A text-book of bacteriology, : a practical treatise for students and practitioners of medicine

Front matter

a

A TEXT - BOOK OF

BACTERIOLOGY

Preface

The volume here presented is primarily a treatise on the fundamental 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, Agricultural, and Industrial — having little in common, except problems of general bacterial physiology and certain fundamental technical procedures.

From no other point of approach, however, is such a breadth of conception attainable, as through the study of bacteria in their relation to disease processes in man and animals. Through such a study one must become familiar not only with the growth characteristics 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 practitioner 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 understanding 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.

H. Z.

August, 1910.

Section I

THE GENERAL BIOLOGY OF BACTERIA AND THE TECHNIQUE OF BACTERIOLOGICAL STUDY

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 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

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

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)

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)

XXX. Bacillus mucosus capsulatus 445

XXXI. Bacillus tetani 454

XXXII. Bacillus of Symptomatic Anthrax, Bacillus of Malignant Edema, Bacillus afrogenes capsulatus, Bacillus botulinus 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

XXXVIII. Bordet-Gengou Bacillus, Morax-Axenfeld Bacillus, Zur

Nedden Bacillus, Ducrey Bacillus 535

XXXIX. The Bacilli of the Hemorrhagic Septicemia Group and

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

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

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

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

70. Staphylococcus colonies 323

71. Micrococcus tetragenus 333

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

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

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

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

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 converging toward a common type, represented by a large group of unicellular 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, locomotion, 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 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 correspond 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 "infusion animalcula " were demonstrable in tartar from the teeth, in intestinal 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 philosophers, and had led, in 1546, to the division of contagious diseases by Fracastor, into those transmitted "per contactum," and those conveyed 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 discovered 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 remarkably 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 established 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 substances, were living beings, which bore a causal relationship to the process 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 existing 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 fermentation.

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 etiological research, it was Henle who formulated the postulates of conservatism, 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 carried out.

It was during this period also that one of the most fundamental 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 fortifying his opinions in favor of spontaneous generation by extensive experimentation. He had placed putrefying material and vegetable infusions 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 experiments 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 produced chemical changes in his solutions which had made spontaneous generation impossible.

The experiments of Schulze, in 1836, who failed to find living organisms 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 sixteenth 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 centuries 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 experiments 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 research 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 cottonwool, innumerable microorganisms were deposited upon the filter. A single shred of such a contaminated filter dropped into a flask of previously 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 contact with air, would remain uncontaminated provided that the entrance 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 contamination, 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 complete 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."

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 fermentations, such as those of lactic and butyric acid, as well as the decomposition 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 microorganism 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 investigations, not only confirmed the observations of the two authors mentioned 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 conditions, 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 Recklinghausen, 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 controversy 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, extending 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.