder of the healthy cow, milk is sterile. If pyogenic or systemic diseases of bacterial origin exist in the cow, the milk may, under certain circumstances, be infected even within the mammary glands. In the milk ducts and in the teats, however, even in perfectly healthy animals, a certain number of bacteria may be found. For this reason, even when all precautionary measures are followed, the milk as received in the pail is usually contaminated. As a matter of fact, the anatomical location of the udder and the mechanical difficulties of milking make it practically impossible to collect milk under absolutely aseptic conditions, and, under the best circumstances, from 100 to 500 microorganisms per c.c. may usually be found in freshly taken milk. Withdrawn under conditions of ordinary cleanliness, the bacterial contents of milk are considerably higher than this. After the proc- ess of milking, in spite of all practicable precautions, the chances for the contamination of milk are considerable; but even could these be eliminated, the bacterial contents of a given sample would ultimately rapidly increase because of the rich culture medium which the milk provides for bacteria. Whether large increases shall take place or not de- pends, in the first place, upon the temperature at which milk is kept, and, in the second place, upon the length of time which intervenes before its consumption. Though fresh milk possesses slight bactericidal powers,1 1 Rosenau and McCoy, Jour. Med. Res., 18, 1908. 681 682 BACTERIA IN AIR, SOIL, WATER, AND MILK these are by no means sufficient to be of practical importance in the inhibition of bacterial growth. Kept at or about freezing-point, the bacterial contents of milk do not appreciably increase. At higher tem- peratures, however, a rapid propagation of bacteria takes place which, especially during the summer months, speedily leads to enormous num- bers. In a case reported by Park,1 where milk, containing at the first examination 30,000 microorganisms per cubic centimeter, was kept at 30° C. (86° F.) for twenty-four hours, the count at the end of this time yielded fourteen billions of bacteria for the same quantity. It is of much importance, therefore, that the cleanliness of dairies, of cattle, and in the handling of milk should be reinforced by the utmost care in chilling and icing during shipment and before sale. Because of its great importance, especially for the health of the chil- dren in large cities during the summer months, the milk question has, of recent years, received much attention from health officers. In the city of New York, the question has been made the subject of many careful studies by Park 2 and his associates. Commissions, working in Chicago,3 Boston,4 and other large towns, have placed the sale of milk under more or less exact bacteriological supervision. Park has de- termined that the milk, as sold in New York stores during the cold weather, not infrequently averages seven hundred and fifty thousand bacteria per cubic centimeter; during the hot summer months, the bacterial contents of similar milk not infrequently average one million and more, for the same quantity.5 In consequence of these and other researches, large dairies have introduced bacteriological precautions into their method of milk production. They have attempted the reduc- tion of the bacterial contents of milk by scrupulous cleanliness of the barns and of the udders and teats of the cow, by the elimination of dis- eased cattle, by sterilization of the vessels in which the milk is received, and of the hands of the milker; also by the immediate filtering and cooling of the milk and the packing of the milk cans in ice, where they remain until delivered to the consumer. In consequence of such measures, it is possible for cities to be supplied with milk containing no more, and often less, than fifty thousand bacteria to the cubic centimeter. A standard of cleanliness has been established in various towns by the » Park, W. H., "Pathogenic Bacteria," New York, 1905, p. 463. 2 Park, Jour, of Hygiene, 1, 1901. 3 Jordan and Heinemann, Rep. of the Civic Federation of Chicago, 1904. 4 Sedgwick and Batchelder, Bost. Med. and Surg. Jour., 126, 1892. 5 Escherich, Fort. d. Medizin, 16 and 17, 1885. BACTERIA IN MILK 683 introduction of the so-called " certified milk/' which, by the New York Milk Commission, is required to contain no more than thirty thousand bacteria per cubic centimeter. Great stress is laid upon such numerical counts simply in that they are approximate estimates of cleanliness. Most of the bacteria, however, contained in milk are non-pathogenic, and numbers much larger than the maximum required for certified milk may be present without actual disease or harm following its consump- tion. The various species of bacteria which may be found in milk include almost all known varieties. Whether there are special, so-called milk bacteria or not is a question about which investigators have expressed widely differing opinions. It is probable that many of the species, formerly regarded as specifically belonging to milk, are there simply by virtue of their habitual presence in fodder, straw, or bedding, or upon cattle. It is likely, furthermore, that some of these species are found with great regularity because of their power to outgrow other species under the cultural conditions offered them in milk. Under normal conditions, milk always undergoes a process which is popularly known as souring and curdling. This is due to the forma- tion of lactic acid from the milk sugar and is the result of the enzymatic activities of several varieties of bacteria commonly found in milk. Most common among these bacteria is the so-called Bacillus lactis aerogenes, an encapsulated bacillus closely related to the colon-bacillus group. (See page 451.) The transformation of the lactose into lactic acid may occur either directly by hydrolytic cleavage: C12 H22 0„ + H20 = 4 C3 H6 03; or indirectly through a monosaccharid, Ca H22 On + H2 O = 2 C6 H12 06 - 4 C3 H6 03. Other microorganisms which may cause lactic-acid fermentation in milk are the so-called Streptococcus lacticus, the common pyogenic streptococcus, the Staphylococcus aureus, Bacillus coli communis and communior, and many other species. Most commonly concerned in lactic- acid production, however, according to Heinemann,1 are the two first- mentioned, Bacillus lactis aerogenes and Streptococcus lacticus. The secret of the regularity with which some of these bacteria are present in sour milk is probably found in the ability of these varieties to withstand a much higher degree of acidity of the culture medium than other species. 1 Heinemann, Jour, of Inf. Dis., 3, 1906. 684 BACTERIA IN AIR, SOIL, WATER, AND MILK In consequence, they are able to persist and develop when cultural con- ditions are absolutely unsuited to other bacteria. Consequent upon acidification of the milk by lactic-acid formation, there is coagulation of casein. Casein precipitation/ however, may also be due to a non-acid coagulation caused by a bacterial ferment. Casein precipitated in this way may be redissolved by a bacterial trypsin or casease, produced by the same or other bacteria, the milk again becoming entirely liquid, transparent, and of a yellowish color. The casein precipitated by lactic-acid formation, however, is never thus redissolved, because the high acidity does not permit the pro- teolytic ferments to act.1 Butyric-acid fermentation in milk, a common phenomenon, is also an evidence of bacterial growth. As a rule, it is produced by the anaerobic bacteria, and is a process developing much more slowly than other fer- mentations. A large number of bacteria have been described which are capable of producing such changes, the chemical process by which they are produced being, as yet, not entirely understood. It is probable that the process takes place after hydrolysis of the disaccharid some- what according to the following formula: C6 H12 0Q = C4 H8 02 + 2 C02 + 2 H2. Special bacteria have been described in connection with this form of milk fermentation,2 most of them non-pathogenic. It is unquestionable, however, that many of the well-known pathogenic bacteria, such as Bacillus aerogenes capsulatus, Bacillus cedematis maligni, possess the power of similar butyric-acid formation. While less commonly observed in milk, because milk is rarely kept long enough to permit of the action or development of these enzymes, the butyric-acid fermentation is of importance in connection with butter, where it is one of the causes pro- ducing rancidity. Alcoholic fermentation may take place in milk as a result of the ac- tivities of certain yeasts. Upon the occurrence of such fermentations depends the production of kefyr, koumys, and other beverages which have been in common use for many years, especially in the region of the Caucasus. The characteristic quality of these beverages is contrib- uted by the feeble alcoholic fermentation produced by members of the saccharomyces group, but side by side with tnis process lactic-acid forma- i Conn, Exper. Stat. Rep., 1892. 2 Schattenfroh und Grasberger, Arch. f. Kyg.', 37, 1900. BACTERIA IN MILK 685 tion also takes place. Beijerinck,1 who has carefully studied the so- called kefyr seeds, used for the production of kefyr in the East, has isolated from them a form of yeast similar in many respects to the ordinary beer yeast, and a large bacillus to which he attributes the lactic-acid formation. Occasional but uncommon changes which occur in milk lead to the formation of the so-called " slimy milk," yellow and green milk, and bitter milk. These may be due to a number of bacteria. A microorgan- ism which is commonly found in connection with the slimy changes in milk is the so-called Bacillus lactis viscosus. According to the researches of Ward,2 this microorganism is frequently derived from water and it is the water supply which should attract attention whenever such trouble occurs in dairies. The so-called blue, green, and yellow changes are usually due to chromogenic bacteria, such as Bacillus cyanogenes, Bacillus prodigiosus, and others. "Bitter milk," a condition which has occasionally been observed epi- demically, is also the consequence of the growth of microorganisms. Conn,3 in 1891, isolated from a specimen of bitter cream a diplococcus which occasionally forms chains and which in sterilized milk develops rapidly, producing an extremely bitter taste. The organism of Conn differs from a similar diplococcus described by Wagmann 4 in that it possesses the ability of producing butyric acid. Milk in Relation to Infectious Disease. — As a source of direct in- fection, milk is second only to water, and deserves close hygienic at- tention. A large number of infectious diseases have been traced to milk, although the actual proof of the etiological part played by it in such cases has often been difficult to adduce and has necessarily been indirect. Nevertheless, even when indirect proof only has been brought, it has been sufficiently convincing to necessitate the most careful investigation into milk supplies whenever epidemics of certain infectious maladies occur. Typhoid-fever epidemics have been frequently traced to milk in- fection, and, in this disease, milk is, next to water, the most frequent etiological factor. Schuder,5 in an analysis of six hundred and fifty typhoid epidemics, found four hundred and sixty-two attributed to 1 Beijerinck, Cent. f. Bakt., vi, 1889. 2 Ward, Bull. 165, Cornell Univ. Agri. Exp. Stat., 1899. 3 Conn, Cent. f. Bakt., ix, 1891. 4 Wagmann, Milchztg., 1890. 5 Schuder, Zeit. f. Hyg., xxxviii. 1901. 686 BACTERIA IN AIR, SOIL, WATER, AND MILK water, one hundred and ten to milk, and seventy -eight to all other causes. Trask l compiled statistics of one hundred and seventy-nine typhoid epidemics supposed to have been caused by milk, in various parts of the world. In all such epidemics the origin of infection was generally trace- able to diseased or convalescent persons employed in dairies, to con- taminated well water used in washing milk utensils, or to the use of cans and bottles returned from dwellings where typhoid fever had existed. Actual bacteriological proof of the infectiousness of milk by the isolation of Bacillus typhosus is rare, but has been accomplished in isolated in- stances. In the case of one epidemic, Conradi 2 isolated the bacillus from the milk on sale at a bakery at which a large number of the infected individuals had purchased their milk. The examination of market milk at Chicago, through a period of eight years, revealed the presence of typhoid bacilli but three times. In spite of the few cases in which actual bacteriological proof has been brought, it is not unlikely that careful and systematic researches would reveal a far greater number, since many writers have shown that typhoid bacilli may remain alive in raw milk for as long as thirty days,3 and may actively proliferate in the milk during this time. One peculiarity of epidemics which may aid in arousing the suspicion that they have originated in milk is that, in such cases, women and children are far more frequently attacked than men.4 A feature which adds considerably to the dangers of milk infection is the unfortunate absence of any gross changes, such as coagulation, by the growth of typhoid bacilli. Scarlet fever,a though as yet of unknown etiology, has in many cases been traced indirectly to milk infection. Trask has collected fifty-one epidemics of scarlet fever presumably due to milk. In one epidemic occurring in Norwalk, Conn.,6 twenty-nine cases were distributed among twenty-five families living in twenty-four different houses. The indi- viduals affected did not attend the same school, and were of entirely different social standing, the only factor common to all of them being the milk supply. i Trask, Bull. No. 41, U. S. Pub. Health and Mar. Hosp. Serv., Wash. 2 Conradi, Cent. f. Bakt.. I, xl, 1905. 3 Heim, Arb. a. d. kais. Gesundheitsamt, v. « Wilckens, Zeit. f. Hyg., xxvii, 1898. 5 Trask, loc. cit. e Herbert E. Smith, Rep. Conn. State Bd. of Health, 1897. BACTERIA IN MILK 687 Diphtheria has been frequently traced to the use of infected milk. In most of the epidemics reported as originating in this way, the proof has been necessarily indirect. In two out of twenty-three epidemics reported by Trask, however, Bacillus diphtherise was isolated from the milk directly. The ability of the Klebs-Loeffler bacillus to proliferate and remain alive for a long while in raw miik has been demonstrated by Eyre 1 and others. Whether or not cholera asiatica may be transmitted by means of milk has been a disputed question. Hesse 2 claims that cholera spirilla die out in raw milk within twelve hours. This statement, however, has not been borne out by other observers.3 Unquestionable cases of direct transmission of cholera by means of milk have been reported by a num- ber of writers, notably by Simpson.4 The relation of milk to the diarrheal diseases of infants has, of late years, received a great deal of attention. In large cities, during the summer months, numerous cases of infantile diarrhea among bottle- fed babies occur, which, in many instances, are attributed to feeding with contaminated milk. Park and Holt,5 who have made extensive re- searches upon this question in New York City, have come to the con- clusion that the harmful effects of contaminated milk upon babies can not be ascribed to any given single microorganism in the milk. Specifically toxic properties were found by these writers for none of the one hundred and thirty-nine different species of bacteria isolated from unsterilized milk. It is unlikely, therefore, that the diarrheal diseases among babies have a uniform bacteriological cause. Whether or not these diarrheal conditions depend entirely upon the bacterial contents of milk or, in a large number of cases at least, upon the inability of the child to digest cows' milk because of chemical conditions, must be left undecided. Park and Holt, in analyzing their extensive data, conclude that milk containing " over one million bacteria to the cubic centimeter is certainly harmful to the average infant. " The significance of the presence of streptococci in milk/as an element of danger, has recently received much attention in the literature. Heine- mann,6 who has made a careful comparison of Streptococcus lacticus » Eyre, Brit. Med. Jour., 1899. 2 Hesse, Zeit. f. Hyg., xvii, 1894. 5 Basenau, Arch. f. Hyg., xxiii, 1895. 4 Simpson, Indian Med. Gaz., 1887. 6 Park and Holt, Arch, of Ped., Dec, 1903. 6 Heinemann, Jour. Inf. Dis., 3, 1906. 688 BACTERIA IN AIR, SOIL, WATER, AND MILK (formerly spoken of as Bacillus acidi Iactici [Kruse] ) , with other strep- tococci, has shown that, essentially, this streptococcus does not differ from streptococci from other sources, and is practically indistinguish- able by cultural methods from Streptococcus pyogenes. Similar com- parisons made by Schottmuller,1 Muller,2 and others have led to like re- sults. Since streptococci may be found in milk from perfectly normal cows and are almost regularly associated with lactic-acid fermenta- tion, it is unlikely that these microorganisms hold any specific relation- ship to disease. The presence of pus cells and leucocytes in milk, together with streptococci, was also formerly regarded as of great importance. Enumerations of leucocytes in milk were first made by Stokes and Weggefarth.3 Their method of enumeration consisted in centrifugaliz- ing a definite volume of milk, spreading the entire sediment over a definite area on a slide, and counting the leucocytes found in a number of fields. Calculations from this may then be made as to the number of leucocytes per cubic centimeter. This method, and modifications of it, have been used by a large number of observers, but the value of the con- clusions drawn from them has been much exaggerated. Normal milk may contain leucocytes in moderate numbers, and importance may be attached to such leucocyte counts only when their number largely ex- ceeds that present in other specimens of perfectly normal milk. When- ever such high leucocyte counts are found, of course, a careful veteri- nary inspection and examination for pyogenic disease should be made upon the cows. Foot-and-mouth disease, an infectious condition prevailing among cattle, characterized by a vesicular rash on the mouth and about the hoofs, has, in a number of cases, been definitely shown to be transmitted to man through the agency of milk. Notter and Firth 4 reported an epidemic occurring among persons supplied with milk from a single dairy in which foot-and-mouth disease prevailed among the cows. In this epidemic, two hundred and five individuals were affected with vesic- lar eruptions of the throat, with tonsillitis and swellings of the cervical lymph nodes. Similar cases have been reported by Pott.5 Although anthrax has never been definitely shown to have been 1 Schottmtiller, Munch, med. Woch., 1903. ^MiXller, Arch. f. Hyg., lvi, 1906. 3 Stokes and Weggefarth, Med. News, 91, 1897. 4 Notter and Firth, quoted from Harrington, " Theory and Practice of Hygiene." 5 Pott, Munch, med. Woch., 1899. BACTERIA IN MILK 689 conveyed by milk, Boschetti 1 has succeeded in isolating living anthrax bacilli from a sample of milk examined two weeks after its withdrawal from the cow. Milk and Tuberculosis. — The question of the conveyance of tuber- culosis by means of milk is a subject which, because of its great importance, has been extensively investigated by bacteriologists. A large number of observers have succeeded in proving the presence of tubercle bacilli in the milk of tuberculous cows by intraperitoneal in- oculation of rabbits and guinea-pigs with samples of milk. Such posi- tive results have been obtained by Bang,2 Hirschberger,3 Ernst,4 and many others. A number of these observers, notably Ernst, have shown that tubercle bacilli may be present in the milk without tuberculous dis- ease of the udders. In an examination of the milk supply of Washington, D. C, by J. G. Anderson,5 six and seventy -two hundreths per cent of the samples of milk examined contained tubercle bacilli. The path of entrance of the bacilli from the cow into the milk has long been a subject of controversy. That the bacilli may easily enter the milk, when tuberculous disease of the udder is present, stands to reason and is universally conceded. It is now believed also, on the basis of much experimentation, that in systemically infected cows tubercle bacilli may pass through the mammary glands into the milk, without evidence of local disease in the secreting gland. An experi- ment performed by the Royal British Tuberculosis Commission 6 illus- trates this point. A cow, injected subcutaneously with tubercle bacilli behind the shoulder, began to discharge tubercle bacilli in the milk within seven days after inoculation and continued to do so until death from generalized tuberculosis. Milk may become indirectly contaminated, furthermore, with tubercle bacilli emanating from the feces of cows. Schroeder and Cotton 7 have shown that tubercle bacilli may be present in the feces of cattle at a stage so early in the disease that diagnosis can be made only by a tuberculin test. Whether or not contaminated milk is common as an etiological 1 Boschetti, Giorn. mecL vet., 1891. 2 Bang, Deut. Zeit. f. Tierchem., xi, 1884. 3 Hirschberger, Deut. Arch. f. klin. Med., xliv, 1889. 4 Ernst, H. C, Amer. Jour. Med. Sci., xcviii, 1890. 5 Anderson, Bull. No. 41, U. S. Pub. Health and Mar. Hosp. Serv., Wash., 1908. 6 Quoted from Mohler, P. H., and Mar. Hosp. Serv. Bull. 41, 1908. 7 Schroeder and Cotton, Bull. Bureau Animal Industry, Wash., 1907. 44 690 BACTERIA IN AIR, SOIL, WATER, AND MILK factor in human tuberculosis, must be considered at present as an un- settled question. Behring, at the Congress of Veterinary Medicine, at Cassel, in 1903, advanced the view that pulmonary tuberculosis in adults may be a late manifestation of a milk infection contracted dur- ing infancy. He stated as his own opinion, moreover, that most cases of tuberculosis in man are traceable to this origin. The problem is as difficult of solution as it is important. In bottle-fed infants, infection by means of milk unquestionably occurs with considerable frequency. Smith,1 Kossel, Weber, and Huess,2 and others, have isolated tubercle bacilli of the bovine type from the mesenteric lymph nodes of many infected children. Animal experimentation has, furthermore, revealed that lesions in the mesenteric nodes, as well as later in the bronchial lymph nodes, may occur as a consequence of feeding tubercle bacilli, without any demonstrable lesions in the intestinal mucosa. It is thus certain that infection by the ingestion of tuberculous milk may occur, especially among young children who, as is well-known, are com- paratively susceptible to bacilli of the bovine type. Whether or not such infection will account for many cases of tuberculosis in adults is a ques- tion which, for final solution, will require much more investigation. The sole reliable method of approaching it lies in determining the type, human or bovine, of the bacilli present in a large number of cases. Ex- perience thus far seems to indicate that the bovine type is comparatively rare in the pulmonary disease of adults. The value of the tuberculin reaction for diagnosis, and the elimination of all cattle showing a positive reaction, for the prevention of tubercu- losis, can not be overestimated. The failure of the test in diseased animals is rare, and an accurate diagnosis can be established in over 90 per cent of diseased animals.3 The assertion that the cattle are permanently injured by tuberculin injections is without scientific basis. If this test were conscientiously carried out, and infected cattle elim- inated, the dangers from bovine bacillus infection would be practically eliminated, for there are but few instances in which science has been able to furnish such definite information for absolute protection. It is need- less to say, however, that the carrying out of such precautions is subject to great expense and great difficulties of organization. Dairy inspection is practiced in the vicinity of many of our larger 1 Smith, Trans. Assn. Amer. Physic, 18, 1S03. 2 Kossel, Weber, and Huess, Tuberkul. Arb. a. d. kais. Gesundheitsamt, 1904, 1905, Hft. 1 and 3. 3 Mohler, loc. cit. BACTERIA IN MILK 691 cities, and the movement is daily gaming ground. Until fully estab- lished, however, upon a financial basis which brings the best products within the means of the poorer classes, other inexpensive measures to render milk safe must often be resorted to. Sterilization by high temperatures is objected to by pediatricians because of the physical and chemical changes produced in the milk which are said to detract from its nutritive value. The development of scurvy and rickets in infants has often been attributed to the use of such milk. These objections, however, do not apply to the use of milk which has been subjected to the process of " pasteurization." By this term is meant the heating of any substance to 60° C. for twenty to thirty minutes. The process, first devised by Pasteur for the purpose of destroying germs in wine and beer in which excessive heating was supposed to injure flavor, brings about the death of all microorganisms which do not form spores — in other words, of all the bacteria likely to be found in milk which can give rise to infection per os. At the same time the chemical and physical constitution of the milk is not appreciably changed, at least not to an extent which renders it less valuable as a food. Statistics by Park and Holt 1 have shown strikingly the advantages of pasteurized over raw milk in infant feed- ing. Of fifty-one children fed with raw milk during the summer months, thirty-three had diarrhea, two died, and only seventeen remained entirely well. Of forty-one receiving pasteurized milk, but ten had diarrhea, one died, and thirty-one remained entirely well throughout the summer. The actual diminution of the living bacterial contents of milk by pasteurization is enormous, the milk so treated often con- taining not more than one thousand, usually less than fifteen thou- sand, living bacteria to each cubic centimeter. Methods of Estimating the Number of Bacteria in Milk. — In estimating the number of bacteria in milk, colony counting in agar or gelatin plates is resorted to. Great care must be exercised in obtaining the specimens. If taken from a can, the contents of the can should be thoroughly mixed, since the cream usually contains many more bacteria than the rest of the milk. The specimen is then taken into a sterile test tube or flask, If the' milk is supplied in an ordinary milk bottle, this should be thoroughly shaken before being opened, and the specimen for exam- ination taken out with a sterile pipette. Dilutions of the specimen can then be made in sterile broth or salt solution. If an initial dilution 1 Park and Holt, loc. cit. G92 BACTERIA IN AIR, SOIL, WATER, AND MILK of 1 : 100 is made, quantities ranging from 1 c.c. to 0.1 c.c. of this will furnish 0.01 c.c. to 0.001 c.c. of the milk, respectively. Inoculation of properly cooled tubes of melted neutral agar and gelatin, with varying quantities of these dilutions, are then made and plates poured. After twenty-four to forty-eight hours at room temperature or in the in- cubator, colony counting is done as described upon page 161, and the proper calculation is made. In samples in which few bacteria are expected, direct transference of 1/20 or 1/40 of a c.c. of the whole milk into the agar may be made. This saves time but is less accurate than the method given above. Bacteria and Butter. — Butter is made from cream separated from milk either by standing or by centrifugalization. After this, the cream is agitated by churning, which brings the small fat-globules into mutual contact, allows them to adhere to each other and form clumps of butter. It has been a matter of common experience, however, that unless the cream is allowed to "ripen" for a considerable period before churning, the resulting butter lacks the particular quality of flavor which gives it its market value. The interval of ripening, at first a necessity upon small farms where cream must be collected and allowed to accumulate, has now been recognized as an essential for the production of the best grades of butter, and it has been shown that the changes taking place in the cream during this period are referable to the action of bacteria. Cream, which before the ripening process contains but 50,000 bacteria to each cubic centimeter, at the end of a period of "ripening " will often contain many millions of microorganisms. At the same time, the cream becomes thick and often sour. The species of bacteria which take part in this process and which, therefore, must determine to a large extent the quality of the end prod- uct, are various and, as yet, incompletely known. Usually some variety of lactic-acid bacilli is present and these, as in milk, outgrow other species and, according to Conn,1 are probably essential for "ripening." It would be of great practical value, therefore, if definite pure cultures of the bacteria which favor the production of agreeable flavors could be distributed among dairies. In Denmark this has been attempt- ed by first pasteurizing the cream and then adding a culture of bacteria isolated from "favorable" cream. These cultures, delivered to the dairyman, are planted in sterilized milk, in order to increase their quan- tity, and this culture is then poured into the pasteurized cream. In 1 Conn, "Agricultural Bacteriology," Phila., 1901. BACTERIA IN MILK 693 most cases, these so-called " starters " are not pure cultures, but mixtures of three or more species derived from the original cream. Adverse accidents in the course of butter-making, such as " souring " or "bittering" of butter, are due to the presence of contaminating, probably proteolytic, microorganisms in the cream during the process of " ripening.' .' As a means of transmitting infectious disease, butter is of importance only in relation to tuberculosis. Obermuller,1 Rabinowitch,2 Boyce,3 and others, have repeatedly found tubercle bacilli in market butter, and Mohler,4 Washburn, and Rogers have recently shown that these bacilli could remain alive and virulent for as long as five months in butter kept at refrigerator temperature. The acid-fast butter bacillus, described by Rabinowitch as similar to the true Bacillus tuberculosis, shows decided cultural and morphological differences from the latter. Bacteria and Cheese. — The conversion of milk products into cheese consists in a process of proteid decomposition which, by its end products, leucin, tyrosin, and ammonia compounds, largely determines the cheese- flavors. The production of cheese, therefore, is due to the action of proteolytic bacterial enzymes 5 and the variety of a cheese is largely determined by the microorganisms which are present and by the cultural conditions prevailing. The sterilization of cream, or the addition of antiseptics, absolutely prevents cheese production. The organisms which are concerned in such processes have been extensively studied and attempts have been made, with moderate success, to produce a definite flavor with pure cultures. In the production of cheese the two varieties, hard and soft cheeses, depend not so much upon the bacterial varieties as upon the differ- ences in the treatment of the curds before bacterial action has begun. In the former case, a complete freeing of the curds from the whey furnishes a culture medium which is comparatively dry and of almost exclusively proteid composition; in the latter, retention of whey gives rise to cultural conditions in which more rapid and complete bacterial action may take place. The holes, which are so often observed in some of the hard cheeses, are due to gas production during the process of " ripening." 1 Obermuller, Hyg. Rundschau, 14, 1897. 2 Rabinowitch, Zeit. f. Hyg., xxvi, 1897. 3 Boyce and Woodhead, Brit. Med. Jour., 2, 1897. * Mohler, U. S. P. H. and Mar. Hosp. Serv. Bull. 41, 1908. 8 Freudenreich, Koch's Jahresbericht, etc., 135, 1891. 694 BACTERIA IN AIR, SOIL, WATER, AND MILK As to the varieties of microorganisms present in various cheeses, much careful work has been done. Duclaux 1 attributed the "ripening" of some of the soft cheeses to a microorganism closely related to Bacillus subtilis. V. Freudenreich 2 in part substantiated this, but laid particular stress upon the action of Oidium lactis, a mold, and upon several varieties of yeast. Conn,3 more recently, in a bacteriological study of Camembert cheese, has demonstrated that the production of this cheese depends upon the united action of two microorganisms, one an oidium, like the Oidium lactis of Freudenreich, which is found chiefly in the interior softened areas, the other a mold belonging to the penicillium variety, found in a matted felt-work over the surface and penetrating but a short distance. In spite of the scientific basis upon which the work of these men and of others has seemed to place cheese production, attempts at uniformity in cheese production have met with almost insuperable obstacles because of the presence of a variety of adventi- tious microorganisms which, depending in species and proportion upon the local conditions under which the various cheeses have been produced, have added minor characteristics of flavor which have determined market value. Occasional failure of good results in cheese production 4 is due to contamination with other chromogenic or putrefactive bacteria. In its relationship to the spread of infectious disease, cheese is relatively unimportant except in regard to tuberculosis. Typhoid and other non-spore forming pathogenic germs can not survive the condi- tions existing during cheese-ripening for any length of time. Tubercle bacilli, both of the human and bovine types, have been found in cheese by Harrison 5 and others, and Galtier has shown experimentally that tubercle bacilli may remain alive and virulent in both salted and un- salted cheese for as long as ten days. THE LACTIC-ACID BACILLI AND METCHNIKOFF'S BACTERIO- THERAPY A problem which has occupied clinical investigation for many years is that of gastrointestinal autointoxication. There are a number of conditions occurring in man, in which symptoms profoundly affecting i Duclaux, "Le Lait," Paris, 1887. 2 V. Freudenreich, Cent. f. Bakt., II, i, 1895. s Conn, Bull. Statis. Agri. Exp. Stat. 35, 1905. 4 Beijerinck, Koch's Jahresber, etc., 82, 189. 5 Harrison and Galtier, quoted from Mohler, U. S. Pub. H. and Mar. Hosp. Serv., Hygiene Lab. Bull. 41, 1908. LACTIC-ACID BACILLI 095 the nervous system, the circulation, and, in a variety of ways, the entire body, can be clinically traced to the intestines, and can, in many cases, be relieved by thorough purgation and careful diet. In some of these conditions, specific microorganisms can be held accountable for the diseases (B. enteritidis, B. botulinus, etc.). In other cases, however, etiological investigations have met with but partial success because of the large variety of microorganisms present in the intestinal tract and because of the complicated symbiotic conditions thereby produced. Intestinal putrefaction, recognized as the cardinal feature of such maladies, has been attributed to Bacillus proteus vulgaris,1 to Bacillus aerogenes capsulatus, to Bacillus putrificus,2 and to a number of other bacteria, but definite and satisfactory proof as to the etiological im- portance of any of these germs has not yet been advanced. The fact remains, however, that, whatever may be the specific cause, the disease itself, a grave and often fatal affliction, may be clinically traced, in a number of cases, to the absorption of poisons from the intestinal canal, and it is more than likely that these poisons are the products of bacterial activity. Reason dictates, furthermore, that the bacteria primarily responsible for the production of these toxic substances do not belong to the varieties which attack carbohydrates only, but must belong to that class of aerobic and anaerobic germs which possess the power of breaking up proteids — in other words, the bacteria of putrefaction. On the basis of the mutual antagonism existing in culture between many acid-producing bacteria and those of putrefaction — a phenomenon recognized by some of the earliest workers in this field, many investigators have suggested the possibility of combating intestinal putrefaction by adding acid-forming bacteria together with carbohydrates to the diet of patients suffering from this condition. The first to suggest this therapy wTas Escherich 3 who proposed the use, in this way, of Bacillus lactis aerogenes; with the same end in view, Quincke,4 a little later, suggested the use of yeasts — Oidium lactis. The reasoning underlying these attempts was meanwhile upheld by experiments carried out both in vitro and upon the living patient. Thus Brudzinski 5 was able to demonstrate that Bacillus lactis aerogenes, in culture, inhibited the development of certain races of the proteus species and succeeded in 1 Lesage, Rev. de med., 1887. 2 Tissier, Ann. de l'inst. Pasteur, 1905. s Escherich, Therapeut. Monatshefte., Oct., 1887. 4 Quincke, Verhandl. des Congress f. Inn. Med., Wiesbaden, 1898. 5 Brudzinski, Jahrbuch f. Kinderheilkunde, 52, 1900 (Erganzungsheft). 696 BACTERIA IN AIR, SOIL, WATER, AND MILK obtaining markedly favorable results by feeding pure cultures of Bacillus lactis aerogenes to infants suffering from fetid diarrhea. Similar ex- periments 1 carried out with the Welch bacillus (aerogenes capsulatus) and Bacillus coli, however, had no such corroboratory results, since this anaerobe possesses a considerable resistance against an acid reaction. In considering the difficulties of the problems involved in this question, y \ v V Fig. 156. — Bacillus bulgaricus. it occurred to Metchnikoff2 that much of the practical failure of therapy, based upon the principles stated above, might be referred to insufficient powers of acid production on the part of Bacillus coli, Bacillus lactis aerogenes, and other germs previously used. In searching for more pow- erful acid producers, his attention was attracted to Bacillus bulgari- 1 Tissier and Martelly, Ann. de l'inst. Pasteur, 1906. 2 Metchnikoff, "Prolongation of Life," G. P. Putnam's Sons, N. Y.; also in "Bac- teriotherapie," etc. " Bibliotheque de therapeutique," Gilbert and Carnot, Paris, 1909. BACTERIA IN THE INDUSTRIES 697 cus, isolated from milk by Massol * and Cohendy2 in 1905. This bacillus, according to the researches of Bertram! and Weisweiller,3 produces as much as 25 grams of lactic acid per liter of milk. In addition to this, it manufactures, from the same quantity of milk, about 50 centigrams of acetic and succinic acids and exerts no putrefactive action upon pro- teids. Added to these characters, it is especially adapted to therapeutic application by its complete lack of pathogenicity. The administration of the bacillus to patients suffering from intestinal putrefaction, first suggested by Metchnikoff in 1906, has, since that time, been extensively practiced and often with remarkable success. In spite of sharp criticism, especially by Luersen and Kiihn,4 who deny much of the antiputrefactive activity of the bacillus, the treatment of Metchnikoff has found many adherents, upon the basis of purely clinical experiment. It is not possible to review completely the already ex- tensive literature. Among the more valuable contributions may be mentioned the articles by Grekoff,5 by Wegele,6 and by Klotz.7 In Metchnikoff s experiments and in the work of his immediate successors, the bacillus was used either in milk culture or in broth in which it was induced to grow in symbiosis with other microorganisms. Recently, North 8 has suggested the use of Bacillus bulgaricus in parts of the body other than the digestive tract. His work was made feasible by the discovery that the bacillus could be cultivated in dex- trose-pepton broth to which calcium carbonate has been added, after the manner recommended by Hiss. With such cultures, applied in the form of a spray, inflammations of the ear, nose, throat, genitourinary tract, etc., have been treated, many of them with success. BACTERIA IN THE INDUSTRIES Bacteria and Tobacco. — In the manufacture of tobacco, the har- vested leaves are first dried and then heaped up in large masses in which the tobacco undergoes fermentation. During this fermentation, which 1 Massol, Revue medicale de la Suisse romande, 1905. 2 Cohendy, Comptes rend, de la soc. de biol., 60, 1906. 3 Bertrand and Weisweiller, Ann. de Tinst. Pasteur, 1906. 4 Luersen and Kiihn, Cent. f. Bakt., II, xx, 1908. 5 Grekoff, "Observations cliniques sur l'effet du lact. agri.," etc., St. Petersburg, 1907. 0 Wegele, Deut. med. Woch., xxxiv, 1908. 7 Klotz, Zentralbl. f. innere Med., 1908. s North, Med. Record, March, 1909. 698 BACTERIA IN AIR, SOIL, WATER, AND MILK goes on at temperatures varying from 50° C. to 60° C, carbohydrates are split up and much nicotin is destroyed.1 The end products consist largely of C02 and various organic acids, butyric, formic, succinic, etc. During the fermentation, bacteria of many varieties are found in the heaps of tobacco leaves and many attempts have been made to deter- mine flavors artificially by inoculating tobacco leaves of a poorer quality with cultures obtained from the finer Havana grades. Suchsland2 and others, who have attempted this, claim to have obtained marked im- provements in domestic products by this method. The bacteria found in tobacco fermentation belong to many varieties. Some, of these are closely related to the proteus and subtilis groups. Others are distinctly thermophilic, an attribute required by the high temperatures attained in the fermenting tobacco leaves. It is probable that the tobacco flavors can not be regulated by bacteriological methods alone, since it has been shown by Loew 3 that an important factor in the tobacco fer- mentation is contributed by the leaf-enzymes, which, of course, depend intimately upon soil and climatic conditions. Opium Productions. — In the preparation' of opium for smoking pur- poses, the raw product is subjected to a prolonged period of fermentation by which the carbohydrates in the material are destroyed. According to various observers, the process is carried out in most cases by a species of aspergillus. Indigo Production. — Indigo, which is obtained from the plants "Isatis tinctoria" and "Indigofer tinctoria," is not present, as such, in the plants. In some of these it is found in the form of indican, in others, as indoxyl. It .has been shown by Alvarez and others that the oxida- tion of indican and indoxyl into indigo-blue is carried out largely by bacterial oxydases. Sterilized indigo plants do not produce the blue color. Alvarez 4 has isolated a bacillus closely related to the Bacillus mucosus capsulatus group, to the action of which he attributes this oxidation. Bacteria in the Tanning of Hides. — Raw animal hides are subject to decomposition until treated by a process known as tanning. This consists first in the depilation of the dried and salted skins, either by partial putrefaction in an atmosphere saturated with water vapor or by chemical treatment with solutions of milk of lime. After this, the 1 Behrens, quoted from Fliigge, "Die Mikroorganismen," Bd. 1, Leipzig, 1896. 2 Suchsland, Ber. der Deut. botan. Ges., ix. 3 Loew, Rep. U. S. Dep. Agriculture, 59, 1899. * Alvarez, Comptes rend, de Tacad. des sci. vol. 105. BACTERIA IN THE INDUSTRIES 699 tanning proper consists in subjecting the skins to prolonged immersion in solutions made up according to a large variety of formulae — the principle of all of which, however, seems to be found in the mixing of various organic ingredients, such as bran-mash, oak-bark, and often dried animal excreta, in which fermentation and acid production occurs. According to Haenlein * this acidification is the essential process by which the leather is sterilized and rendered soft and pliable. This author has described a microorganism, Bacillus corticalis, which he found regu- larly present in fir-tree bark and to which he ascribes the acid fermenta- tion occurring in tanning liquors in which this ingredient is employed. Wood,2 who has worked extensively upon the subject, has attempted with some success to substitute pure cultures for the old uncertain chance mixtures employed. In spite of these investigations, however, while we must acknowledge the probable importance of bacteria in the tanning process, the subject is by no means upon a scientific or exact basis. 1 Haenlein, Cent. f. Bakt. II, i, 1895. 2 Wood, Jour. Soc. Chem. Industry, 1895, 1899. INDEX OF AUTHORS Abbott, 97, 326 Abel, 189, 448, 450, 549 Abel and Claussen, 575 achard and bensaude, 430 Adami, 392 Adami and Chapin, 677 Agramonte, 655, 656 Albrecht and Ghon, 375, 377, 378, 547, 551 Alvarez, 698 Alvarez and Tavel, 497 Anderson, J. G., 689 Andrewes and Horder, 350 Aristotle, 2 Arloing, 63 Arloing and Chauveau, 469 Arloing, Cornevin and Thomas, 466 Arloing, Leclainche and Vallee, 466 Arning, 502 Aronson, 90, 347, 348, 349, 350 Arrhenius and Madsen, 211 Arthus, 297 Arustamoff, 607 ASAKAWA, 214 ASCOLI AND FlGARI, 201 axenfeld, 538 Babes, 11, 503, 506, 542, 601, 602, 611, 636 Babes and Lepp, 197 Baginsky, 344, 458, 511 Baginsky and Sommerfeld, 344, 345, 662 Bail, 292, 293, 330 Bail and Petterson, 292 Bail and Weil, 293 Baldwin, 489 Bandelier and Roepke, 492 Bandi and Simonelli, 586 Bang, 689 Banzhaf, 219, 220 Barker and Cole, 424 Bartel, 484 Basenau, 430, 687 Bauer, 268 Baumgarten, 106, 477, 483 Baumler, 532 Beck, 488, 494, 506 Beck, M., Kolle and Wassermann, 509 Beckmann, 78 Behrens, 698 v. Behring, 76, 77, 78, 79, 195, 205, 221, 295, 484, 485, 559 v. Behring and Kitasato, 196, 198 v. Behring and Kitashina, 295 v. Behring and Wernicke, 196, 198 Beijerinck, 26, 27, 685, 694 Belfanti, 456 Belfanti and Carbone, 200 Beljaeff, 198 Beraneck, 487 Berestnew, 606, 608, 614 Bertarelli, 579, 592 Berthelot, 54 Bertrand and Weisweiller, 697 Besredka, 298, 302, 346, 416 Besredka and Steinhardt, 299 Bezancon, 335 Bezancon, Griffon and Le Sourd, 541 Bienstock, 393, 479 Biggs and Park, 231 Billroth, 7 Binaghi, 368 Birch-Hirschfeld, 192, 392 Bitter, 31, 168, 497 Blaise and Sambac, 64 Bloodgood, 472 Blumer, 411 bogart and bernard, 201 Boland, 569 Bollinger, 610 Bolton, 24 701 702 INDEX OF AUTHORS Bordet, 200, 224, 225, 228, 232, 236, 240, 242, 243, 338, 347, 367, 537 Bordet and Gay, 261 Bordet and Gengou, 245, 261, 262, 535 Bordoni-Uffreduzzi, 359 Borrissow, 328 Borsiekow, 139 Boschetti, 689 BoSTROEM, 612 boyce and woodhead, 693 Brau and Denier, 579 Brauell, 6 Brieger, 185, 195, 415 Brieger and Boer, 459, 460, 514 Brieger and Cohn, 459, 460 Brieger and Frankel, 513 Brieger and Kempner, 476 Brill and Libman, 569 Bruce, 542 Bruck, 215 Brudzinski, 695 v. Brunn, 89 Buchner, 22, 63, 152, 186, 198, 203, 224, 225, 389, 673 Buchner and Hahn, 487 Buchner and Meisenheimer, 50 Budd, 399 BtDINGER, 327 Buerger, 99, 350, 351, 355 BUFFON, 4 Bullock and Atkin, 282, 284 Bullock and Hunter, 571 Bullock and Western, 282 Bumm, 380, 381 bunge and trautenroth, 106 burkholtz, 63 Busse, 619 bctschli, 11 Buxton, 428, 429 Buxton and Coleman, 180, 431 Cagniard-Latour, 3 Calkins, 638, 645 Calmette, 199, 203, 488 Calmette and Guerin, 647, 649 Canalis, 189 Cantacuzene, 280 Cantani, 531 Capaldi, 135 Carlo and Rattone, 454 Carnot and Fournier, 363 Carriere, 47 Carroll, 657 Carter, 615 Caste llani, 234, 405, 603 Certes, 35 Chamberland 4.nd Roux, 194, 559, 561 Chantemesse, 412, 424 Chantemesse and Widal, 418 Charrin, 567, 568 Charrin and Roger, 228 Chauveau, 195, 564 • Christen, 67, 69 Christmas, 385 Chudiakow, 27 Citron, 273, 294 Clairmont, 449 Clarke, 645 Class,, 662 COBBETT, 516 COHENDY, 697 Cohn, 5, 36 Coleman \nd Buxton, 405 Coles, 498 Conn, 415, 684, 685, 692, 694 Conradi, 138, 405, 441, 686 CONRADI AND DrIGALSKI, 135 copeland, 679 Cornet, 344, 483 Cornil and Babes, 655 Councilman, 645 Councilman, Mallory and Wright, 374, 376, 377 courmont and doyen, 461 Cramer, 21 Creite, 458 Curtis, 619 Cushing, 431 cushing and llvingood, 392 D'Arsonville and Charrin, 65 Davaine, 6, 553 Dean, 283 Debrand, 459 Delezenne, 201 Deneke, 581 INDEX OF AUTHORS 703 Denys, 347, 348, 485, 487, 492 Denys and Leclef, 281, 347 Denys and Marchand, 347 Denys and Van de Velde, 331 De Schweinitz and Dorset, 22, 484 Deslongchamps, 326 De Toma, 482 Deutsch and Feistmantel, 292 Dieudonne, 64, 216, 556 Dobbin, 472 Doerr, 186, 299, 441 doerr and russ, 303 donath and landsteiner, 248 Donitz, 188, 218 doutrelepont, 498 Dreyer and Madsen, 209, 215 Drigalski, 677 Drigalski and Conradi, 406 dubarre and terre, 495 Duclaux, 694 Ducrey, 540 v. Dungern, 201, 242, 411 Dunham, 386, 470, 471, 574 Durham, 428, 432 Durham and Myers, 655 Dusch, 4 dutton and todd, 597 Eberth, 399 Ehrenberg, 2 Ehrlich, 7, 104, 187, 193, 199, 203, 204, 205, 206, 209, 210, 213, 238, 461, 479 Ehrlich, Kossel and Wassermann, 205 Ehrlich and Morgenroth, 226, 241, 242, 243, 246 Ehrlich and Sachs, 242, 243 Eichstedt, 627 Eisenberg, 570 ElSENBERG AND VoLK, 420 v. elsler and pribram, 457 Ejkmann, 47, 49 Elser, 377 Elser and Huntoon, 376, 378, 379, 387 Elsner, 408 Endo, 136 Engelmann, 27, 61 Eppinger, 563, 608 Epstein, 77, 178, 607 van Ermengem, 14, 101, 430, 473 Ernst, 11, 569, 689 Escherich, 344, 389, 394, 451, 507, 682, 695 v. Esmarch, 68, 147, 149 Evans and Russell, 91 Ewing, 586, 645 Eyre, 166, 687 Eyre and Washburn, 360 Farnet, 413 Faure-Beaulieu, 384 Fehleisen, 336, 342, 343 Fermi and Pernossi, 460 Ferran, 28, 195, 456, 579 Ferri and Celli, 64 Ficker, 62, 231, 423 Field, 663 Finger, 522, 527 Finger and Landsteiner, 592 FlNKELSTEIN, 569 FlNKLER AND PRIOR, 581 FlNLAY, 656 Firth and Horrocks, 669 Fischer and Proskauer, 86 Fisher, 9, 56, 59, 325 Fisher, A., 10, 19, 23, 24 Flexner, 197, 378, 413, 434, 442, 608 Flexner and Jobling, 378 Flexner and Lewis, 652 Flexner and Noguchi, 460 Flugge, 81, 83, 198, 325, 385, 575 Foa, 360 Foa and Carbone, 362 v. Fodor, 198, 224 Foges, 458 Foote, 415 Forneaca, 335 Forster, F., 32, 576 Fracastor, 2 Frankel, A., 353, 355, 360, 362, 404, 412, 421, 445, 470, 498, 560, 607, 668 Franzott, 326 Freudenreich, 693, 694 Friedberger and Hartoch, 303 Friedemann, 303 Friedlander, 352, 445 704 INDEX OF AUTHORS v. Frisch, 449 Frosch and Kolle, 340, 368 fuhrmann, 44 Fuller, 674 furbringer, 86 Gabbet, 105, 479 Gaffky, 333, 399, 400, 467 Gaffky, Pfeiffer, Sticker, and Dieu- donne, 552 Galtier, 482, 527 Gamaleia, 580 Garre, 327 Gartner, 429 Gay, 243, 244, 444 Gay and Southard, 299, 300, 302 van Gehuchten, 636 Gengou, 240, 244, 273 Geppert, 82 Gessard, 567, 569 Gheorghiewski, 571 Ghon and Pfeiffer, 376, 386 Ghon and Preyss, 531 Gibson, 219 Gibson and Collins, 219 GlEMSA, 108 VAN GlESON, 637 Gilbert, 430 Gilchrist, 619 Globig, 33 Goldhorn, 588 Goodwin and v. Sholly, 376 Gordon, 350 Gorgas, 656 Gottschlich, 23, 549 Gram, 102 Gran, 49 Grancher and Ledoux-Lebard, 482 Grassberger, 531 Grawitz, 629 Grekoff, 697 Gruber, 67, 78, 421 Gruber and Durham 200, 228, 234 Gruby, 632 Grt'nhagen, 644 Guarnieri, 357, 359, 360, 644 Guerin, 649 Guiteras, 659 Gumprecht, 461 Glnther, 352 Gwyn, 430, 473 Haas, 66 Haenlein, 699 Haffkine, 579 Hahn, 190, 191, 416, 487 Hallier, 7 Hamburger, 303 Hammerschlag, 22, 484 HaMMERSTEN, 46 Hanel, 326 Hankin and Leumann, 20 Hankin and Wesbrook, 168 Hansen, 499, 619 Harrington, 91 Harrison, 425 Harrison and Galtier, 694 Hartman, 341 Hauser, 151, 452 Heim, 686 Heiman, 381, 384 Heinemann, 683, 687 Hektoen, 283, 661 Hektoen and Ruediger, 282, 283 Hellmich, 22 Hellriegel and Wilfarth, 55, 56 Henle, 3 Henrijean, 457 Hericourt and Richet, 296 Herter, 177, 394, 473 Hesse, 27, 133, 482, 687 HlLBERT, 31, 512 Hill, 84, 94, 676 Hirsch, 546, 576 Hirschberger, 689 Hiss, 13, 98, 133, 251, 289, 338, 339, 341, 348, 350, 351, 354, 355, 356, 357, 358, 364, 365, 369, 407, 408, 411, 437, 438 Hiss and Atkinson, 198, .219 Hiss and Russell, 436 Hiss and Zinsser, 291, 366, 378 Hoffman, 233 Hoffmann- We llenhoff, 514 Hogyes, 639, 642, 643 Holst, 430 Home, 661 INDEX OF AUTHORS 705 Horton-Smith, 407, 411 Houston, 668, 679 Howard, 449 Howard and Perkins, 351, 369 Huber, 530, 532 huddleston, 649 Hueppe, 543 Hueppe and Wood, 565 Hughes, 542 Huntemuller, 673 Hunter, 198 Irons, 680 ISAEFF, 363 Israel, 610 Iwanow, 559 Jackson, 138, 678, 680 Jackson and Melia, 133, 138 Jager, 373, 449 Jenner, 108, 193, 646 Joachim, 198 Jones, 75 Joos, 233 Jordan and Heinemann, 682 Jordan and Irons, 675 Jordan, Russell and Zeit, 414 jorgensen and madsen, 233 Kamen, 394, 534 Kappes, 21 Karlinsky, 411 Kelly, 344 Kempner, 199 Kempner and Pollack, 476 Kempner and Schepilewsky, 215 KlRCHER, 1 Kister and Wolff, 237 Kitasato, 454, 457, 460, 463, 547, 549 KlTASATO AND WEYL, 27 Kitt, 466 Kitt and Mayr, 546 Klebs, 7, 352, 487, 504, 655 Klein, 176, 404, 424, 478 Klemperer, 497 Klemperer, G. and F., 362 klingmi ller and baermann, 593 Klotz, 697 45 Knapp, 517, 534 Knoepfelmacher, 652 Knorr, 205, 295, 342 Kobert and Stillmarck, 204 Koch, 7, 63, 73, 77, 82, 83, 87, 321, 336, 352, 467, 477, 480, 485, 486, 487, 488, 491, 492, 493, 534, 553, 556, 559, 560, 572, 577, 597, 598, 678 Koch and Petruschky, 347 Koch and Rabinovitsch, 495 Koch and Wolffhugel, 65, 560 Koch, Gaffky and Loeffler, 65, 564 Kolle and Hetsch, 583 Koi,le and Otto, 331 Kolle and Wassermann, 189, 190, 191, 378 Korn, 496 Korschun, 291 Kossel, 204, 532 kossel and overbeck, 551 Kossel, Weber and Heuss, 494, 690 Kotjar, 628 Kraunhals, 569 Kraus, 186, 200, 235, 237, 328, 329, 423, 441, 444, 579, 580 Kraus and Doerr, 441, 444 Kraus and Low, 232, 397 Kraus and v. Pirquet, 236 Kraus and Stenitzer, 416 Kresling, 524 Kretz, 217 Kronig and Paul, 74, 77, 78, 80, 83 Kruse, 292, 435, 442, 444, 532, 544 Kurth, 431 Kutscher, 343 kutscher and meinicke, 432 kutschert and neisser, 517 Lachner-Sandoval, 606 Landsteiner, 201 Landsteiner and Jagic, 240 Landsteiner and Levaditi, 652 Landsteiner and Popper, 651 Landsteiner, Muller and Poetzl, 263 Lanz and Tavel, 344 Lassar, 592 Laveran, 582 Laws and Anderson, 677 706 INDEX OF AUTHORS Leclainche and Vallee, 465 Ledderhose, 568 Leeuwenhoek, 1 Leichtenstern, 372 Leishman, 281 Lembke, 392 Lentz, 436 Lepierre, 377 Le Roy, des Barres and Weinberg, 368 Lesage, 695 Leuchs, 137 Levaditi, 277, 280, 283, 589 Levaditi and Inmann, 283 Levaditi and Manouelian, 589, 605 Levaditi and Petresco, 586 Lewith, 66 Libman, 340, 344, 431 Libman and Rosenthal, 370 Liborius, 149 v. Lingelsheim, 328, 331, 338, 341, 347, 349, 387 Linossier and Roux, 629 Lister, 6 Loeb, 324 Loeffler, 14, 100, 110, 136, 504, 512, 514, 520, 522 Loeffler and Frosch, 663 Loeffler and Schutz, 520 Loew, 25, 698 Lohlein, 282 Lowenstein, 495 lubarsch, 190 Lubbert, 326 Luersen and Kuhn, 697 Lustgarten, 497, 584 Maassen, 53 Macfadyen, 363 Macfadyen and Rowland, 416 Madsen, 218, 513 Mafucci, 478, 485, 495 Mallory, 611, 662 Mallory and Wright, 110, 112, 150, 452 Mann, 96, 472 Maragliano, 492 Marbaix, 342, 343, 349 Marchiafava and Celli, 371 Marchoux and Salimbeni, 603 Marchoux and Simond, 660 Marchoux, Salimbeni and Simond, 659 Marie and Morax, 461 Marinesco, 476 Marmier, 345 Marmorek, 345, 350, 493 Marschal, 371 Martin, L., 465, 513 Martin and Cherry, 204 Martini and Lentz, 436 Marx, 348, 641 Massol, 697 Mennes, 362, 366 Mesnil, 232 Messea, 15 Metchnikoff, 188, 200, 201, 224, 225, 228, 232, 275, 577, 592, 696 Metchnikoff and Roux, 591 Metchnikoff, Roux and Salimbeni, 579 Meyer, 187 Meyer and Ransom, 461 Michaelis, 96, 253, 263 Michel, 508 Michelson, 607 Mignesco, 63 Migula, 12, 36, 325, 470 Mikulicz, 450 Miller, 411 Miquel, 32, 672 Moeller, 98 Mohler, 490, 663, 689, 690, 693, 694 Moll, 198 moltschanoff, 385 Momont, 560 Monti, 645 Morax, 538 Morax and Marie, 460 Morgenroth, 202 morgenroth and sachs, 265 Moro, 489 Morpurgo, 189 Moser, 662 Mouton, 275 Moxter, 241 MUHLSCHLEGEL, 17 INDEX OF AUTHORS 707 Muller, 334, 498, 688 Muller, Fr., 325 Muller, Otto Friedrich, 2 Muller, P. Th., 243, 254, 255, 272 Mulzer, 586 Muntz, 25 muntz and schlossing, 57 Myers, 215, 253 Naegeli, 483 Nakanishi, 10, 11, 16, 17 Nastjukoff, 531 Needham, 4 Negri, 636 Neisser, 107, 329, 380, 499, 506 Neisser and Sachs, 246, 273 Neisser and Shiga, 441 Neisser and Wechsberg, 201, 244, 329, 330, 331 Neisser, Baermann and Halber- stadter, 603 Nencki and Scheffer, 21 Neufeld, 347, 364, 365, 369, 412 Neufeld and Hune, 283 Neufeld and Rimpau, 282, 366 Neufeld and Topfer, 283 Neumann, 411, 569 Nicolaier, 454 Nicolle, 233, 298, 300, 500, 592 Nicolle and Thenel, 233 Nielsen, 479 Nikati and Rietsch, 577 NlKOLAYSEN, 385 Nissen, 76 Nocard, 430, 495, 522, 608 Nocard and Roux, 10, 478, 495 Noguchi, 264, 265, 267, 270 Norris, 237, 253, 424 Norris and Larkin, 608, 609 Norris, Pappenheimer and Flour- noy, 594 North, 697 NOTTER AND FlRTH, 688 Novy and Freer, 64 Novy and Knapp, 582, 594, 598 Nuttall, 198, 224, 237, 241, 253, 551 Nuttall and Thierfelder, 392 Obermeier, 6, 593 Obermuller, 693 Oehlecker, 484 Ogston, 321, 336 Ohno, 437, 444 Omelianski, 49, 58 Omeltschenko, 79 Ophuls, 619, 620 Oppenheimer, 43, 47 Otto, 297, 298, 300, 655, 660 Ottolenghi, 359 Overton, 187 Pane, 232, 362, 366 Papasotiriu, 391 Pappenheim, 106, 480 Parietti, 677 Park, 87, 216, 217, 412, 682 Park and Carey, 437 Park and Dunham, 436 Park and Holt, 687, 691 Park and Throne, 220 Park and Williams, 366, 513 Pasquale, 338, 580 Passet, 326, 337 Pasteur, 5, 41, 189, 192, 193, 194, 196, 321, 352, 466, 544, 545, 563, 619, 635 Pasteur and Chamberland, 122 Pasteur, Chamberland and Roux, 194, 561 Paul, 646, 647, 648, 650 Peabody and Pratt, 137, 409 Pearce, 202 Perkins, 448 Perrone, 339, 344 Petri, 496, 669 Petruschky, 343, 404, 411, 426, 606, 608 Petterson, 291 Pfaundler, 231, 232, 234 Pfeffer, 54, 56 Pfeiffer, 195, 199, 230, 255, 279, 416, 528, 529, 533, 577, 578, 644 Pfeiffer and Beck, 528, 533 Pfeiffer and Friedberger, 241 Pfeiffer and Isaeff, 199, 224 Pfeiffer and Kolle, 231, 418, 425 Pfeiffer and Nocht. 577, 580 70S IXDEX OF AUTHORS Pfeiffer and "Wassermann, 579 Pfluger, 59 Pfuhl, 453, 532 Pick and Yamanouchi, 300 PlERRALLINI, 276 PlORKOWSKI, 134 V. PlRQUET, 489 v. plrquet and schick, 296, 301 Pitt, 14 Plato, 381 Plaut, 599, 631 Plexciz, 2 poels and dhont, 430 poels and nolen, 368 Pollack, 427 pollender, 6 porges and meier, 263 PORTIER AND RlCHET, 296 Pott, 688 Pratt, 412 Prescott, 391, 679 Preuser, 524 Proscher, 331, 332 Proskauer and Beck, 29 Prudden, 404, 485 Prudden and Hodenpyl, 485 Pryor, 483 Quincke, 695 Rabixovttsch, 496, 622, 693 Radziewsky, 363 Ransom, 461 Ransome and Fullerton, 86 Rayexel, 494, 560 v. Recklinghausen, 7 Redtenbacher, 407 Reed, 656 Reed and Carroll, 430 Reed, Carroll and Agramonte, 656 Reed, Carroll, Agramonte and La- zear, 655, 656, 660 Richardson, 64, 411, 412 Richet, 296, 302 RlCHET and Hericourt, 197, 331 Rickets, 621 Rideal and Walker, 80 Rieder, 64 RlNDFLEISCH, 7 RlXFORD AND GlLCHRIST, 620 Roger, 345 Rohner, 569 Romer, 474 Rosenau and Anderson, 222, 297, 298, 512 Rosenau and McCoy, 681 Rosenbach, 321, 337 Rosenthal, 441 Ross and Milne, 597 Rost, 500, 502 rothberger, 403 Roux, 64, 149, 203 Roux and Chamberland, 469 Roux and Nocard, 525 Roux and Yersin, 107, 504, 512, 514 RUBNER, 24 Ruppel, 22, 486 Russell and Fuller, 676 Sabouraud, 632 Sacharoff, 605 Sachs, 188, 199, 202 Sachse, 54 Sahli, 487 Salmon, 544 Salmon and Smith, 196 Sanarelli, 655 Sanfelice, 469, 620 Sauerbeck, 294 Saul, 77 Savage, 231 SCHAEFFER AND StEINSCHNEIDER, 384 SCHAFER, 31 Schattenfroh, 291, 328 schattenfroh and grasberger, 684 schaudinn, 582 schaudinn and hoffmann, 584 SCHELL AND FlSCHER, 482 SCHERESCHEWSKY, 590 SCHERING, 91 SCHEUERLEN AND SPIRO, 74, 78 SCHILD, 392 schimmelbusch, 327 schlesinger, 346 Schlossmann, 88, 90 Schneider, 325 INDEX OF AUTHORS 709 SCHNITZLER, 453 Scholtz, 380 schottelius, 392 Schottmuller, 338, 345, 349, 351, 369, 405, 431, 688 schreiber, 546 schroeder, 4 schroeder and colton, 689 schroeter, 60 Schcder, 414, 685 schuller, 655 SCHULZE, 4 Schutz, 343, 368 Schutze, 202, 236 Schwann, 3, 4 Sclavo, 357, 564 Sedgwick and Batchelder, 682 Sharnosky, 103 Shattock, 22 Shiga, 433, 436, 440, 442, 444 SlEDENTOPF, 587 SlLBERSCHMIDT, 453 Simon and Lamar, 286 Simon, Lamar and Bispham, 286 Simond, 551 Simpson, 687 Simpson and Hewlett, 80 Smith, 101, 494, 690 Smith, Graham, 517 Smith, Herbert E., 686 Smith, Th., 27, 34, 297, 481, 482, 493, 512, 513, 680 Smith, Th., and Kilbourne, 195 Smith, Th., and Moore, 430 SxMiTH, Th., Brown and Walker, 456 Sobernheim, 553, 564 sobernheim and tomasczewski, 586 sommerville, 80 Spallanazani, Abbe, 4 Spengler, 487 Spilker and Gottstein, 65 Spitzer, 586 Spronck, 435 Spronk, 513 Stern, 237, 421 Stern and Korte, 259, 418 Sternberg, 34, 325, 341, 352, 359, 508, 655, 674 Stevens and Myers, 204 Sticker, 501, 633 Stoker and Weggefarth, 688 Strauss, 524 Strauss and Gamaleia, 485, 495 Strauss and Huntoon, 652 Strelitz, 511 Stricht, 476 Strong, L. W., 447 Strong and Musgrave, 434 Suchsland, 698 SURMONT, 201 SURMONT AND ArNOULD, 560 Tacke, 54 Talamon, 353 Tavel and Krumbein, 368 Tedesco, 532, 533 Terrin, 327 Thayer, 655 Thomas, 654 Tissier, 695 Tissier and Martelly, 392, 696 Tizzoni, 458 Todd, 329, 441 Tokishige, 620 Tomasczewski, 586 Torini, 47 Torrey, 385 Totsuka, 419 Toussaint, 194, 561 Trask, 686 Trillat, 87 Tschistovitch, 236 Tsiklinski, 32 Tunnicliff, 600, 602 Turnbull, 598 Uhlenhuth, 237 Ullmann, 384 Unna, 628 Uschinsky, 28, 126, 514 Vagedes, 494 Vaillard and Dopter, 441 Vaillard and Rouget, 457 Vaillard and Vincent, 459 Valleri-Radot, 4 Vallet, 678 710 INDEX OF AUTHORS Van der Loeff, 644 Van de Velde, 329, 330, 343, 348 Vaughn, 298, 417 Vaughn and AVheeler, 302 Yedder and Duval, 436 Veeder, 415 Veillon, 333 di Vestea and Zagari, 635 VlGNAL, 607 Villemin, 477 Vincent, 599 Voges, 29 Vom dem Borne, 603 vottaler, 455 Wadsworth, 99, 249, 355, 357, 359, 361, 364, 365 Wagmann, 685 Waldeyer, 7 Walker, 419 Ward, 64, 685 Washburn, 366 Wassermann, 195, 199, 204, 236, 242, 383, 385, 569, 570 Wassermann and Bruck, 246, 262 Wassermann and Citron, 294 Wassermann and Proskauer, 514 Wassermann and Schutze, 237 Wassermann and Takaki, 188, 214, 279, 461 Wassermann, Neisser and Bruck, 262 Wassermann, Neisser, Bruck and Schucht, 593 Wassilieff, 524 Wechsberg, 242 Weeks, 534 Weeny, 419 Wegele, 697 Weichselbaum, 333, 353, 372, 376, 386, 445, 448, 532 Weichselbaum and Mt'ller, 534 Weigert, 7, 111, 214, 479 Weill-Halle and Lemaire, 300 Weis, A. H., 328 Welch, 98, 129, 355, 357, 395, 470 Welch and Blachstein, 404 Welch and Flexner, 472 Welch and Nuttall, 177, 469 Wernicke, 551 Wertheim, 382 Wesenberg, 453 Westphal and Uhlenhut, 501 Weyl, 22, 479, 484 WlCKMAN, 651 WlDAL, 421 WlDAL AND NOBE COURT, 430 WlDAL AND SlCARD, 421 WlLCKENS, 686 Wilde, 449, 450, 451 Williams and Lowden, 638 Willson, 678 WlLTSCHOUR, 407 WlNOGRADSKY, 14, 54, 57 Wladimiroff, 525 wolbach and ernst, 494 Wolff, 294, 396 Wolff and Israel, 612 Wolff-Eisner, 301, 488 wolffhugel, 86 Wollstein, 533, 537, 538 Wood, 95, 109, 359, 699 Woronin, 55 Wright, 108, 150, 153, 195, 286, 288, 425, 611, 612, 615 Wright and Douglas, 281, 282 Wright and Lamb, 542 Yersin, 547 Yersin, Calmette and Roux, 552 Zeit, 64 Zettnow, 10, 11, 12, 594 Ziehl, 97, 105, 479 Zinsser, 16, 155, 291, 412, 413, 440, 512, 517, 619 INDEX OF SUBJECTS Abbott's method of staining spores, 97 Absorption method in study of agglu- tination reaction, 234 Achorion Schoenleinii, 630 cultivation of, 631 morphology of, 630 varieties of, 631 Acid formation by bacteria, 166 Acid-fast bacteria, stains for, 104 Acquired immunity, 192 definition of, 192 Actinomyces, 610 cultivation of, 611 discovery of, in cattle, 610 in man, 610 morphology of, 607, 610 pathogenicity of, 613 in animals, 613 in man, 613 parts of body infected in, 613 staining of, 611 varieties of, 614 Actinomycosis, 613 Active immunity. See under Immunity Aerobic organisms, facultative, 26 obligatory, 25 non-infectiousness of, 183 respiratory processes of, 27 Air, bacteria in, 665 dryness and high winds favorable for increase of, 665 estimation of numbers of, 666 occurrence of, in inhabited places, 665 scarcity ot, in places high above earth, 666 settling of, with rain, snow, etc., 66G infectious material carried by, 666 Agar for culture media, 127 lactose-litmus, 129 Agar for culture media, meat extract, 127 meat infusion, 128 Agar slants, cultivation of anaerobic bacteria on, 153 Agglutination reaction, 228 between agglutinin and agglutinin^ stimulating substances, 233 clinical diagnosis by, in typhoid, 229 concentrated agglutinin in, 235 differentiation of bacterial species by, 229 diluted agglutinins in, 235 group agglutination in, 234 immune or chief agglutinin in, 234 macroscopical observation of, 230 for bacterial differentiation, 231 major agglutinin in, 234 microscopical observation of, 229 for clinical diagnosis, 231 minor agglutinins in, 234 nature of, 228 partial agglutinins in, 234 absorption method in study of, 234 proagglutinoid zone in, 235 proagglutinoids in, 235 pseudo-clumping in, 231 specificity of, 234 " thread-reaction " in, 231 upon dead bacteria, 231 upon living bacteria, 231 Agglutination tests, technique of, 250 macroscopic, 252 microscopic, 251 Agglutinins, 200, 228 action of, 240 agglutinin-stimulating substances and, 233 quantitative relations between, 233 reaction between, 233 711 712 INDEX OF SUBJECTS Agglutinins, bactericidal substances com- pared with, 231 cell receptors in, 238 concentrated, failure of, to produce agglutination, 235 diluted, agglutination reaction with, 235 effect of heat on, 232 experimentation with, 231 in agglutination reaction, chief or immune, 234 major, 234 minor, 234 partial, 234 in serum of glanders, 527 in staphylococcus immune sera, 331 nature of, 231 normal, 232 partial, absorption method in study of, 234 production of, 232 in sera of animals, by injection of bacteria, 233 of culture extracts, 233 time of, 233 reaction of. See Agglutination reac- tion specificity of, 234 structure of (Ehrlich), 238 theoretical considerations concerning, 238 " thread-reaction " in, 231 Agglutinogen, 233 Aggressins (Bail's theory), 291 action of, 293 immunization with, 293 nature of, 293 occurrence of, 293 opposition to Bail's theory of, 294 Alcohol, as fixative in staining, 110 Alcoholic fermentation, 51 by yeasts, 52 in milk, 684 process of, 51 Alcohols as disinfectants, 77 Alexin, 198 action of, in blood serum, 224, 225 Alkali formation by bacteria, 166 Allantiasis, 475 Amboceptor and complement, quanti- tative relationship between, 244 Amboceptors, filtration of, 244 multiplicity of, in normal sera, 241 Amylase, 48 action of, 49 occurrence of, 49 Amylolytic ferment. See Amylase Anaerobic cultivation of bacteria, 148. See also under Cultivation of bacteria Anaerobic organisms, facultative, 26 Gram-positive, in feces, 177 infectiousness of, 183 non-invasion of blood stream by, 183 obligatory, 26 respiratory processes of, 26 Anaphylactin, 302 Anaphylaxis, 295 autopsy findings in, 299 definition of, 297 experimentation in, early, 295 immunity after, 299 in diphtheria antitoxin injections, 296 incubation during, 299 inherited, 300 observations in, fundamental, 296 by Arthus, 297 by Besredka, 298, 299 by Besredka and Steinhardt, 299 by Doerr, 299 by Gay and Southard, 299, 300 by Hericourt and Richet, 296 by Nicolle, 298, 300 by Otto, 297, 300 by Pick and Yamanouchi, 300 by Portier and Richet, 296 by Rosenau and Anderson, 297, 298, 300 by Th. Smith, 297 by Vaughn and Wheeler, 298 by Weill-Halle and Lemaire, 300 passive, 300 phenomena of, 295 " phenomenon of Arthus" in, 297 proteid injections in, 295 mode of giving, 298 quantity of, 298 INDEX OF SUBJECTS 713 Anaphylaxis, symptoms in, 299 theories concerning, 301 based on Ehrlich's receptor over- production theory, 301 of Besredka, 302 of Doerr and Russ, 303 of Friedberger and Hartoch, 303 of Gay and Southard, 302 of Hamburger, 303 of v. Pirquet and Schick, 301 of Richet, 302 of Wolff-Eisner, 301 Animal alkaloids, 45 Animal experimentation, 169 animals used in, 169 cages for, 173 autopsies in, 173 inoculations in, 170 Antagonism of bacteria, 31 Anthrax, 553 bacterial causation of, 6 occurrence of, 553 Anthrax bacillus, 553 action of, 561 bacilli resembling, 565 Bacillus anthracoides, 565 Bacillus radicosus, 565 Bacillus subtilis, 565 biology of, 559 cultivation of, 556 early investigation of, 553 experimental inoculation with, 561 immunization against, 563 active (Pasteur's method), 564 attenuation in, 563 passive (Sobernheim's method), 564 in milk, 689 infection with, 562 by inhalation, 562 cutaneous, 562 pulmonary, 563 spontaneous, 562 through alimentary canal, 563 isolation of, 555 morphology of, 554 pathogenicity of, 550 prophylaxis against, 563 resistance of, 560 Anthrax bacillus, staining of, 555 susceptibility of animals to, 560 virulence of, 560 Anthrax, symptomatic, bacillus of, 463 cultivation of, 463 immunization against, 466 vaccines used in, 466 morphology and staining of, 463 occurrence of, 463 pathogenicity of, 464 autopsy findings in, 465 toxins of, 465 Anthropoid apes, blood of, distinguished from human, 237 Antiaggressins, 293 Antiamboceptors, 242 Antianaphylaxis, 299 Antibodies, 197 experimentation and discovery of, 197 alexin, 198 agglutinins, 200 antiferments, 202 antitoxin, 198-199 bacteriolysins, 200 cytotoxins, 201 precipitins, 200 facts and theories concerning, 241 in sera, determination of, by comple- ment fixation. See under Comple- ment fixation Anticomplements, 242 Antiferments, 202 Antigen, definition of, 202 Antilab, 202 Antilactase, 202 Antileucocidin, 331 Antipepsin, 202 Antiricin, discovery and experimenta- tion with, 204 "Antisensibilisin," 302 Antiseptics, inhibition strengths of va- rious, 81 values of, determination of, 80 table of, 80 Antistaphylolysin, 331 Antisteapsin, 202 Antistreptococcic sera, 347 714 INDEX OF SUBJECTS Antitoxic sera, 196 Antitoxin, 198-199 diphtheria. See Diphtheria anti- toxin establishing standard unit for, 205 normal, 205 production of, a final test between toxin and endotoxin, 187 stability of, 206 tetanus. See Tetanus antitoxin unit of, 205 valency of, for toxin, 210 Antivenin, 198 Arthrospores, 16 Ascospores, 618, 626 Ash in bacterial cell, 23 Asiatic cholera. See Cholera Aspergillus, reproduction in, 625 Attenuated cultures in active immuni- zation, 193 Autoclave, 71 technical details of, 72 Trillat, 87 Autointoxication, gastrointestinal, 694 bacteria causing, 695 experimental combating of, by acid-producing bacilli, 695 Metchnikoff's treatment of, by means of Bacillus bulgaricus, 696-7 Autolysins, 248 Autopsies of infected animals, 173 Avian tuberculosis, bacillus of. See Tubercle bacillus, bacilli related to Babes-Ernst granules, 11 Bacilli intermediate between typhoid and colon organisms, 428 bacterial correlation of, 431 bacilli of colon-like morphology in, 432 of typhoid-like morphology in, 432 non-motile bacilli in, 432 classification of, 431 differentiation of, from typhoid and colon groups, 428 by cultural characteristics, 428 by morphology, 428 Bacilli intermediate between typhoid and colon organisms, differentiation of, by motility, 428 hog-cholera bacilli in, 430 meat-poisoning bacilli in, 429 Bacillus enteritidis in, 429 Bacillus icteroides in, 430 Bacillus Morseele in, 430 paratyphoid bacilli in, 430 Bacillus psittacosis in, 430 "Muller" bacillus, 431 paracolon bacillus in, 430 "Seeman" bacillus, 431 pathogenicity of, 431 toxic products of, 431 Bacillus, 37 general description of, 9 Bacillus aerogenes capsulatus, 469 and pernicious anemia, 177 biological considerations of, 472 cultivation of, 471 isolation of, 472 morphology of, 470 occurrence of, 470, 473 pathogenicity of, 472 - staining of, 471 Bacillus anthracoides, 565 Bacillus avisepticus, 544 Bacillus botulinus, 473 antitoxin for, 199 cultivation of, 474 morphology of, 474 pathogenicity of, 475 staining of, 474 toxins of, 476 Bacillus bulgaricus, use of, by Metch- nikoff for treatment of gastrointes- tinal autointoxication, 697 Bacillus butyricus, 496 Bacillus coli communior, 398 Bacillus coli communis, 389 agglutinins for, 396 in immune serum, 396 in normal serum, 397 bladder diseases due to, 395 cholera infantum attributed to, 394 cholera nostras attributed to, 394 cultivation of, 390 INDEX OF SUBJECTS 715 Bacillus coli communis, differentiation of, from meat-poisoning and para- typhoid bacilli, 428 distribution of, 391 in animals, 392 in man, 392 in milk, 391 in nature, 391 in feces, 177 in water, 679 immunization with, 395 inflammatory conditions of liver and gall-bladder attributed to, 395 isolation of, 391 morphology of, 389 pathogenicity of, 393 peritonitis following perforation at- tributed to, 394 septicemia due to, 394 staining of, 389 toxic products of, 395 varieties of, 397 Winckel's disease in the newborn due to, 394 Bacillus diphtherise. See Diphtheria bacillus Bacillus enteritidis, discovery and char- acteristics of, 429 Bacillus fecalis alkaligenes, 426 differentiation of, from typhoid ba- cillus, 427 in feces, 177 Bacillus Hoffmanni. See Diphtheria bacillus, bacilli similar to Bacillus icteroides, 430 Bacillus influenzae. See Influenza bacil- lus Bacillus lactis aerogenes, 451 cultivation of, 451 morphology of, 451 occurrence of, 451 in feces, 177 pathogenicity of, 451 Bacillus leprae. See under Leprosy Bacillus mallei, 520. See also Glanders biological characteristics of, 522 cultivation of, 520 immunity against, 527 Bacillus mallei, morphology of, 520 pathogenicity of, 522 bacteriological diagnosis in, 524 in horses, 522 in man, 524 nodules in, 524 spontaneous infection by, 522 staining of, 520 toxin of, 524 action of, 525 diagnostic use of, 525 directions of U. S. Government for, 526 obtaining and preparation of, 525 Bacillus melitensis. See Micrococcus melitensis Bacillus mesentericus in feces, 177 Bacillus Morseele, discovery and charac- teristics of, 430 Bacillus mucosus capsulatus, 445 association of, with pneumonia, 448 with other diseases of mucous lin- ings, 448-9 cultivation of, 446 cultural characteristics of, 447 immunization against, 449 inoculation of animals with, 449 morphology of, 445 pathogenicity of, 448 staining of, 446 Bacillus murisepticus, 534 Bacillus ozaenae, 450 Bacillus pestis. See Plague bacillus Bacillus proteus vulgaris, 452 cultivation of, 452-3 Bacillus prodigiosus, quantitative chem- ical analysis of, 21 Bacillus psittacosis, 430 Bacillus pyocyaneus, 567 antitoxin against products of, 199 cultivation of, 567 favorable conditions for, 567 pigment in, 568 fluorescent variety of, 569 pyocyanin in, 568 immunization against, 570 filtrates of old cultures in, 570 pyocyanase in, 570 716 INDEX OF SUBJECTS Bacillus pyocyaneus, immunization against, true toxin in, 570 morphology of, 567 occurrence of, in lesions and inflam- matory affections, 569 pathogenicity of, 569 staining of, 567 susceptibility of animals to, 570 toxins of, 570 leucocyte-destroying ferment in, 571 pyocyanase in, 570 pyocyanolysin in, 570 true, 570 from filtrates of old cultures, 570 virulence of, 569 Bacillus radicicola, 55 Bacillus radicosus, 565 Bacillus rhusiopathise, 534 Bacillus smegmatis. See Smegma ba- cillus Bacillus subtilis, 565 Bacillus suisepticus, 545 Bacillus tetani. See Tetanus, bacillus of Bacillus tuberculosis. See Tubercle bacillus Bacillus typhi abdominalis, 399 Bacillus typhi murium, 430 Bacillus typhosus. See Typhoid fever, bacillus of Bacillus xerosis. See Bacillus diph- therise, bacilli similar to Bacteria (see also Bacterial cell) : acid and alkali formation by, 166 acid-fast, stains for, 104 action of, in the body, 184 anabolic or synthetic activities of, 54 in root tubercles, 55 in soil, 54 animal experimentation with, 169 antagonism of, 31 biological activities of, 40, 164 chemical agents injurious to, 73. See also Disinfectants classes of, 182 bacilli, 9 cocci, 9 spirilla, 9 Bacteria, classification of, 35 based on organs of motility, 15 by Bail with regard to aggressins, 294 by Gram stain, 104 by Migula, 37 counting of, 161 cultivation of, 141 by anaerobic methods, 148. See also under Cultivation of bacteria inoculation of media in, 141 dead, in active immunization, 193 degenerative forms of, 20 denitrifying, 52 destruction of. See Destruction of bacteria differentiation of, by fermentation, 48 enzymes produced by, 168 diastatic, 169 inverting, 169 proteolytic, 168 gas formation by, 164. See also Gas formed by bacteria Gram-negative, 104 Gram-positive, 104 in air. See Air, bacteria in in industries, 697 in milk. See Milk, bacteria in in soil. See Soil, bacteria in in tissues, staining of, 110 in water. See Water, bacteria in incubation of, 156. See also under In- cubation of cultures indol production by, 167 isolation of, methods of, 142 katabolic activities of, 41-53 by bacterial enzymes or ferments, 42 varieties of, 48 by denitrifying bacteria, 52 by fat-splitting enzymes, 47 by lab enzymes, 46 by proteolytic enzymes, 43 liberation of energy by, 58 light production of, 59 microscopic study of. See Microscopic study nitrifying. See Nitrifying bacteria INDEX OF SUBJECTS 717 Bacteria, nutrition of, 25. See also under Nutrition of bacteria occurrence of, in the body, 181 parasitic, 29 definition of, 182 pathogenic, 182 phenol production by, 167 physical agents injurious to, 62 pigment formation by, 59 protozoa and, differentiation of, 1 putrefactive, quantitative chemical analysis of, 21 reducing powers of, 167 relation of, to moisture, 35 to physical environment, 31 to pressure, 35 relationship of, to other plants, 35 reproduction of, 18 rate of, 18 varieties of, 18 saprophytic, 29 definition of, 182 size of, 9 staining of, methods of. See Staining of bacteria sulphur. See Sulphur bacteria symbiosis of, 31 thermal death points of, 34 variations in forms of, 19 virulence of, and infectiousness, 183 virulent, sublethal doses of, in active immunization, 195 Bacteriaceae, 37 Bacterial cell, ash in, 23 Babes-Ernst granules in, 11 capsule of, 12 carbohydrates in, 23 chemical constituents of, 21 quantitative analysis of, 21 varieties of, 21 fats in, 22 globulin in, 22 membrane of, 12 metachromatic granules in, 11 morphology of, 100 motility of, 13 Brownian, 14 by flagella, 14 Bacterial cell, motility of, effect of tem- perature on, 15 molecular, 14 true, 14 nucleus in, 10 organs of locomotion of, 13 classification of bacteria based on, 15 osmotic properties of, 23 permeability of membrane of, 23 plasmolysis of, 23 plasmoptysis of, 24 polar bodies in, 11 proteids in, 22 refractive index of parts of, 24 specific gravity of forms of, 24 water in, 21 Bacterial enzymes or ferments, 42 action of, 42 environmental conditions on, 43 reversible, 43 similarity of, to ferments of special- ized cells of higher plants and ani- mals, 43 Bacterial forms, variations of, 19 Bacterial poisons, 184 action of, 187 ptomains and, 185 resistance of, to chemical action and heat, 187 summary of, 305 varieties of, 185 endotoxins, 185 proteins, 186 true toxins, 185 Bacterial products in active immuniza- tion, 195 Bacterial proteins, 186 Bacterial spores, 15 formation of, 15 germination of, 17 position of, 17 varieties of, 15 arthrospores, 16 true or endospores, 16 vegetative forms from, 17 Bactericidal action of blood serum, 224 Bactericidal strengths of common dis- infectants, 83 718 INDEX OF SUBJECTS Bactericidal substances compared with agglutinins, 231 Bactericidal tests, 257 in test tube, 257 technique of, 258 for typhoid fever, 258 in vivo, 255-7 Bacteriemia, definition of, 184 Bacteriological examination of blood cultures, 178 choice of media for, 179 results of, estimation of, 180 technique of obtaining material for, 178 from typhoid patients, 180 of exudates, 175 of feces, 176 of material from patients, 174 technique of collecting, 174 of urine, 176 Bacteriology, development and scope of, 1-8 Bacteriolysins, 200 immune, 225 Bacteriolytic tests, 255 Pfeiffer's test in, 255 determination of bacteriolytic power of serum against a known micro- organism in vivo by, 255 identification of microorganism in known immune serum in vivo by, 257 Bacteriotropins, 282 Bacterium, 37 Bail, aggressin theory of, 292 opposition to, 294 Barsiekow's medium for colon-typhoid differentiation, 139 Bauer's modification of Wassermann test for syphilis, 268 "Bazillenemulsion," 487 Beggiatoa, genus, 38 Beggiatoaceae, 38 Berkefeld filter, 120, 121 Bile medium for colon-typhoid differen- tiation, 138 Bile-salt agar, MacConkey's, for colon- typhoid differentiation, 138 Bitter milk, bacteria causing, 685 Black Death, 546 Bladder diseases due to colon bacillus, 395 Blastomycetes. See Yeasts and Yeast cells Blood, laked, 225 the seat of immunizing agencies, 198 Blood corpuscles, red, in Ehrlich's the- ory of lytic process in blood serum, 227 Blood cultures, bacteriological examina- tion of, 178 results of, estimation of, 180 choice of media for, 179 technique of obtaining material for, 178 from typhoid patients, 180 Blood media, method of obtaining, 140 Blood serum, bactericidal action of, 224 Bordet's interpretation of lytic proc- esses of, 225 immune, 198 reactivation of bactericidal power of, by normal serum, 225 reactivation of bacteriolytic powers of, by normal serum, 226 lytic processes of, 224 normal, 198 Bordet's lytic theory of constituents of, 225 alexin in, 225 "sensitizing substance" in, 225 obtaining of, from animals, 249 from man, 249 reactions with. See Serum reactions Blood serum media, method of obtain- ing, 139 Bollinger, discovery of actinomyces of cattle by, 610 Bordet and Gengou, discovery of whoop- ing-cough bacillus by, 535 Bordet-Gengou bacillus, 535 cultivation of, 536 compared with that of influenza bacillus, 537 technique of, 536-7 morphology of, 535 INDEX OF SUBJECTS 719 Bordet-Gengou bacillus, morphology of, compared with that of influenza bacillus, 537 pathogenicity of, 537 staining of, 535 toxins of, 537 Botulism, 475 Bouillon, malachite green, for colon- typhoid differentiation, 137 Bouillon filtre (Denys), 487 Bovine tuberculosis, bacillus of. See Tubercle bacillus, bacilli related to Broth used for culture media, 124 calcium carbonate, 126 glycerin, 125 meat extract, 124 meat infusion, 124 nitrate, 126 pepton-salt, 126 sugar-free, 125 Uschinsky's proteid-free, 126 Bruce, discovery of Malta fever bacil- lus by, 542 Buboes, 540 Buchner, discovery of Bacillus coli com- munis by, 389 Buchner's method of pyrogallic absorp- tion of oxygen in cultivation of anaerobic bacteria, 152 Wright's modification of, 153 Buerger's method of staining capsules, 99 Butter, making of, 692 bacteria aiding, 692 transmission of infection by, 693 tubercle bacilli in, 693 Butter bacillus, 496 Butyric-acid fermentation in milk, 684 Cadaverin, 45 Cages for animals, 171, 172, 173 Calcium-carbonate broth, 126 Capaldi's medium for colon-typhoid dif- ferentiation, 135 Capsule stains in staining of bacteria, 98 Carbohydrates in bacterial cell, 23 Carbolic acid as disinfectant, 77 Carbolic-acid coefficient, 80 Carbon dioxid formed by bacteria, 164 Carbon in nutrition of bacteria, 25 Casein, coagulation of, in milk, 684 Castelli, discovery of Spirochete per- tenuis by, 603 Cell-receptors, three forms of, in expla- nation of all known antibodies (Ehrlich): hap tines of the first order, 240 haptines of the second order, 240 haptines of the third order, 240 Cellulase, 49 Charbon. See Anthrax. Charbon symptomatique. See Anthrax, symptomatic, bacillus of Cheese, making of, 693 bacteria aiding, 693, 694 pathogenic organisms in, 694 Chemotaxis, negative and positive, defi- nition of, 277 Chicken cholera bacillus, 544 cultivation of, 544 immunization with, 545 morphology and staining of, 544 occurrence of, in animals, 544 pathogenicity of, 544 Chicken-pox, relation of, to smallpox, 647 Chlamydobacteriacese, 38, 606 classification of, 606 morphology of various forms of, 606 Chloride of lime as disinfectant, 76 Chlorine as disinfectant, 86 Cholera, Asiatic, 572 diagnosis of, 574 epidemics of, 576 immunization in, 579 active, 579 protective inoculation in, 579 in animals, 577 infection in, 576 pathological findings in, 576 spirillum of, 572 biological considerations of, 576 cultivation of, 573 diagnosis of, by " cholera-red " reaction, 574 hygienic considerations of, 578 in feces, 177 isolation of, 575 720 INDEX OF SUBJECTS Cholera, spirillum of, isolation of, from feces, 575 from water, 575, 678 morphology of, 572 pathogenicity of, 576 in animals, 577 in man, 576 resistance of, 576 spirilla resembling, 580 Spirillum Deneke, 581 Spirillum Massaua, 580 Spirillum Metchnikovi, 580 Spirillum of Finkler-Prior, 581 staining of, 573 toxin of, 578 traced to milk, 687 Cholera, fowl, bacillus of, 7 Cholera infantum attributed to colon bacillus, 394 Cholera nostras attributed to colon bacillus, 394 Cholera-red reaction, 574 Chromo-bacteria, 59 Chromogenic Gram-negative cocci, 387 Cladothrix, 38, 607 morphology of, 606 Clostridium Pasteurianum, 54 Clearing of culture media, 119 by filtering, 120 with eggs, 119 Coagulins, 235 Cobra poison and its antitoxin, experi- mentation with, 204 Coccaceae, 37 Cocci, description of, 9 Coefficient of inhibition, 80 Colon bacillus. See Bacillus coli communis Colon bacillus group, 389 Colon test, for analysis of water, 679 Colon-typhoid differentiation, media for, 133 Barsiekow's, 139 bile, 138 Capaldi's, 135 Conradi-Drigalski, 135 Endo's, 136 Hesse's, 133 Hiss' plating, 133 Colon-typhoid differentiation, media for, Hiss' tube, 133 Jackson's lactose-bile, 138 Loeffler's malachite green, 136 Leuchs' modification of, 137 MacConkey's bile-salt, 138 malachite green bouillon, 137 neutral-red, 139 Piorkowski's urine gelatin, 134 Colon-typhoid-dysentery group, bacilli of, 388 characteristics of, 388 Colonies in agar, 146 Colony-fishing, 146 Colony study of bacteria, 161 Color indicator in titration, 117 Comma bacillus. See Spirillum cholerae asiaticse Complement, deviation of, 244 filtration of, 244 fixation of. See Complement fixation in Ehrlich's theory of lytic process in blood serum, 226 in normal blood serum, 226 multiplicity of, in normal sera, 242 Complement fixation, action in, 245 by precipitates, 244 determination of antigen by, in serum reactions, 271 of antibodies in sera by, 261 principles of, 261 reaction in, 261 Wassermann test for. See Was- sermann test for diagnosis of syphilis proteid differentiation by, 273 Complementoids, 243 Conjonctivite subaigue, 538 Conradi-Drigalski medium, for colon- typhoid differentiation, 135 isolation of typhoid bacillus in stools by, 408 Cotton used in filtering culture media, 120 Counting of bacteria, 161 Cowpox, relation of, to smallpox, 646 use of, in immunization against small- pox, 646 Crenothrix, 38 INDEX OF SUBJECTS 721 Creolin, 78 Cultivation of bacteria, anaerobic, 148 by absorption of oxygen by pyro- gallic acid in alkaline solu- tions, 152 Buchner's method, 152 Wright's modification of, 153 combined with air exhaustion and hydrogen replacement, 153 on agar slants, 153 without use of hydrogen, 155 by displacement of air with hydro- gen, 151 by mechanical exclusion of air, 148 Esmarch's method, 149 fluid media covered with oil, 150 Liborius' method, 149 Roux's method, 149 Wright's method, 150 Zinsser's apparatus for, 156 colony study in, 161 counting of bacteria in, 161 incubation in, 156. See also under Incubation of cultures Culture media, 124 agar for, 127 lactose-litmus, 129 meat extract, 127 meat infusion, 128 blood for, 140 blood serum for, 139 broth for, 124 calcium carbonate, 126 glycerin, 125 meat extract, 124 meat infusion, 124 nitrate, 126 pepton-salt, 126 sugar-free, 125 Uschinsky's proteid-free, 126 clearing of, 119 by filtering through cotton, 120 through paper, 121 with eggs, 119 Dorsett egg, 130 enriching substances used in, 139, 140 fluid, covered with oil, in anaerobic cultivation of bacteria, 150 46 Culture media, for colon-typhoid differ- entiation, 133 glassware preparation in, 113 glycerin for, 126 ingredients of, 115 choice of, 116 milk, 130 potato for, 130 glycerin, 130 preparation of, 113 general technique of, 113 process of, 124-40 serum, 131 Hiss' serum water, for fermenta- tion tests, 132 Loeffler's, 131 slanting of, 123 special, 133 sterilization of, 121 filtration in, 122 heat in, 121, titration of, 117 color indicator in, 117 process of, 117 for alkaline media, 118 reaction of, 117 adjustment of, 119 tubing of, 121 Cultures, attenuated, in active immu- nization, 193 incubation of. See under Incubation of cultures Cytase, 279 Cytoryctes variolar, 645 Cytotoxins, 201 specific, injury to organs by, 201 Decay, action of, 44 Defensive factors of animal organism, 189 Degenerative forms of bacteria, 20 Denitrifying bacteria, 52 activities of, 53 occurrence of, 53 Destruction of bacteria, 62 by chemical agents, 73. See also Dis- infectants gaseous, 86 722 INDEX OF SUBJECTS Destruction of bacteria, by chemical agents, in solution, 73 inorganic, 73 organic, 77 by physical agents, 62 drying, 62 electricity, 65 heat, 65 light, 63 Diarrheal diseases traced to milk, 687 Diastase. See Amylase Differential stains in staining of bac- teria, 102 Diphtheria, tracing of, to milk, 687 Diphtheria antitoxin, 216 anaphylaxis in injections of, 296 normal, 205 production of, 216 horses used in, 216-17 technique of, 217 toxin for, 216 stable, 206 standardization of, 218 concentration and purifying in, 219 technique of, 218 unit for, 205 Diphtheria bacillus, 504 - bacilli similar to, 514 Bacillus Hoffmanni, 514 cultivation of, 516 morphology of, 515 staining of, 515 Bacillus xerosis, 517 cultivation of, 518 differentiation of, from other ba- cilli, 578 other bacilli, 519 biological characteristics of, 507 cultivation of, 508 degenerative forms of, 19 differentiation of, from other forms, 107, 514 et seq. grouping of, 507 isolation of, 509 morphology of, 505 "ground" type in, 506 occurrence of, in body, 510-511 pathogenicity of, 510 Diphtheria bacillus, pathogenicity of, in animals, 511 "pseudo-membranes " in, 510 resistance of, 508 staining of, 506 Gram's method of, 507 polar or Babes-Ernst bodies in, 506 Neisser's stain for, 506 Roux's stain for, 507 toxin of, 512. See also Diphtheria toxin chemical and physical properties of, 512 production of, 512 media employed in, 512 Park- Williams Bacillus No. 8 in, 512 resistance of, 514 Diphtheria toxin, analysis of (Ehrlich), 205-15 method of partial absorption in, 209 side-chain theory of, 212 summary of, 215 constitution of (Ehrlich), 210 graphic form of (Ehrlich), 211 views of Arrhenius and Madsen on, 212 epitoxoid in, 208 molecule of, haptophore group in, 207 toxophore group in, 207 normal solution of, 205 partial absorption of, 209 standardization of, Limes death in, 208 Limes zero in, 207 time changes in, 206 toxoid form of, 207 protoxoids in, 209 syntoxoids in, 209 toxon in, 209 unit for, 205 Diplococcus gonorrhoeae, 380 cultivation of, 381 conditions favorable to, 383 "Wertheim's medium for, 381-2 differentiation of, from Micrococcus catarrhalis, 386 early work in, 380 INDEX OF SUBJECTS 723 Diplococcus gonorrhoeae, infection of, in man, 384 morphology of, 380 pathogenicity of, in animals, 385 in man, 384 resistance of, 384 staining of, 381 Diplococcus lanceolatus. See Diplococ- cus pneumoniae Diplococcus mucosus, 387 Diplococcus pneumoniae, 352 cultivation of, 355 differentiation of, from streptococcus, 357, 367 cultural, 368 morphological, 367 by bile test, 369 by capsule, 367 by fermentations, 369 by grouping, 367 by growth in blood media, 367, 369 by shape, 367 immunization against, 363 active, methods of, 363 agglutinins in immune sera in, 364 table of, 365 technique of, 364 opsonins in immune sera in, 366 precipitins in immune sera in, 365 passive, with immune sera, 366 isolation of, 358 modes of inoculation with, 361 morphology of, 353 capsules in, 353-4 lancet-shape of cocci in, 353 pairing of cocci in, 353 pathogenicity of, 361 pneumonic complications and, 362 resistance, of , 358 staining of, 355 susceptibility of animals to, 360 toxic products of, 362 virulence of, 360 in animals, 360 in man, 361 Disinfectants, 73 bactericidal strengths of common, 83 Disinfectants, gaseous, 86 chlorine, 86 formaldehyde, 87 oxygen, 86 ozone, 86 sulphur dioxide, 86 testing of efficiency of, 79 carbolic-acid coefficient in, 80 determination of antiseptic values in, 80 of disinfectant values in, 82 table of, 83 factors in, 79 used in solution, 73 inorganic, 73 acids, bases, and salts, 74 halogens and derivatives, 75 chlorid of lime, 76 tetrachlorid of iodin, 76 oxydizing agents, 76 hydrogen peroxid, 76 potassium permanganate, 76 organic, 77 alcohols, 77 carbolic acid, 77 cresol group, 78 essential oils, 78 formaldehyde, 78 iodoform, 77 Disinfection, practical, 84 of feces, 85 of hands, 85 of instruments, 85 of linen, etc., 85 of rooms, etc., 86 of sputum, 84 of urine, 85 Dissociation, electrolytic, 74 relation between degree of, and bactericidal powers of solutions, 74 Dorsett egg medium, 130 Drying in destruction of bacteria, re- sistance to, 62 DT^M250, definition of, 205 Ducrey bacillus, 540 cultivation and isolation of, 541 discovery of, 540 724 INDEX OF SUBJECTS Ducrey bacillus, infection with, 540 pathogenicity of, 541 Dysentery, autopsy findings in, 440 occurrence of, 440 symptoms of, 440 Dysentery bacilli, 433 biological considerations of, 439 differentiation of, by Hiss, through agglutination tests, 438 through fermentation tests, 437 immunization with, 442 active, 442 agglutinins in, 442 bactericidal substances in, 442 true toxins in, 443 passive, 443 in feces, 178 investigation of : by Flexner, 434 by Hiss and Russell, 436 by Kruse, 435 by Lentz, 436 by Martini and Lentz, 436 by Ohno, 437 by Park and Carey, 437 by Park and Dunham, 436 by Shiga, 433 by Spronck, 435 by Strong and Musgrave, 434 by Vedder and Duval, 435 pathogenicity of, 439 pseudo-dysentery bacillus and, 435, 436 Shiga's bacillus in, 433 cultural characteristics of, 434 morphology of, 433 toxi? products of, 440 action of, on animals, 441 obtaining of, from cultures, 440 "Y" bacillus in, 436 Eberth, discovery of typhoid bacillus by, 399 Edema, malignant, bacillus of. See under Malignant edema Eel-blood serum, toxic, 205 Eggs used in clearing culture media, 119 Ehrlich's analysis of diphtheria toxin, 205-215. See also under Diphtheria toxin Eichstedt, discovery of Microsporon fur- fur by, 627 Electricity in destruction of bacteria, 65 Electrolytic dissociation, 74 relation between degree of and bac- tericidal powers of solutions, 74 Elser and Huntoon, discovery of pseudo- meningococcus by, 379 Eisner's potato-extract gelatin, isola- tion of typhoid bacillus in stools by, 407 Emphysematous gangrene, isolation of Bacillus aerogenes capsulatus from, 472 Endolysins, 291 Endo's fuchsin-agar, isolation of ty- phoid bacillus in stools by, 409 Endo's medium for colon-typhoid dif- ferentiation, 136 Endospores, 16 Endotoxins, 185, 306 compared with pigments, 186, 309 of Staphylococcus pyogenes aureus, 328 of Streptococcus pyogenes, 345 summary of, 306 toxins distinguished from, 186 Environment and bacteria, 31 Enzymes, definition of, 42 katalyzers and, analogy between, 42 produced by bacteria, 168 diastatic, 169 inverting, 169 proteolytic, 168 varieties of. See under specific names Epitoxoid in toxin, 208 Erlenmeyer flask, 114 van Ermengem, discovery of Bacillus botulinus by, 473 van Ermengem's method of staining flagella, 101 Escherich, discovery of Bacillus lactis aerogenes by, 451 Esmarch roll tubes in isolation of bac- teria, 147 INDEX OF SUBJECTS 725 Esmarch's method of anaerobic culti- vation of bacteria, 149 Essential oils as disinfectants, 78 Exudates, bacteriological examination of. See Bacteriological examination Eumycetes. See Hyphomycetes Facultative aerobes, 26 Facultative anaerobes, 26 Farcy, 523 Fat-splitting enzymes, 47 action of, methods of investigating, 47 varieties of, 47 Fats in bacterial cell, 22 Favus, 630 Feces, bacteriological examination of, 176 disinfection of, 85 number of bacteria in, 176 varieties of bacteria in, 177 Fermentation, alcoholic, 51 in milk, 684 butyric-acid, in milk, 684 enzymes of, 48, 52 in development of bacteria, 26 inversion in, 42 lactic-acid, 50, 683 process of, 48 Fermentation tests, serum media for, 132 Filtering, in clearing of culture media, 120 through cotton, 120 through paper, 121 in sterilization of culture media, 122 Filters, varieties of: Berkefeld, 120, 121 Kitasato, 123 Maassen, 124 Reichel, 122 Filtration of immune body and comple- ment, 244 Fish tuberculosis, bacillus of, 495 Fishing, colony, 146 Fixation of complement, action in, 246 by precipitates, 244 "Fixator," 279 Flagella, arrangement of, 15 structure of, 14 varieties of, 14 Flagella stains in staining of bacteria, 100 Florence flask, 114 Fluid media covered with oil, used in cultivation of bacteria, 150 Foot-and-mouth disease, 663 etiological factor of, 663 immunity in, 664 in milk, 688 pathology of, 663 transmission of, 663 Formaldehyde as disinfectant, 78, 87 generation of, by breaking up solid polymer with heat, 90 by Breslau method, 87 by direct evaporation, 87 by glycerin addition, 88 by lime method, 91 by potassium-permanganate meth- od, 91 by Trillat method, 87 gauging amount of formalin in, 91 Trillat autoclave for, 87 Fractional sterilization, 70 at high temperatures, 70 at low temperatures, 71 Frambcesia tropica, 603 Friedlander, discovery of Bacillus mu- cosus capsulatus by, 445 Friedlander bacillus. See Bacillus mu- cosus capsulatus v. Frisch, discovery of Bacillus of rhi- noscleroma, 449 Fusiform bacilli: from carious teeth, 602 from noma, 602 from scurvy, 602 of Vincent's angina, 600 Gaffky, discovery of Micrococcus tetra- genus by, 334 Gartner, discovery of Bacillus enteri- tidis by, 429 Gas formed by bacteria, 164 analysis of, 164 carbon dioxide in, 164 726 INDEX OF SUBJECTS Gas formed . by bacteria, analysis cf, hydrogen in, 165 hydrogen sulphide in, 165 quantitative, 166 Gas-pressure regulators for incubators, 160 Moitessier's, 160 Gastrointestinal autointoxication, 694 bacteria causing, 695 experimental combating of, by acid- producing bacilli, 695 Metchnikoff's therapy of, by means of Bacillus bulgaricus, 696-7 Gelase, 49 Gessard, discovery of Bacillus pyocaneus by, 568 Glanders, bacteriological diagnosis of, 524 immunity in, 527 in horses, 522 acute form, 522 chronic form, 523 in man, 524 nodules of, 524 spontaneous infection in, 522 Glanders bacillus. See Bacillus mallei Globulin in bacterial cell, 22 Glycerin, use of, for culture media, 126 meat extract, 126 meat infusion, 127 Glycerin broth, 125 Glycerin potato, 130 Gonococcus. See Diplococcus gonor- rhoeae Gram-negative bacteria, 104 Gram-negative cocci, chromogenic, 387 Gram-negative micrococci, table of di- agnosis of, by differential value of sugar fermentation, 387 Gram-positive bacteria, 104 in feces, 177 Gram's method of staining bacteria, 102 classification of pathogenic bacteria by, 104 Paltauf's modification of, 103 Group agglutination, 234 Gruber-Widal reaction, 250 Gruby, discovery of Trichophyton ton- surans by^ 631-2 Guarnieri's medium, Welch's modifica- tion of, 129 Halogens as disinfectants, 75 "Hanging block" method in study of bacteria, 94 "Hanging drop" method in study of bacteria, 93 Hansen, discovery of lepra bacillus by, 499 Haptophore group in toxin molecule, 207 in toxon molecule, 209 Haptophore groups in immune body, 228 complementophile, 228 cytophile, 228 Hauser, discovery of Bacillus proteus vulgaris by, 452 Heat, in destruction of bacteria, 65 dry and moist, comparison of, 66 effect of various degrees of, 65 moist, advantages of, 66 penetrating power of, 67 in sterilization of culture media, 121 Heat sterilization, 68 dry heat, 68 by burning, 68 by hot air, 68 moist heat, 69 by boiling, 69 by fractional sterilization, 70 by live steam, 60 by steam under pressure, 71 Hektoen and Ruediger on structure of opsonins, 283 Hemagglutinins, 235 Hematogen, 530 Hemolysin, immune, 226 Hemolysins of Staphylococcus pyogenes aureus, 329 of Streptococcus pyogenes, 345 specificity of, 247 varieties of: autolysins, 247 heterolysins, 247 isolysins, 247 Hemolysis, 201, 225 INDEX OF SUBJECTS 727 Hemolytic tests, 259 obtaining blood for, 259 in large quantities, 260 in small quantities, 259 standard concentration of red blood cells for, 259 Hemolytic unit, definition of, 265 Hemorrhagic septicemia bacilli, 543 morphology of, 543 staining of, 543 varieties of: bacillus of chicken cholera, 544 Bacillus pestis, 546 bacillus of swine plague, 545 Hepatotoxin, 201 Hesse's medium for colon-typhoid differ- entiation, 133 Heterolysins, 247 Hides, tanning of, bacteria in, 698 Hiss' agar-gelatin medium, isolation of typhoid bacillus in stools by, 407 Hiss' leucocyte extract theory and therapy, 289 Hiss' methods of staining capsules, 98 Hiss' plating media for colon-typhoid differentiation, 133 Hiss' tube medium for colon-typhoid differentiation, 133 Hiss' serum water media for fermenta- tion tests, 132 "Hog-cholera" bacilli, 430 differentiation of, from swine plague bacillus, 546 Hogyes, dilution method of, in treat- ment of rabies, 643 Horse meat, detection of, by precipitin tests, 254 Horses used in production of diphtheria antitoxin, 216-17 of tetanus antitoxin, 221 Howard and Perkins, discovery of Strep- tococcus mucosus by, 351 Hydrogen, formation of, by bacteria, 165 in nutrition of bacteria, 28 Hydrogen peroxid as disinfectant, 76 Hydrogen sulphid formed by bacteria, 165 Hydrophobia. See Rabies Hypersusceptibility. See Anaphylaxis Hyphomycetes, 623 conditions favorable to growth of, 626 diseases caused by, 627 favus, 630 pityriasis versicolor, 627 ringworm, 631 thrush, 629 diseases sometimes accompanied by, 633 morphology of, 623 reproduction of, 623 mycomycetes in, 624 Aspergillus variety in, 625 Penicillium variety in, 625 sporulation by other methods in, 625 phycomycetes in, Mucorinse vari- ety of, 623 sexual reproduction of, 624 structural classification of, 623 varieties of, 623 mycomycetes, 623 morphology of (typical), 623, 624 ascospores in, 626 conidia (or spores) in, 626 conidiophores in, 625 hyphal branches (septate) in, 625 mycelial threads (septate) in, 625 sterigmata (septate) in, 625 morphology of less frequent forms of, 625 ascospores in, 625 chlamydospores in, 626 phycomycetes, 623 morphology of (typical), 623-4 columella in, 624 hyphal branches in, 624 mycelial threads (non-septate) in, 624 sporangium in, 624 spores in, 624 morphology of sexual reproduc- tion forms of, 624 gametophores, in, 624 zygospores in, 624 728 INDEX OF SUBJECTS "Immune body" in blood serum, 226 in Ehrlich's theory of lytic process in blood serum, 226 haptophore groups of, 228 complementophile, 228 cytophile, 228 Immunity, absolute, 190 acquired, 192 definition of, 190, 192 active, 193 artificial, 193 definition of, 193 experimentation with attenuated cultures for, 193 with bacterial products for, 195 with dead bacteria for, 195 with sublethal doses of fully viru- lent bacteria for, 195 definition of, 189 natural, 190 individual, 191 of races, 191 of species, 190 passive, 196 relation of, to phagocytotic powers in animals, 279 Immunization, blood the seat of, 198 Incubation of cultures, 156 incubators in, 158 gas-pressure regulators for, 160 thermo-regulators for, 159 Indigo production, bacteria in, 698 Individual immunity, 191 in higher animals, 192 in lower animals, 191 Indol production by bacteria, 167 Industries, bacteria in, 697 Infection, definition of, 181 fundamental factors in, 181 paths of, 183 inner, 184 outer, 183 Infectiousness, definition of, 182 due to number of bacteria, 182 due to variations in virulence, 183 Influenza, epidemic of, in 1889-90, 528 organs attacked in, 532 Influenza bacillus, 528 bacteria related to, 533 Bacillus murisepticus, 534 bacillus of pleuro-pneumonia, 534 Bacillus rhusiopathia?, 534 Koch- Weeks bacillus, 534 pseudo-influenza bacillus, 533 biology of, 532 isolation and cultivation of, 529 on agar and gelatin, 529 blood added in, 529 hemoglobin added in, 529 hematogen added in, 530 morphology and staining of, 528 pathogenicity of, 532 in experimental inoculation of ani- mals, 533 organs attacked in, 532 susceptibility of animals to, 533 Inhibition, coefficient of, 80 Inhibition strengths of various antisep- tics, 81 Inoculation of animals, 170 intraperitoneal, 172 intravenous, 172 subcutaneous, 171 Inoculation of media, 141 technique of transferring bacteria in, 141 virus used in transferring bacteria in, 141 Insusceptibility of cold-blooded animals, 190 Intravital method of Nakanishi study of bacteria, 94 Invasion, paths of, 183 inner, 184 outer, 183 Inversion by fermentation, 42 Invertase, 49 Iodoform as disinfectant, 77 Iodin, tetrachlorid of, as disinfectant, 76 Iron compounds in nutrition of bacteria, 29 Isolation of bacteria, 142 early methods of, 143 present methods of, 143 in INDEX OF SUBJECTS 729 Isolation of bacteria, present methods of, Esmarch roll tubes in, 147 Koch's plates in, 144 surface streaking in, 148 Isolysins, 247 Israel, discovery of actinomyces of man by, 610 Jackson's lactose-bile medium for colon- typhoid differentiation, 139 Jenner's discovery of immunization in smallpox by vaccinia, 646 Katalyzers, definition of, 42 enzymes and, analogy between, 42 Kefyr, 684 Kitasato, discovery of Bacillus tetani by, 454 discovery of plague bacillus by, 546 Kitasato filter, 123 Klebs, discovery of diphtheria bacillus by, 504 Koch, discovery of cholera spirillum by, 572 discovery of tubercle bacillus by, 477 Koch plates in isolation of bacteria, 144 Koch- Weeks bacillus, 534 Koumys, 684 L + , definition of, 208 L0, definition of, 207 Lab enzymes, 46 Lactase, 50 Lactic-acid bacilli in therapy of gastro- intestinal auto-intoxication, 696-7 Lactic-acid fermentation, 50 bacteria of, 50 in milk, 683 Lactose-bile medium, Jackson's, for colon-typhoid differentiation, 138 Lactose-litmus agar, 129 Laked blood, 225 Langenbeck, discovery of Oi'dium albi- cans by, 629 Lautenschlager's thermo-regulator, 158, 159 Leprolin, 502 Leprosy, 499 Leprosy, bacillus of, 496, 499 cultivation of, 500 differentiation of, from tubercle ba- cillus by staining, 106, 500 inoculation with, 500, 502 morphology of, 499 occurrence of, in body, 501 pathogenicity of, 500 relation of, to tubercle bacillus, 502 staining of, 499 toxic products of, 502 clinical varieties of, 501 contagiousness of, 502 occurrence of, 500 tuberculin administered in, results of, 503 Leptothrix, 607 morphology of, 606 Leuchs' modification of Loeffler's mala- chite-green medium, 137 Leucocidin of Staphylococcus pyogenes aureus, 329 action of, upon leucocytes, 330 discovery of, 329 leucotoxin differentiated from, 331 obtaining of, 330 Leucocyte extract, 289 effect of, upon infections in animals, 290 in man, 291 experimentation with, 291 obtaining of, 290 Leucotoxin, 201, 331 Liborius' method of anaerobic cultiva- tion of bacteria, 149 Light in destruction of bacteria, 63 action of, 64 resistance to, 63 varieties of, 64 Lime, chlorid of, as disinfectant, 76 Limes death, definition of, 208 Limes zero, 207 Linen, etc., disinfection of, 85 von Lingelsheim, discovery of Diplococ- cus mucosus by, 387 discovery of Micrococcus pharyngis siccus by, 387 Lipase, 47 730 INDEX OF SUBJECTS Liver and gall-bladder, inflammatory conditions of, attributed to colon bacillus, 395 Lobar pneumonia, infectiousness of, 352 Loeffler and Schutz, discovery of glan- ders bacillus by, 520 Loeffler' s malachite-green media, isola- tion of typhoid bacillus in stools by, 409 Loeffler's method of staining flagella, 100 Loeffler's serum medium, 131 Lustgarten, discovery of smegma bacil- lus by, 497 Lysins, 224 action of, Bordet's interpretation of, 225 compared with Ehrlich's, 225, 228 summary of, 228 Ehrlich's theory of, 226 compared with Bordet's, 226 complement in, 226 immune body in, 226 formation of, 226 haptophore groups in, 228 complementophile, 228 cytophile, 228 red corpuscles in, 227 side chains or receptors in, 226 over-reproduction of, 226 in blood serum, 225 experimentation in, 224 in immune blood serum, reactivated by normal serum, 226 in normal blood serum, Bordet's theory of, 225 alexin in, 225 sensitizing substance in, 225 investigation of, by Ehrlich, 226 Lysol, 78 Lyssa. See Rabies Maassen filter, 124 MacConkey's bile-salt agar, for colon- typhoid differentiation, 138 Macrophages, definition of, 276 Madura foot. See Mycetoma Malachite-green media, for colon-ty- phoid differentiation, 136 bouillon, 137 Loeffler's, for isolation of typhoid bacillus in stools, 409 Leuchs' modification of Loeffler's, 137 Malignant edema, bacillus, of 466 cultivation of, 468 early investigation of, 467 immunity in, 469 morphology of, 467 pathogenicity of, 468 staining of, 468 Mallein, 524 action of, 525 diagnostic use of, 525 directions of U. S. government for, 526 obtaining and preparation of, 525 Malta fever, 524 Maltase, 50 Maragliano's serum for tuberculosis, 492 Marchiafava and Celli, discovery of meningococcus by, 371 Marmorek's serum for tuberculosis, 493 Measles, 661 investigation for virus of, 661 by Hektoen, 661 by Home, 661 Meat, determination of nature of, by precipitin tests, 254 used for culture media, 115 soluble, 116 Meat extract, 116 Meat-extract agar, 127 Meat-extract broth, 124 Meat-extract gelatin, 126 Meat-infusion agar, 128 Meat-infusion broth, 124 Meat-infusion gelatin, 127 Meat-poisoning bacilli, 429, 473 Meningitis, microorganisms causing, 37] primary, 371 secondary, 371 serum therapy of, 378 INDEX OF SUBJECTS 731 Meningococcus. See Micrococcus intra- cellularis meningitidis Metachromatic granules, 11 Metacresol as disinfectant, 77 Metchnikoff's therapy of gastrointestinal auto-intoxication by means of Bacil- lus bulgaricus, 696-7 by means of lactic-acid bacilli, 697 "Microbe de la coqueluche," 535 Micrococci, 321. See also Staphylo- cocci Micrococcus, 37 Micrococcus catarrhalis, 385 differentiation of, from gonococcus, 386 from meningococcus, 386 Micrococcus intracellularis meningitidis, 371 cultivation of, 374 oxygen in, 375 viability of organism in, 375 differentiation of, from Micrococcus catarrhalis, 386 early observation of, 371-2 immunization against, 378 agglutinins in immune sera in, 378 modes of inoculation of, 377 morphology of, 373 pathogenicity of, 376 in animals, 377 in man, 376 pseudomeningococcus differentiated from, 379 resistance of, 376 staining of, 374 susceptibility of animals to, 377 viability of, 375 Micrococcus melitensis, 542 cultivation of, 542 morphology and staining of, 542 Micrococcus pharyngis siccus, 387 Micrococcus tetragenus, 333 cultivation of, 333 pathogenicity of, 333 Microorganisms, discovery of, 1 pathogenic, 321 Microphages, definition of, 276 Microscopic study of bacteria, 93 in fixed preparations, 94 process of, 94. See also under Staining in living state, 93 by hanging block method, 94 by hanging drop method, 93 by intravital method of Nakanishi, 94 Microspira, 37 Microsporon furfur, 627 clinical picture of infection of, 627 cultivation of, 628 morphology of, 627-8 Milk, alcoholic fermentation in, 684 anthrax bacilli in, 689 bacteria in, 681 butter a means of transmitting, 693 butter-making aided by, 692 cheese-making aided by, 693 numbers of, 682 estimating of, 691 propagation of, 682 sources of, 681 under ordinarily hygienic condi- tions, 681 varieties of, 683 " bitter," 685 butyric-acid fermentation in, 684 certified, 683 cholera traced to, 687 coagulation of casein in, 684 color changes in, 685 diarrheal diseases traced to, 687 diphtheria traced to, 687 foot-and-mouth disease virus in, 688 lactic-acid fermentation in, 683 pasteurization of, 691 pus cells and leucocytes in, 688 relation of, to infectious diseases, 685 scarlet fever traced to, 686 "slimy," 685 streptococci in, 687 supervision of supply of, 682 tubercle bacilli in, 689 infection from, 690 precautions against, 690 dairy inspection in, 690 732 INDEX OF SUBJECTS Milk, tubercle bacilli in, precautions against, tuberculin test of cows in, 690 transmission of, from cow, 689 typhoid fever epidemics traced to, 685 Milk media, 130 Milzbrand, 553 Moeller's method of staining spores, 98 Moitessier's gas-pressure regulator, 169 Molds. See Hyphomycetes Morax-Axenfeld bacillus, 538 cultivation of, 538 morphology and staining of, 538 pathogenicity of, 539 Motility of bacteria, 14 by flagella, 14 Brownian, 14 effect of temperature on, 15 molecular, 14 organs of, 13 true, 14 Mucorinse, reproduction in, 623-4 Muguet. See Thrush Multiplicity of amboceptors in normal sera, 241 of complement in normal sera, 242 Mycetoma, clinical picture of, 615 granules in, 615 melanoid, 615 cultivation of, 615-16 morphology of, 615 ochroid, 615 Mycomycetes, 623 Nakanishi, "intravital" staining method of, in study of bacteria, 94 Negri bodies in central nervous system in rabies, 636 demonstration of, 637 diagnosis of rabies by, 638 explanation of, 638 staining of, 108 Neisser, discovery of Diplococcus gon- orrhoeae by, 380 discovery of lepra bacillus by, 499 Nephrotoxin, 201 Neufeld and Rimpau's discovery of opsonic substances, 282 Neurotoxin, 201 Neutral-red medium for colon-typhoid differentiation, 139 "New tuberculin" (Koch), 486 "New tuberculin-bacillary emulsion," 487 Nitrate-solution broth, 126 Nitrifying bacteria, 57 action of, 58 agricultural importance of, 58 Nitrogen fixation by bacteria, 54 microorganism of, 54 in root tubercles, 55 experimentation on, 56 microorganism of, 55 process of, 56 in soil, 54 Nitrogen in nutrition of bacteria, 28 sources of supply of, 28 Noguchi's modification of Wassermann test for syphilis, 270 Novy jar, 154 Nucleus in bacterial cell, 10 Nutrient media. See Culture media Nutrition of bacteria, 25 carbon in, 25 hydrogen in, 28 nitrogen in, 25 oxygen in, 25 salts in, 29 Obermeier, discovery of spirochsetae of relapsing fever by, 593 Obligatory aerobes, 25 Obligatory anaerobes, 26 O'idium albicans, 628 discovery of, 629 morphology of, 629 varieties of, 629 "Old tuberculin" (Koch), 485 Opium production, bacteria in, 698 "Opsonic coefficient of extinction," 286 Opsonic index, finding of, 286 Opsonic test, Wright's, 284 obtaining of bacterial emulsion for, 284 of blood serum for, 284 of leucocytes for, 284 INDEX OF SUBJECTS 733 Opsonic test, Wright's, opsonic index in, finding of, 286 parallel control test on normal serum in, 285 "pool" in, 285 technique of, 284 Simon, Lamar and Bispham's tech- nique of, 286 dilutions in, 286 opsonic coefficient of extinction in, 286 Opsonins, 281 decrease of phagocytic power upon introduction of bacteria without, 282 definition of, 282 increase of phagocytic power upon in- troduction of, 283 Neufeld and Rimpau's discovery of, 282 normal and immune, 282-3 specificity of, 282 structure of, according to Hektoen and Ruediger, 283 Wright's test of. See Opsonic test Wright's theory of, 282 Orthocresol as disinfectant, 78 Osmotic properties of bacterial cell, 23 Oxydases, 50 Oxygen as disinfectant, 86 in development of bacteria, 25 free, 26 absence of, 26 indirect supply of, 26 in nutrition of bacteria, 25 Ozone as disinfectant, 86 Paltauf's modification of Gram's stain, 103 Pancreascytotoxin, 201 Paper used in filtering culture media, 121 Paracolon bacillus, 430 Paracresol as a disinfectant, 78 Parasites, bacterial, 29 facultative, 30 media for growth of, 29 definition of, 182 infectiousness of, 182 pathogenicity of, 182 Passive immunity. See under Immunity Passive immunization, definition of, 196 Pasteur, discovery of bacillus of chicken cholera by, 544 discovery of bacillus of malignant edema by, 466 discovery of Diplococcus pneumoniae by, 353 technique of, in rabies therapy, 639 Pasteurization of milk, 691 Pathogenic bacteria, 182, 321 Pathogenicity, fundamental factors of, 181 of bacteria, 182 Penicillium, reproduction in, 625 Pepton-salt solution broth, 126 Pericardial exudates, bacteriological ex- amination of, 175 Peritoneal exudates, bacteriological ex- amination of, 175 Peritonitis following perforation attrib- uted to colon bacillus, 394 Perlsucht, 493 Permanganate of potassium as disin- fectant, 76 Pernicious anemia and Bacillus aerogenes capsulatus, 177 Peroxid of hydrogen as disinfectant, 76 Petri dish, 115, 144 Petruschky, discovery of Bacillus fecalis alkaligenes by, 426 Pfeiffer, discovery of influenza bacillus by, 528 discovery of Micrococcus catarrhalis by, 385 discovery of pseudo-influenza bacil- lus by, 528 Phagocytic index, 286 Phagocytosis, 275 cells active in, 276 "fixed," 276 macrophages, 276 microphages, 276 "wandering," 276 cells of animal origin in, 278 chemotaxis in, 277 complement or " cytase " in, 279 definition of, 275 734 INDEX OF SUBJECTS Phagocytosis, dependence of, on opso- nins, 281-2 diminution of, upon introduction of bacteria without opsonic serum, 282 immune body or " fixator" in, 279 immunity and, 279 in higher animals, 276 in protozoa, 275 increase of, upon the introduction of opsonic substances in serum, 282 macrophages in, 276 Metchnikoff 's theory of, 276 opposition to, 279 microphages in, 276 process of, in the body upon introduc- tion of bacteria, 277 upon introduction of nutrient broth, 276 susceptibility of various microorgan- isms to, 278 variety of phagocyte in, determined by the bacterium, 278 Phenol production by bacteria, 167 Phosphates in the nutrition of bacteria, 29 Phosphorescence produced by bacteria, 59 Phragmidiothrix, 38 Phycomycetes, 623 Pigment, formation of, by bacteria, 59 chemical nature of, 59 cultural conditions on, 60 Piorkowski's urine gelatin for colon-ty- phoid differentiation, 134 Pityriasis versicolor, 627 clinical picture of, 627 microorganism causing. See Micro- sporon furfur occurrence of, 627 Plague, bacillus of, 546 biology of, 549 degeneration forms of, 20 immunization against, 551 active, 552 isolation and cultivation of, 548 morphology of, 547 Plague, bacillus of, pathogenicity of, 549 resistance of, 549 staining of, 548 transmission of, 551 toxins of, 551 epidemics of, 546 in animals, 550 inoculation in, 550 spontaneous infection in, 551 in man, 549 autopsy findings in, 550 bacteriological diagnosis of, in life, 550 infection in, 549, 550 localized form of, 549, 550 pneumonic form of, 550 transmission of, 551 Planococcus, 37 Planosarcina, 37 Plasmolysis of bacterial cell, 23 Plasmoptysis of bacterial cell, 24 Plating in isolation of bacteria, 143 Pleural exudates, bacteriological exami- nation of, 175 Pleuro-pneumonia, organism of, 534 Pneumobacillus. See Bacillus mucosus capsulatus. Pneumococcus, discovery of, 7 Pneumococcus. See Diplococcus pneu- moniae Pneumonia, complications of, 362 lobar, infectiousness of, 352 Poisons, bacterial. See Bacterial poi- sons Polar bodies, 11 special stains for, 107 Poliomyelitis, acute anterior, 651 immunity in, 653 infectiousness of, 651 inoculation of animals with spinal substance of, 651 by Flexner and Lewis, 652, 653 by Knoepfelmacher, 652 by Landsteiner and Levaditi, 652 by Landsteiner and Popper, 652 resistance of virus of, 653 Polychrome stains in staining of bac- teria, 107 INDEX OF SUBJECTS 735 Potassium permanganate as disinfect- ant, 76 Potato media, 130 glycerin, 130 Pour plate, technique of making, 145 Precipitin tests, 252 bacterial nitrates for, 254 technique of, 255 determining nature of meat by, 254 precipitating anti-sera for, 252 against albumin solutions, 253 technique of production of, 252 proteid solutions to be tested by, 254 Precipitins, 200, 235 agglutinins and, structure of (Ehr- lich), 238 cell-receptors in, 238 theoretical considerations concern- ing, 238 bacterial differentiation by, 237 differentiation of proteids by, 237, 254 distinguishing blood of animal species by, 237 effect of heat on, 236 experimentation in, 235 group reaction of, 237 nature of, 236 specificity of, 237 Proagglutinoids, 235 Proteid differentiation by complement fixation, 273 substances necessary for, 273 technique of, 274 by precipitins, 237, 254 Proteid injections, anaphylaxis in, 295. See also Anaphylaxis Proteids in bacterial cell, 22 Proteins, bacterial, 186 Proteolytic enzymes, 43 action of, 44 in breaking down animal excreta, 46 bacteria producing, 44 proteids necessary to, 44 ptomains produced by, 45 Proteus group, bacilli of, 452 cultivation of, 452 morphology and staining of, 452 occurrence of, 452 pathogenicity of, 453 Protozoa, and bacteria, differentiation of, 1 staining of, 108 Pseudo-dysentery bacillus, 435, 436 Pseudo-influenza bacillus, 533 Pseudo-membranes in diphtheria, 510 Pseudomeningococcus, 379 Pseudomonas, 37 Ptomains, 45, 185, 306 bacterial poisons and, 185 discovery of, 185 occurrence of, 185 toxins distinguished from, 45 varieties of, 45 Pus, bacteriological examination of, 175 Pus cells and leucocytes in milk, 688 Putrefaction, action of, 44 Putrefactive bacteria, quantitative an- alysis of, 21 Putrescin, 45 Pyemia, definition of, 184 Pyocyanase, 570 immunizing powers of, 570 Pyocyanin, 568 Pyocyanolysin, 571 Pyrogallic acid, use of, in cultivation of anaerobic bacteria, 152 Rabies, 634 course of, 635 in animals, 635 in men, 636 diagnosis of, by presence of Negri bodies in central nervous system, 636-638 experimental infection of, 634 incubation in, 635 Negri bodies in central nervous sys- tem of, 636 demonstration of, 637 by van Gieson's method, 637 by Williams and Lowden's meth- od, 638 736 INDEX OF SUBJECTS Rabies, Negri bodies in, demonstration of, staining in, 637 Mann's method of, 637 diagnosis by, 636-8 occurrence of, 634 pathology of, 636 specific therapy of (Pasteur's tech- nique), 639 attenuation and preparation of virus fixe in, 639 inoculation of rabbits with virus fixe in, 639 spinal cord of inoculated rabbits in, desiccation of, 640 emulsification of, 641 treatment of cases with injections of spinal-cord solution in, 641 Hogyes dilution method in, 643 scheme of, used at Pasteur In- stitute, 641 used in New York Department of Health, 642 virulence of virus of, 635 Racial immunity, 191 "Rage." See Rabies Rauschbrand. See Bacillus of sympto- matic anthrax Receptors of toxin molecule in side- chain theory, 213 chemical action of, 213 over-production of, 214 Red blood cells, antibodies produced by, 200 Reducing powers of bacteria, 167 Refractive index of parts of bacterial cell, 24 Reichel filter, 122 Reichert's thermo-regulator, 158, 159 Relapsing fever, 593 immunity in, 599 symptoms of, 597 transmission of, 598 varieties of, 597 Relapsing fever spirochaete, 593 cultivation of, 594 morphology and staining of, 593 pathogenicity of, 595 in animals, 595 Relapsing fever spirochaete, pathogeni- city of, in man, 597 symptoms of, 597 transmission of, 598 varieties of, 597 Reproduction of bacteria, 17 Resistance, definition of, 189 Rhinoscleroma, bacillus of, 449 Ricin, experimentation with, 204 Ringworm, 631 Root tubercles, 55 microorganism of nitrogen fixation in, 55 Roux's method of anaerobic cultivation of bacteria, 149 Sacchromycetes. See Yeasts and Yeast cells Salts in nutrition of bacteria, 29 Saphrophytes, bacterial, 29, 30 definition of, 182 Sarcina, 37 Sarcophysematos bovis. See Bacillus of symptomatic anthrax Scarlatina. See Scarlet fever Scarlet fever, 662 favorable influence of streptococcus antisera in, 662 streptococci present in, 662 traced to milk, 686 Schaudinn and Hoffmann, discovery of Spirochaete pallida by, 585 Schizomycetes, 37 Schweineseuche, 545 Scorpion poison, antitoxin for, 199 "Sensibilisin," 302 "Sensibilisinogen," 302 Septicemia, definition of, 184 diagnosis of, by isolation of bacteria from the blood, 178 due to colon-bacillus infection, 394 hemorrhagic. See Hemorrhagic sep- ticemia Serum media, 131 Loefiier's, 131 Serum reactions, technique of, 249. See also under individual tests agglutination tests in, 250 INDEX OF SUBJECTS 737 Serum reactions, antigen determined in, by complement fixation, 271 for typhoid fever, 271 obtaining of material for, 271 test in, 272 bactericidal and bacteriolytic tests in, 255 complement, fixation in, for deter- mination of antibodies, 261 for determination of antigen, 271 for proteid differentiation, 273 hemolytic tests in, 259 precipitin tests in, 252 proteid differentiation by comple- ment fixation in, 273 substances necessary for, 273 test in, 274 Wassermann test in, 262 modifications of, 268 "Serum sickness," 296 Serum water media for fermentation tests, 132 Shiga, discovery of dysentery bacillus by, 433 Shiga's bacillus, 433 cultural characteristics of, 434 morphology of, 433 Side-chain theory of toxin-antitoxin reaction, 212 chemical action in, 213 elements of molecules in, 213 atom group, 213 side chains or receptors, 213 over-production of receptors in, 214 Side chains, action of, in Ehrlich's theory of lytic process in blood serum, 226 Slanting of culture media, 123 " Slimy " milk, bacteria causing, 685 Smallpox, 644 etiological factor of, 644 immunization in, 645 by vaccination, 646, 650 Jenner's discovery of, 646 technique of, 650 value of, 650 production of vaccine for, 647. See also under Vaccine occurrence of, 644 47 Smallpox, protozoan incitant of, research for, 644 relation of chicken-pox to, 647 relation of cowpox to, 646 transmission of, 645 vaccine bodies in, discovery of, 644 explanations for, 645 Ewing's, 645 Smegma bacillus, 496, 497, 584 cultivation of, 498 morphology of, 497 occurrence of, 497 staining of, 498 identification of bacillus by, 498 tubercle bacillus and, differentiation between, by stains, 106 Smith's modification of Pitfield's method of staining flagella, 101 Snake poison, antitoxin for, 199 Soil, bacteria in, 667 from burial of infected cadavers, 669 in agricultural regions, 667 numerical estimation of, 669 pathogenic, in surface layers, 668 Solutions, saturated, for staining of bacteria, 95 staining-power of, 96 Soor. See Thrush Species immunity, 190 differences in, 190 Specific gravity of forms of bacterial cell, 24 "Specific precipitates," 236 Spider poison, antitoxin for, 199 Spinal fluid, bacteriological examination of, 176 Spirillacese, 37 Spirillum, 38 description of, 9 Spirillum cholerse asiaticae. See under Cholera Spirillum Deneke, 581 Spirillum of Finkler-Prior, 531 Sprillum Massaua, 580 Spirillum Metchnikovi, 580 Spirochete, 38 Spirochete anserina, 605 Spirochete Duttoni, 598 738 INDEX OF SUBJECTS Spirochete gallinHrum, 603 immunization against, 605 similarity of, to Spirochete anserina, 605 transmission of, 604, 605 Spirochete of relapsing fever. See Re- lapsing fever, spirochete of Spirochete of Vincent's angina. See Vincent's angina, spirochete of Spirochete pallida, 583 animal pathogenicity of, 591 cultivation of, 590 demonstration of, 586 in living state, 586 in smears, 587 by India-ink preparation, 588 by Goldhorn's method of stain- ing, 588 by Schaudinn and Hoffmann's method of staining, 587 by Wood's method of staining, 588 in tissues, 588 by Levaditi's method, 589 by Levaditi and Manouelian's method, 589 immunization against, 592 active, 592 passive, 593 morphology of, 585 observation of, 586-7 occurrence of, in syphilis cases, 585-6 staining of, 108 Spirochete pertenuis, 603 morphology of, 603 similarity of, to Spirochete pallida, 603 " Spirochete refringens," 585 Spirochetes, cultivation of, 583 differentiation of, from spirilla, 583 diseases caused by, 582. See also under specific names reproduction in, 582 structure of, 582 Spiroso'ma, 37 Spore stains in staining of bacteria, 97 Spores, bacterial, 15 formation of, 15 germination of, 17 Spores, bacterial, position of, 17 varieties of, 16 arthrospores, 16 true or endospores, 16 vegetative forms from, 17 Sporulation, physiological significance of, 17 process of, 16 Sputum, disinfection of, 84 Stable antitoxin, 206 Staining of bacteria, chemical principles in process of, 96 acid-fast bacteria stains, 104 Baumgarten's method, 106 Bunge and Trautenroth's method, 106 Ehrlich's method, 104 Gabbet's method, 105 Pappenheim's method, 106 Ziehl-Neelson method, 105 capsule stains, 98 Buerger's method, 99 Hiss' methods, 98 copper sulphate, 98 potassium carbonate, 98 Wadsworth's method, 99 Welch's method, 98 differential stains, 102 Gram's method, 102 classification by, 104 Paltauf 's modification of, 103 riagella stains, 100 van Ermengem's method, 101 Loefner's method, 100 Smith's modification of Pitfield's method, 101 polychrome stains, 107 Giemsa's method, 108 Jenner's method, 108 Wright's modification of Irish- man's method, 108 Wood's method, 109 special stains for polar bodies, 107 Neisser's method, 107 Roux's method, 107 spore stains, 97 Abbott's method, 97 Moeller's method, 98 INDEX OF SUBJECTS 739 Staining of bacteria, chemical principles in process o", staining in tis- sues, 110 for actinomyces in sections, 112 for Gram-positive bacteria, 111 Gram-Weigert method, 111 in celloidin sections, 111 in paraffin sections, 111 for tubercle bacilli in sections, 112 in celloidin sections, 112 in paraffin sections, 112 Loeffler's method, 112 saturated solutions used in, 95 staining solutions in, power of, 96 steps in process of: (1) smearing, 94 (2) drying, 95 (3) fixing, 95 (4) staining, 95 (5) washing, 95 (6) blotting, 95 (7) mounting, 95 Standardization of diphtheria antitoxin, 218 of tetanus antitoxin, 221 Staphylococci, 321. See also under individual staphylococci definition of, 321 in feces, 177 Staphylococcus epidermidis albus, 332 Staphylococcus pyogenes albus, 332 Staphylococcus pyogenes aureus, 322 cultural characters of, 323 immunization against, 331 active, 332 agglutinins in, 331 modes of inoculation with, 327 morphology of, 322 pathogenicity of, 326 in animals, 327 in man, 327 pigment formation of, 325 resistance of, 325 to chemicals, 326 to desiccation, 326 to heat and cold, 325 staining of, 322 susceptibility of animals to, 326 Staphylococcus pyogenes aureus, sus- ceptibility of man to, 327 thermal death point of, 325 toxic products of, 328 endotoxins, 328 hemolysins, 328 leucocidin, 329. See also under Leucocidin virulence of, 326 Staphylococcus pyogenes citreus, 332 Steam in sterilization, 67 live, 69 saturated, 68 superheated, 68 Stegomyia fasciata, 659 Sterilization of culture media, 121 filtration in, 122 heat in, 121 Sternberg, discovery of Diplococcus pneumoniae by, 353 Stimulins, 281 Streaking, surface, in isolation of bac- teria, 148 "Street virus," 635 Streptococci, 37, 336 capsulated, description of organisms reported as, 367-369 classification of, 349 by carbohydrate fermentation pow- ers, 350 by reactions to immune sera, 350 morphological, 349 Streptococcus longus seu erysi- pelatos in, 349 Streptococcus minor seu viridans in, 350 Streptococcus mucosus in, 350 definition of, 336 differentiation of, from pneumococci, 357, 367 cultural, 368 morphological, 367 in feces, 177 in milk, 687 preparation of, for agglutination test, 251 pyogenic. See Streptococcus pyo- genes 740 INDEX OF SUBJECTS Streptococcus erysipelatis, 343 Streptococcus longus seu erysipelatos, 349 Streptococcus mitior seu viridans, 350 Streptococcus mucosus, 350, 351 Streptococcus pyogenes, 336 brevis, 338, 339 cultivation of, 338 early experimentation with, 336 immunization against, 346 immune sera of infected animals in, 347 agglutinins in, 348 precipitins in, 349 specificity of, 348 standardization of, 348 leucocyte extracts in, 348 technique of, 347 longus, 338 modes of inoculation with, 342 in animals, 342 in man, 343 morphology of, 338 pathogenicity of, 341 in animals, 342 in man, 343 resistance of, 341 staining of, 338 susceptibility of animals to, 342 toxic products of, 344 endotoxins, 345 hemolysins, 345 virulence of, 341 Streptothrix, 38, 607 cultivation of, 609 morphology of, 607, 609 Sublethal doses of virulent bacteria in active immunization, 195 Sugar-free broth, 125 Sulphates in nutrition of bacteria, 29 Sulphur bacteria, 60 physiology of, 61 spectroscopic examination of, 61 varieties of, 60 Sulphur dioxid as disinfectant, 86 Sulphuretted hydrogen. See Hydrogen sulphid Suprarenal cytotoxin, 201 Swine-plague bacillus, 545 differentiation of, from hog-cholera bacillus, 546 immunization against, 545 morphology of, 545 pathogenicity of, 545 Symbiosis of bacteria, 31 Symptomatic anthrax, bacillus of. See Anthrax, symptomatic Syphilis, 583 microorganism of. See Spirochete pallida Tanning of hides, bacteria in, 698 Temperature, attained by application of various degrees of pressure, 72 effect of, on activity of bacteria, 15 high, 34 low,34 relation of, to bacteria, 31 maximum, 32 minimum, 32 optimum, 32 to cultures with spores, 33 to vegetative forms, 33 "Tetanolysin," 205, 461 "Tetanospasmin," 461 Tetanus antitoxin, 220 production of, 220 horses used in, 221 technique of, 221 toxin for, 220 standardization of, 222 unit of (Society of American Bacteri- ologists), 222 Tetanus bacillus, 454 autopsy findings in infections of, 458 biological characteristics of, 456 cultivation of, 456 distribution of, 455 early observation of, 454 favorable conditions for growth of, 457 incubation of, 458 isolation of, by Kitasato, 454 morphology of, 454 pathogenicity of, 457 following wounds, 458 INDEX OF SUBJECTS 741 Tetanus bacillus, pathogenicity of, rela- tion of spores to, 457 resistance of, 457 staining of, 455 toxin of, 458 central nervous system attacked by, 461 mode of reaching, 461 incubation period of, 461 isolation of, 459 by chemical reaction, 460 by filtration, 459 by precipitation, 459 production of, 459 resistance of, 460 strength of, 460 susceptibility of animals to, 460 Thermal death points, 34 Thermo-regulators for incubators, 159 Lautenschager's, 158, 159 Reichert, 158, 159 Thiothrix, 38 Thrush, 629 microorganism causing, 629 Timothy, bacillus of, 496 Tissue sections, method of staining, 111 Gram-Weigert, 111 in celloidin sections, 111 in paraffin sections, 111 staining of bacteria in, 110 Titration of culture media, 117 color indicator in, 117 process of, 117 for alkaline media, 118 reaction of, 117 adjustment of, 119 Tobacco industry, bacteria in, 697 Torulae, 617 Toxin, constitution of (Erlich), 210 graphic form of (Ehrlich), 211 views of Arrhenius and Madsen on, 212 diphtheria. See under Diphtheria toxin endotoxin distinguished from, 186 epitoxoid in, 208 in side-chain theory, cell-nutrition in, 213 Toxin, in side-chain theory, chemical action of, 213 elements of, 213 atom group, 213 side chains or receptors, 213 over production of receptors in, 214 molecule of, haptophore group in, 207 toxophore group in, 207 partial absorption of, 209 standardization of, 207 Limes death in, 208 Limes zero in, 207 time changes in, 206 toxoid form of, 207 protoxoids in, 209 syntoxoids in, 209 toxon and, difference in action of, 209 toxon in, 209 valency of antitoxin for, 210 used for production of diphtheria antitoxin, 216 Toxin-antitoxin reaction, 203 side-chain theory in, 212 summary of, 215 theories as to process of, 203 by destruction of toxin by its specific antitoxin, 203 by direct union of toxin and anti- toxin, 203 through mediation of tissue cells, 203 time element in, 204 Toxin solution, normal, 205 Toxin unit, 205 Toxins, 185 compared with pigments, 186, 309 summary of, 305 Toxoid form of diphtheria toxin, 207 Toxoids, varieties of, 209 epitoxoid form in, 208 protoxoids, 209 syntoxoids, 209 Toxon, in diphtheria toxin, 209 toxin and, difference in action of, 209 Toxon molecule, 209 haptophore group in, 209 toxophore group in, 209 742 INDEX OF SUBJECTS Toxophore group in toxin molecule, 207 in toxon molecule, 209 Trichomycetes. See Chlamydobacteri- aceae Trichophyton tonsurans, 631 cultivation of, 633 demonstration of, 632 morphology of, 632 occurrence of, 631 Tricresol, 78 Trillat autoclave, 87 Tubing of culture media, 121 "Typhoplasmin," 416 Typhoid bacillus. See under Typhoid fever Typhoid fever, bacillus of, 399 biological conditions favorable to, 403 cultivation of, 399 differentiation of, from Bacillus fecalis alkaligenes, 427 from meat-poisoning and para- typhoid bacilli, 428 discovery of, 7, 399 immunization against. See under Typhoid fever, immunization in in blood during disease, 405 obtaining cultures of, 405 in feces, 177 in gall-bladder, 411 in rose spots, 412 in sputum, 412 in stools, 406 examination in, 406 isolation of, 407 on Conradi-Drigalski medium, 408 on Eisner's potato-extract gel- atin, 407 on Endo's fuchsin-agar, 409 on Hiss' agar-gelatin media, 407 on Loeffler's malachite-green media, 409 time of appearance in, 406 in urine, 411 inoculation of animals with, 404 with endotoxin of, 417 isolation of, 403 Typhoid fever, bacillus of, in water, 676 morphology of, 399 pathogenicity of, 404 in animals, 404 in man, 404 staining of, 399 suppurative lesions due to, 412 toxic products of, 415 obtaining of, 417 varieties of: endotoxins, 415 true toxins, 416 typhoplasmin, 416 diagnosis in, by agglutinins in blood serum, 420 Widal test in, 421 obtaining blood for, 422 by bactericidal substances in blood serum, 419 by bactericidal tests in vivo, 258 by opsonic index, 424 epidemics of, traced to milk, 685 hygienic considerations in, 413 immunization in, 417 by inoculation with typhoid bacilli, 417 active, 424 technique of Pfeiffer and Kolle in, 425 of Wright in, 425 substances found in blood after, 418 agglutinins in, 419 chief or major, 420 group, 420 bactericidal, 419 bacteriolytic, 418 opsonins in, 424 precipitins in, 423 obtaining blood cultures in, 180 prophylactic measures in, 414 specific therapy in, 424 transmission of, 413, 414 by flies, 415 from milk, 414 from oysters, 415 from water supply, 414 INDEX OF SUBJECTS 743 Typhoid fever, without intestinal le- sions, 413 Tubercle bacillus, 477 bacilli related to, 493 Bacillus butyricus, 496 bacillus of avian tuberculosis, 494 cultivation of, 495 discovery of, 484 morphology and staining of, 495 susceptibility of animals to, 495 bacillus of bovine tuberculosis, 493 early investigation of, 493 cultivation of, 493 differentiation of, from human type, 494 morphology of, 493 bacillus of fish tuberculosis, 495 bacillus of leprosy, 496, 502 bacillus of timothy, 496 Bacillus smegmatis, 496 biological considerations of, 482 chemical analysis of, 484 cultivation of, 480 " Nahrstoff Heyden " in, 482 media for, 480 discovery of, 7 early investigation of, 477 examination for, by animal inoculation, 175 by Ziehl-Neelson staining method, 176 in feces, 178 in milk, 691 isolation of, 480 leprosy bacillus and, differentiation between, by stains, 106 methods of staining, 104, 105, 106 in sections, 112 celloidin, 112 paraffin, 112 morphology of, 477 pathogenicity of, 483 frequency in, 483 mode of infection in, 484 mortality in, 483 preparation of, for agglutination test, 251 quantitative analysis of, 22 Tubercle bacillus, smegma bacillus and, differentiation between, by stains, 106 staining of, 478 differentiation of, from acid-fast group by Pappenheim's method of, 480 Ehrlich's anilin-water-gentian- vio- let solution in, 479 Gabbet's decoloration and coun- terstaining in, 479 Ziehl's carbol-fuchsin solution in, 479 toxins of, 485 endotoxins in, 485 tuberculins in, 485 bouillon nitre (Denys), 487 "new tuberculin-bacillary emul- sion" (Koch), 487 "new tuberculin" (Koch), 486 original method of making of, 486 present method of making of, 486 "old tuberculin" (Koch), 485 " tuberculoplasmin ' ' (Buchner andHahn), 487 Tuberculin. See under Tubercle bacillus, toxins of " Tuberculoplasmin " (Buchner and Hahn), 487 Tuberculosis, frequency of, 483 immunization in, passive, 492 Maragliano's serum in, 492 Marmorek's serum in, 493 mode of infection in, 484 mortality of, 483 tuberculin in, diagnostic use of, 487 cutaneous reaction in, 489 in cattle, 489 ophthalmo reaction in, 488 subcutaneous injection of, 487 dosage and reaction in, 488 therapeutic uses of, 491 original, 491 present, 491 dosage in, 492 preparations employed in, 492 744 INDEX OF SUBJECTS Urine, bacteriological examination of, 176 Urobacillus liquefaciens, 453 Uschinsky's proteid-free medium, 126 Vaccine production, for immunization in smallpox, 647 animals used in, 647 calves used for, 647 cleanliness observed in stabling of, 647 material used for vaccination of, 648 vaccination in, 648 preparation of field in, 648 scarifications in, 648 vaccinia vesicles developed in, 648 obtaining of vaccine from, 649 by curettage, 649 by ivory tips, 649 testing of vaccine in, for bacteria, 650 for efficiency, 649 Vaccine therapy of Wright, 286 dosage for, 288 opsonic curve in, 288 production of vaccines- in, 286 standardization of emulsion in, 287 enumeration of bacteria against red blood cells in, 287-8 sterilization of vaccine in, 288 Variola. See Smallpox Vegetative forms from bacterial spores, 17 "Vibrion septique." See Malignant edema, bacillus of Vincent's angina, 599 spirochete of, 599 cultivation of, 601 fusiform variety of, 600 bacilli of other diseases resem- bling, 602. See also under Fu- siform bacilli other bacilli accompanying, 602 spirillum variety of, 601 symptoms of, 599 Vincent's spirilla, staining of, 108 Virulence, definition of, 183 variations in, and infectiousness, 183 Virulent bacteria, sublethal doses of, in immunization, 195 Virus fixe, in specific therapy of rabies, 639 Wadsworth's method of staining cap- sules, 99 Wassermann test for diagnosis of syph- ilis, 262 antigen for, 262 determination of necessary quantity of, 264 obtaining of, from alcoholic ex- tracts of syphilitic organs, 263 from alcoholic solution of normal organs, 263 from salt-solution of syphilitic liver, 263 of syphilitic spleen, 262 preparation of, by Noguchi meth- od, 264 complement in, 266 hemolytic serum in, 265 obtaining of, 265 potency of, 265 quantity of, 265 unit in, definition of, 265 determination of, 266 modifications of, 268 Bauer's, 268 Noguchi's, 270 preparation for, 262 serum to be tested for syphilitic anti- body in, 267 sheep corpuscles in, 267 technique of, 267 Water, bacteria in, 671 in ground waters, 673 in perennial springs, 674 in wells, 673 in rain and snow, 672 in surface waters, 672 influence of rain on, 672 light and temperature factors in purification of, 673 self-purification in, 672 pathogenic, 671 of cholera, 671 of diarrheal diseases, 671 of typhoid fever, 671 INDEX OF SUBJECTS 745 Water, bacteria in, qualitative analysis of, 676 isolation of cholera vibrio in, 678 Koch's method of, 678 isolation of colon bacillus in, 679 isolation of typhoid bacillus in, 676 Adam's and Chapin's method of, 676 Drigalski's method of, 677 Parietti's method of, 677 Vallet's method of, 677 quantitative estimations of, 674 collecting of specimens for, 674 colon bacilli in, 680 colon test in, 679 counting of bacilli in, 680 counting in, 676 incubation of specimens in, 676 plating of specimens in, 675 value of, 676 in bacterial cell, 21 Welch, discovery of Bacillus aerogenes capsulatus by, 469 Welch's method of staining capsules, 98 Welch's modification of Guarnieri's medium, 129 Wertheim's medium for cultivation of gonococcus, 381-2 Winckel's disease in the newborn due to colon bacillus, 394 Wires used in transfering bacteria, 141 WolfThugel counting plate, 162 Wool-sorter's disease, 563 Wright's method of anaerobic cultiva- tion of bacteria, 150 Wright's modification of Buchner's pyro gallic method of cultivation of anaerobic bacteria, 153 Wright's theory of opsonins, 282 Wright's vaccine therapy. See Vaccine therapy of Wright Yaws, 603 Yeast cells, cultivation of, 621 demonstration of, 620 morphology of, 617 reproduction in, by budding, 617 by spore formation, 618 Yeasts, 617 differentiation of, from other microor- ganisms, 617 fermentation by, 618 industrial employment of, for fermen- tative purposes, 52 infection of, in animals, 620 in man, 619 clinical picture of, 620 pathogenic varieties of, 621 Yellow fever, 654 clinical picture of, 654 distribution of, 654 etiology of, 654 immunity in, 660 investigation of, by Guiteras and Mar- choux, Salimbeni and Simond, 659 results of, 659 by Reed, Carroll, Agramonte, and Lazear, 656 results of, 659 microorganism of, biological proper- ties of, 655 research for, 654 by Cornil and Babes, 654 by Sanarelli, 655 by Sternberg, 655 Stegomyia fasciata in, 659 description of, 659 power of transmission of infection by, reasons for, 660 tropical countries most favorable for, 660 transmission of, 656 by mosquitoes, 656 discovery of, by Finlay, 656 investigation and confirmation of, by United States Commission, 656 Yersin, discovery of plague bacillus by, 547 Zur Nedden's bacillus, 539 cultivation of, 539 morphology and staining of, 539 pathogenicity of, 540 Zymase, 51, 618 N