(Blasgow Portland's Students Manua Is. MANUAL OF BACTERIOLOGY. NUNQUAM AL1UD NATURA, AL1UD SAPIENTIA DICIT. MANUAL OF ACT E RIO LOGY BY ROBERT MUIR, M.A., M.D., F.R.C.P.Ed. PROFESSOR OF PATHOLOGY, UNIVERSITY OF GLASGOW AND JAMES RITCHIE, M.A., M.D., B.Sc. LECTURER IN PATHOLOGY, UNIVERSITY OF OXFORD SECOND EDITION WITH ONE HUNDRED TWENTY-SIX ILLUSTRATIONS EDINBURGH AND LONDON YOUNG J. PENTLAND 1899 EDINBURGH : PRINTED FOR YOUNG J. PENTLANU, II TEVIOT PLACE, AND 38 WEST SMITH FIELD, LONDON, E.C., BY R. AND R. CLARK, LIMITED All rights reserved. PREFACE TO SECOND EDITION. In preparing this- edition we have made no change in the original plan of the book. The text, however, has been carefully revised, and the results of the more recent re- searches have been incorporated. Some parts have been condensed, but, in consequence of the introduction of new subject-matter and of additional illustrations, the size of the book as a whole has been considerably increased. We trust that these alterations will be found to be in the direction of improvement. May 1899. PREFACE. The science of Bacteriology has, within recent years, become so extensive, that in treating the subject in a book of this size we are necessarily restricted to some special departments, unless the description is to be of a superficial character. Accordingly, as this work is intended primarily for students and practitioners of medicine, only those bacteria which are associated with disease in the human subject have been considered. 'We have made it a chief endeavour to render the work of practical utility for beginners, and, in the account of the more important methods, have given elementary details which our experience in the practical teaching of the subject has shown to be necessary. In the systematic description of the various bacteria, an attempt has been made to bring into prominence the evidence of their having an etiological relationship to the corresponding diseases, to point out the general laws governing their action as producers of disease, and to con- sider the effects in particular instances of various modifying circumstances. Much research on certain subjects is so recent that conclusions on many points must necessarily V1U PREFACE. be of a tentative character. We have, therefore, in our statement of results aimed at drawing a distinction between what is proved and what is only probable. In an Appendix we have treated of four diseases ; in two of these the causal organism is not a bacterium, whilst in the other two its nature is not yet determined. These diseases have been included on account of their own import- ance and that of the pathological processes which they illustrate. Our best thanks are due to Professor Greenfield for his kind advice in connection with certain parts of the work. We have also great pleasure in acknowledging our indebtedness to Dr. Patrick Manson, who kindly lent us the negatives or preparations from which Figs. 119- 124 have been executed. As we are convinced that to any one engaged in practical study, photographs and photomicrographs supply the most useful and exact information, we have used these almost exclusively in illustration of the systematic description. These have been executed in the Pathological Laboratory of the University of Edinburgh by Mr. Richard Muir. The line drawings were prepared for us by Mr. Alfred Robinson, of the University Museum, Oxford. To the volume is appended a short bibliography, which, while having no pretension to completeness, will, we hope, be of use in putting those who desire further information on the track of the principal papers which have been pub- lished on each of the subjects considered. June 1897. CONTENTS. CHAPTER I. General Morphology and Biology. Introductory— Terminology— Structure of the bacterial cell— Repro- duction of bacteria— Spore formation— Motility— Minute structure of bacterial protoplasm — Chemical composition of bacteria — Classification— Food supply — Relation of bacteria to moisture, gaseous environment and temperature — Conditions affecting bacterial motility — Effects of bacteria in nature — Methods of bacterial action — Variability among bacteria — Death of bacteria Page i CHAPTER II. Methods of Cultivation of Bacteria. Introductory — Methods of sterilisation — The preparation of culture media — The use of the culture media — The methods of the separation of aerobic organisms — The principles of the culture of anrerobic organisms — Miscellaneous methods — General laboratory rules • . . ■ • ■ • •'• • «34 CHAPTER III. Microscopic Methods — General Bacteriological Diagnosis— Inoculation of Animals. The microscope — Examination of hanging-drop cultures — Film pre- parations— Examination of bacteria in tissues — The cutting of X CONTENTS. sections— Staining principles— Mordants and decolorisers— For- mulas of stains— Gram's method and its modifications— Contrast stains— Stain for tubercle and leprosy bacilli— Staining of spores and flagella— Observation of agglutination and sedimentation- Routine bacteriological examination— Methods of inoculation — Autopsies on animals page CHAPTER IV. Non-Pathogenic Micro-organisms — Fungi. Mucorinag — Ascomycetse — Perisporiacese — Yeasts and toruke . 133 CHAPTER V. Relations of Bacteria to Disease — The Pro- duction OF TOXINES BY BACTERIA. Introductory — Conditions modifying pathogenicity — Susceptibility and resistance — Modes and effects of bacterial action— Tissue changes produced by bacteria — Local lesions — General lesions — The production of toxines by bacteria and the nature of these — Allied vegetable and animal poisons — The theory of toxic action . . . . . , , . • 139 CHAPTER VI. Suppuration and Allied Conditions. Nature of suppuration —The bacteria of suppuration — Experimental inoculation — Virulence of streptococcus pyogenes — Suppuration without bacteria — Lesions in the human subject — Mode of en- trance and spread of pyogenic bacteria— Ulcerative endocarditis — Acute suppurative periostitis — Erysipelas — Methods of examina- tion for pyogenic bacteria . . . . . .163 CHAPTER VII. Gonorrhoea, Soft Sore, Syphilis. The gonococcus — Microscopical characters — Cultivation — Relations to disease — Its toxine — Distribution — Gonococcus in joint affections — Methods of diagnosis — Soft sore — Syphilis . . .189 CONTENTS. xi CHAPTER VIII. Acute Pneumonia. Introductory— Historical— Bacteria in pneumonia— Fraenkel'spneumo- coccus— Friedlander's pneumobacillus— Distribution of pneumo- bacteria — Experimental inoculation — Etiological relations _ to pneumonia— The toxines of the pneumococcus— Immunisation against pneumococcus— Methods of examination . Page 204 CHAPTER IX. Tuberculosis. Historical— Tuberculosis in animals— The tubercle bacillus —Staining reactions— Cultivation of tubercle bacillus— Powers of resistance —Action on the tissues— Histology of tubercular nodules— Dis- tribution of bacilli— Bacilli in tubercular discharges— Experi- mental inoculation— Avian tuberculosis— Action of dead tubercle bacilli — Sources of human tuberculosis — Koch's tuberculin— Toxines of the tubercle bacillus — Anti-tubercular serum— Active immunisation against tubercle — The smegma bacillus — Methods of examination . . . • • • • • 224 CHAPTER X. Leprosy. Pathological changes — Bacillus of leprosy — Distribution in lesions — Relations to disease — Methods of diagnosis . . .258 CHAPTER XI. Glanders and Rhinoscleroma. The natural disease — The bacillus of glanders— Cultivation of glanders bacillus — Powers of resistance — Experimental inoculation — Action on the tissues — Mode of spread — Mallein and its preparation — Methods of examination — Rhinoscleroma . . . 268 Xll CONTENTS. CHAPTER XII. Actinomycosis. Characters of the actinomyces — Tissue lesions— Distribution of lesions — Cultivation of actinomyces — Experimental inoculation — Methods of examination and diagnosis — Madura disease . . Page 282 CHAPTER XIII. Anthrax. Historical summary — Bacillus anthracis — Appearances of cultures — Biology — Sporulation — Natural anthrax in animals — Experi- mental anthrax — Anthrax in man — Pathology — Toxines of the bacillus anthracis — Mode of spread in nature— Immunisation against anthrax — Methods of examination . . . 295 CHAPTER XIV. Typhoid Fever — Bacilli Allied to the Typhoid Bacillus. Historical summary — The bacillus typhosus — Morphological characters — Characters of cultures — Bacillus coli communis — Reactions of B. typhosus and B. coli — Bacillus enteritidis (Gaertner) — Psitta- cosis bacillus — Pathological changes in typhoid fever — Suppura- tions in typhoid fever — Pathogenic effects produced in animals — Toxines of B. typhosus — Immunisation of animals — Relations of bacilli to the disease — Serum diagnosis — Vaccination against typhoid — Methods of examination . . . . 317 CHAPTER XV. Diphtheria. Historical — General facts — Bacillus diphtheria: — Microscopical char- acters— Distribution — Cultivation — Inoculation experiments — The toxines of diphtheria — Variations in virulence of bacilli — Summary of pathogenic action — Methods of diagnosis . 353 CONTENTS. xin CHAPTER XVI. Tetanus. Introductory-Historical^^^ -Characters of cultures -Conditions of growth-Pa thogen c effects-Experimental inoculation— Tetanus toxmes-Antitetanic serum — Methods of examination -Malignant oedema— Char- acters of bacillus— Methods of diagnosis-Quarter evil 1 age CHAPTER XVII. Cholera. Introductory— The cholera spirillum— Distribution of the spirilla- Cultivation— Powers of resistance — Experimental inoculation— Toxines of cholera spirillum— Inoculation of human subject- Modes of distinguishing the spirillum— General summary— Spirilla resembling the cholera organism— Methods of diagnosis — Metchnikoff's spirillum— Fihkler and Prior's spirillum— Deneke s spirillum . • n CHAPTER XVIII. Influenza, Plague, Relapsing Fever, Malta Fever, Yellow Fever. Influenza bacillus— Microscopical characters— Cultivation— Distribu- tion Experimental inoculation— Methods of examination — Bacillus of plague— Microscopical characters— Distribution — Cultivation — Experimental inoculation — Immunity — Relapsing fever— Characters of the spirillum— Relations to the disease- Malta fever — Micrococcus melitensis — Relations to the disease — Methods of diagnosis— Yellow fever— Bacteria in yellow fever- Immunity against B. icteroides . . • • 43 1 CHAPTER XIX. Immunity. Introductory — Acquired immunity — Artificial immunity — Varieties — Active immunity — Methods of production — Attenuation and exaltation of virulence — Passive immunity — Action of the serum —Antitoxic serum — Standardising of toxines and of antisera — XIV CONTENTS. Nature of antitoxic action — Antimicrobic serum — Lysogenic and agglutinative actions— Pfeiffer's phenomenon — Therapeutic effects of antisera — Theories as to acquired immunity — Natural im- munity— Natural bactericidal powers . . . Page 458 APPENDIX A. Smallpox and Vaccination. Jennerian vaccination — Relations of smallpox and cowpox — Bacteria in smallpox — The nature of vaccination .... 497 APPENDIX B. Hydrophobia. Introductory — Pathology — The hydrophobic virus — Prophylaxis — Antirabic serum — Methods ...... cos APPENDIX C. Malarial Fever. Characters of the malarial parasite — Varieties of the malarial parasite — Relations to the disease — Methods of examination . . 515 APPENDIX D. Dysentery. Amoebic dysentery — Characters of the amoeba — Distribution of the amoeba — Relations to disease — Varieties of dysentery — Methods of examination ....... 527 Bibliography . 535 Index 553 LIST OF ILLUSTRATIONS. : ♦ PAGE FIG. 1. Forms of bacteria . • • • ■ l7 2. Hot-air steriliser . 3. Koch's steam steriliser • 38 4. Autoclave . • • • • 39 5. Steriliser for blood serum . . . • .40 6. Meat press . . • • • 42 7. Hot-water funnel . . . • • . • 47 8. Blood-serum inspissator . . . • • 51 9. Potato jar . . ■ • 54 10. Cylinder of potato cut obliquely . . . -54 11. Ehrlich's tube containing piece of potato . . -54 12. Apparatus- for filling tubes . . • -57 13. Tubes of media . . . • • -57 14. Platinum wires in glass handles . . . • 5^ 15. Method of inoculating solid tubes . . 59 16. Rack for platinum needles . . . .60 17. Petri's capsule . . • ■ • .62 18. Koch's levelling apparatus for use in preparing plates . 64 19. Koch's levelling apparatus, showing transference of plate . 65 20. Tube for Esmarch's roll culture . . . .65 21. Apparatus for supplying hydrogen for anaerobic cultures . 69 22. Esmarch's roll-tube adapted for culture of anaerobes . 70 23. Flask for anaerobes in liquid media . . .72 24. Flask arranged for culture of anaerobes which develop gas . 73 25. Tubes for anaerobic cultures on the surface of solid media . 74 26. Slide for hanging-drop cultures . . . -75 27. Graham Brown's chamber for anaerobic hanging-drops . 76 28. Apparatus for counting colonies . . . -77 29. Geissler's vacuum pump for filtering cultures . .81 30. Chamberland's bougie with flask arranged for filtration . 81 31. Chamberland's bougie, with lamp funnel . . .82 x v ' LIST OF ILL US Hi A TIONS. F1G- PAGE 32. Bougie inserted in rubber stopper . . . .82 33. Muencke's modification of Chamberland's filter . . 83 34. Flask for filtering large quantities of fluid . . -83 35. Tubes for demonstrating gas-formation by bacteria . 85 36. Reichert's gas regulator . . . . .89 37. Hearson's incubator for use at 37" C. . . .90 38. Cornet's forceps for holding cover-glasses . . -95 39. Needle for manipulating paraffin sections . . . 101 40. Syphon wash-bottle ..... 105 41. Tubes used in testing agglutinating and sedimenting pro- perties of serum . . . . .119 42. Test-tube and pipette for obtaining fluids containing bacteria 122 43. Curved needle for intraperitoneal inoculations . .129 44. Forms of fungi . . . . . 135 45. Staphylococcus pyogenes aureus, young culture on agar. x 1000 ...... 166 46. Two stab cultures of staphylococcus pyogenes aureus in gelatine . . . . . .167 47. Streptococcus pyogenes, young culture on agar. x 1000 . 168 48. Stroke culture of the streptococcus pyogenes on sloped agar 169 49. Micrococcus tetragenus. x 1000 . . .171 50. Streptocecci in acute suppuration. x 1000 . .179 5 1 . Minute focus of commencing suppuration of the brain. x 50 . . . . . . .181 52. Secondary infection of a glomerulus of kidney by the staphylo- coccus aureus. x 300 . . . .183 53. Section of a vegetation in ulcerative endocarditis. x 600. 185 54. Portion of film of gonorrhceal pus. x 1000 . .190 55. Gonococci from a pure culture on blood agar. x 1000 . 192 56. Film preparation of pneumonic sputum. x 1000. . 208 57. Stroke culture of FraenkePs pneumococcus on agar . 210 58. Fraenkel's pneumococcus from a pure culture on blood agar of twenty-four hours' duration. x 1000 . .211 59. Stab culture of Friedlander's pneumobacillus . .212 60. Friedlander's pneumobacillus from a young culture on agar. x 1000 . . . • • .212 61. Capsulated pneumococci in blood taken from the heart of a rabbit. x 1000 . . . • .216 62. Tubercle bacilli from a pure culture on glycerine agar. x 1000 22& 63. Tubercle bacilli in phthisical sputum. x 1 000 . . 229 64. Cultures of tubercle bacilli on glycerine agar . .231 65. Tubercle bacilli in section of human lung in acute phthisis. x 1000 23° 66. Tubercle bacilli in giant cells. x 1000 . • 239 67. Tubercle bacilli in urine. x 1000 . ■ ■ 24° LIST OF ILLUSTRATIONS. fio- , PAGE 256 71 72 68. Smegma bacilli. x 1000 . ■ ■ • 69. Section through leprous skin, showing the masses of cellular granulation tissue in the cutis, x 80 . . • 59 70 Superficial part of leprous skin, showing the cells of the granulation tissue filled with bacilli. x 500 . . 202 High-power view of portion of leprous nodule. x 1 100 . 203 Glanders bacilli among broken-down cells. xiooo . 271 73. Glanders bacilli from a pure culture. x 1000 . • 272 74. Actinomycosis of human liver. x 500 . . • 2 4 75. Actinomyces with clubs in human kidney. x 500 . 2»o 76. Cultures of the actinomyces on glycerine agar • • 289 77. Actinomyces from a culture on glycerine agar. x 1000 . 290 78. Surface colony of the anthrax bacillus. x 30 • 29b 79. Anthrax bacilli arranged in chains. x 1000 . • 2gb 80. Stab culture of the anthrax bacillus . • • 299 81. Anthrax bacilli containing spores. x 1 000 . • 301 82. Scraping from spleen of guinea-pig dead of anthrax, x 1000 304 83. Portion of kidney of guinea-pig dead of anthrax. x 300 . 306 84. Large clump of typhoid bacilli in a spleen. x 500 . 3*9 85. Typhoid bacilli from a young culture on agar. x 1000 . 320 86. Typhoid bacilli from a young culture on agar, showing flagella. x 1000 ....>• 322 87. Stab and stroke cultures of typhoid bacillus and bacillus coh 323 88. Bacillus coli communis. x 1000 . • • 325 89. Film preparation from diphtheria membrane. x 1000 . 355 90. Section through a diphtheritic membrane in trachea, show- ing diphtheria bacilli in clumps. x 1000 . -357 91. Cultures of the diphtheria bacillus on an agar plate . 3°° 92. Diphtheria bacilli from a twenty-four hours' culture on agar. x 1000 . ■ • • • ■ 93. Diphtheria bacilli of larger size than in previous figure. x 1000 . • • • • 94. Involution forms of the diphtheria bacillus. x 1000 . 361 95. Film preparation of discharge from wound in a case of tetanus. x 1000 . 96. Tetanus bacilli, showing flagella . . . -379 97. Spiral composed of numerous twisted flagella of the tetanus bacillus . • • • • .380 98. Tetanus bacilli, some of which possess spores. x 1000 . 380 99. Stab culture of the tetanus bacillus. (Kitasato) . . 382 100. Film preparation in a case of malignant oedema in the human subject. x 1000 .... 396 101. Bacillus of malignant cedema. x 1000 . ■ . 397 102. Stab cultures in agar of tetanus bacillus, malignant cedema and quarter-evil ..... 398 103. Bacillus of quarter-evil, showing spores. x 1000 . 401 360 361 361 378 xvu 1 LIST OF ILL USTRA TIONS. FIG. I'AGU 104. Cholera spirilla. x 1000 105. Cholera spirilla stained to show the terminal flagella. x 1000 405 106. Cholera spirilla from an old agar culture. x 1000 . 406 107. Stab culture of the cholera spirillum . . . 4Qg 108. Colonies of the cholera spirillum in a gelatine plate ; three days' growth . . ... . .409 109. Metchnikoff's spirillum. x 1000 . . 426 110. Stab cultures of Metchnikoff's and of Finkler and Prior's spirillum ...... 427 111. Finkler and Trior's spirillum from an agar culture. x 1 000 428 112. Influenza bacilli from a culture on blood agar. x 1000 113. Bacillus of plague from a young culture. x 1000 114. Bacillus of plague in chains. x 1000 115. Spirilla of relapsing fever in human blood (after Koch) x about 1000 . . 116. Micrococcus melitensis, from a two days' culture on agar at 37° C. x 1000 • . . . , 117. Bacillus icteroides, from a young culture on agar (Sanarelli) x 1000 118. Culture of bacillus icteroides on agar, showing the char acteristic appearance when incubated at the two tempera- tures mentioned (Sanarelli). Natural size . . 454 1 1 9- 1 24. From dried blood-films, showing parasites of malarial fever. x about 1000 . . . . • 519 125. Amoebse of dysentery . . . . .528 126. Section of wall of liver abscess, showing an amoeba of spherical form with vacuolated protoplasm. x 1000 . 530 431 437 438 444 449 454 MANUAL OF BACTERIOLOGY. MANUAL OF BACTERIOLOGY. CHAPTER I. GENERAL MORPHOLOGY AND BIOLOGY. Introductory.— At the bottom of the scale of living things there exists a group of organisms to which the name of bacteria is usually applied. These are apparently of very simple structure and may be subdivided into two sub-groups, a lower and simpler and a higher and better developed. The lower forms are the more numerous, and consist of minute unicellular masses of protoplasm devoid of chloro- phyll, which multiply by simple fission. Some are motile, others non-motile. Their minuteness may be judged of by the fact that in one direction at least they usually do not measure more than i /z, (^o inch)- These forms can be classified according to their shapes into three main groups — (i) A group in which the shape is globular. The members of this are called cocci. (2) A group in which the shape is that of a straight rod — the proportion of the length to the breadth of the rod varying greatly among the different members. These are called bacilli. (3) A group in which the shape is that of a curved or spiral rod. These are called spirilla. The fuller description of the characters of these - GENERAL MORPHOLOGY AND BIOLOGY. groups will be more conveniently taken later (p. 16). In some cases, especially among the bacilli, there may occur under certain circumstances changes in the protoplasm whereby a resting stage or spore is formed. The higher forms show advance on the lower along two lines, (i) On the one hand they consist of filaments made up of simple elements such as occur in the lower forms. These filaments may be more or less septate, may be pro- vided with a sheath, and may show branching either true or false. The minute structure of the elements comprising these filaments is analogous to that of the lower forms. Their size, however, is often, somewhat greater. The lower forms sometimes occur in filaments, but here every member of the filament is independent, while in the higher forms there seems to be a certain interdependence among the individual elements. For instance, growth may occur only at one end of a filament, the other forming an attachment to some fixed object. (2) The higher forms, moreover, present this further development that in certain cases some of the elements may be set apart for the reproduction of new individuals. Terminology. — The term bacterium of course in strict- ness only refers to the rod-shaped varieties of the group, but as it has given the name bacteriology to the science which deals with the whole group, it is convenient to apply it to all the members of the latter, and to reserve the term bacillus for the rod-shaped varieties. Other general words, such as germ, microbe, micro-organism, are often used as synonymous with bacterium, though, strictly, they include the smallest organisms of the animal kingdom. While no living organisms lower than the bacteria are known, the upper limits of the group are difficult to define, and it is further impossible in the present state of our knowledge to give other than a provisional classification of the forms which all recognise to be bacteria. The division into lower and higher forms, however, is fairly well marked, and we shall therefore refer to the former as the lower bacteria, and to the latter as the higher bacteria. STRUCTURE OF THE BACTERIAL CELL. 3 Morphological Relations.— The relations of the bacteria to the animal kingdom on the one hand and to the vegetable on the other constitute a somewhat difficult question. The occurrence of spore formation among the lower forms is analogous to what takes place m certain unicellular organisms— the. flagellata— which, though some of the members contain chlorophyll, are usually ranked in the animal kingdom with the protozoa. On the other hand, sporulation as it occurs in the yeasts resembles more closely what takes place among the bacteria, and further, the fact that many bacteria can derive the carbon they require for their nourishment from tartrates and their nitrogen from ammonia or its salts, makes it natural that they should be ranked in the vegetable kingdom with other non-chlorophyllous plants as fungi. Such an association is further borne out by the fact that while the higher fungi present many analogies with the higher algae, both probably having a common descent, there is a group of lower algae the members of which morphologically are analogous to the bacteria. These algae are unicellular masses of protoplasm, having generally the same shapes as the bacteria, and largely multiply by fission. Endo- genous sporulation, however, does not occur, nor is motility associated with the possession of flagella. Also their protoplasm differs from that of the bacteria in containing chlorophyll and another blue-green pigment called phycocyan. From the morphological resemblances, however, between these algae and the bacteria, and from the fact that fission plays a predominant part in the multiplication of both, they have been grouped together in one class as the Schizophyta or splitting plants (German, Spaltpflanzen). And of the two divisions forming these Schizophyta the splitting algae are denominated the schizophyceae (German, Spaltalgen), while the bacteria or splitting fungi are called the schizomycetes (German, Spaltpilzen). The bacteria are, therefore, in proper scientific nomenclature, to be spoken of as the schizomycetes. Certain bacteria which have been described as containing chlorophyll ought probably to be grouped among the schizophyceae. General Morphology of the Bacteria. The Structure of the Bacterial Cell. — On account of the minuteness of bacteria the investigation of their structure is attended with great difficulty. When examined under the microscope, in their natural condition, e.g., in water, they appear merely as colourless refractile bodies of the different shapes named. Spore formation and motility, when these exist, can also be observed, but little else can be made out. For their proper investigation advantage is always taken of the fact of their affinities for various dyes, especially those 4 GENERAL MORPHOLOGY AND BLOLOGY. which are usually chosen as good stains for the nuclei of animal cells. Certain points have thus been determined The bacterial cell consists of a sharply contoured mass of protoplasm which reacts to, especially basic, aniline dyes like the nucleus of an animal cell. From this fact it has been deduced that there is probably a close relationship between the protoplasm of bacteria and the chromatin of the nuclear protoplasm. Our knowledge of micro-chemistry is, however, too scanty to justify any definite conclusion being drawn on this point. To speak generally, a healthy bacterium when stained presents the appearance of a finely granular or almost homogeneous structure. The protoplasm of the bacterial cell is surrounded by an envelope which can in some cases be demonstrated by overstaining a specimen with a strong aniline dye, when it will appear as a halo round the bacterium. This envelope may sometimes be seen to be of considerable thickness. Its innermost layer is probably of a denser consistence, and sharply contours the contained protoplasm, giving the latter the appearance of being surrounded by a membrane. It is only, however, in some of the higher forms that a true membrane occurs. Sometimes the outer margin of the envelope is sharply defined, in which case the bacterium appears to have a distinct capsule, and is known as a capsulated bacterium (vide Fig. i, No. 4; and Fig. 56). The cohesion of bacteria into masses depends largely on the character of the envelope. If the latter is glutinous, then a large mass of the same species may occur, formed of individual bacteria embedded in what appears to be a mass of jelly. When this occurs, it is known as a zooglcea mass. On the other hand, if the envelope has not this cohesive property the separation of individuals may easily take place, especially in a fluid medium in which they may float entirely free from one another. Many of the higher bacteria possess a sheath which, though probably a cellular excretion, has a much more definite structure than is found among the lower forms. It resists external influences, possesses elasticity, and serves to bind the elements of the organism together. REPRODUCTION AMONG THE LOWER BACTERIA. 5 Eeproduction among the Lower Bacteria. — When a bacterial cell is placed in favourable surroundings it multi- plies ; as has been said, this, in the great majority of cases, takes place by simple fission. In the process a constriction appears in the middle and a transverse unstained line develops across the protoplasm at that point. The process goes on till two individuals can be recognised, which may remain for a time attached to one another, or become separate, according to the character of the envelope, as already explained. In most bacteria growth and multiplica- tion go on with great rapidity. A bacterium may reach maturity and divide in from twenty minutes to half an hour. In the latter case a simple calculation will show that, at the end of twenty-four hours, from one individual 17,000,000 similar individuals will be produced. As shown by the results of artificial cultivation, others, such as the tubercle bacillus, multiply much more slowly. Sometimes division proceeds so rapidly that the young individuals do not reach the adult size before multiplication again occurs. This may give rise to anomalous appearances. When bacteria are placed in unfavourable conditions as regards food, etc., growth and multiplication take place with difficulty. In the great majority of cases this is evidenced by changes in the appearance of the protoplasm. Instead of its maintain- ing the regularity of shape seen in healthy bacteria, various aberrant appearances are presented. This occurs especially in the rod- shaped varieties, where flask-shaped or dumb-bell- shaped individuals may be seen. The regularity in structure and size is quite lost. The appearance of the protoplasm also is often altered. Instead of, as formerly, staining well, it does not stain readily, and may have a uniformly pale, homogeneous appearance, while in an old culture only a small proportion of the bacteria may stain at all. Some- times, on the other hand, a degenerated bacterium contains intensely stained granules or globules which may be of large size. Such aberrant and degenerate appearances are referred to as involution forms. That these forms really betoken degenerative changes is shown by the fact that, on their GENERAL MORPHOLOGY AND BIOLOGY. being again transferred to favourable conditions, only slight growth at first takes place. Many individuals have un- doubtedly died, and the remainder which live and develop into typical forms may sometimes have lost some of their properties. Reproduction among the Higher Bacteria. — Most of the higher bacteria consist of threadlike structures more or less septate and often surrounded by a sheath. The organism is frequently attached at one end to some object or to another individual. It grows to a certain length and then at the free end certain cells called gonidia are cast off from which new individuals are formed. These gonidia may be formed by a division taking place in the terminal element of the filament such as has occurred in the growth of the latter. In some cases, however, division takes place in three dimensions of space. The gonidia have a free existence for a certain time before becoming attached, and in this stage are sometimes motile. They are usually rodlike in shape, some- times pyriform. They do not possess any special powers of resistance. Spore Formation. — In certain species of the lower bacteria, under certain circumstances, changes take place in the protoplasm which result in the formation of bodies called spores, to which the vital activities of the original bacteria are transferred. Spore formation occurs chiefly among the bacilli and in some spirilla. Its commencement in a bacterium is indicated by the appearance in the proto- plasm of a minute highly refractile granule unstained by the ordinary methods. This increases in size, and assumes a round, oval, or short rod-shaped form, always shorter but often broader than the original bacterium. In the process of spore formation the rest of the bacterial protoplasm may remain unchanged in appearance and staining power for a considerable time {e.g., B. tetani), or, on the other hand, it may soon lose its power of staining and ultimately disappear, leaving the spore in the remains of the envelope {e.g., B. anthracis). This method of spore formation is called endogenous. Bacterial spores are always non-motile. The spore may appear in the centre of the bacterium, or it may be at one extremity, or a short distance from one extremity (Fig. i, No. n). In structure the spore consists of amass of protoplasm surrounded by a dense membrane. This SPORE FORMATION. 7 can be demonstrated by methods which will be described, the underlying principle of which is the prolonged applica- tion of a powerful stain. The membrane is supposed to confer on the spore its characteristic feature, namely, great capacity of resistance to external influences such as heat or noxious chemicals. Koch, for instance, in one series of experiments, found that while the bacillus anthracis in the unspored form was killed by a two minutes' exposure to i per cent carbolic acid, spores of the same organism re- sisted an exposure of from one to fifteen days. When a spore is placed in suitable surroundings for growth it again assumes the original bacillary or spiral form. The capsule dehisces either longitudinally, or terminally, or transversely. In the last case the dehiscence may be partial, and the new individual may remain for a time attached by its ends to the hinged spore-case, or the dehis- cence may be complete and the bacillus grow with a cap at each end consisting of half the spore-case. Sometimes the spore-case does not dehisce, but is simply absorbed by the developing bacterium. It is important to note that in the bacteria spore forma- tion is rarely,, if ever, to be considered as a method of multiplication. In at least the great majority of cases only one spore is formed from one bacterium, and only one bacterium in the first instance from one spore. Sporulation is to be looked upon as a resting stage of a bacterium, and is to be contrasted with the stage when active multiplication takes place. The latter is usually referred to as the vegeta- tive stage of the bacterium. Regarding the signification of spore formation in bacteria there has been some difference of opinion. According to one view it may be regarded as representing the highest stage in the vital activity of a bacterium. There is thus an alternation between the vegetative and spore stage, the occurrence of the latter being necessary to the maintenance of the species in its greatest vitality. Such a rejuvenescence, as it were, through sporulation, is known in many algae. - In support of this view there are certain facts. In many cases, for instance, 8 GENERAL MORPHOLOGY AND BIOLOGY. spore formation only occurs at temperatures specially favourable for growth and multiplication. There is often a temperature below which, while vegetative growth still takes places, speculation will not occur, and in the case of B. anthracis, if it be kept at a temperature above the limit at which it grows best, not only are no spores formed, but the species may lose the power of sporulation. Furthermore, in the case of bacteria preferring the presence of oxygen for their growth, an abundant supply of this gas may favour sporulation. It is probable that even among bacteria pre- ferring the absence of oxygen for vegetative growth, the presence of this gas favours sporulation. Most bacterio- logists are, however, of opinion that when a bacterium forms a spore, it only does so when its surroundings, especially its food supply, become unfavourable for vegetative growth ; it then remains in this condition until it is placed in more suitable surroundings. Such an occurrence would be analogous to what takes place under similar conditions in many of the protozoa. Often sporulation can be prevented from taking place for an indefinite time if a bacterium is constantly supplied with fresh food (the other conditions of life being equal). The presence of substances excreted by the bacteria themselves plays, however, a more important part in making the surroundings unfavourable than the mere exhaustion of the food supply. A living spore will always develop into a vegetative form if placed in a fresh food supply. With regard to the rapid formation of spores when the conditions are favourable for vegetative growth, it must be borne in mind that in such circumstances the conditions may really very quickly become unfavourable for a continu- ance of growth, for not only will the food supply around the growing bacteria be rapidly exhausted, but the excre- tion of effete and inimical matters will be all the more rapid. We must note that the usually applied tests of a body developed within a bacterium being a spore are (i) its staining reaction, namely, resistance to ordinary staining fluids, but capacity of being stained by the special methods MOTILITY. 9 devised for the purpose {vide p. 114); (2) the fact that the bacterium containing the spore has higher powers of re- sistance against inimical conditions than a vegetative form. It is important to bear these tests in mind, as in some of the smaller bacteria especially, it is very difficult to say whether they spore or not. There may appear in such organisms small unstained spots the significance of which it is very difficult to determine. The Question of Arthrosporous Bacteria.— It is stated by Hueppe that among certain organisms, e.g., some streptococci, certain indi- viduals may without endogenous spoliation take on a resting stage. These become swollen, stain well with ordinary stains, and they are stated to have higher power of resistance than the other forms ; further, when vegetative life again occurs it is from them that multiplication is said to take place. From the fact that there is no new formation within the protoplasm, but that it is the whole of the latter which participates in the change, these individuals have been called arthro- spores. The existence of such special individuals amongst the lower bacteria is extremely problematical; They have no distinct capsule, and they present no special staining reactions, nor any microscopic features by which they can be certainly recognised, while their alleged increased powers of resistance are very doubtful. All the phenomena noted can be explained by the undoubted fact that in an ordinary growth there is very great variation among the individual organisms in their powers of resistance to external conditions. Motility. — As has been stated, many bacteria are motile. Motility can be studied by means of hanging drop prepara- tions (vide p. 74). The movements are of a darting, roll- ing or vibratile character. The degree of motility depends on the temperature, on the age of the growth, and on the medium in which the bacteria are. Sometimes the move- ments are most active just after the cell has multiplied, sometimes it goes on all through the life of the bacterium, sometimes it ceases when sporulation is about to occur. Motility is associated with the possession of fine wavy thread-like appendages called flagella, which for their demonstration require the application of special staining methods (vide Fig. 1, No. 12 ; and Fig. 86). They have been shown to occur in many bacilli and spirilla, but only in a few species of cocci. They vary in length, but may 10 GENERAL MORPHOLOGY AND BIOLOGY. be several times the length of the bacterium, and may be at one or both extremities or all round. When terminal they may occur singly or there may be several. The nature of these flagella has been much disputed. Some have held that, unlike what occurs in many algae, they are not actual prolongations of the bacterial protoplasm, but merely appendages of the envelope, and have doubted whether they are really organs of locomotion. There is now, however, little doubt that they belong to the protoplasm.' By appropriate means the central parts of the latter can be made to shrink away from the peripheral {vide infra, " plasmolysis "). In such a case movement goes on as before, and in stained preparations the flagella can be seen to be attached to the peripheral zone. It is to be noted that flagella have never been demonstrated in non-motile bacteria, while, on the other hand, they have been observed in nearly all motile forms. There is little doubt, however, that all cases of motility among the bacteria are not de- pendent on the possession of flagella, for in some of the special spiral forms, and in most of the higher bacteria, motility is probably due to contractility of the protoplasm itself. The Minuter Structure of the Bacterial Protoplasm. — Many attempts have been made to obtain deeper informa- tion as to the structure of the bacterial cell, and especially as to its behaviour in division. These have largely turned on the interpretation to be put on certain appearances which have been observed. These appearances are of two kinds. First, under certain circumstances irregular deeply- stained granules are observed in the protoplasm, often, when they occur in a bacillus, giving the latter the appear- ance of a short chain of cocci. They are often called metachromatic granules {vide Fig. i, No. 16) from the fact that by appropriate procedure they can be stained with one dye, and the protoplasm in which they lie with another ; sometimes, when a single stain is used, such as methylene blue, they assume a slightly different tint from the protoplasm. STRUCTURE OF BACTERIAL PROTOPLASM. ii For the demonstration of the metachromatic granules two mettods have been advanced. Ernst recommends that a few drops of Loffler s methylene blue {vide p. 108) be placed on a ™-^P^™^ and he latter passed backwards and forwards over a Bunsen flame for half a minute after steam begins to rise. The preparation is then washed in water and counter-stained for one to two minutes m watery Bismarck-brown. The granules are here stained blue, the protoplasm brown. Neisser stains a similar preparation in warm carbol-fuchsin, washes with i per cent sulphuric acid and counter-stains with Loffler s blue Here the granules are magenta, the protoplasm blue. Hie general character of the granules thus is that they retain the first stain more intensely than the rest of the protoplasm does. A second appearance which can sometimes be seen in specimens stained in ordinary ways is the occurrence of a concentration of the protoplasm at each end of a bacterium, indicated by these parts being deeply stained. These deeply -stained parts are sometimes called polar granules {vide Fig. I, No. 16, the bacillus most to the right), (German, Polkornchen or Polkorner). Both the metachromatic and the polar granules have been looked upon by different observers as spores. Against this view, however, is the fact that growths in which they exist show no higher degree of resistance than growths in which they cannot be observed. Further, they do not react to the strict methods of spore staining. _ Some have considered the metachromatic granules to be evidences of the process of division in the bacterial protoplasm, i.e., of a kind of mitosis. If such is the case they ought to be observed in some members of a growth where active multiplication is going on, and this is not so. The con- ditions in which they occur are in growths where the food material is becoming exhausted, or in growths which have been subjected to unfavourable conditions. Thus they have been observed in bacteria which have been grown for a few days at the most favourable temperature, and thereafter allowed to develop further at less suitable temperatures. It is therefore very probable that the occur- rence of metachromatic granules in a bacterium indicates the onset of degenerative changes. In perfectly healthy and young bacteria, moreover, appearances of granule formation and of vacuolation may be accidentally produced by physical means in the occur- GENERAL MORPHOLOGY AND BIOLOGY. rence of what is known as plasi?wlysis. To speak generally, when a mass of protoplasm surrounded by a fairly firm envelope of a colloidal nature is placed in a solution con- taining salts in greater concentration than that in which it has previously been living, then by a process of osmosis the water held in the protoplasm passes out through the membrane, and, the protoplasm retracting from the latter, the appearance of vacuolation is presented. Now in mak- ing a dried film for the microscopic examination of bacteria the conditions necessary for the occurrence of this process may be produced, and the appearances of vacuolation and of polkorner may thus be brought about. Plasmolysis in bacteria has recently been extensively investigated,1 and has been found to occur in some species more readily than in others. We may conclude that such appearances as vacuolation of the bacterial protoplasm and polkorner are either signs of degeneration, like the metachromatic granules, or are artificially produced. All of them are most frequently observed in old or otherwise enfeebled cultures. Butschli has published interesting observations on the minute struc- ture of some large sulphur-containing bacteria. These were found to consist of an outer membrane enclosing the protoplasm, which was divided into two parts — an outer protoplasmic network containing bac- terio-purpurin, and an inner part, the greater portion of the latter being stained blue with htematoxylin more deeply than the outer, in specimens out of which the bacterio-purpurin had been dissolved. In this central part thus stained there were red granules, which Butschli regards as the metachromatic granules of Ernst. The bacilli in the specimens he examined seem, however, to have been healthy. Butschli looks upon the outer part of the central body as corresponding to the protoplasm of an ordinary cell, the inner part as corresponding to the nucleus. In one smaller bacterium he found evidence of the former only at the end of the cell. He therefore thinks that the greater part of the bacterial cell may correspond to a nucleus, and that this is surrounded by a thin layer of protoplasm, which in the smaller bacteria probably escapes notice, unless when it is specially abundant at the ends. This terminal plasma has also been found by Wager in another bacterium. 1 Consult Fischer, " Untersuchungen tiber Bakterien," Berlin, 1894; " Ueber den Bau der Cyanophyceen und Bakterien," Jena, 1897. CHEMICAL COMPOSITION OF BACTERIA. 13 Among the bacteria BUtschli further finds confirmation of his views on the mesh-like structure of protoplasm generally. A bacillus, he finds, consists of four or five more or less square meshes laid end to end, and he gives microphotographs of bacilli presenting such appearances (vide Fig. I, Nos. i$a and b). With regard to these observations of BUtschli, Fischer holds that the appearances seen were due to plasmolysis, and considers that there is no evidence of differentiation between protoplasm and nucleus in the bacterial cell. The Chemical Composition of Bacteria. — In the bodies of bacteria many definite substances occur. Some bacteria have been described as containing chlorophyll, but these are properly to be classed with the schizophycese. Sulphur is found in some of the higher forms, and starch granules are also described as occurring. Many species of bacteria, when growing in masses, are brilliantly coloured. Com- paratively few bacteria, however, associated with the pro- duction of disease give rise to pigments. In some of the organisms classed as bacteria a pigment named bacterio- purpurin has been observed in the protoplasm, and similar intracellular pigments probably occur in some of the larger forms of the lower bacteria and may occur in the smaller. Exact observation is, however, a work of great difficulty, and in the majority of the smaller forms it is impossible to determine whether the pigment occurs inside or outside the protoplasm. In many cases, for the free production of pigment abundant oxygen supply is necessary. On the other hand, sometimes, as in the case of spirillum rubrum, the pigment is best formed in the absence of oxygen. Sometimes the faculty of forming it may be lost by an organism for a time, if not permanently, by the conditions of its growth being altered. Thus, for example, if the B. pyocyaneus be exposed to the temperature of 42 0 C. for a certain time, it loses its power of producing its bluish pigment. Pigments formed by bacteria often diffuse out into, and colour, the medium for a considerable distance around. Comparatively little is known of the nature of bacterial pigments. Zopf, who has devoted much attention to the pigments occurring in the 14 GENERAL MORPHOLOGY AND BIOLOGY. lower plants, has found that many of them belong to a group of colouring matters which occur widely in the %'egetable and animal kingdoms, viz. the lipochromes. These lipochromes, which get their name from the colouring matter of animal fat, include the colouring matter in the petals of Ranunculaceae, the yellow pigments of serum and of the yolks of eggs, and many bacterial pigments. Among the latter is a lipochrome carotin, which is also the pigment in carrots and tomatoes. The lipochromes are characterised by their solubility in chloroform, alcohol, ether, and petroleum, and by their giving indigo- blue crystals with strong sulphuric acid, and a green colour with iodine dissolved in potassium iodide. Though crystalline compounds of these have been obtained, their chemical constitution is entirely unknown and even their percentage composition is disputed. Some observations have been made on the chemical structure of bacterial protoplasm. Nencki precipitated the bodies of putrefactive bacteria with 2-3 per cent hydrochloric acid, filtered them off, extracted with alcohol and ether, and dissolved the residue with .5 per cent potassium hydrate solution. This solution contained an albumin which was fairly constant in its percentage composition in samples obtained from different mixtures of these bacteria, and which Nencki named mycoprotein ; it was soluble in water, acids, and alkalies, insoluble in solutions of neutral salts. The albuminoid constituents of bacteria, however, vary, for from anthrax spores Nencki obtained an albumin which he calls anthraxprotein, and which differs from mycoprotein in its being insoluble in water, acetic acid, and dilute mineral acids. Both differ from nucleo-albumin, a constituent of the nuclei of higher cells, in containing no phosphorus. Other observers have isolated similar bodies having, however, different percentage compositions from those given by Nencki. Buchner isolated a series of bodies from different species of bacteria by dissolving in weak alkali and precipitating the resultant with acid. These he also calls proteins, and adduces some evidence to show that it is to them that the affinity of bacteria for basic aniline dyes is due. They differ from Nencki's proteins in containing phosphorus. According to some recent results the amount of nitrogenous material present varies according to the temperature at which growth THE CLASS1FICA TION OF BA C TERIA . 1 5 has taken place, according to the age of the culture, and also according to the medium used. Besides nitrogenous material, salts of sodium, potassium, magnesium, and phos- phorus may be present in the bacterial protoplasm. In certain cases traces of cellulose or of substances resembling cellulose, and also fatty bodies have been isolated. It is probable that the composition of bacteria varies somewhat in different species. The Classification of Bacteria. — There have been numerous schemes set forth for the classification of bac- teria, the fundamental principle running through all of which has been the recognition of the two sub-groups and the type forms mentioned in the opening paragraph above. In the attempts to still further subdivide the group, scarcely two systematists are agreed as to the characters on which sub-classes are to be based. Our present knowledge of the essential morphology and relations of bacteria is as yet too limited for a really natural classification to be attempted. To prepare for the elaboration of the latter, Marshall Ward suggests that in every species there should be studied the habitat, best food supply, condition as to gaseous environment, range of growth, temperature, morphology, and life history, special properties and patho- genicity. We must thus be content with a provisional and in- complete classification. We have said that the division into lower and higher bacteria is recognised by all, though, as in every other classification, there occur transitional forms. In subdividing the bacteria further, the forms they assume constitute at present the only practicable basis of classifica- tion. The lower bacteria thus naturally fall into the three groups mentioned, the cocci, bacilli, and spirilla, though the higher are more difficult to deal with. Subsidiary, though important, points in still further subdivision are the planes in which fission takes place and the presence or absence of spores. The recognition of actual species is often a matter of great difficulty. The points to be observed in this will be discussed later (p. 125). 16 GENERAL MORPHOLOGY AND BIOLOGY. I. The Lower Bacteria.1 — These, as we have seen, are minute unicellular masses of protoplasm surrounded by an envelope, the total vital capacities of a species being repre- sented in every cell. They present three distinct type forms, the coccus, the bacillus, and the spirillum, and endogenous sporulation may occur. They may also be motile. r. The Cocci. — In this group the cells range in different species from .5-2 /a in diameter, but most measure about 1 jx. Before division they may increase in size in all directions. The species are usually classified according to the method of division. If the cells divide only in one axis, and through the consistency of their envelopes remain attached, then a chain of cocci will be formed. A species in which this occurs is known as a streptococcus. If division takes place irregularly the resultant mass may be compared to a bunch of grapes, and the species is often called a staphy- lococcus. Division may take place in two axes at right angles to one another, in which case cocci adherent to each other in packets of four (called tetrads) or sixteen, may be found, the former number being the more frequent. To all these forms the word micrococcus is often generally applied. The individuals in a growth of micrococci often show a tendency to remain united in twos. These are spoken of as diplococci, but this is not a distinctive character, since every coccus as a result of division becomes a diplococcus, though in some species the tendency to remain in pairs is well marked. The adhesion of cocci to one another depends on the character of the capsule. Often this has a well-marked outer limit (micrococcus tetragenus), sometimes it is of great extent, its diameter being many times that of the coccus (streptococcus mesenteriodes). It is especially among the streptococci and staphylococci that the pheno- menon of the formation of arthrospores is said to occur. In none of the cocci have endogenous spores been certainly observed. The number of species of the streptococci and 1 For the illustration of this and the succeeding systematic paragraphs, vide Fig. 1. Fig. i. — I. Coccus. 2. Streptococcus. 3. Staphylococcus. 4. Capsulatecl diplo- coccus. 5. " Biscuit "-shaped coccus. 6. Tetrads. 7. Sarcina form. 8. Types of bacilli (1-8 are diagrammatic). 9. Non-septate spirillum X 1000. 10. Ordinary spirillum— («) comma-shaped element; (i) formation of spiral by comma-shaped elements X 1000. 11. Types of spore formation. 12. Flagellated bacteria. 13. Changes in bacteria produced by plasmolysis (after Fischer). 14. Bacilli with terminal protoplasm (Biitschli). 15. (a) Bacillus composed of five protoplasmic meshes ; (b) protoplasmic network in micrococcus (Biitschli). 16. Bacteria con- taining metachromatic granules (Ernst, Neisser) — some contain polar granules. 17. Beggiatoa alba. Both filaments contain sulphur granules — one is septate. 18. Thiothnx tenuis (Winogradski). 19. Leptothrix innominata (Miller). 20. Clado- thrix dichotoma (Zopf). 21. Streptothrix actinomyces (Bostrom), {a) colony under low power ; ) filament showing true branching ; (t) filament containing coccus-like bodies ; {(f) filament with club at end. 2 1 8 GENE RA L MORPHOL OGY AND BIOL OGY. staphylococci probably exceeds 150. Besides those men- tioned there are cocci which divide in three axesatright angles to one another. These are usually referred to as sarcince. If the cells are lying single they are round, but usually they are seen in cubes of eight with the sides which are in contact slightly flattened. Large numbers of such cubes may be lying together. The sarcinae are, as a rule, rather larger than the other members of the group. Most of the cocci are non-motile, but a few motile species possessing flagella have been described. 2. Bacilli. — These consist of long or short cylindrical cells, with rounded or sharply rectangular ends, usually not more than 1 /a broad, but varying very greatly in length. They may be motile or non-motile. Where flagella occur, these may be distributed all round the organism, or only at one or both of the poles (pseudomonas). Several species are provided with sharply-marked capsules (B. pneumoniae). In many species endogenous sporulation occurs. The spores may be central or terminal, round, oval, or spindle- shaped. Great confusion in nomenclature has arisen in this group in conse- quence of the different artificial meanings assigned to the essentially synonymous terms bacterium and bacillus. Migula, for instance, applies the former term to non-motile species, the latter to the motile. Hueppe, on the other hand, calls those in which endogenous sporula- tion does not occur, bacteria, and those where it does, bacilli. In the ordinary terminology of systematic bacteriology the word bacterium has been almost dropped, and is reserved, as we have done, as a general term for the whole group. It is usual to call all the rod-shaped varieties bacilli. 3. Spirilla. — These consist of cylindrical cells more or less spiral or wavy. Of such there are two main types. In one there is a long non-septate, usually slender, wavy or spiral thread (e.g., spirillum of relapsing fever, Fig. 1, No. 9). In the other type the unit is a short curved rod (often referred to as of a "comma" shape). When two or more of the latter occur, as they often do, end to end with their curves alter- nating, then a wavy or spiral thread results. An example of this is the cholera microbe (Fig. 1, No. 10). This latter THE HIGHER BACTERIA. 19 type is of much more frequent occurrence, and contains the more important species. Motility among the first group is often not associated, as far as is known, with the possession of flagella. The cells here apparently move by an undu- lating or screw-like contraction of the protoplasm. Most of the motile spirilla, however, possess flagella. Of the latter there may be one or two, or a bunch containing as many as twenty, at one or both poles. Division takes place as among the bacilli, and in some species endogenous sporulation has been observed. Three terms are used in dividing this group, to which different authors have given different meanings. These terms are spirillum, spirochseta, vibrio. Migula makes "vibrio" synonymous with " microspira," which he applies to members of the group which possess only one or two polar flagella; "spirillum" he applies to similar species which have bunches of polar flagella, while " spiro- chseta " is reserved for the long unflagellated spiral cells. Hueppe applies the term " spirochseta " to forms without endospores, "vibrio" to those with endospores in which during sporulation the organism changes its form, and " spirillum " to the latter when no change of form takes place in sporulation. Fliigge, another systematise applies "spirochseta" and "spirillum" indiscriminately to any wavy or corkscrew form, and "vibrio" to forms where the undulations are not so well marked. It is thus necessary, in denominating such a bacterium by a specific name, to give the authority from whom the name is taken. II. The Higher Bacteria. — These show advance on the lower in consisting of definite filaments branched or un- branched. In most cases the filaments at more or less regular intervals are cut by septa into short rod-shaped or curved elements. Such elements are more or less inter- dependent on one another, and special staining methods are often necessary to demonstrate the septa which demar- cate the individuals of a filament. There is further often a definite membrane or sheath common to all the elements in a filament. Not only, however, is there this close organic relationship between the elements of the higher bacteria, but there is also interdependence of function ; for example, one end of a filament is frequently concerned merely in attaching the organism to some other object. The greatest 2o GENERAL MORPHOLOGY AND BIOLOGY. advance, however, consists in the setting apart among most of the higher bacteria of the free terminations of the filaments for the production of new individuals, as has been described (p. 6). There are various classes under which the species of the higher bacteria are grouped ; but our knowledge of them is still somewhat limited, as many of the members have not yet been artificially cultivated. The beggiatoa group consists of free swimming forms, motile by undulating contractions of their protoplasm. For the demonstration of the rod- like elements of the filaments special staining is necessary. The filaments have no special sheath, and the protoplasm contains sulphur granules. The method of reproduction is doubtful. The thiothrix group resembles the last in structure, and the protoplasm also contains sulphur granules ; but the filaments are attached at one end, and at the other form gonidia. The leptothrix group resembles closely the thio- thrix group, but the protoplasm does not contain sulphur granules. In the cladothrix group there is the appearance of branching, which, however, is of a false kind. What happens is that a terminal cell divides. It divides again, and pushes the product of its first division to one side. There are thus two terminal cells lying side by side, and as each goes on dividing, the appearance of branching is given. Here, again, there is gonidium formation; and while the parent organism is in some of its elements motile, the gonidia move by means of flagella. The highest development is in the streptothrix group, to which belongs the streptothrix actinomyces, or the actinomyces bovis, an important pathogenic agent. Here the organism consists of a felted mass of non- septate filaments, in which true dichotomous branching occurs. Under certain circum- stances threads grow out, and produce chains of coccus- like bodies from which new individuals can be reproduced. Such bodies are often referred to as spores, but they have not the same staining reactions nor resisting powers of so high a degree as ordinary bacterial spores. Some- times too the protoplasm of the filaments breaks up into bacillus - like elements, which may also have the FOOD SUPPLY OF BACTERIA. 21 capacity of originating new individuals. In the strepto- thrix actinomyces there may appear a club-shaped swelling of the membrane at the end of the filament, which has by some been looked on as an organ of fructification, but which is most probably a product of a degenerative change. The streptothrix group as a whole is a link between the bacteria on the one hand, and the lower fungi on the other. Like the latter, the streptothrix forms show the felted mass of non-septate branching filaments, which is usually called a mycelium. On the other hand, the breaking up of the protoplasm of the streptothrix into coccus- and bacillus-like forms, links it to the other bacteria. General Biology of the Bacteria. There are five prime factors which must be considered in the growth of bacteria, namely, food supply, moisture, relation to gaseous environment, temperature, and light. Food Supply. — The great function performed by bacteria in nature is the breaking up into simpler constituents of the complicated organic substances which form the bodies of dead plants" and animals, or which are excreted by the latter while they are yet alive. The natural food of bacteria is therefore of an extremely complex nature. Not only is it so to start with, but seeing that, as a general rule, many bacteria grow side by side, the food supply of any particular variety is, relatively to it, altered by the growth of the other varieties present. It is thus impossible to imitate the natural food environment of any species. The artificial media used in bacteriological work may therefore be poor substitutes for the natural supply. In certain cases, however, the conditions under which we grow cultures may be better than they naturally are. For while one of two species of bacteria growing side by side may favour the growth of the other, it may also in certain cases hinder it, and, therefore, when the latter is grown alone it may grow better. Most bacteria seem to produce excretions which are unfavourable to their own vitality, for 22 GENERAL MORPHOLOGY AND BLOLOGY. it is a frequent experience that, when a species is sown on a mass of artificial food medium, it does not in the great majority of cases go on growing till the food supply is exhausted, but soon ceases to grow. Effete products diffuse out into the medium and prevent growth. Such diffusion may be seen when the organism produces pig- ment, which frequently can be observed in a transparent medium far beyond the limit of the growth of the organism, e.g., B. pyocyaneus growing on gelatine. In supplying artificial food for bacterial growth, the general principle ought to be to imitate as nearly as possible the natural surroundings, though it is found that there exists a con- siderable adaptability among organisms. With the patho- genic varieties it is usually found expedient to use media derived from the fluids of the animal body, and in cases where bacteria growing on plants are being studied, in- fusions of the plants on which they grow are frequently used. With some bacteria special substances are necessary to support life. Thus some species, in the protoplasm of which sulphur granules occur, require sulphuretted hydrogen to be present. In nature the latter is usually provided by the growth of other bacteria. When the food supply of a bacterium fails, it degenerates and dies. The proof of death lies in the fact that when it is transferred to fresh and good food supply it does not multiply. If the bacterium spores, it may then survive the want of food for a very long time. It may here be stated that the reaction of the food medium is a matter of great importance. Most bacteria prefer a slightly alkaline medium, and some, e.g., the cholera vibrio, will not grow in the presence of the smallest amount of free acid. Moisture. — The presence of water is necessary for the continued growth of all bacteria. The amount of drying which bacteria in the vegetative stage will resist varies very much in different species. Thus the cholera spirillum is killed by two or three hours' drying, while the staphylo- coccus pyogenes aureus will survive ten days' drying, and the bacillus diphtherias still more. In the case of spores RELATION OF BACTERIA TO TEMPERATURE. 23 the periods are much longer. Anthrax spores will survive drying for several years, but here again moisture enables them to resist longer than when they are quite dry When organisms have been subjected to such hostile influences, even though they survive, it by no means follows that they retain all their vital properties. m Relation to Gaseous Environment.— The relation ot bacteria to the oxygen of the air is such an important factor in the life of bacteria that it enables a biological division to be made among them. Some bacteria will only live and grow when oxygen is present. To these the title of obligatory aerobes is given. Other bacteria will only grow when no oxygen is present. These are called obligatory ancerobes. In still other bacteria the presence or absence of oxygen is a matter of indifference. This group might theoretically be divided into those which are preferably aerobes, but could be anaerobes, and those which are pre- ferably anaerobes, but could be aerobes. As a matter of fact such differences are manifested to a slight degree, but all such organisms are usually grouped as facultative ancerobes, i.e., preferably aerobic but capable of existing without oxygen. Examples of obligatory aerobes are B. proteus vulgaris, B. subtilis j of obligatory anaerobes, B. tetani, B. cedematis maligni, while the great majority of pathogenic bacteria are facultative anaerobes. With regard to anaerobes, hydrogen and nitrogen are indifferent gases. Many anaerobes, however, do not flourish well in an atmo- sphere of carbon dioxide. Very few experiments have been made to investigate the action on bacteria of gas under pressure. A great pressure of carbon dioxide is said to make the B. anthracis lose its power of sporing, but it seems to have no effect on its vitality nor on that of the B. typhosus. With the bacillus pyocyaneus, however, it is said to destroy life. Temperature. — For every species of bacterium there is a temperature at which it grows best. This is called the "optimum temperature." There is also in each case a maximum temperature above which growth does not take 24 GENERAL MORPHOLOGY AND BIOLOGY. place, and a minimum temperature below which growth does not take place. As a general rule the optimum tem- perature is about the temperature of the natural habitat of the organism. For organisms taking part in the ordinary processes of putrefaction the temperature of warm summer weather (200 to 240 C.) may be taken as the average optimum, while for organisms normally inhabiting animal tissues 350 to 390 C. is a fair average. The lowest limit of ordinary growth is from 120 to 14° C, and the upper is from 420 to 440 C. In exceptional cases growth may take place as low as 5° C, and as high as 700 C. It is to be noted that while growth does not take place below or above a certain limit it by no means follows that death takes place outside such limits. Organisms can resist cooling below their minimum or heating beyond their maximum without being killed. Their vital activity is merely paralysed. Especially is this true of the effect of cold on bacteria. The results of different observers vary ; but if we take as an example the cholera vibrio, Koch found that while the minimum temperature of growth was 1 6° C, a culture might be cooled to - 320 C. without being killed. With regard to the upper limit, few ordinary organisms in a spore-free condition will survive a tempera- ture of 570 C, if long enough applied. Many organisms lose some of their properties when grown at unnatural temperatures. Thus many pathogenic organisms lose their virulence if grown above their optimum temperature, and some chromogenic forms, most of which prefer rather low temperatures, lose their capacity of producing pigment, e.g., spirillum rubrum. Some organisms which grow best at a temperature of from 6o° to 70° C. have been isolated from dung, the intestinal tract, etc. These have been called thermophilic bacteria. Effect of Light. — Of recent years much attention has been paid to this factor in the life of bacteria. Direct sunlight is found to have a very inimical effect. One observer found that an exposure of dry anthrax spores for one and a half hours to sunlight killed them. When CONDITIONS AFFECTING BACTERIAL MOTILITY. 25 they were moist, a much longer exposure was necessary. Typhoid bacilli were killed in about one and a half hours, and similar results have been obtained with many other organisms. In such experiments the thickness of the medium surrounding the growth is an important point. Death takes place more readily if the medium is scanty or if the organisms are suspended in water. Any fallacy which might arise from the effect of the heat rays of the sun has been excluded, though light plus heat is more fatal than light alone. In direct sunlight it is chiefly the green, violet, and, it may be, the ultra-violet rays which are fatal. Diffuse daylight has also a bad effect upon bacteria, though it takes a much longer exposure to do serious harm. A powerful electric light is as fatal as sunlight. Here, as with other factors, the results vary very much with the species under observation, and a distinction must be drawn between a mere cessation of growth and the condition of actual death. Conditions affecting the Movements of Bacteria. — In some cases differences are observed in the behaviour of motile bacteria, contemporaneous with changes in their life history. Thus, in the case of bacillus subtilis, movement ceases when sporulation is about to take place. On the other hand, in the bacillus of symptomatic anthrax, move- ment continues while sporulation is progressing. Under ordinary circumstances motile bacteria appear not to be constantly moving but occasionally to rest. In every case the movements become more active if the temperature be raised. Most interest, however, attaches to movements which, from the use of an unscientific terminology, are often described as if they were purposive, that is when the bacilli are attracted to certain substances and repelled by others. Schenk, for instance, observed that motile bacteria were attracted to a warm point in a way which did not occur when the bacteria were dead and therefore only subject to physical conditions. His method was to intro- duce the upturned point of a copper rod into a drop of fluid containing the bacteria and suspended from the lower GENERAL MORPHOLOGY AND BLOLOGY. surface of a cover-glass. On the outer end of the rod being warmed, heat waves were, of course, conducted up to the point and the bacteria swarmed round the latter. Most important observations have been made on the attraction and repulsion exercised on bacteria by chemical agents, which have been denominated respectively positive and negative chemiotaxis. Pfeiffer investigated this subject in many lowly organisms, including bacterium termo and spirillum undula. The method was to fill with the agent a fine capillary tube, closed at one end, to introduce it into a drop of fluid containing the bacteria under a cover-glass, and to watch the effect through the microscope. Fallacies due to the passing of the fluid out of the tube otherwise than by diffusion, to temperature changes, and to vibration, seem to have been excluded, and control experiments were performed with dead bacteria. The general result was to indicate that motile bacteria may be either attracted or repelled by the fluid in the tube. The effect of a given fluid differs in different organisms, and a fluid chemiotactic for one organism may not act on another. Degree of con- centration is important, but the nature of the fluid is more so. Of inorganic bodies salts of potassium are the most powerfully attracting bodies, and in comparing organic bodies the important factor is the molecular constitution. These observations have been confirmed by Ali-Cohen, who found that while the vibrio of cholera and the typhoid bacillus were scarcely attracted by chloride of potassium they were powerfully influenced by potato juice. Further, the filtered products of the growth of many bacteria have been found to have powerful chemiotactic properties. It is evident that all these observations have a most important bearing on the action of bacteria, though we do not yet know their true significance. Corresponding chemiotactic phenomena are shown also by certain animal cells, e.g., leucocytes, to which reference is made below. The Parts played by Bacteria in Nature. — As has been said, the great function of bacteria is to break up into more simple combinations the complex molecules of the organic EFFECTS OF BACTERIA IN NATURE. 27 substances which form the bodies of plants and animals, or which are excreted by them. In some cases we know some of the stages of disintegration, but in most cases we know only general principles and sometimes only results In the case of milk, for instance, we know that lactic acid is produced from the lactose by the action of the bacillus acidi lactici and of other bacteria. From urea we know that ammonium carbonate is produced by the micrococcus ureffi. That the very complicated process of putrefaction is due to bacteria is absolutely proved, for any organic substance can be preserved indefinitely from ordinary putre- faction by the adoption of some method of killing all bacteria present in it, as will be afterwards described. This state- ment, however, does not exclude the fact that molecular changes take place spontaneously in the passing of the organic body from life to death. Many processes not usually referred to as putrefactive are also bacterial in their origin. The souring of milk, already referred to, the becoming rancid of butter, the ripening of cream and of cheese, are all due to bacteria. A certain comparatively small number of bacteria have been proved to be the causal agents in some disease processes occurring in man, animals, and plants. This means that the fluids and tissues of living bodies are, under certain circumstances, a suitable pabulum for the bacteria involved. The effects of the action of these bacteria are analogous to those taking place in the action of the same or other bacteria on dead animal or vegetable matter. The complex organic molecules are broken up into simpler products. We shall study these processes more in detail later. Meantime we may note that the disease-producing effects of bacteria form the basis of another biological division of the group. Some bacteria are harmless to animals and plants, and apparently under no circumstances give rise to disease in either. These are known as sapro- phytes. They are normally employed in breaking up dead animal and vegetable matter. Others normally live on or in the bodies of plants and animals and produce disease. GENERAL MORPHOLOGY AND BIOLOGY. These are known as parasitic bacteria. Sometimes an attempt is made to draw a hard and fast line between the saprophytes and the parasites, and obligatory saprophytes or parasites are spoken of. This is an erroneous distinc- tion. Some bacteria which are normally saprophytes can produce pathogenic effects (e.g., bacillus oedematis maligni), and it is consistent with our knowledge that the best- known parasites may have been derived from sapro- phytes. On the other hand, the fact that most bacteria associated with disease processes, and proved to be the cause of the latter, can be grown in artificial media, shows that for a time at least such parasites can be saprophytic. As to how far such a saprophytic existence of disease- producing bacteria occurs in nature, we are in many instances still ignorant. The Methods of Bacterial Action. — The processes which bodies being split up by bacteria undergo depend, first, on the chemical nature of the bodies involved and, secondly, on the varieties of the bacteria which are acting. The destruction of albuminous bodies which is mostly involved in the wide and varied process of putrefaction can be undertaken by whole groups of different varieties of bacteria. The action of the latter on such substances is analogous to what takes place when albumins are subjected to ordinary gastric and intestinal digestion. In these circumstances, therefore, the production of albumoses, peptones, etc., similar to those of ordinary digestion, can be recognised in putrefying solutions, though the process of destruction always goes further, and still simpler substances, e.g., indol, are the ultimate results. The process is an exceedingly complicated one when it takes place in nature, and different bacteria are probably concerned in the different stages. Many other bacteria, e.g., some pathogenic forms, though not concerned in ordinary putrefactive processes, have a similar digestive capacity. When carbohydrates are being split up, then various alcohols, ethers, and acids are pro- duced. During bacterial growth there is not unfrequently the abundant production of such gases as sulphuretted ACTION OF BACTERIAL FERMENTS. ■ 29 hydrogen, carbon dioxide, methane, etc. For an exact knowledge of the destructive capacities of any particular bacterium there must be an accurate chemical examination of its effects when it has been grown in artificial media the nature of which is known. The precise substances it is capable of forming can thus be found out. Many sub- stances, however, are produced by bacteria, of the exact nature of which we are still ignorant, for example, the toxic bodies which play such an important part in the action of many pathogenic species. Many of the actions of bacteria depend on the produc- tion by them of ferments of a very varied nature and com- plicated action. Thus the digestive action on albumins depends on the production of a peptic ferment analogous to that produced in the animal stomach. Ferments which invert sugar, which split sugars up into alcohols or acids, which coagulate casein, which split up urea into ammonium carbonate, have all been isolated from different bacteria. Such ferments may be diffused into the surrounding fluid, or be retained in the cells where they are formed. Sometimes the breaking down of the organic matter appears to take place within, or in the immediate proximity of, the bacteria, sometimes wherever the soluble ferments reach the organic substances. And in certain cases the ferments diffused out into the surrounding medium probably break down the latter to some extent, and prepare it for a further, probably intracellular, disintegration. Thus in certain putrefactions of fibrin, if the process be allowed to go on naturally, the fibrin dissolves and ultimately great gaseous evolution of carbon dioxide and ammonia takes place, but if the bacteria, shortly after the process has begun, are killed or paralysed by chloroform, then only a peptonisation of the fibrin occurs, without the further splitting up and gaseous production being observed. That a purely intracellular digestion may take place is illustrated by what has been shown to occur in the case of the micrococcus ureas, which from urea forms ammonium carbonate by adding water to the urea molecule. Here, if after the action has com- 30 GENERAL M ORPHOL OGY AND BIOL OGY. menced, the bacteria are filtered off, no further production of ammonium carbonate takes place, which shows that no ferment has been dissolved out into the urine. If now the bodies of the bacteria be extracted with absolute alcohol or ether, which of course destroy their vitality, a substance is obtained of the nature of a ferment, which, when added to sterile urine, rapidly causes the production of ammonium carbonate. This ferment has evidently been contained within the bacterial cells. As has been said, some bacteria seem to be capable of building up out of simple chemical compounds bodies which are more complex. This function is best illustrated in a group of bacteria which probably play a most important economic function in fertilising the soil by con- verting ammonia compounds into nitrites and nitrates, and thus making the nitrogen more available for plant nutrition. These so-called nitri- fying organisms have been investigated by Professor and Mrs. Frankland, by Professor Warington, and by Winogradski. Their isolation pre- sented great difficulties, none of the ordinary methods being available, as the organisms sought were quickly overgrown by the ordinary bacteria of the soil. Winogradski, however, succeeded in getting fairly pure cultures by taking advantage of the fact that they were capable of growing in the entire absence of organic matter, to exclude which he took most elaborate precautions. The media used contained potassium phosphate, sulphate of magnesium, sulphate of ammonium, basic carbonate of magnesium, and water. An inorganic gelatinous substance was sometimes added, namely, hydrate of silica. On such a medium ordinary bacteria could not develop to any extent. The nitrifying organisms flourished, and there was evidence of abundant oxidation of ammonia and the formation of nitrites, and to a less extent of nitrates. Not only so, but these organisms could derive their carbon from the carbonates present. There is evidence that the nitrifying organisms consist of two groups, one of which forms nitrites from ammonia compounds, the other forming nitrates from these nitrites. It is also known that other organisms exist which are capable of forming compounds by taking up the free nitrogen of the air. On the roots of all leguminous plants small nodules, usually called tuber- cles, are found. These are not developed if the plant is growing in soil free from bacteria, and plants thus grown are not so vigorous as those which grow in ordinary soil. Further, in the interior of these tubercles bacteria-like bodies are observed. There is a good deal of evidence that these are either bacteria or allied organisms, that they take up free nitrogen from the air, and make it available for the nutrition of the plant. If this be the case, a reason is found for the VARIABILITY AMONG BACTERIA. 31 idea long held by agriculturists that the growth of a crop of beans or peas fertilises the soil and improves subsequent crops. The Occurrence of Variability among Bacteria. — The question of the division of the group of bacteria into definite species has given rise to much discussion among vegetable and animal morphologists. In 1872 Cohn stated the opinion that bacteria showed as distinct species as the other lower plants and animals. He recognised the great divisions into which bacteria naturally fall when the forms under which they appear are considered, but he carefully guarded himself against the error that mere considerations of form are sufficient for a proper natural classification of the group. In such a classification the history of the whole life cycle of an organism, and especially the course of its development, must be taken into account. Variations in form occurring in particular cases have, however, been made the basis for criticism of the statement that the large numbers of bacteria which have been identified are really to be looked upon as distinct species. The extreme case for the existence of variability was put by Nageli, who held that "the same species in the course of generations might present different morphological and physiological forms which might give rise at one time to the souring of milk, at another to butyric acid fermentation, at another to the putrefaction of albuminous matter, at another to diphtheria, at another to typhoid, at another to cholera." Such an extreme view was advanced before the elaboration by Koch of methods by which growths of a single kind of bacteria without admixture of any other variety can be obtained. Undoubtedly many of the earlier observations were made on mixtures of different organisms. Thus the upholders of the occurrence of great variability founded their view very largely on observations of a group of organisms closely •allied to the bacteria which, from a peculiar pigment called " purpurin " in their protoplasm, have been called the purpurin bacteria (German, Purpurbakterien). From a more recent study of the group by Winogradski, however, these seem to be a mixture of many different species, each of which maintains during multiplication its characteristic form. Practically no one at the present day holds that an organism can appear now as a bacillus, now as a coccus, now as a spirillum. Nor is such a view in any way supported by the occurrence in isolated cases among the higher bacteria of coccus-like and bacillus-like seg- mentation, such as we have seen to take place in the streptothrix group. With regard to the bacteria as a whole we may say that each variety tends to conform to the definite type of structure 32 GENERAL MORPHOLOGY AND BIOLOGY. and function which is peculiar to it. On the other hand, slight variations from such type can occur in each. The size may vary a little with the medium in which the organism is growing, and under certain similar conditions the adhesion of bacteria to each other may also vary. Thus cocci, which are ordinarily seen in short chains, may grow in long chains. The capacity to form spores may be altered, and such properties as the elaboration of certain ferments or of certain pigments may be impaired. Also the characters of the growths on various media may undergo variations. As has been remarked, variation as observed consists largely in a tendency in a bacterium to lose properties ordinarily possessed, and all attempts to transform one bacterium into an apparently closely allied variety (such as the B. coli into the B. typhosus) have failed. This of course does not preclude the possibility of one species having been originally derived from another or of both having descended from a common ancestor, but we can say that only variations of an unimportant order have been observed to take place, and here it must be remembered that in many cases we can often have forty-eight or more generations under observa- tion within twenty-four hours. If we accept De Bary's definition of a species, we can have little difficulty in saying that species exist among the bacteria. The definition is : " By the term species we mean the sum total of the separate individuals and generations which, during the term afforded for observations, exhibit the same periodically repeated course of development within certain empirically determined limits of variation." The Death of Bacteria. — The death of bacteria is usually judged of by the fact that, when they are trans- ferred to a fresh quantity of an artificial medium in which they previously grew, no growth takes place. Under the microscope the counterpart of this of course would be the cessation of division when surrounded by such a medium. All bacteria can be killed by heat, drying, starvation, and chemical agents, as we have seen. Great attention has been paid to the latter, which are usually called antiseptics, though THE DEATH OF BACTERIA. 33 germicides would be a more proper term to apply. The action of such agents depends on the variety of bacterium to be killed, on its state of nutrition, whether it is in a vegetative or a spored condition, on the temperature at which the agent acts, on the medium in which it acts, and on the nature of the chemical agent itself. Among inorganic bodies the salts of the metals with high atomic weights act more potently than those with lower, and the most powerful antiseptic bodies are probably the perchloride and periodide of mercury. The reaction of the agent is a point of great importance ; as a general rule, the more powerful an acid is, the greater is its capacity as a germicide. The importance of oxidising and reducing agents as germicides has probably been overestimated. Among organic bodies the members of the aromatic series are all more or less potent — the favourite member for practical use being carbolic acid. In comparing the action of antiseptic agents the all-important point is their relative molecular constitution. From the number of conditions we have enumerated, which must be considered in estimating antisepticity, it is evidently impossible to make definite statements as to the value of particular agents unless all the conditions are stated. As a general rule, however, the two solutions most commonly used, which will kill the greatest variety of bacteria in the shortest time, are a i in 20 solution of carbolic acid and a 1 in 1000 solution of perchloride of mercury. 3 CHAPTER II. METHODS OF CULTIVATION OF BACTERIA. Introductory. — In order to study the characters of any species of bacterium it is necessary to have it growing apart from every other species. In the great majority of cases where bacteria occur in nature, this condition is not fulfilled. In the general processes of putrefaction many different species occur all mingled with each other. Only in the blood and tissues in some diseases do particular species occur singly and alone. We usually have, therefore, to remove a bacterium from its natural surroundings and grow it on an artificial food medium. When we have succeeded in separating it, and have got it to grow on a medium which suits it, we are said to have obtained a pure culture^ These pure cultures are absolutely necessary for the proper study of bacteria ; for, when many individuals ofa^pafticular species are growing together^ e mass forme^by their aggregation frequently presents chaYacteri^trc^appearances which con- stitute specific differences^ The recognition of different species of bacteria depends, in fact, far more on the char- acters presented by pure cultures and their behaviour in different food media, than on microscopic examination. The latter in most cases only enables us to refer a given bacterium to its class. For the greater number of specific characters we rely on the observation of pure cultures. Again, in inquiring as to the possible possession of patho- genic properties by a bacterium, the obtaining of pure THE METHODS OF STERILISATION. 35 cultures is absolutely essential. If two or more different organisms be present together, we cannot say that any one of them is the cause of the disease in question. To obtain pure cultures, then, is the first requisite of bacteriological research. Now, as bacteria are practically omnipresent, we must first of all have means of destroying all extraneous organisms which may be present in the food media to be subsequently used for growing the bacteria we wish to study, in the vessels in which the food media are contained, and on all instruments which are to come in contact with our cultures. The technique of this destructive process is called sterilisation. We must therefore study the methods of sterilisation. The growth of bacteria in other than their natural surroundings involves further the prepara- tion of sterile artificial food media, and when we have such media prepared we have still to look at the technique of the separation of micro-organisms from mixtures of these, and the maintaining of pure cultures when these have been obtained. We shall here find that different methods are necessary according as we are dealing with aerobes or anozrobes. Each of these methods will be considered in turn. The Methods of Sterilisation. To exclude extraneous organisms, all food materials, glass vessels containing them, wires used in transferring bacteria from one culture medium to another, instruments used in making autopsies, etc., must be sterilised. These objects being so different, various methods are necessary. The foods comprise meat infusions, jellies, potatoes, etc., and a method suitable for their sterilisation evidently may not be suitable for the sterilisation of, say, a glass flask. Bacteria may be killed by various methods. Many chemicals will kill them, but the difficulty of subsequently removing such chemicals, so that they may not interfere with the growth of the microbes we wish to cultivate, makes their use inapplicable. We therefore in practice take advantage of the principle that all bacteria are destroyed by heat. 36 METHODS OF CULTIVATION OF BACTERIA. The temperature necessary varies with different bacteria, and the vehicle of heat is also of great importance. The two vehicles employed are hot air and hot water or steam. The former is usually referred to as " dry heat," the latter as " moist heat." As showing the different effects of the two vehicles, Koch found, for instance, that the spores of bacillus anthracis, which were killed by moist heat at ioo° C, in one hour, required three hours' dry heat at 140° C. to effect death. Both forms of heat may be applied at different temperatures — in the case of moist heat above ioo° C, a pressure higher than that of the atmosphere must of course be present. A. Sterilisation by Dry Heat. A. (1) Red Heat or Dull Red Heat.— Red heat is used for the sterilisation of the platinum needles which, it-wttise found, are -so constantly in use. A dull heat is used for cauteries, the points of for- ceps, and may be used for the incidental sterilisation of small glass objects (cover- slips, slides, occasionally when necessary even test- tubes), care of course being taken not to melt the glass. The heat is obtained by an ordinary Bunsen burner. A. (2) Sterilisation by Dry Heat in a Hot -Air Chamber. — The chamber (Fig. 2) consists of an outer and inner case of sheet iron. In the bottom of the outer there is a large hole. A Bunsen is lit beneath this, and thus plays on the bottom of the inner case, round all of the sides of which the hot air rises and escapes through holes m the top Fig. 2.- -Hot-air steriliser. STERILISATION BY MOIST HEAT. 37 of the outer case. A thermometer passes down into the interior of the chamber, half-way up which its bulb should be situated. It is found as a matter of experience, that an exposure in such a chamber for one hour to a temperature of 1700 C, is sufficient to kill all the organisms which usually pollute articles in a bacteriological laboratory, though circumstances might arise where this would be insufficient. H This means of sterilisation is used for the, glass flasks, d test-tubes, plates, Petri's dishes, the use of which will be described. Such pieces of apparatus are thus obtained sterile and dry. It is advisable to put glass vessels into the chamber before heating it, and to allow them to stand in it after sterilisation till the temperature falls. Sudden heating or cooling is apt to cause glass to crack. The y^J^M method; is unsuitable for food media. Sc^id-^sdiar-would be scorched by such a tempej^tuee^stmMImd media would not reach it at the ordinary which this nutrient gelatine melts, and therefore another j gelatinous substance called agar, which does not melt \ below 980 C, was substituted. Bouillon made from meat extract, gelatine, and agar media, and the modifications of these, constitute the chief materials in which bacteria are grown. Preparation of Meat Extract. The flesh of the ox, calf, or horse is usually employed. Horse-flesh has the advantage of being cheaper and con- taining less fat than the others ; though generally quite 42 METHODS OF CULTIVATION OF BACTERIA. 4- suitable, it has the disadvantage for certain purposes of containing a larger proportion of fermentable sugar. The flesh must be freed from fat, and finely minced. To a pound of mince add iooo c.c. distilled water, and mix thoroughly in a shallow dish. Set aside in a cool place for twenty-four hours. Skim off any fat present, removing the last traces by stroking the surface of the fluid with pieces of filter paper. Place a clean linen cloth over the mouth of a large filter funnel, and strain the fluid through it into a flask. Pour the minced meat into the cloth, and gather- ing up the edges of the latter in the left hand, squeeze out the juice still held back in the contained meat. Finish this expression by putting the cloth and its contents into a meat press (Fig. 6), similar to that used by pharmacists in preparing extracts ; thus squeeze out the last drops. The resulting sanguineous fluid contains the soluble albumins of the meat, the soluble salts, extractives, and colouring matter, chiefly haemoglobin. It is now boiled thoroughly for two hours, by which process the albumins coagulable by heat are coagulated. Strain now through a clean cloth, boil for another half-hour, and filter through white Swedish filter paper (best, C. Schleicher u. Schull, No. 595). Make up to 1000 c.c. with distilled water. The resulting fluid ought to be quite transparent, of a yellowish colour without any red tint. If there is any redness, the fluid must be reboiled and filtered till this colour disappears, otherwise in the later stages it will become opalescent. A large quantity of the extract may be made at a time, and what is not immediately required is put into a large flask, the neck plugged with cotton wool, and the whole sterilised by methods B (2) or (3). This extract contains very little albuminous matter, and consists chiefly of the soluble salts of the muscle, certain extractives, and altered colouring Fig. 6. — Meat press. PEPTONE BOUILLON MEDIA. 43 matters, along with any slight traces of soluble proteid not coagulated by heat. It is of acid reaction. We have now to see how, by the addition of proteid and other matter, it may be transformed into proper culture media. i. Bouillon Media. — These consist of meat extract with the addition of certain substances to render them suitable for the growth of bacteria. i '(a). Peptone Broth or Bouillon.— Add to the meat extract .5 per cent sodium chloride and 1 per cent peptone albumin. Boil till both are quite dissolved, and neutralise with a 4 per cent solution of sodium hydrate. Add the latter drop by drop, shaking thoroughly between each drop and testing the reaction by means of litmus paper. Go on till the reaction is slightly but distinctly alkaline. Neutral- isation must be practised with great care, as under certain circumstances, depending on the relative proportions of the different phosphates of sodium and potassium, what is known as the amphoteric reaction is obtained, i.e. red litmus is turned blue, and blue red, by the same solution. The sodium hydrate must be added till red litmus is turned slightly but distinctly blue, and blue litmus is not at all tinted red. After alkalinisation, allow the fluid to become cold, filter through Swedish filter paper into flasks, make up to original volume with distilled water, plug the flasks with cotton wool, and sterilise by methods B (2) or (3), PP- 37> 39- This method of neutralisation is to be recom- mended for all ordinary work. In this medium the place of the original albumins of the meat is taken by peptone, a soluble proteid not coagulated by heat. Here it may be remarked that the commercial peptone albumin is not pure peptone, but a mixture of albumoses (see footnote, p. 154) with a variable amount of pure peptone. The addition of the sodium chloride is necessitated by the fact that alkalinisation precipitates some of the phosphates and carbonates present. Experience has shown that sodium chloride can quite well be substituted. The reason for the alkalinisation is that it is found that most bacteria grow best on a medium slightly alkaline to litmus. Some, e.g. the cholera vibrio, wilr*not grow at all on even a slightly acid medium. Standardisation of Reaction of Media. — While the 44 METHODS OF CULTIVATION OF BACTERIA. above procedure of dealing with the reaction of a medium is sufficient for ordinary work it is important to have a more exact method for making media to be used in growing organisms, the growth characteristics of which are to be described for systematic purposes. Such a method should also be used in studying the changes in reaction produced in a medium by the growth of bacteria. It, however, involves considerable difficulty, and should not be under- taken by the beginner. It entails the preparation of solutions of acid and alkali which may be used for deter- mining the original reaction of the medium, and for accurately making it of a definite degree of alkalinity. Normal 1 and decinormal solutions of sodium hydrate and hydrochloric acid are used. Preparation of Standard Solutions. — These cannot be accurately made by direct methods. Sodium hydrate takes up water from the air while being weighed, and the accurate estimation of the strength of hydrochloric acid from the specific gravity requires the most delicate chemical manipulations. To prepare these solutions the first require- ment is a deci-normal solution of silver nitrate. This salt can be most accurately weighed in a chemical balance. Its molecular weight is 170, therefore a deci-normal solution will have 17 grs. dissolved in one litre of distilled water. From this a normal solution of hydrochloric acid can be derived by titration, potassium chromate being used as an indicator. From this a deci-normal acid solution can be made. Corresponding standard (normal and deci-normal) solutions of soda (NaOH) are prepared by titrating against the acid solutions, phenol- phthalein being here used as the indicator. The above standard solutions having been obtained, the reaction of a given medium is found by titration. Since the original reaction of meat extract ought to be acid, as 1 A "normal" solution of any salt is prepared by dissolving an "equivalent " weight in grammes of that salt in a litre of distilled water. If the metal of the salt be monovalent, i.e., if it be replaceable in a com- pound by one atom of hydrogen (e.g. sodium), an equivalent is the mole- cular weight in grammes. In the caseof NaCl, it would be 58.5 grammes (atomic weight of Na = 23, of 0 = 35.5). If the metal be bivalent, i.e., requiring two atoms of H for its replacement in a compound (e.g., cal- cium), an equivalent is the molecular weight in grammes divided by two. Thus in the case of CaCl2 an equivalent would be 55.5 grammes (atomic weight of Ca = 40, of Cla = 7*)- STANDARDISING THE REACTION OF MEDIA. 45 stated above, the deci-normal soda solution will be em- ployed The indicator must always be phenol-phthalein, (. per cent in 50 per cent alcohol). Commencing alkalinity— the point to be aimed at— is shown by the appearance of a pink colour. It has been found that when a medium such as bouillon reacts neutral to litmus, the addition on the average of 2 5 per cent of normal soda solution is necessary before it reacts neutral to phenol-phthalein. Now as litmus was originally introduced by Koch, and as nearly all bacterial research has been done with media tested by litmus, it is evidently difficult to say exactly what precise degree of alkalinity is the optimum for bacterial growth, for it is evident from what has been said that such precision is only attained by the use of phenol-phthalein. It is prob- ably safe to say, however, that when a medium has been rendered neutral to this indicator by the addition of NaOH, the optimum degree is attained by the addition of 1.5 per cent normal HC1, the medium being then slightly alkaline to litmus. In other words, the optimum reaction for bacterial growth lies, as Fuller has pointed out, about midway between the neutral point indicated by phenol-phthalein and the neutral point indicated by litmus. The only objection to the use of phenol-phthalein is that its action is somewhat vitiated if free C0.2 be present. This can be completely obviated as follows. Before test- ing any medium it is boiled in the porcelain dish into which titration takes place. The soda solutions are best stored in bottles such as that shown in Fig. 40, having on the air inlet a little bottle filled with soda lime with tubes fitted as in the large one. The C02 of the air which passes through is thus removed. Method. — The practical application of these principles is as follows. Take the medium with all its constituents dissolved and filter it. Place 5 c.c. in a porcelain dish, add 50 c.c. of distilled water and 1 c.c. phenol-phthalein, and boil. Run in deci-normal soda till neutral point is 46 METHODS OF CULTIVATION OF BACTERIA. reached. Repeat process thrice and take mean to obtain amount of soda required. From this calculate acidity of medium per litre, and neutralise with normal soda solution. Check calculation by a fresh titration of 5 c.c. of the neutralised medium. Steam for half an hour, and take reaction again to see that it is constant. Now add normal HC1 in the ratio of 1.5 c.c. per cent. The gelatine and agar media {vide infra) are treated in the same way. 1 (p). Glucose Broth. — To the other constituents of 1 (a) there is added 1 or 2 per cent of grape sugar. The steps in the preparation are the same. Glucose being a reducing agent, no free oxygen can exist in a medium containing it, and therefore glucose broth is used as a culture fluid for anaerobic organisms. 1 (f). Glycerine Broth. — The initial steps are the same as in 1 (a), but after filtration 6 to 8 per cent of glycerine (sp. grav. 1.25) is added. This medium is especially used for growing the tubercle bacillus when the soluble products of the growth of the latter are required. 2. Gelatine Media. — These are simply the above broths, with gelatine added as a solidifying body. 2 (a). Peptone Gelatine. — Take of meat extract say 1000 c.c, add 5 grammes sodium chloride, 10 grammes peptone, and from 100 to 150 grammes gelatine (the "gold label " gelatine of Coignet et Cie, Paris, is the best). The gelatine is cut into small pieces, and added with the other constituents to the extract ; they are then thoroughly melted on a sand bath, or in the " Koch." The fluid medium is then rendered slightly alkaline, as in 1 (a), and filtered through filter paper. As the medium must not be allowed to solidify during the process, it must be kept warm. This is effected by putting the flask and funnel into a tall Koch's steriliser, in which case the funnel must be supported on a tripod, as there is great danger of the neck of the flask breaking if it has to support the funnel and its contents. The filtration may also be carried out in a hot-water funnel (Fig. -f)-. — This consists of an PEPTONE GELATINE. 47 Fig. 7. — Hot-water funnel. outer tin funnel, the neck of which is fitted with a perforated corkAthrough which is placed the stem of an inner glass funnel. The inter- space between trie two /funnels is filled with wateAwhiofi is kept hot by a Bunsen \mder a side arm let into the oyfiter funnel. Whichever instrument be used, before filtering shake up the melted medium, as it is apt while melting to have settled into layers of different density. Sometimes what first comes through is turbid. If so, replace it in the unfiltered part : often the subsequent filtrate in such circumstances is quite clear. A litre flask of the finished product ought to be quite transparent. If instead it is partially opaque, add the white of an egg and boil thoroughly over the sand bath. The consequent coagulation of the albumin carries down the opalescent material, and on making up with distilled water to the original quantity and refiltering, it will be found to be clear. The flask containing it is then plugged with cotton wool and sterilised, best by method B (2), p. 37. If the autoclave be used the temperature employed must not be above 1050 C, and exposure not more than a quarter of an hour on three successive days. Too much boiling, or boiling at too high a temperature, as has been said, causes a gelatine medium to lose its property of solidification. This transparent solid gelatine medium is that chiefly employed for the culture of aerobic bacteria at ordinary temperatures. The exact percentage of gelatine used in its preparation depends on the temperature at which growth is to take place. Its firmness is its most valuable characteristic, and to maintain this in summer weather, 15 parts per 100 are necessary. A limit is placed on higher percentages by the fact that, if the gelatine be too stiff, it will split on the perforation of its substance by the 48 METHODS OF CULTIVATION OF BACTERIA. platinum needle used in inoculating it with a bacterial growth; 15 per cent gelatine melts at about 24° C. 2 (b). Glucose Gelatine. — The constituents are the same as 2 (a), with the addition of 1 to 2 per cent of grape sugar. The method of preparation is identical. This medium is used for growing anaerobic organisms at the ordinary temperatures. 3. Agar Media (French, "gelose ").— The disadvantage of gelatine is that at the blood temperature (38° C), at which most pathogenic organisms grow best, it is liquid. To get a medium which will be solid at this temperature, agar is used as the stiffening agent instead of gelatine. Unlike the latter, which is a proteid, agar is a carbohydrate. It is derived from the stems of various sea-weeds grow- ing in the Chinese seas, popularly classed together as "Ceylon Moss." The best for bacteriological purposes is that consisting of the thin dried stem of the sea-weed itself. 3 (a). "Ordinary" Agar. — Prepare peptone bouillon, medium 1 (a), up to the stage of sterilisation. For every 100 c.c. take 1.5 grammes agar. Cut it up into very fine fragments (in fact till it is as nearly as possible dust), add to the bouillon and allow to stand all night. Then boil' gently in a water bath for two or three hours, till the agar is thoroughly melted. Test with litmus to see that reaction is still slightly alkaline, make up to original volume with distilled water, and filter. Filtration here is a very slow process and must be carried out in a tall Koch's steriliser. In doing this, it is well to put a glass plate over the filter funnel to prevent condensation water from dropping off -j the roof of the steriliser into the medium. If a slight degree of turbidity may be tolerated, it is sufficient to filter through a felt bag or jelly strainer. Plug the flask con- taining the filtrate, and sterilise either in autoclave for fifteen minutes or in Koch's steriliser for one and a half hours. Agar melts just below ioo° C, and on cooling solidifies about 39° C. 3 (b). Glycerine Agar. — To 3 (a) after filtration add AGAR MEDIA. 4') 6 to S per cent of glycerine and sterilise as above. This is used especially for growing the tubercle bacillus. 3 (c). Glucose Agar. — Prepare as in 3 (a), but add 1 to 2 per cent of grape sugar along with agar. This medium is used for the culture of anaerobic organisms at temperatures above the melting-point of gelatine. It is also a superior culture medium for some aerobes, e.g. the B. diphtherias. These bouillon, gelatine, and agar preparations constitute the most frequently used media. Growths on bouillon do not usually show any characteristic appearances which facilitate classification, but such a medium is of great use in investigating the soluble toxic products of bacteria. The most characteristic developments of organisms take place on the gelatine media. These have., however, the dis- advantage of not being available when growth is to take place at any temperature above 24° C. For higher temperatures agar must be employed. Agar is, how- ever, never so transparent. Though quite clear when fluid, on solidifying it always becomes slightly opaque. Further, growths upon it are never so characteristic as those on gelatine. It is, for instance, never liquefied, whereas some organisms, by their growth, liquefy gelatine and others do not — a fact of prime importance. Agar smeared with Blood. — This method was introduced by Pfeiffer for growing the influenza bacillus, and it has been used for the organisms which are not easily grown on the ordinary media, e.g. the gonococcus and the pneumo- coccus. Human blood or the blood of animals may be used. "Sloped tubes" {vide p. 58) of agar are employed (glycerine agar is not so suitable). Purify a finger frrst with 1- 1 000 corrosive sublimate, dry, and then washfwith absolute alcohol to remove the sublimate. Allow the alcohol to evaporate. Prick with a needle sterilised by heat, and, catching a drop of blood in the loop of a sterile platinum wire {vide p. 58), smear it on the surface of the agar. The excess of the blood runs down and leaves a film on the surface. Cover the tubes with india-rubber caps, and incubate them for one to two days at 3 8° C. before 4 U f c 5o ME THODS OF C UL TT VA TJON OF BAC TFRIA . use, to make certain that they are sterile. Agar poured out in a thin layer in a Petri dish may be smeared with blood in the same way and used for cultures. Peptone Solution. A simple solution of peptone (Witte) constitutes a suitable culture medium for many bacteria. The peptone in the proportion of 1-2 per cent, along with .5 per cent NaCl is dissolved in distilled water by heating. The fluid is then filtered, placed in tubes and sterilised. The re- action is usually distinctly alkaline, which condition is suitable for most purposes. For special purposes the reaction may be standardised. In such a solution the cholera vibrio grows with remarkable rapidity. It is also much used for testing the formation of indol by a particular bacterium ; and by the addition of one of the sugars to it the fermentative powers of an organism may be tested. Litmus may be added to show any change in reaction. Lit?nus Media. — To any of the above media litmus (French, tournesol) may be added to show change in re- action during bacterial growth. The litmus is added, before sterilisation, as a strong watery solution in sufficient quantity to give the medium a distinctly bluish tint. During the development of an acid reaction the colour changes to a pink and may subsequently be discharged. Blood Serum. Koch introduced this medium, and it is prepared as follows : Plug the mouth of a tall cylindrical glass vessel (say a 1000 c.c. measure) with cotton wool, and sterilise by steaming it in a Koch's steriliser for one and a half hours. Take it to the place where a horse, ox, or sheep is to be killed. When the artery or vein of the animal is opened, allow the first blood which flows, and which may be contaminated from the hair, etc., to escape ; fill the vessel with the blood sub- BLOOD SERUM. 5' sequently shed. Carry carefully back to the laboratory without shaking, and place for twenty-four hours in a cool place, preferably an ice chest. The clear serum will separate from the clotted blood. With a sterile 10 c.c. pipette, transfer this quantity of serum to each of a series of test-tubes which must previously have been sterilised by dry heat. The serum may, with all precautions, have been contaminated during the manipulations, and must be sterilised. As it will coagulate if heated above 68° C, advantage must be taken of the intermittent process of sterilisation at 57° C. « (method B (4)). It is therefore kept for one hour at this temperature ^^^afl X tion " is probably an initial stage of coagulation, and is effected by keeping the serum at 650 C. till it stiffens. This temperature is just below the coagulation point of the serum. The more slowly the operation is performed the clearer will be the serum. The apparatus used is seen in Fig. 8. It consists of a rectangular, shallow, covered, hot-water jacket, which can be rapidly heated by an S-shaped Bunsen con- * taining many lateral perforations, from each of which a '■ flame issues. The apparatus rests on four legs, the front two of which can be shortened, and thus the whole tilted ; forward. Tubes containing a suitable quantity of serum ' can thus be laid on their sides without the contents reach- ing as high as the plug. The serum tubes being thus on each of eight succes- sive days. It is always well to incubate it for a day at 370 C. before use, to see that the result is successful. After steri- lisation it is " inspis- sated," by which process a clear solid medium is obtained. " Inspissa- 52 METHODS OF CULTIVATION OF BACTERIA. placed, and the temperature being raised to 65° C, the contents solidify in a sloped position in the interior. It is well not only to have the jacket filled with water, but also to put some water in the trough in which the tubes lie, and also to have a thermometer in the water. This pre- vents cooling of the tubes when the lid is raised to see if the process is complete. As is evident, the preparation of this medium is tedious, but its use is necessary for the observation of particular characteristics in several patho- genic bacteria, notably the tubercle bacillus. Pleuritic and other effusions may be prepared in the same way, and used as media, but care must be taken in their use, as we have no right to say that pathological effusions have the same chemical composition as normal serum. If blood be collected with strict aseptic precautions, then sterilisation of the serum is unnecessary. To this end the mouth of the cylinder used for collecting the blood, instead of being plugged with wool, has an india-rubber bung inserted in it through which two bent glass tubes pass. The outer end of one of these is of convenient length and, before sterilisation, a large cap of cotton wool is tied over it ; the other tube is plugged with a piece of cotton wool. In the slaughter-house the cap is removed and the tube is inserted into the blood vessel as a cannula. The cylinder is thus easily filled. Another method is to conduct the blood to the cylinder by means of a sterilised cannula and india-rubber tube, the former being inserted in the blood vessel. The serum obtained under such circumstances must be incubated before use, to make sure that it is sterile. Loffler's Blood Serum.— This is the best medium for the growth of the B. diphtheria? and may be used for other organisms. It has the following composition. Three parts of calfs or lamb's blood serum are mixed with one part ordinary neutral peptone bouillon made from veal with 1 per cent of grape sugar added to it. Though this is the original formula it can be made from ox or sheep serum and beef bouillon without its qualities being markedly impaired. Sterilise by method B (4) as above.* SERUM MEDIA. 53 Alkaline Blood Serum (Lorrain Smith's Method). — To each 100 c.c. of the serum obtained as before, add 1-1.5 c-c- of a 10 per cent solution of sodium hydrate and shake it gently. Put sufficient of the mixture into each of a series of test-tubes, and laying them on their sides, sterilise by method B (2). If the process of sterilisation be carried out too quickly, bubbles of gas are apt to form before the serum is solid, and these interfere with the usefulness of the medium. Dr. Smith informs us that this can be obviated if the serum be solidified high up in the Koch's steriliser, in which the water is allowed only to simmer. In this case sterilisation ought to go on for one and a half hours. A clear solid medium (consisting practically of alkali-albumin) is thus obtained, and he has found it of value for the growth of the organisms for which Koch's serum is used, and especially for the growth of the B. diphtherias. Its great advantage is that aseptic precautions in obtaining blood from the animal are not necessary, and it is easily sterilised. Marmorek's Serum Media. — There has always been a difficulty in maintaining the virulence of cultures of the pyogenic streptococci, but Marmorek has succeeded in doing so by growing them on the following media, which are arranged in the order of their utility : — 1. Human serum 2 parts, bouillon 1 part. 2. Pleuritic or ascitic serum 1 part, bouillon 2 parts. 3. Asses' or mules' serum 2 parts, bouillon 1 part. 4. Horse serum 2 parts, bouillon 1 part. Human serum can be obtained from the blood shed in venesection, the same precautions being taken as in the case of that got in the slaughter-house. In the case of these media, sterilisation is effected by method B 4, and they are used fluid. Potatoes as Culture Material. (a) In Potato J ars. — The jar consists of a round, shallow, glass vessel with a similar cover {vide Fig. 9). It is washed 54 METHODS OF CULTIVATION OF BACTERIA. Fig. 9. — Potato jar. with 1-1000 corrosive sublimate, and a piece of circular filter paper, moistened with the same, is laid in its bottom. On this latter are placed four sterile watch glasses. Two firm, healthy, small, round potatoes, as free from eyes as possible, and with the skin whole, are scrubbed well with a brush under the tap and steeped for two or three hours in 1- 1 000 corrosive sublimate. They are steamed in the Koch's steriliser for thirty minutes or, longer, or in the autoclave for a quarter of an hour. When cold, each is grasped between the left thumb and forefinger (which have been sterilised with sublimate) and cut r„i,„,w nf ' . , Mg. 10. —Cylinder ol through the middle with potato cut obnqueiy. a sterile knife. It is best to have the cover of the jar raised by an assistant, and to perform the cutting beneath it. Each half is put in one of the watch glasses, the cut surfaces, which are then ready for inoculation with a bacterial growth, being uppermost. Smaller jars, each of which holds half of a potato, are also used in the same way and are very convenient. (b) By Slices in Tubes. —This method, introduced by Ehrlich, is the best means of utilising potatoes as a medium. A large, long potato is well washed and scrubbed, and -EhrHch's peeled with a clean knife. A cylinder is then tube contain- DOreci fr0m its interior with an apple corer or ing piece of & cork borer) and is cut obliquely, as P0tat°' in Fig. 10. Two wedges are thus obtained, each of which is placed broad end down in a test-tube of special form (see Fig. n). In the wide part at the bottom MILK AS A CULTURE MEDIUM. 55 of this tube is placed a piece of cotton wool, which catches any condensation water which may form. The wedge rests on the constriction above this bulbous portion. The tubes, washed, dried, and with cotton wool in the bottom and in the mouth, are sterilised before the slices of potato are in- troduced. After the latter are inserted, the tubes are . steamed in the Koch steam steriliser for one hour. An . ordinary test-tube may be used with a piece of sterile • absorbent wool in its bottom, on which the potato may % rest. The use of the potato as a medium is very important, as in certain cases the growths of bacteria on it are very characteristic. Potatoes ought not to be prepared long before being used, as the surface is apt to become dry and discoloured. It is well to take the reaction of the potato with litmus before sterilisation, as this varies ; normally in young potatoes it is weakly acid. The reaction of the potato may be more accurately estimated by steaming the potato slices for a quarter of an hour in a known quantity of distilled water and then estimating the reaction of the water by phenol-phthalein. The required degree of acidity or alkalinity is obtained by adding the necessary quantity of HC1 or NaOH solution (p. 45) and steaming for other fifteen minutes. The water is then poured off and steri- lisation continued for another half hour. Potatoes before being inoculated ought always to be incubated at 370 C. for a night, to make sure that their sterilisation has been successful. Qlul*** Milk as a Culture Medium. • This is a convenient medium for observing the effects of bacterial growth in changing the reaction, in coagulating the soluble albumin and in fermenting the lactose. It is prepared as follows : fresh milk is taken, preferably after having had the cream " separated " by centrifugalisation, as is practised in the best dairies, and is steamed for fifteen minutes in the Koch, it is then set aside in an ice chest or cool place over night to facilitate further separation of 56 METHODS OF CULTIVATION OF BACTERIA. cream. The milk is siphoned off from beneath the cream. The reaction of fresh milk is alkaline. If great accuracy is necessary any required degree of reaction may be ob- tained by the titration method. It is then placed in tubes and sterilised by methods B (2) or B (3). Bread Paste. This is useful for growing torulae, moulds, etc. Some ordinary bread is cut into slices, and then dried in an oven till it is so dry that it can be pounded to a fine powder in a mortar, or rubbed down with the fingers and passed through a sieve. Some 100 c.c. flasks are washed, dried, and sterilised, and a layer of the powder half an inch thick placed on the bottom. Distilled water, sufficient to cover the whole of it, is then run in with a pipette held close to the surface of the bread, and, the cotton-wool plugs being replaced, the flasks are sterilised in the Koch's steriliser by method B (2). The reaction is slightly acid. The Use of the Culture Media. The culture of bacteria is usually carried on in test- tubes conveniently 6 x § in. If new, these ought to be carefully washed and dripped, and their mouths are plugged with pledgets of plain cotton wool. They are then sterilised for one hour at 1 700 C. The reason is that the glass, being usually packed in straw, is covered with the extremely resist- ing spores of the bacillus subtilis. Tubes which have been in use are merely well washed, dried thoroughly, and plugged. Cotton-wool plugs are universally used for protecting the sterile contents of flasks and tubes from contamination with the bacteria of the air. The contained air passes through the plug during sterilisation \ what passes back on cooling is filtered free of germs by the wool. A medium thus protected will remain sterile for years. Whenever a protecting plug is removed for even a short time, the sterility of the contents may be endangered. It is well to THE USE OF CULTURE MEDIA. 57 place the bouillon, gelatine, and agar media in the test-tubes directly after filtration. The media can then be sterilised in the test-tubes. In filling tubes, care must be taken to run the liquid down the centre, so that none of it drops on the inside of Fig. 12. — Apparatus which may be used for filling tubes. The apparatus explains itself. The india-rubber stopper with its tubes ought to be sterilised before use. rfjf * , Fig. 13. — Tubes of media. Ordinary upright tube. b. Sloped tube. c. Deep " tube for cultures of anserobes. the upper part of the tube with which the cotton -wool plug will be in contact, otherwise the latter will subsequently stick to the glass and its removal will be difficult. The tubes may, when filled, be placed in cages made of fine wire gauze and sterilised. If all the contents of a flask of medium be not filled into tubes, the remainder must be 58 METHODS OF CULTIVATION OF BACTERIA. re-sterilised before being stored. In the case of liquid media, test-tubes are filled about one-third full. With the solid media the amount varies. In the case of gelatine media, tubes filled one-third full and allowed to solidify while standing upright, are those commonly used. With organisms needing an abundant supply of oxygen the best growth takes place on the surface of the medium, and for practical purposes the surface ought thus to be as large as possible. To this end " sloped " agar and gelatine tubes are used. To prepare these, tubes are filled only about one-sixth full, and after sterilisation are allowed to solidify, lying on their sides with their necks supported so that the Fig. 14. — Platinum wires in glass handles. a. Straight needle for ordinary puncture inoculations, b. " Platinum loop. ' c. Long needle for inoculating "deep" tubes. contents extend 3 to 4 inches up, giving an oblique sur- face when held upright after solidification. Thus agar is commonly used in such tubes (less frequently gelatine is also " sloped "), and this is the position in which blood serum is inspissated. Tubes, especially those of the less commonly used media, should be placed in large jars provided with stoppers, otherwise the contents are apt to evaporate. A tube of medium which has been inoculated with a bacterium, and on which growth has taken place, is called a " culture." A " pure culture " is such that only one organism is present. The methods of obtaining pure cultures will presently be described. They vary according as we are dealing with aerobic or anaerobic organisms. When a fresh tube of medium is inoculated from an already INOCULATION BY PLATINUM WIRES. 59 existing culture, the resulting growth is said to be a " sub- culture" of the first. All manipulations involving the transference of small portions of growth either from one medium to another, as in the inoculation of tubes, or, as will be seen later, to cover-glasses for microscopic examina- tions, are effected by pieces of platinum wire (Nos. 24 or 27 Birmingham wire gauge — the former being the thicker) fixed in glass rods 8 inches long. Every worker should have three such wires. Two are 2 J inches long, one of these being straight (Fig. 14, a), and the other having a loop turned upon it (Fig. 14, b). The latter is referred to as the platinum "loop '•' or platinum "eyelet," and is used for many purposes. " Taking a loopful " is a phrase con- stantly used. The third wire (Fig. 14, c) ought to be 4^ inches long and straight. It is used for making anaerobic cultures. Cultures on a solid medium are referred to (1) as " puncture " or "stab" cultures (German, Stichkultur), or (2) as "stroke" cultures (Strichkultur), according as they are made (1) on tubes solidified in the upright posi- tion, or (2) on' sloped tubes. To inoculate say one ordinary up- right gelatine tube from another, the two tubes are held in an inverted posi- tion between the forefinger and thumb of the left hand with their mouths towards the person holding Fig. 15.— Another method of inoculating them ; the plugs solid tubes, are twisted round once or twice to make sure they are not adhering to the glass. The short, straight platinum wire is then heated to redness from point to insertion, and 2 to 3 inches of 6o METHODS OF CULTIVATION OF BACTERIA. the glass rod are also passed two or three times through the Bunsen flame. It is held between the right fore and middle fingers, with the needle projecting backwards, i.e. away from the right palm. Remove plug from culture with right forefinger and thumb, and continue to hold it between the same fingers, by the part which projected beyond the mouth of the tube. Now touch the culture with the platinum needle, and, withdrawing it, replace plug. In the same way remove plug from tube to be inoculated, and plunge platinum wire down the centre of the gelatine to within half an inch of the bottom. It must on no account touch the glass above the medium. The wire is then immediately sterilised. A variation in detail of this method is to hold the plug of the tube next the thumb between the fore and middle fingers, and the plug of the other between the middle and ring fingers, then to make the inoculation (Fig. 15). The sub-culture is labelled, and in a bacteriological laboratory a label should never be licked. If a tube contain a liquid medium, it must be held in a sloping position between the same fingers, as above. When a stroke culture is made the same manipulations are gone through. Here the platinum loop is used, and a little of the culture is smeared in a line along the surface of the medium from below upwards. In inoculating tubes, it is always well, on remov- ing the plugs to make sure that no strands of cotton fibre are adhering to the inside of the necks. As these might be touched with the charged needle and the plug thus be con- taminated, they must be removed by heating the inoculating needle red- Fig. 16. -Rack for platinum needles. hot and scorching them off with it. When the platinum wires are not in use they may be laid in a rack made by bending up the ends of a piece of tin, as in THE SEP A RA TION OF A ERQBIC BA C TERIA. 6 i Fig. 1 6. Before commencing inoculation manipulations, this rack ought to be sterilised by passing it several times through the flame. To prevent contamination of cultures by bacteria falling on the plugs while these are exposed to the air during inoculation manipulations, some v bacteriologists singe the plugs in the flame before replacing. This is, however, in most cases a needless precaution. • If the top of a plug be dusty it is best to singe it before extraction. The Methods of the Separation of Aerobic Organisms. The general principle underlying the methods of separa- tion is the dilution of the bacterial mixture, till each microbe is sufficiently separated from its neighbours to allow it to multiply into a growth (called a " colony "), without the latter coming in contact with the colonies pro- duced by other microbes present. In order to render the colonies easily accessible, the medium is made to solidify in as thin a layer as possible, by being poured out on glass plates. As the optimum temperatures of organisms vary, it is necessary to adapt to the process a low melting-point medium, 'such as gelatine, and a high melting-point medium, such as agar. Many pathogenic organisms, e.g., pneumo- coccus, B. diphtherias, etc., grow too slowly on gelatine to allow its ready use. On the other hand, many organisms, e.g., some occurring in water, do not develop on agar incubated at 370 C. Separation by Gelatine Media. — With both the gelatine and agar media the fluid medium containing bacilli is poured out on plates of glass, and, therefore, when growth takes place, " plate cultures " are said to be obtained. As the naked-eye and microscopic appearances of colonies are often very characteristic, plate cultures, besides use in separation, are often taken advantage of in the description of individual organisms. The plate- culture method can 62 METHODS OF CULTIVATION OF BACTERIA. also be used to test whether a tube culture is or is not pure. The suspected culture is plated (three plates being 1 prepared, as will be described). If all the colonies are the same, then the cultures may be held to be pure. Either simple plates of glass 4 inches by 3 inches are used, or, what are more convenient, circular glass cells I with similar overlapping covers. The latter are known as Petri's - dishes or capsules (Fig. 17). They are usually 3 inches in diameter and half an inch deep. The advantage of these is that they do not require to be kept Fig. 17.— Petri's capsule. jev£i ^y a Special apparatus (Cover shown partially raised.) ^ ^ .g ing, and can be readily handled afterwards without admitting impurities. Whether plates or capsules are used, they are washed, dried with a clean cloth, and sterilised for one hour in dry air at 1700 C, the plates being packed in sheet-iron boxes made for the purpose (see Fig. 18). 1. Glass Capsules.— -While in certain circumstances, as when the number of colonies has to be counted, it is best to use plates, in the usual laboratory routine Petri's cap- j sules are to be preferred for the above reasons. The contents of three gelatine tubes, marked a, b, c, are liquefied by placing in a beaker of water at any temperature between 25° C. and 38° C Inoculate a with the bacterial mixture. The amount of the latter to be taken varies. If the microscope shows enormous numbers of different kinds of bacteria present, just as much as adheres to the point of a straight platinum needle is sufficient. If the number of bacilli is small one to three loops of the mixture may be transferred to the medium. Shake a well, but not so as to cause many fine air-bubbles to form. Transfer two loops of 1 For .narking glass vessels it is convenient to use the red, blue, or yellow pencils made for the purpose by Faber. PETRI'S CAPSULES. 63 gelatine from a to b. Shake b and transfer two loops to c. The plugs of the tubes are in each case replaced and the tubes returned to the beaker. The contents of the three tubes are then poured out into three capsules. In doing so the plug of each tube is removed and the mouth of the tube passed two or three times through the Bunsen flame, the tube being meantime rotated round a longitudinal axis. Any organisms on its rim are thus killed. The capsules are labelled and set aside till growth takes place. The colonies appear as minute rounded points, whitish 1 or variously coloured. Their characters can be more J minutely studied by means of a hand-lens or by inverting the capsule on the stage of a microscope and examining with a low power through the bottom. From their charac- ters, colour, shape, contour, appearance of surface, lique- faction or non- liquefaction of the gelatine, etc., the colonies | can be classified into groups. Further aid in the grouping of the varieties is obtained by making film preparations and examining them microscopically. Gelatine or agar tubes may then be inoculated from a colony of each variety, 1 and the growths obtained are then examined both as to their purity and as to their special characters, with a view to their identification (p. 123). 2. Glass Plates (Koch). — When plates of glass are to be used, an apparatus on which they may be kept level while the medium is solidifying is, as has been said, necessary. An apparatus devised by Koch is used (Figs. 1 8, 1 9). This consists of a circular plate of glass (with the upper surface ground, the lower polished) on which the plate used for pouring out the medium is placed. The latter is protected from the air during solidification by a bell jar. The circular plate and bell jar rest on the flat rim of a circular glass trough, which is filled quite full with a mixture of ice and water to facilitate the lowering of the temperature of whatever is placed beneath the bell jar. The glass trough •rests on corks on the bottom of a large circular trough, which catches any water that may be spilled. This trough in turn rests on a wooden triangle with a foot at 64 METHODS OF CULTIVATION OF BACTERIA. each corner, the height of which can be adjusted, and which thus constitutes the levelling apparatus. A spirit level is placed where the plate is to go, and the level of the ground glass plate thus assured. There is also prepared a "damp chamber," in which the plates are to be stored after being made. This consists of a circular glass trough with a similar cover. It is sterilised by being washed outside and inside with perchloride of mercury i-iooo, and a circle of filter paper moistened with the same is Fig. i 8.— Koch's levelling apparatus for use in pre- paring plates. Hands shown in first position for transferring sterile plate from iron box to beneath bell jar, where it subsequently has the medium poured out upon it. laid on its bottom. Glass benches on which the plates may be laid are similarly purified. To separate organisms by this method three tubes, a, b, c, are inoculated as in using Petri's capsules. The hands having been washed in perchloride of mercury i-iooo and dried, the plate box is opened, and a plate lifted by its opposite eds*es and transferred to the levelled ground glass (as in Fig/ 1 9). The bell jar of the leveller being now lifted a little, the gelatine in tube a is poured out on the surface of the sterile plate, and while still fluid, is spread by stroking with the rim of the tube. The plate is now trans- AVON'S METHOD OF PREPARING PLATES. 65 ferred to the moist chamber as rapidly as possible, so as to avoid atmospheric contamination. To do this, one of the benches is put on the top of the chamber. The top is then lifted off and placed on the table near the leveller. The plate is then quickly transferred to the bench. The latter is lifted by its ends and placed at the bottom of the moist Fig. 19. — Koch's levelling apparatus. Hands shown in second position just as the plate is lowered on to the ground glass surface. By executing the transference of the plate from the box in this way, the surface which was undermost in the latter is uppermost in the leveller, and thus never meets a current of air which might contaminate it. Fig. 20. — Esmarch's tube for roll culture. chamber, the top of which is now replaced. Tubes b and c are similarly treated, and the resulting plates stacked in series on the top of a. The chamber is labelled and set aside for a few days till the colonies appear in the gelatine plates. The further procedure is of the same nature as with Petri's capsules. 3. Esmarch's Roll Tubes. — Here the principle is that of dilution as before. In each of three test-tubes il or xl 5 66 METHODS OF CULTIVATION OF BACTERIA. inch in diameter, gelatine to the depth of f of an inch is placed. These are sterilised. The gelatine is melted and inoculated with the bacterial mixture as in making plate cultures, but instead of being poured out it is rolled in a nearly horizontal position under a cold tap or on a block of ice till it solidifies as a uniformly thin layer on the inside of the tube. Practically we deal with a cylindrical plate of gelatine instead of a flat one. A convenient form of tube for this method is one with a constriction a short distance below the plug of cotton wool (Fig. 20). The great disadvantage of the method is, that if organisms lique- fying the gelatine be present, the liquefied gelatine con- taminates the rest of the gelatine. Separation by Agar Media. — 1. Agar Plates. — The only difference between the technique here and that with gelatine, depends on the difference in the melting-points of the two media. Agar, we have said, melts at 98° C, and becomes again solid a little under 400 C. As it is dangerous to expose organisms to a temperature above 420 C, it is neces- sary in preparing tubes of agar to be used in plate cultures to first melt the agar, by boiling in a vessel of water for a few minutes, and then to cool them to about 420 C. before inoculating. The manipulation must be rapidly carried out, as the margin of time, before solidification occurs, is narrow ; otherwise the details are the same as for gelatine. Esmarch's tubes are not suitable for use here, as the agar does not adhere well to the sides. If to the agar 2 per cent of a strong watery solution of pure gum arabic is added, Esmarch's tubes may, however, be used. 2. Separation by Stroking Mixture on Surface of Agar Media. — The bacterial mixture, instead of being mixed in the medium, is spread out on its surface. The method may be used both when the bacteria to be separated are in a fluid, and when contained in a fairly solid tissue or substance, such as a piece of diphtheritic membrane. In the case of a tissue, for example, a small portion entangled in the loop of a platinum needle is stroked in successive parallel longitudinal strokes on sloped agar, the same SEPARA TION- OF PA THOGENIC BACTERIA. 67 aspect being brought in contact with the agar in all the strokes. Three strokes may be made on each . tube, and three tubes are usually sufficient. In this process the organisms on the surface of the tissue are gradually rubbed off, and when growth has taken place it will be found that in the later strokes the colonies are less numerous than in the earlier, and sufficiently far apart to enable parts of them to be picked off without the needle touching any but one colony. When, as in the case of diphtheritic membrane, putrefactive organisms are likely to be present on the surface of the tissue, these can be in great part removed by washing it well in cold water previously sterilised {vide Diphtheria). In the case of liquids, the loop is charged and similarly stroked. Tubes thus inoculated must be put in the in- cubator in the upright position and must be handled care- fully so that the condensation water, which always is present in incubated agar tubes, may not run over the surface. Agar, poured out in a Petri's capsule and allowed to stand till firm, may be used instead of successive tubes. Here a sufficient number of strokes can be made in one capsule. Sloped blood-serum tubes may be used instead of agar. The method is rapid and easy, and gives good results. Separation of Pathogenic Bacteria by Inoculation of Animals. — It is found difficult and often impossible to separate by ordinary plate methods certain pathogenic organisms, such as B. tuberculosis, B. mallei, and the pneumococcus, when such occur in conjunction with other bacteria. These grow best on special media, and the first two (especially the tubercle bacillus) grow so slowly that the other organisms present outgrow them, cover the whole plates, and make separation impossible. The method adopted in such cases is to inoculate an animal with the mixture of bacilli, wait until the particular disease develops, kill the animal, and with all aseptic precautions {vide p 131) inoculate tubes of suitable media from characteristic lesions situated away from the seat of inoculation, e.g. from spleen ■n the case of B. tuberculosis, spleen or liver in the case of mallei> and heart blood in the case of pneumococcus 68 METHODS OF CULTIVATION OF BACTERIA. Separation by killing Non-spored Forms by Heat. — This is a method which has a limited application. As has been said, the spores of a bacterium resist heat more than the vegetative forms. When a mixture contains spores of one bacterium and vegetative forms of this and other bacteria, then if the mixture be boiled for a few minutes all the vege- tative forms will be killed, while the spores will remain alive and will develop subsequently. This method can be easily tested in the case of cultivating B. subtilis from hay infusion. A little chopped-up hay is placed in a flask of water, which is boiled for about ten minutes. On this being allowed to cool and stand, in a day or two a scum forms on the surface, which is found to be a pure culture of the bacillus subtilis. The method is also often used to aid in the separation of B. tetani, vide infra. The Principles of the Culture of Anaerobic Organisms. All ordinary media, after preparation, may contain traces of free oxygen, and will absorb more from the air on standing. (i) For the growth of anaerobes this oxygen may be expelled by the prolonged passing of an inert gas, such as hydrogen, through the medium (liquefied if necessary). Further, the medium must be kept in an atmosphere of the same gas, while growth is going on. (2) Media for anaerobes may be kept in contact with the air, if they contain a reducing agent which does not inter- fere with bacterial growth. Such an agent takes up any oxygen which may already be in the medium, and prevents further absorption. The reducing body used is generally glucose, though formate of sodium may be similarly em- ployed The preparation of such media has already been described (pp. 46-49). *n this case the medium ought to be of considerable thickness. The Supply of Hydrogen for Anaerobic Cultures.— I he gas is generated in a large Kipp's apparatus from pure SEPARATION OF ANsE ROBES. 69 sulphuric acid and pure zinc. It is passed through three wash-bottles as in Fig. 21. In the first is placed a solu- tion of lead acetate (1 in 10 of water) to remove any traces of sulphuretted hydrogen. In the second is placed a 1 in 10 solution of silver nitrate to remove any arsenietted hydrogen which may be present if the zinc is not quite pure. In the third is a 10 per cent solution of pyrogallic acid in caustic potash solution (1 : 10) to remove any traces of oxygen. The tube leading from the last bottle to the Fig. 21.— Apparatus for supplying hydrogen for anasrobic cultures. a. Kipp's apparatus for manufacture of hydrogen, b. Wash-bottle containing i-io solution oflead acetate, c. Wash-bottle containing i-io solution of silver nitrate, d. Wash-bottle containing i-io solution of pyrogallic acid, (b, c, and d are intentionally drawn to a larger scale than a to show details.) vessel containing the medium ought to be sterilised by passing through a Bunsen flame, and should have a small plug of cotton wool in it to filter the hydrogen germ-free. Separation of Anaerobic Organisms.— (a) In glucose gelatine. A x\ inch test-tube has as much gelatine put into it as would be used in the Esmarch roll-tube method. It is corked with an india-rubber stopper having two tubes passing through it, as in Fig. 22. The ends of the tubes are partly drawn out as shown, and covered with pluo-s of cotton wool. Three such test-tubes are prepared, and 7o METHODS OF CULTIVATION OF BACTERIA. they are sterilised in the steam steriliser (p. 38). After sterilisation the gelatine is melted and one tube inoculated with the mixture containing the anaerobes ; the second is inoculated from the first, and the third from the second, as in making ordinary gelatine plates. After inoculation the gelatine is kept liquid by the lower ends of the tubes being placed in water at about 30° C, and hydrogen is passed in through tube x for twenty minutes. The gas-supply tubes are then completely sealed off at x and i, and each test-tube is rolled as in Esmarch's method till the gelatine solidifies as a thin layer on the internal surface. A little hard paraffin may be run between the rim of the test-tube and the stop- per, and round the perforations for the gas-supply tubes, to ensure that the apparatus is air-tight. The gelatine is thus in an atmosphere of hydrogen in which the colonies may develop. The latter may be examined and isolated in a way which will be presently described. The method is ad- mirably suited for all anaerobes which grow at the ordinary temperature. (b) In glucose agar (Vignal's method). Three pieces of ordinary quill-tubing (bore about \ inch) about a foot long are taken, the ends are tapered in the gas flame, and they are then sterilised. Three ordinary test-tubes, a, b, of glucose agar are now boiled in a beaker, cooled to 42° C, and inoculated with the bacterial mixture as for plate cultures, i.e., b from a and c from b. A current of hydrogen being passed through the pieces of quill-tubing to expel the air, the contents of each of the test-tubes are now sucked up' with the mouth into one of the quill-tubes and both the ends of the latter sealed off in the flame. The agar will solidify in situ and the tubes may be incubated. The Fig. 22. — Esmarch's roll- tube adapted for culture con- taining anaerobes. CULTURES OF A NM ROBES. 7i- colonies will be observed as small grey specks. The tube in which these are farthest apart must be chosen, the glass notched with a file opposite to a colony to be examined, and the quill-tube broken. A tube of a suitable medium may now be inoculated with the growth. If there is any appearance of gas formation in the agar the tubes must be broken carefully, otherwise the contents may be ejected ; for this reason it is desirable to take for examination a tube containing very few colonies. It is even better to use four tubes and make the dilution more complete. It is often advisable in dealing with material suspected to contain anaerobes to inoculate an ordinary deep glucose agar tube with it, and incubating for 24 or 48 hours, to then apply an anaerobic separation method to the resultant growth. Sometimes the high powers of resistance of spores to heat may be taken advantage of in aiding the separation {vide Tetanus). Cultures of Anaerobes. — When by one or other of the above methods separate colonies have been obtained, growth may be maintained on media in contact with ordinary air. The media generally used are those which contain reducing agents, and the test-tubes containing the medium must be filled to a depth of 4 inches. They are sterilised as usual and are called "deep" tubes. The long straight platinum wire is used for inoculating from a colony on a glucose gelatine roll-tube or a glucose agar quill-tube, and it is plunged well down into the "deep" tube. A little air gets into the upper part of the needle track, and no growth takes place there, but in the lower part of the needle track growth occurs. The needle may be pre- vented from carrying down air by melting the upper half- inch of the medium. This is easily effected with gelatine, but in the case of agar care must of course be taken to cool the part down to 42° C. In the case of agar it is better to liquefy the whole tube and cool the lower part until it is solid by putting it in water. From such " deep " cultures growths may be maintained indefinitely by succes- sive sub-cultures in similar tubes. Even ordinary gelatine 72 METHODS OF CULTIVATION OF BACTERIA. and agar can be used in the same way if the medium is heated to boiling-point before use to expel any absorbed oxygen. Cultures of Anaerobes in Liquid Media. — It is necessary to employ such in order to obtain the toxic products of the growth of anaerobes. Glucose broth is most convenient. It is placed either (i) in a conical flask with a lateral open- ing and a perforated india-rubber stopper, through which a bent glass tube passes, as in Fig. 23, «, by which hydrogen may be delivered, or (2) in a conical flask with a rubber Fig. 23. a. Flask for anserobes in liquid media. Lateral nozzle and stopper fitted for hydrogen supply. b. A stopper arranged for a flask without lateral nozzle. stopper furnished with two holes, as in Fig. 23, b, through a tube in one of which hydrogen is delivered, while through the tube in the other the gas escapes. The inner end of the gas delivery tube must in either case be below the surface of the liquid ; the inner end of the lateral nozzle in the one case, and the inner end of the escape tube in the other, must of course be above the surface of the liquid. The single tube in the one case and the two tubes in the other ought to be partially drawn out in a flame to facilitate subsequent complete sealing. The ends of the tubes through which the gas is to pass are previously protected by pieces of cotton wool tied on them- It is well previously CULTURES OF ANsRROBES IN LIQUID MEDIA. 73 to place in the tube, through which the hydrogen is to be delivered, a little plug of cotton wool. The flask being thus prepared, it is sterilised by methods B (2) or B (3). On cooling it is ready for inoculation. In the case of the flask with the lateral nozzle, the cotton-wool covering having been momentarily removed, a wire charged with the organ- ism is passed down to the bouillon. In the other kind of flask the stopper must be removed for an instant to admit the wire. The flask is then connected with the hydrogen apparatus by means of a short piece of sterile india-rubber tubing, and hydrogen is passed through for half an hour. In the case of flask (1), the lateral nozzle is plugged with molten paraffin covered with alternate layers of cotton wool and paraffin, the whole being tightly bound on with string. The entrance tube is now completely drawn off in the flame before being disconnected from the hydrogen ap- paratus. In the case of flask (2), first the exit tube and then the en- trance tube are sealed off in the flame before the flask is disconnected from the hydrogen ap- paratus. It is well in the case of both flasks to run some melted paraffin all over the rubber stop- per. Sometimes much gas is evolved by anas- robes, and in dealing with an organism where this will occur, provision must be made for its escape. This is conveniently done by leading down the exit tube, and letting the end just dip into a trough of mercury (Fig. 24), or into mercury in a little bottle tied on to the end of the exit tube. The pressure of gas within causes an escape at the mercury contact, which at the same time acts as an efficient valve. a h Fig. 24. — Flask arranged for culture of anterobes which develop gas. b is a trough of mercury into which exit tube dips. 74 METHODS OF CULTIVATION OF BACTERIA. The method of culture in fluid media is used to obtain the soluble products of such anajrobes as the tetanus bacillus. When it is desired to grow anaerobes on the surface of a solid medium such as agar, tubes of the form shown in r — \ '■ J — i _e_-— — " :% Yig. 25. Tubes for anaerobic cultures on the surface of solid media. Fig. 25, a and b, may be used. A stroke culture having been made, the air is replaced by hydrogen as just described, and the tubes are fused at the constrictions. Such a method is of great value when it is required to get the bacteria free from admixture of medium, as in the case of staining flagella. Miscellaneous Methods. Hanging-drop Cultures.— It is often necessary to observe micro-organisms alive, either to watch the method and rate of their multiplication, or to investigate whether or not they are motile. This is effected by making hanging -drop cultures The method in the form to be described is only suitable for aerobes. For this special slides are necessary Two forms are in use and are shown in Fig. 26. In A there is ground out on one surface a hollow having a diameter of about half an inch. That shown in B explains itself The slide to be used and a cover-glass are sterilised bv hot air in a Petri's dish, or simply by being heated in a Bunsen and laid in a sterile Petri to cool. In the case of A one or other of two manipulation methods may be employed. (1) If the organism be growing in a liquid HANGING-DROP PREPARATIONS. 75 culture, a loop of the liquid is placed on the middle of the under surface of the sterile cover-glass, which is held in forceps, the points of which have been sterilised in a Bunsen flame. If the organism be growing in a solid medium, an eyelet of sterile bouillon is placed on the cover-glass in the same position, and a very small quantity of the culture (picked up with a platinum needle) is rubbed up in the bouillon. The cover is then carefully lowered over the cell on the slide, the drop not being allowed to touch the wall or the edge of the cell. The edge of the cover-glass is A Fig. 26. A. Hollow-ground slide for hanging-drop cultures shown in plan and section. B. Another form of slide for similar cultures. covered with vaseline, and the preparation is then complete and may be placed under the microscope. If necessary, it may be first incubated and then examined on a warm stage. (2) The sterile cover-glass is placed on a sterile plate (an ordinary glass plate used for plate cultures is convenient). The drop is then placed on its upper surface, the details being the same as in the last case. The edge of the cell in the slide is then painted with vaseline, and the slide, held with the hollow surface downwards, is lowered on to the cover-glass, to the rim of which it of 76 METHODS OF CULTIVATION OF BACTERIA. course adheres. The slide with the cover attached is then turned right side up, and the preparation is complete. In the case of B the drop of fluid is placed on the centre of the table x. The drop must be thick enough to come in contact with the cover-glass when the latter is lowered on the slide, and not large enough to run over into the surrounding trench y. The cover-glass is then lowered on to the drop, and vaseline is painted along the margin of the cover-glass. The method of microscopic examination is described on page 94. Ancerobic Hanging- drop Cultures. — The growth and examination of bacteria in hanging-drops under anaerobic conditions involve considerable difficulty, but may be carried out in an apparatus devised by Graham Brown (Fig. 27). It consists of two brass plates (a and a) which can be approximated by screws, and which have two rounded apertures in their middle f in. in diameter. These support two rubber rings, an upper thinner one (b) and a lower thick one (d), their inner diameter being the same as that of the apertures in the plates. Between b and d is placed a stout cover-glass of suitable size (c) ; d is separated from the plate a by a square plate of glass (e) (a portion of an ordinary glass-slide for microscopical purposes does well). Two small metal tubes (J) are inserted through the rubber d. THE COUNTING OF COLONIES. 77 Method of use : — Fix up the apparatus as shown above, the screws being just tight enough to keep the parts in position, and sterilise in the steam steriliser. Screw up more firmly so as to make the rubber bulge slightly. Fill a hypodermic syringe with some sterile glucose bouillon, push the needle through the rubber d and, tilting the point of the needle against the glass c, slowly inject enough to form a drop on the under surface of c. Withdraw the syringe and inoculate its point with the bacterium, again introduce and inoculate the drop. Pass hydrogen through one of the tubes for fifteen minutes, close the ends of the tubes, and incubate at the required temperature. The apparatus can be put on the stage of a microscope and examined from time to time. The Counting of Colonies. — An approximate estimate of the number of bacteria present in a given amount of a fluid (say, water) can be arrived at by counting the number of colonies -which develop when that amount is added to a tube of suitable medium, and the latter plated and incubated. An or- dinary plate should fig. 28. -Apparatus for counting colonies, be used in such a case, and the medium poured out in as rectangular a shape as possible. For the counting, an apparatus such as is shown in Fig. 28 is employed. This consists of a sheet of glass ruled into squares as indicated, and sup- ported by its corners on wooden blocks. The table to which these blocks are attached has a dark surface. The plate-culture containing the colonies is laid on the top of the ruled glass. The numbers of colonies in, say, twenty of the smaller squares are then counted, and an average struck. The total number of squares covered by the 78 ME THODS OF CUL TI VA T10N OF BA C TERIA. medium is then taken, and by a simple calculation the total number of colonies present can be obtained. Plate- cultures in Petri's dishes are sometimes employed for pur- poses of counting. The bottoms of such dishes are, how- ever, never flat, and the thickness of the medium thus varies in different parts. If these dishes are to be used, a circle of the same size as the dish can be drawn with Chinese white on a black card, the circumference divided into equal arcs, and radii drawn. The dish is then laid on the card, the number of colonies in a few of the sectors counted, and an average struck as before. In counting colonies it is always best to aid the eye with a small hand lens. The Bacteriological Examination of the Blood. — This is usually done by taking a small drop from the skin surface, e.g., the lobe of the ear. The part should be thoroughly washed with i-iooo corrosive sublimate and dried with sterile cotton wool. It is then washed with absolute alcohol to remove the antiseptic, drying being allowed to take place by evaporation. A prick is then made with a sterile surgical needle, the drop of blood is caught with a sterile platinum loop and smeared on the surface of agar or blood serum. It is rare to obtain growths from the blood of the human subject {vide special chapters), and if colonies appear the procedure should be repeated to exclude the possibility of accidental contamination. When larger quantities of blood are obtainable, e.g., by venesection, suitable animals may be inoculated with several cubic centimetres. In this way infection may be produced from even human blood, e.g., when the subject is suffering from the effects of the pathogenic streptococci. In examining the blood of the spleen a portion of the skin over the organ is sterilised in the same way, a few drops are withdrawn from the organ by a sterile hypodermic syringe and cultures made. (For microscopic methods, vide p. 96.) . y 1 The Bacteriological Examination of Urine.— In sucn an examination care must be taken to prevent the con- BACTERIOLOGICAL EXAMINATION OF WATER. 79 tamination of the urine by extraneous organisms. In the male it is usually sufficient to wash thoroughly the glans penis and the meatus with i-iooo corrosive sublimate — the tips of the meatus being everted for more thorough cleansing. The urine is then passed into a series of sterile flasks, the first of which is rejected in case contamination has occurred. In the female, after similar precautions as regards external cleansing, the catheter must be used. The latter must be boiled for half an hour, and anointed with olive oil sterilised by half an hour's exposure in a plugged flask to a temperature of 1200 C. Here again, it is well to reject the urine first passed. It is often advisable to allow the urine to stand in a cool place for some hours, to then withdraw the lower portion with a' sterile pipette, to centrifugalise this, and to use the urine in the lower parts of the centrifuge tubes for microscopic examination or culture. The Bacteriological Examination of Water. — This may be undertaken with a view to finding either the number of bacteria present or the varieties present. In either case a small quantity (| to 1 c.c.) is taken in a sterile pipette and added to a tube of gelatine, which is then plated and incubated at the room temperature. In the case of water taken from a house tap, it should be allowed to run for several hours before the sample is taken, as water standing in pipes in a house is under very favourable conditions for multiplication of bacteria taking place, and if this precau- tion be not adopted, an altogether erroneous idea of the number present may be obtained. In the case of the examination of river water, the gelatine plates ought to be prepared on the spot ; at any rate the time elapsing between the sample being taken and the plates being pre- pared must be as short as possible, otherwise the bacteria will multiply, and again an erroneous idea of their number be obtained. When samples have to be taken for transport to the laboratory, these are best collected in four- ounce, wide -mouthed, stoppered bottles, which are to be sterilised by dry heat (the stopper must be sterilised separately from 8o ME THODS OF CULT1 VA 71 ON OF BA C 7 'E7\7A . the bottle and not inserted in the latter till both are cold, otherwise it will be so tightly held as to make removal very difficult). In using such a bottle it is best to immerse it in the water, and then remove the stopper with forceps. Care must be taken not to touch the water-bed as the vegetable matter covering it contains a large number of organisms. Plates must be prepared from the samples as ' soon as possible. Filtration of Cultures. — For many purposes it is neces- sary to filter all the organisms from fluids in which they may have been growing. This is especially done in obtain- ing the soluble toxic products of bacteria. The only filter capable of keeping back such minute bodies as bacteria, is that formed from a tube of unglazed porcelain as intro- duced by Chamberland. There are several filters, differing slightly in detail, all possessing this common principle. Sometimes the fluid is forced through the porcelain tube. In one form the filter consists practically of an ordinary tap screwed into the top of a porcelain tube. Through the latter the fluid is forced and passes into a chamber formed by a metal cylinder which surrounds the porcelain tube. The fluid escapes by an aperture at the bottom. Such a filter is very suitable for domestic use, or for use in surgical operating-theatres. As considerable pressure is necessary, it is evident it must be put on a pipe leading directly from the main. Sometimes, when fluids to be filtered are very albuminous, they are forced through a porcelain cylinder by compressed carbonic acid gas. In ordinary bacteriological work, however, it is usually more convenient to suck the fluid through the porcelain by exhausting the air in the receptacle into which it is to flow. This is conveniently done by means of a Geissler's water -exhaust pump (Fig. 29, g), which must be fixed to a tap leading directly from the main. The connection with the tap must be effected by means of a piece of thick-walled rubber-tubing as short as possible, wired on to tap and pump, and firmly lashed externally with many turns of strong tape. Before lashing with the tape the tube may be strengthened by fixing round THE FILTRATION OF CULTURES. 8 1 it with rubber solution strips of the rubbered canvas used for mending punctures in the outer case of a bicycle tyre. A manometer tube (b) and a receptacle (c) (the latter to catch any back flow of water from the pump if the filter accidentally breaks) are intercepted be- tween the filter and the pump. These are usually arranged on a board a, as in Fig. 29. Between the tube / and the pump g, and be- tween the tube d and the filter, it is convenient to insert Fig. 29. — Geissler's vacuum pump arranged lengths of flexible w'tn manometer for filtering cultures. (The tap lead - tubing con and pump are intentionally drawn to a larger o " scale than the manometer board to show details. ) nected up at each- end with short, stout-walled rubber-tubing. Various modifications of the filter are used, (a) An ap- paratus is arranged as in Fig. 30. The fluid to be filtered is placed in the cylindrical ves- sel a. Into this a " candle " or "bougie" of porcelain dips. From the upper end of the bougie a glass tube with thick Fig. 30. — Chamberiand's rubber connections, as in Fig candle and flask arranged for j„ . a , , °, filtration. 3°> pioceeds to flask b and passes through one of the two perforations with which the rubber stopper of the flask is furnished. Through the other opening a similar tube 6 82 METHODS OF CULTIVATION OF BACTERIA. proceeds to the exhaust -pump. When the latter is put into action the fluid is sucked through the porcelain and passes over into flask b. This apparatus is very good, but not suitable for small quantities of fluid. {!>) A very good apparatus can be arranged with a lamp funnel and the porcelain bougie. These may be fitted up in two ways, (i) An india-rubber washer is placed round the bougie c at its glazed end {vide Fig. 31). On this the narrow end of the funnel d, which must, of course, be of Fig. 31. — Chamberland's bougie nrranged with lamp funnel for filtering a small quantity of fluid. Fig. 32. — Bougie inserted through rubber stopper for same purpose as in Fig. 31. an appropriate size, rests. A broad band of sheet rubber is then wrapped round the lower end of the funnel, and the projecting part of the bougie. It is firmly wired to the funnel above and to the bougie below. The extreme point of the latter is left exposed, and the whole apparatus, being supported on a stand, is connected by a glass tube with the lateral tube of the flask b; the tube a is connected with the exhaust-pump. The fluid to be filtered is placed between the funnel and the bougie in the space e, and is sucked through into the flask b. (2) This modification is shown muencke's filter. 83 of the funnel an india- in Fig. 32. Into the narrow part rubber stopper is fitted, which has a perforation in it sufficiently large to receive the candle, which it should grasp tightly. (c) Muencke's modi- fication of the Cham- berland principle is seen in Fig. 33. It consists of a thick -walled flask, a, the lower part conical, the upper cylindrical, Fig. 33. — Muencke's modification of Chamberland's filter. with a strong flange on the lip. There are two lateral tubes, one horizontal to con- nect with exhaust-pipe, and one sloping, by which the contents may be poured out. Passing into the upper cylindrical part of the flask is a hollow porcelain cylinder b, of less diameter than the cylindrical part of flask a. It is closed below, open above, and rests by a pro- jecting rim on the flange of the flask, an asbestos washer, c, being interposed. The fluid to be filtered is placed in the porce- lain cylinder, and the whole top covered, as shown at f, with an india-rubber cap with a central perforation ; the tube d is connected with the exhaust-pump and the Fig. 34. — Flask tube £ plugged with a rubber stopper. porcelain When a large quantity of fluid is to be l^geiequantitiesni(rf filtered a receptacle such as that shown fluid, in Fig. 34 may be used. The tap in its 84 ME THODS OF C UL TI VA 7' JON OF BA CTERIA . bottom enables the nitrate to be removed without the apparatus being unshipped. Before any one of the above apparatus is used, it ought to be connected up as far as possible and sterilised in the Koch's steriliser. The ends of any important unconnected parts ought to have pieces of cotton-wool tied over them. After use the bougie is to be sterilised in the autoclave, and after being dried is to be passed carefully through a Bunsen flame, to burn off all organic matter. If the latter is allowed to accumulate the pores become filled up. The success of filtration must be tested by inoculating tubes of media from the filtrate, and observing if growth takes place, as there may be minute perforations in the candles sufficiently large to allow bacteria to pass through. Filtered fluids keep for a long time if the openings of the glass vessels in which they are placed are kept thoroughly closed, and if these vessels be kept in a cool place in the dark. Sometimes the fluids may be evaporated to dryness in vacuo over sulphuric acid, and kept in an air-tight bottle in a dry state. Instead of being filtered off, the bacteria may be killed by various antiseptics, chiefly volatile oils, such as oil of mustard (Roux). These oils are stated to have no injurious effect on the chemical substances in the fluid, and they may be subsequently removed by evaporation. It is not prac- ticable to kill the bacteria by heat when their soluble pro- ducts are to be studied, as many of the latter are destroyed by a lower temperature than is required to kill the bacteria themselves. The Observation of Bacterial Fermentations in Sugars. The capacity of certain species of bacteria to originate fermentations in sugars constitutes an important biological factor. The end products of such fermentations may be various. They differ according to the sugar employed and according to the species under observation, and frequently a species which will ferment one sugar has no effect on another. The substances finally produced, speaking roughly, may be alcohols, acids, or gaseous bodies (chiefly BACTERIAL FERMENTATIONS IN SUGARS. 85 carbon dioxide, hydrogen, and methane). For the estima- tion of the two former groups complicated chemical pro- cedure may be necessary. The formation of gases is, however, usually taken as the criterion of the possession of fermentative properties. Generally speaking, it is reliable, and the methods to be pursued are simple. It must not be forgotten, however, that some organisms give rise to FlG- 35- — Tubes for demonstrating gas-formation by bacteria. «, tube with "shake" culture. Durham's fermentation tube. c, ordinary form of fermentation tube. sulphuretted hydrogen by breaking up the proteid present. The formation of this gas can be detected by the blackening of lead acetate when it is added to the gas-containing medium. The following are the chief methods for detecting the formation of gas : — (1) Gelatine Shake Cultures (Fig. 35 a). — The gelatine in the tube is melted as for making plates ; while liquid it is inoculated with the growth to be observed, and shaken to distribute the organisms throughout the jelly. It is then allowed to solidify, and is set aside at a suitable tempera- S6 METHODS OF CULTIVATION OF BACTERIA. ture. If the bacterium used is a gas-forming one, then, as growth occurs, little bubbles appear round the colonies. These frequently coalesce to form bubbles of a larger size, and those which are superficial in process of time diffuse out of the medium. This method is very frequently used for studying gas formation by B. coli. (2) Durham's Tubes (Fig. 35 b). — The plug of a tube which contains about one-third more than usual of a liquid medium is taken, and a small test-tube is slipped into it mouth downwards. The plug is replaced and the tube sterilised thrice at ioo° C. The air remaining in the smaller tube is thereby expelled. The tube is then inocu- lated with the bacterium to be tested. Any gas developed collects in the upper part of the inner tube. (3) The Fermentation Tube (Fig. 35 c). — This consists of a tube of the form shown, and the figure also indicates the extent to which it ought to be filled. It is inoculated in the bend with the gas -forming organism, and when growth occurs the gas collects in the upper part of the closed limit, the medium being displaced into the bulb. The composition of the medium is, of course, of great importance, and in testing the effect of a bacterium on a given sugar it is essential that this sugar alone be present. The first method is usually used with ordinary gelatine, and the gas-formation in most cases results from fermentation of the glucose naturally present in the medium from trans- formation of the glycogen of muscle. (It is only a more delicate method of demonstrating what sometimes occurs along the line of growth in an ordinary gelatine stab-culture.) The amount of glucose naturally present, however, vanes much, and therefore glucose should be added to the medium if the effects on this sugar are to be observed. When other sugars — lactose, mannite, etc. — are to be tested, these should be added either to a simple peptone solution as Durham recommends, or to bouillon previously freed from dextrose as described below — fermentation being observed by either of the methods (2 ) or (3). OBSER VA TION OF IND OL- FORMA 7 ION. 87 To obtain a "dextrose-free " bouillon it is usual to inoculate ordinary bouillon with some organism, such as B. coli, which is known to ferment dextrose, and allow the latter to act for 48 hours. The bouillon is then filtered and resterilised. A sample is tested for another period of 48 hours with B. coli, to make certain that all the dextrose has been removed. If no fresh gas formation is observed, then to the remainder of the bouillon the sugar to be investigated may be added. It is preferable that the addition should be made in the form of a sterile solution. If the raw crystals be placed in the bouillon and this then sterilised, there is the danger that chemical changes may take place in the sugar, in consequence of its being heated in the presence of sub- stances (such as the alkali) which may act deleteriously upon it. The Observation of Indol-formation by Bacteria. — The formation of indol from albumin by a bacterium sometimes constitutes an important specific characteristic. To observe indol production the bacterium is grown preferably at incubation temperature on a fluid medium containing peptone. The latter may either be ordinary bouillon or peptone solution (see p. 50). Indol production is recog- nised by the fact that when acted on by nitric acid in the presence of nitrites > a nitroso-indol compound is produced, which has a rosy red colour. Some bacteria {e.g. the cholera vibrio) produce nitrites as well as indol, but often, in making the test (e.g. in the case of B. coli), the nitrites must be added. This may be effected by using yellow nitric acid (which of course contains nitrous acid) for the test, or by adding to an ordinary tube of medium 1 c.c. of a .02 per cent solution of potassium nitrite, and testing with pure nitric or sulphuric acid. In any case only a drop of the acid need be added to say 10 c.c. of medium. If no result be obtained at once it is well to allow the tube to stand for an hour, as sometimes the reaction is very slowly produced. The amount of indol produced by a bacterium seems to vary very much with certain unknown qualities of the peptone. It is well therefore to test a series of peptone with an organism (such as the B. coli) known to produce indol, and noting the sample with which the best reaction is obtained, to reserve it for making media to be used for the detection of this product. 88 METHODS OF CULTIVATION OF BACTERIA. The Storing and Incubation of Cultures. — Gelatine cultures must be grown at a temperature below their melting-point, i.e. for 10 per cent gelatine, below 22" (J. They are usually kept in ordinary rooms, which vary, of course, in temperature at different times, but which have usually a range of from about 120 C. to 180 C. Agar and serum media are usually employed to grow bacteria at a higher temperature, corresponding to that at which the organisms grow best, usually 370 C. in the case of pathogenic organisms. For the purpose of maintaining a uniform temperature incubators are used. These vary much in the details of their structure, but all consist of a chamber with double walls between which some fluid (water or glycerine and water) is placed, which, when raised to a certain tem- perature, ensures a fairly constant distribution of the heat round the chamber. The latter is also furnished with double doors, the inner being usually of glass. Heat is supplied from a burner fixed below. These burners vary much in design. Sometimes a mechanism devised in Koch's laboratory is affixed, which automatically turns off the gas if the light be accidentally extinguished. Between the tap supplying the gas, and the burner, is interposed a gas regulator. Such regulators vary enormously in design, but for ordinary chambers which require to be kept at a constant temperature, Reichert's is as good and simple as any and is not expensive. It is shown in Fig. 36. It consists of a long tube f closed at the lower end, open at the upper, and furnished with two lateral tubes. The lower part is filled with mercury up to a point above the level of the lower lateral tube. The end of the latter is closed by a brass cap through which a screw d passes, the inner end of which lies free in the mercury. The height of the latter in the perpendicular tube can thus be varied by increasing or decreasing the capacity of the lateral tube by turning the screw a few turns out of or into it. Into the upper open end of the perpendi- cular tube fits accurately a bent tube, g, drawn out below to a com- paratively small open point, c, and having in its side a little above the point a minute needle-hole called the peephole or bye-pass e. To fix the apparatus the long mercury bulb is placed in the jacket of the chamber to be controlled, tube a is connected to gas supply, tube b with the burner. The upper level of the mercury should be some STORING AND INCUBATION OF CULTURES. 89 distance below the lower open end of tube c. The burner is now lit. The gas passes in at a through c and e and out at b to the burner. W hen the thermometer in the interior of the chamber indicates that the desired temperature S~ has been reached, the screw d is turned till the mercury reaches the end of the tube c. Gas can only now pass through the peephole e, and the flame goes down. The contents of the jacket cool, the mercury contracts off the end of tube c, and the flame rises. This alterna- tion going on, the temperature of the chamber is kept very nearly constant. If the mercury cuts off the gas supply before the desired temperature is reached, and the screw d is as far out as it will go, then some of the mercury must be removed. Similarly, if when the desired temperature is reached and the screw d is as far in as it can go, the mercury does not reach c, some more must be introduced. If the amount of gas which passes through the peephole is sufficient still to raise the temperature of the chamber when c is closed by the rise of the mercury, then the peephole is too large. Tube c must be unshipped and e plastered over with sealing-wax, which is pricked, while still soft, with a very fine needle. The gas flame, when only the peephole is supplying gas, ought to be sufficiently large not to be blown out by small currents of air. If the pressure of gas supplied to a regulator varies much in the 24 hours a pressure regulator ought to be interposed between the gas tap and the instrument. Several varieties of these can be obtained. In all cases g ought to be fixed to b with a turn of wire. Fig. 36. — Reichert's gas regulator. The varieties of incubators are, as we have said, numerous. The most complicated and expensive are made by German manufacturers. Many of these are unsatisfactory. They easily get out of order and are difficult to repair. We have found those of Hearson of London extremely good, and in proportion to their size much cheaper than the German articles. They are fitted with an admirable regulator. It is preferable in using an incubator to connect the regulator with the gas supply and with the Bunsen by flexible metal tubing. It is necessary go METHODS OF CULTIVATION OF BACTERIA. to see that there is not too much evaporation from the surface of cultures placed within incubators, otherwise they may quickly dry up. It is thus advisable to raise the amount of water vapour in the interior by having in the bottom of the incubator a flat dish full of water from which evaporation may take place. Tubes which will require to be long in the incubator should have their plugs covered either by india-rubber caps or by pieces of sheet rubber tied over them. These caps should be previously sterilised in pIG- j7. — Hearson's incubator for use at 37° C. 1-1000 corrosive sublimate and then dried. Before they are placed on the tubes the cotton-wool plug ought to be well singed in a flame. " Cool " incubators are often used for incubating gelatine at 210 to 220 C. Here again Hearson's design is as good as any in the market. Permanent Preservation of Cultures. — This may be conveniently effected by means of formalin vapour. The cotton-wool of the tube containing the culture to be preserved is removed and soaked in formalin (40 per cent formol aldehyde). It is then replaced and covered with an GENERAL LABORATORY RULES. 9i india-rubber cap. After exposure to the vapour in this way for two or three days the culture will be found to be dead. The final stage in the process is to close the open end of the tube so as to prevent evaporation.. Melted sealing-wax or other substance may be poured over the cotton-wool, which is first burned off down to the tube, the whole being then covered by an india-rubber cap, or the upper end of the tube may be melted in a Bunsen flame and thus sealed. In the latter case tubes longer than those generally em- ployed should be used, so as to leave a longer portion at the top beyond the medium, otherwise in sloped tubes part of the medium is apt to be melted. Cultures preserved in this way maintain their form practically unchanged for several years, though many coloured growths are apt to lose the colour. Liquefied gelatine usually becomes solidified by the action of formalin vapour, so that the tubes can be freely handled. In the case of agar tubes any water of condensation should first of all be carefully poured off. General Laboratory Rules. — On the working bench of every bacteriologist there should be a large dish of 1-1000 solution of mercuric chloride in water. Into this all tubes, vessels, plates, hanging-drop cultures, etc., which have con- tained bacteria and with which he has finished, ought to be at once plunged (in the case of tubes the tube and plug should be put in separately). On no account whatever are such infected articles to be left lying about the laboratory. The basin is to be repeatedly cleaned out. All the glass is carefully washed in repeated changes of tap water to remove the last trace of perchloride of mercury, a very minute quantity of which is sufficient to inhibit growth. Old cultures which have been stored for a time and from which fresh sub-cultures have been made ought to be steamed in the Koch's steriliser for two or three hours, or in the autoclave for a shorter period and the tubes thoroughly washed out. Besides a basin of mercuric chloride solution for infected apparatus, etc., there ought to be a second reserved for the worker's hands in case of any accidental contamination. In making examinations 92 METHODS OF CULTIVATION OF BACTERIA. of organs containing virulent bacteria, the hands should be previously dipped in i-iooo mercuric chloride and allowed to remain wet with this solution. No food ought to be partaken of in the laboratory, and pipes, etc., are not to be laid with their mouth-pieces on the bench. No label is to be licked with the tongue. Before leaving the laboratory the bacteriologist ought to wash the hands and forearms with i-iooo mercuric chloride and then with yellow soap. In the case of any fluid containing bacteria being accidentally spilt on the bench or floor, i-iooo mercuric chloride is to be at once poured on the spot. The air of the laboratory ought to be kept as quiet as possible. CHAPTER III. MICROSCOPIC METHODS — GENERAL BAC- TERIOLOGICAL DIAGNOSIS— INOCULATION OF ANIMALS. The Microscope. — For ordinary bacteriological work a good microscope is essential. It ought to have a heavy stand, with rack and pinion and fine adjustment, a double mirror (fiat on one side, concave on the other), a good condenser, with an iris diaphragm, and a triple nose-piece. It is best to have three objectives, either Zeiss A, D, and inch oil immersion, or the lenses of other makers corre- sponding to these.- The oil immersion lens is essential. It is well to have two eye-pieces, say Nos. 2 and 4 of Zeiss or lenses of corresponding strengths. The student must be thoroughly familiar with the focussing of the light on the lens by means of the condenser, and also with the use of the immersion lens. It may here be remarked that when it is desired to bring out in sharp relief the margins of unstained objects, e.g., living bacteria in a fluid, a narrow aperture of the diaphragm should be used, whereas, in the case of stained bacteria, when a pure colour picture is desired, the diaphragm ought to be widely opened. The flat side of the mirror ought to be used along with the condenser. When the observer has finished for the time being with his immersion lens he ought to wipe off the oil with a piece of silk or very fine washed linen. If the oil has dried on the lens it may be moistened with xylol— never with alcohol, 94 MICROSCOPIC METHODS. which will dissolve the material by which the lens is fixed in its metal carrier. Microscopic Examination of Bacteria, i. Hanging- drop Preparations. — Micro-organisms may be examined : (i) alive or dead in fluids ; (2) in film preparations ; (3) in sections of tissues. In the two last cases advantage is always taken of the affinity of bacteria for certain stains. When they are to be examined in fluids a drop of the liquid may be placed on a slide and covered with a cover- glass.1 It is more usual, however, to employ hanging-drop preparations. The technique of making these has already been described (p. 74). In examining them microscopic- ally, it is necessary to use a very small diaphragm. It is best to focus the edge of the' drop with a low-power objective, and, arranging the slide so that part of the edge crosses the centre of the field, to clamp the preparation in this position. A high-power lens is then turned into position and lowered by the coarse adjustment to a short distance above its focal distance; it is now carefully screwed down by the fine adjustment, the eye being kept at the tube meanwhile. The shadow of the edge will be first recognised, and then the bacteria must be carefully looked for. Often a dry lens is sufficient, but for some purposes the oil immersion is required. If the bacteria are small and motile a beginner may have great difficulty in seeing them, and it is well to practise at first on some large non-motile form such as anthrax. In fluid preparations the natural appearance of bacteria may be studied, and their rate of growth determined. The great use of such preparations, however, is to find whether or not the bacteria are motile, and for determining this point it is advisable to use either broth or agar cultures not more than twenty-four hours old. In the latter case a small fragment of growth is broken down in broth or in sterile water. Sometimes it is an advantage to colour the solution in which the 1 In bacteriological work it is essential that cover -glasses of No j thickness {i.e. , . 14 mm. thick) should be used, as those of greater th.ckness, are not suitable for a in. lens. FIL M PR EPA PA TIONS. 95 hanging-drop is made up with a minute quantity of any aniline dye, say a small crystal of gentian violet to 100 c.c. of bouillon. Such a degree of dilution will not have any effect on the vitality of the bacteria. Ordinarily, living bacteria will not take up a stain, but even though they do not, the contrast between the unstained bacteria and the tinted fluid will enable the observer more easily to recognise the former. 2. Film Preparations, (a) Dry Method. — This is the .most extensively applicable method of microscopically examining bacteria. Fluids containing bacteria, such as blood, pus, scrapings of organs, can be thus investigated, as also cultures in fluid and solid media. The first requisite is a perfectly clean cover-glass. Many methods are recommended for ob- taining such. The test of this being accomplished is that, when the drop of fluid containing the bac- glass, it can be uniformly spread with the platinum needle all over the surface without showing any tendency to retract into drop- lets. The best method is that recommended by Van Ermengem. The cover-glasses are placed for some time in a mixture of concentrated sulphuric acid 6 parts, potassium bichromate 6 parts, water ioo parts, then washed thoroughly in water and stored in absolute alcohol. For use, a cover-glass is either dried by wiping with a clean duster or is simply allowed to dry. This method will amply repay the trouble, and really saves time in the end. A clean cover having been obtained, the film preparation can now be made. If a fluid is to be examined a loopful may be placed on the cover-glass, and either spread out over the surface with the needle, or another clean cover may be placed on the top of the first, the drop thus spread out between them and the two then drawn apart. When a culture on a solid medium is to be examined a loopful of teria is placed upon the Fig. 38. — Cornet's forceps for holding cover-glasses. 96 MICROSCOPIC METHODS. distilled water is placed on the cover-glass and a minute particle of growth rubbed up in it and spread all over the glass. The great mistake made by beginners is to take too much of the growth. The point of the straight needle should just touch the surface of the culture, and when this is rubbed up in the droplet of water and the film dried, there should be an opaque cloud just visible on the cover- glass. When the film has been spread it must next be dried by being waved backwards and forwards at arm's- length above a Bunsen flame. The film must then be fixed on the glass by being passed three or four times slowly through the flame. In doing this a good plan is to hold the cover-glass between the right forefinger and thumb ; if the fingers just escape being burned no harm will accrue to the bacteria in the film. In making films of a thick fluid such as pus it is best to spread it out on one cover with the needle. The result will be a film of irregular thickness, but sufficiently thin at many parts for proper examination. Scrapings of organs are very convenient if only the presence or absence of organisms is inquired after. Such scrapings may be smeared directly on the cover-glasses with or without the addition of sterile distilled water. In the case of blood, a fairly large drop should be allowed to spread itself between two cover-glasses, which are then to be slipped apart, and being held between the forefinger and thumb are to be dried by a rapid to-and-fro movement in the air. A film prepared in this way may be too thick at one edge, but at the other is beautifully thin. If it is desired to preserve the red blood corpuscles in such a film it must be fixed by one of the following methods : by being placed (a) in a hot-air chamber at 1200 C. for half an hour, (6) in a mixture of equal parts of alcohol and ether for half an hour, then washed and dried, or (c) in a saturated solution of corrosive sublimate for two or three minutes, then washed for half an hour in running water and dried. (Fig. 61 shows a film prepared by the latter method.) In the case of urine, the specimen must be FIL M PRE PAR A TIONS. 97 allowed to stand, and films made from any deposit which occurs ; or, what is still better, the urine is centrifugalised, and films made from the deposit which forms. After dried films are thus made from urine it is an advantage to place a drop of distilled water on the film and heat gently to dissolve the deposit of salts ; then wash in water and dry. In this way a much clearer picture is obtained when the preparation is stained. Films dried and fixed by the above methods are now ready to be stained by the methods to be described below. (b) Wet Method.— -If it is desired to examine the fine histological structure of the cells of a discharge as well as to investigate the bacteria present, it is advisable to substitute " corrosive " films for the " dried " films, the preparation of which has been described. The initial stages in the pre- paration of corrosive films are the same as for other films, but instead of being dried in air they are placed, while still wet, film downwards on a saturated solution of perchloride of mercury in .75 per cent sodium chloride, in which they are allowed to remain for five minutes. They are then placed for half an hour, with occasional gentle shaking, in .75 per cent sodium chloride solution to wash out the corrosive sublimate. They are then placed in successive strengths of methylated spirit, being allowed to remain a few minutes in each. After this treatment they are stained and treated as if they were sections. The nuclear structure, mitotic figures, etc., are by this method well preserved' whereas these are considerably distorted in dried films. Another excellent method of fixing film preparations is that devised by Gulland. The fixing solution has the composition — absolute alcohol, 25 c.c, pure ether, 25 c.c, alcoholic solution of corrosive sublimate (2 grm. in 10 c.c' of alcohol), about 5 drops. The films are placed, while still wet, in this solution for five minutes or longer. They are then washed well in water, and are ready for staining. A contrast stain can be applied at the same time as the fixing solution, by saturating the 25 c.c. of alcohol with 7 98 MICROSCOPIC METHODS. eosin before mixing. Thereafter the bacteria, etc., may be stained with methylene -blue or other stain, as described below. This method has the advantage over the previous that, as a small amount of corrosive sublimate is used, less washing is necessary to remove it from the preparation, and deposits are less liable to occur. 3. Examination of Bacteria in Tissues. — For the examin- ation of bacteria in the tissues, the latter must be fixed and hardened, in preparation for being cut with a microtome. Fixation consists in so treating a tissue that it shall per- manently maintain, as far as possible, the condition it was in when removed from the body. Hardening consists in giving such a fixed tissue sufficient consistence to enable a thin section of it to be cut. A tissue, after being hardened, may be cut in a freezing microtome, but far finer results can be obtained by embedding the tissue in solid paraffin and cutting with some of the more delicate microtomes of which, for pathological purposes, the small Cambridge rocker is by far the best. For bacteriological purposes embedding in celloidin is not advisable, as the celloidin takes on the aniline dyes which are used for staining bacteria, and is apt thus to spoil the preparation, and besides thinner' sections can be obtained by the paraffin method. The Fixation and Hardening of Tissues. — Absolute alcohol may be used for the double purpose of fixing and hardening. If the piece of tissue is not more than £ inch in thickness it is sufficient to keep it in this reagent for one or two days. If the pieces are thicker a longer exposure is necessary, and in such cases it is better to change the alcohol at the end of the first twenty-four hours, as the first alcohol is diluted by water which comes out of the fluids of the tissue. The tissue must be tough without being hard, and the necessary consistence, as estimated by feeling with the fingers, can only be judged of after some experience. If the tissues are not to be cut at once, they may be pre- served in 50 per cent spirit. Corrosive sublimate is an excellent fixative agent. It is best used as a saturated solution in .75 per cent sodium THE CUTTING OF SECTIONS. 99 chloride solution. Dissolve the sublimate in the salt solution by heat. The separation of crystals on cooling shows that the solution is saturated. For small pieces of tissue \ inch in thickness, twelve hours' immersion is sufficient. If the pieces are larger, twenty-four hours is necessary. It is very important for the success of the subsequent procedures that the corrosive sublimate should be now thoroughly washed out of the tissues. They should be tied up in a piece of gauze, and this placed in a stream of running water for from twelve to twenty -four hours, according to the size of the pieces. They are then placed for twenty-four hours in each of the following strengths of methylated spirit (free from naphtha1): 30 per cent, 60 per cent, and 90 per cent. Finally they are placed in absolute alcohol for twenty-four hours and are then ready to be prepared for cutting. If the tissue is very small, as in the case of minute pieces removed for diagnosis, the stages may be all compressed into twenty-four hours. In fact after fixation in corrosive the tissue may be transferred directly to absolute alcohol, the perchloride of mercury being removed after the sections are cut, as will be after- wards described. Methylated Spirit. — Small pieces of tissue may be placed in methylated spirit, which is to be changed after the first day. In six to seven days they will be hardened. If the pieces are large, a longer time is necessary. The Cutting of Sections.— 1. By Means of the Freezing Microtome.— Pieces of tissue hardened by any of the above methods must have all the alcohol removed from them by washing in running water for twenty-four hours. They are then placed for from twelve to twenty-four hours (according to their size) in a thick syrupy solution containing two parts of gum arabic and one part of sugar. They are then cut 1 Ordinary commercial methylated spirit has wood naphtha added to it to discourage its being used as a beverage. The naphtha being insoluble m water a muky fluid results from the dilution of the spirit Bylaw Chennsts can only sell 8 ounces of pure spirit at a time. Most patho- IX'tuXr ai'C' h°WeVer> HcCnSCd by the Ekdse to P-espirit 100 MICROSCOPIC METHODS. on a freezing microtome (of which Cathcart's is a good example) and placed for a few hours in a bowl of water so that the gum and syrup may dissolve out. They are then stained or they may be stored in methylated spirit. 2. Embedding and Cutting in Solid Paraffin. — This method gives by far the finest results, and should always be adopted when practicable. The principle is the impregna- tion of the tissue with paraffin in the melted state. This paraffin when it solidifies gives support to all the tissue elements. The method involves that, after hardening, the tissue shall be thoroughly dehydrated, and then thoroughly permeated by some solvent of paraffin which will expel the dehydrating fluid and prepare for the entrance of the paraffin. The solvents most in use are chloroform, cedar oil, xylol, and turpentine ; of these chloroform and cedar oil are the best, the former being preferred as it permeates the tissue more rapidly. The more gradually the tissues are changed from reagent to reagent in the processes to be gone through, the more successful is the result. A necessity of the process is an oven with hot-water jacket, in which the paraffin can be kept at a constant temperature just above its melting-point, a gas regulator, e.g., Reichert s, being of course necessary. The tissues occurring in patho- logical work have a tendency to become brittle if overheated, and therefore the best results are not obtained by using paraffin melting about 5 8° C, such as is employed in most biological laboratories. We have used for some years a mixture of one part of paraffin, melting at 48 , and two parts of paraffin melting at 5 4° G This mixture has a melt- ing-point between 52c and 53° C, and it serves all ordinary purposes well. An excellent quality of paraffin is that known as the " Cambridge paraffin," but many scientific, instrument makers supply paraffins which for ordinary purposes, are quite as good, and much cheaper. The successive steps in the process of paraffin embedding are as follows : — Pieces of tissue, however hardened are placed alcohol for twenty-four hours in order to their complete dehydration. THE CUTTING OF SECTIONS. 101 2. Transfer now to a mixture of equal parts of absolute alcohol and chloroform for twenty-four hours. 3. Transfer to pure chloroform for twenty-four hours. At the end of this time the tissues should sink or float heavily. 4. Transfer now to a mixture of equal parts of chloroform and paraffin and place on the top of the oven for from twelve to twenty-four hours. If the temperature there is not sufficient to keep the mixture melted then they must be put inside. 5. Place in pure melted paraffin in the oven for twenty-four hours. For holding the paraffin containing the tissues, small tin dishes such as are used by pastry-cooks will be found very suitable. There must be a considerable excess of paraffin over the bulk of tissue present, other- wise sufficient chloroform will be present to vitiate the final result and not give the perfectly hard block obtained with pure paraffin. With experience, the persistence of the slightest trace of chloroform can be recognised by smell. In the case of very small pieces of tissue the time given for each stage may be much shortened, and where haste is desirable Nos. 2 and 4 may be omitted. Otherwise it is better to carry out the process as described. 6. Cast the tissues in blocks of paraffin as follows : Pairs of L-shaped pieces of metal made for the purpose by instrument makers must be at hand. By laying two of these together on a glass plate, a rectangular trough is formed. This is filled with melted paraffin taken from a stock in a separate dish. In it is immersed the piece of tissue which is lifted out of its pure paraffin bath with heated forceps. The direction in which it is to be cut must be noted before the paraffin becomes opaque. When the paraffin has begun to set, the glass plate and trough have cold water run over them. When the block is cold, the metal L's are broken off and, its edges having been pared, it is stored in a pill-box. The Cutti?ig of Paraffin Sections. — Sections must be cut as thin as possible, the Cambridge rocking microtome Fig. 39.— Needle with square of paper on end for manipulating paraffin sections. being, on the whole, most suitable. They should not exceed 8 p in thickness, and ought, if possible, to be about 4 For their manipulation it is best to have two needles on handles, two camel's-hair brushes on handles, and a 102 MICROSCOPIC METHODS. needle with a rectangle of stiff writing paper fixed on it as in the diagram (Fig. 39). When cut, sections are floated on the surface of a beaker of water kept at a tempera- ture about io° C. below the melting-point of the paraffin. On the surface of the warm water they become perfectly flat. Fixation on Ordinary Slides, (a) Gulland's Method. — A supply of slides well cleaned being at hand, one of them is thrust obliquely into the water below the section, a corner of the section is fixed on it with a needle and the slide withdrawn. The surplus of water being wiped off with a cloth, the slide is placed on a support, with the section downwards, and allowed to remain on the top of the paraffin oven or in a bacteriological incubator for from twelve to twenty-four hours. It will then be sufficiently fixed on the slide to withstand all the manipu- lations necessary during staining and mounting. (£>) Fixation by Mann's Method. — This has the advantage of being more rapid than the previous one. A solution of albumin is prepared by mixing the white of a fresh egg with ten parts of distilled water and filtering. Slides are made perfectly clean with alcohol. One is dipped into the solution and its edge is then drawn over one surface of another slide so as to leave on it a thin film of albumin. This is repeated with the others. As each is thus coated, it is leant, with the film downwards, on a ledge till dry and then the slides are stored in a wide stoppered jar till needed. The floating out is performed as before. The albuminised side is easily recognised by the fact that if it is breathed on, the breath does not condense on it. The great advantage of this method is that the section is fixed after twenty to thirty minutes' drying at 37° C. If the tissue has been hardened in any of the bichromate solutions and embedded in paraffin, this or some corre- sponding method of fixing the sections on the slide must be used. Preparation of Paraffin Sections for Staining. — Before staining, the paraffin must be removed from the section. This is best done by dropping on xylol out of a drop bottle. When the paraffin is dissolved out, the superfluous xylol is wiped off with a cloth and a little absolute alcohol dropped on. When the xylol is removed the superfluous alcohol is wiped off and a little 50 per cent methylated spirit dropped on During these procedures sections must on no account be' allowed to dry. The sections are now ready to be stained Deposits of crystals of corrosive sublimate often occur in sections which have been fixed by this reagent. THE S TAIN1NG OF BA C TERIA . 103 These can be readily removed by placing the sections, before staining, for a few minutes in equal parts of Gram's iodine solution (p. in) and water, and then washing out the iodine with methylated spirit. To save repetition we shall in treating of stains suppose that, with paraffin sections, the above preliminary steps have already been taken, and further that sections cut by a freezing microtome are also in spirit and water. The Staining of Bacteria. Staining Principles. — To speak generally, the protoplasm of bacteria reacts to stains in a manner similar to the nuclear chromatin, though sometimes more and sometimes less actively. The bacterial stains par excellence are the basic aniline dyes. These dyes are more or less compli- cated compounds derived from the coal-tar product aniline (C6H5 . NH2). Many of them have the constitution of salts. Such compounds are divided into two groups according as the staining action depends on the basic or the acid portion of the molecule. Thus the acetate of rosaniline derives its staining action from the rosaniline. It is there- fore called a basic aniline dye. On the other hand, ammonium picrate owes its action to the picric acid part of the molecule. It is therefore termed an acid aniline dye. These two groups have affinities for different parts of the animal cell. The basic stains have a special affinity for the nuclear chromatin, the acid for the protoplasm and various formed elements. Thus it is that the former — the basic aniline dyes — are especially the bacterial stains. The number of basic aniline stains is very large. The following are the most commonly used : 1 — Violet Stains. — Methyl-violet, R-5R (synonyms : Hoffmann's violet, dahlia). Gentian-violet (synonyms : benzyl-violet, Pyoktanin). Crystal violet. 1 For further information on this subject the student is referred to Rawitz, " Leitfaden fur histologische Untersuchungen," Jena, 1895, from which the synonyms used in the text are taken. 104 MICROSCOPIC METHODS. Blue Stains. — Methylene-blue 1 (synonym : phenylene-blue). Victoria-blue. Thionin-blue. Red Stains. — Basic fuchsin (synonyms : basic rubin, magenta). Safranin (synonyms : fuchsia, Girofie). Broivn Stain. — Bismarck -brown (synonyms : vesuvin, phenylene- brown). It is of the greatest importance that the stains used by the bacteriologist should be good, and therefore it is advisable to obtain those prepared by Griibler of Leipzig. One is then perfectly sure that one has got the right stain. Of the stains specified, the violets and reds are the most intense in action, especially the former. It is thus easy in using them to overstain a specimen. Of the blues, methylene-blue probably gives the best differentiation of structure, and it is difficult to overstain with it. Thionin- blue also gives good differentiation and does not readily overstain. Its tone is deeper than that of methylene-blue and it approaches the violets in tint. Bismarck-brown is a weak stain, but is useful for some purposes. Formerly it was much used in photomicrographic work, as it was less actinic than the other stains. It is not, however, needed now, on account of the improved sensitiveness of the plates. It is most convenient to keep saturated alcoholic solu- tions of the stains made up, and for use to filter a little into about ten times its bulk of distilled water in a watch- glass. A solution of good body is thus obtained. Most bacteria (except those of tubercle, leprosy, and a few others) will stain in a short time in such a fluid. Watery solutions may also be made up, e.g. a saturated watery solution of methylene-blue or a i per cent solution of gentian-violet. Stains must always be filtered before use. Otherwise there may be deposited on the preparation granules which it is impossible to wash off. The violet stains in solution in 1 This is to be distinguished from methyl-blue, which is a different com- pound. THE STAINING OF COVER- GLASS FILMS. 105 water have a great tendency to decompose. Only small quantities should therefore be prepared at a time. The Staining of Cover-glass Films. — Films are made from cultures as described above. The cover-glass may be floated on the surface of the stain in a watch-glass, or the cover- glass held in forceps with film side uppermost may have as much stain poured on it as it will hold. When the preparation has been exposed for the requisite time, usually a few minutes, it is well washed in tap water in a bowl, or with distilled water with such a simple contrivance as that figured (Fig. 40). The figure explains itself. When the film has been washed the surplus of water is drawn off with a piece of filter-paper, the preparation is carefully dried high over a flame, a drop of xylol balsam is applied, and the cover- glass mounted on a slide. Xylol balsam must be used for mounting all bacterial preparations. The reason is that xylol causes the colour FlG- 4°-— Syphon wash- . r 1 1 „ , bottle for distilled water used to fade less than any other solvent in washing preparations, of balsam. It is sometimes ad- vantageous to examine films in a drop of water in place of balsam. The films can be subsequently dried and mounted permanently. In the case of tubercle, special stains are necessary (p. 113), but with this exception, practically all bacterial films made from cultures can be stained in this way. Some bacteria, e.g., typhoid, glanders, take up the stains rather slowly, and for these the more intensive stains, red or violet, are to be preferred. Films of fluids from the body (blood, pus, etc.) can be generally stained in the same way, and this is often quite sufficient for diagnostic purposes. The blue dyes are io6 MICROSCOPIC METHODS. here preferable, as they do not readily overstain. In the case of such fluids, if the histological elements also claim attention it is best first to stain the cellular protoplasm with a one to two per cent watery solution of eosin (which is an acid dye), and then to use a blue which will stain the bacteria and the nuclei of the cells. In the case of films made from urine, where there is little or no albuminous matter present, the bacteria may be imperfectly fixed on the slide, and are thus apt to be washed off. In such a case it is well to modify the staining method. A drop of stain is placed on a slide, and the cover -glass, film -side down, lowered upon it. After the lapse of the time necessary for staining, a drop of water is placed at one side of the cover- glass and a little piece of filter-paper at the other side. The result is that the stain is sucked out by the filter- paper. By adding fresh drops of water and using fresh pieces of filter -paper, the specimen is washed without any violent application of water, and the bacteria are not displaced. For the general staining of films a saturated watery solution of methylene-blue will be found to be the best stain to commence with. The Use of Mordants and Decolorising Agents.— In films of blood and pus, and still more so in sections of tissues, if the above methods are used, the tissue elements may be stained to such an extent as to quite obscure the bacteria. Hence many methods have been devised in which the general principle may be said to be (a) the use of substances which, while increasing the staining power, tend to fix the stain in the bacteria, and (b) the subsequent treatment by substances which decolorise the overstained tissues to a greater or less extent, while they leave the bacteria coloured. The staining capacity of a solution may be increased — (a) By the addition of substances such as carbolic acid, aniline oil, or metallic salts, all of which probably act as mordants. DEHYDRATION AND CLEARING. 107 (/>) By the addition of alkalies, such as caustic -potash or ammonium carbonate, in weak solution. (c) By the employment of heat. (d) By long duration of the staining process. As decolorising agents we use chiefly mineral acids (hydrochloric, nitric, sulphuric), vegetable acids (especially acetic acid), alcohol (either methylated spirit or absolute alcohol), or a combination of spirit and acid, e.g., methyl- ated spirit with a drop or two of hydrochloric acid added, also various oils, e.g., aniline, clove,, etc. In most cases about thirty drops of acetic acid in a bowl of water will be sufficient to remove the excess of stain from over-stained I films and sections. More of the acid may, of course, be added if necessary. Hot water also decolorises to a certain extent ; over- stained films can be readily decolorised by placing a drop of water on the film and heating gently over a flame. When preparations have been sufficiently decolorised by j an acid, they should be well washed in tap water, or in dis- J tilled water with a little lithia carbonate added. The methods embracing the use of a stain with a mor- I dant, and a decoloriser, are very numerous, and we can only enumerate the best of them. Dehydration and Clearing. — It is convenient, first of all, to indicate the final steps to be taken after a specimen is stained. We have already described the mounting of film preparations. Sections after being stained must be de- hydrated, cleared, and then mounted in xylol balsam. Dehydration is most commonly effected with absolute alcohol. Alcohol, however, sometimes decolorises the stained organisms more than is desirable, and therefore Weigert devised the following method of dehydrating and clearing by aniline oil, which, though it may decolorise somewhat, does not do so to the same extent as alcohol. As much as possible of the water being removed, the section is placed in aniline oil ; or if it has been cut in paraffin, some aniline oil is placed on the section, and 1 MICROSCOPIC ME TIIODS. the slide moved to and fro. The section is dehydrated and becomes clear. The process may be accelerated by heating gently. The preparation is then treated with a mixture of two parts of aniline oil and one part of xylol, and then with xylol alone, after which it is mounted in xylol balsam. Sections stained for bacteria should always be cleared, at least finally, in xylol, for the same reason that xylol balsam is to be used for mounting films, viz. that it dissolves out aniline dyes less readily than such clearing reagents as clove oil, etc. Xylol, however, requires the previous de-' hydration to have been more complete than clove oil, which will clear a section readily when the dehydration has been only partially effected by, say, methylated spirit. If a little decolorisation of a section is still required before mounting, clove oil may be used to commence the clearing, the process being finished with xylol. We sometimes have to deal with bacteria which show a; special. tendency to be decolorised. This tendency can be obviated by adding a little of the stain to the alcohol, or aniline oil, employed in dehydration. In the latter case a little of the stain is rubbed down in the oil. The mixture is allowed to stand. After a little time a clear layer forms on the top with stain in solution, and this can be drawn off! with a pipette. • When methylene-blue, methyl-violet, or gentian-violet are the stains being used they can, after the proper degree of decolorisation has been reached, be fixed in the tissues by treating for a minute with ammonium molybdate (2^ per cent in water). The Formulae of some of the more commonly used Stain Com- binations. 1 . LoffleSs Methylene-blue. Saturated solution of methylene-blue in alcohol • 3° c-c- Solution of potassium hydrate in distilled water (1-10,000) 100 c.c. (This dilute solution may be conveniently made by adding 1 c.c. of a 1 per cent solution to 99 c.c. of water.) FORMULAE OF STAINS. 109 Sections may be stained in this mixture for from a quarter of an hour to several hours. They do not readily overstain. The tissue con- taining the bacteria is then decolorised if necessary with \-\ per cent acetic acid, till it is a pale blue-green. The section is washed in water, rapidly dehydrated with alcohol or aniline oil, cleared in xylol, and mounted. The tissue may be contrast-stained with eosin. If this is desired, after decolorisation wash with water, place for a few seconds in 1 per cent solution of eosin in absolute alcohol, rapidly complete dehydration with pure absolute alcohol, and proceed as before. Films may be stained with Loffler's blue by five minutes' exposure or longer in the cold. They usually do not require decolorisation, as the tissue elements are not. overstained. 2. Kiihne's Methylene-blue. Methylene-blue . . . 1.5 gr. Absolute alcohol . ■. . 10 c.c. Carbolic acid solution (1-20) . . 100 c.c. Stain and decolorise as with Loffler's blue, or decolorise with very weak hydrochloric acid (a few drops in a bowl of water). 3. Carbol- Thionin-blue. — Make up a stock solution consisting of 1 gramme of thionin-blue dissolved in 100 c.c. carbolic acid solution (1-40). For use, dilute 1 volume with .3 of water and filter. Stain sections for five minutes or upwards. Wash very thoroughly with water, otherwise a deposit of crystals may occur in the subsequent stages. Decolorise with very weak acetic acid. A few drops of the acid added to a bowl of water is quite sufficient. Wash again thoroughly with water. Dehydrate with absolute alcohol or aniline oil. Thionin-blue stains more deeply than methylene-blue, and gives equally good differentiation. It is very suitable for staining typhoid and glanders bacilli in sections. Cover-glass preparations stained by this method do not usually require decolorisation. 4. Gentian-violet in Aniline Oil Water.- — Two solutions have here to be made up. (a) Aniline oil water. Add about 5 c.c. aniline oil to 100 c.c. distilled water in a flask, and shake violently till as much as possible of the oil has dissolved. Filter and keep in a covered bottle to prevent access of light, (b) Make a saturated solution of gentian-violet in alcohol. When the stain is to be used, 1 part of (b) is added to 10 parts of (a), and the mixture filtered. The mixture should be made not more than twenty-four hours before use. Stain sections for a few minutes ; then decolorise with methylated spirit. Sometimes it is advantageous to add to the methylated spirit a little hydrochloric acid (2-3 minims to 100 c.c). This staining solution is not so much used by itself, as in Gram's method, which is presently to be described. Instead of aniline oil water, carbolic acid solution (1-20) may be used in the same way. I IO MICROSCOPIC METHODS. 5. Carbol-Fuchsin (see p. 1 13).— This is a very powerful slain, and, when used in the undiluted condition, \-\ minute's staining is usually sufficient. It is better, however, to dilute with five to ten times its volume of water and stain, for a few minutes. In this form it has a very wide application. Methylated spirit with or without a few drops of acetic acid is the most convenient decolorising agent. Then dehydrate thoroughly, clear, and mount. Various other staining combinations might be given, | but the above are the best and most widely used. If the reader has thoroughly grasped the remarks made above on the general principles which underlie the stain- ing of bacteria, he will be able to use any combination to which his attention may be directed. We may only add here that different organisms take up and hold different stains with different degrees of intensity, and thus duration of staining and degree of decolorisation must be varied. It may be laid down as a general rule that, so long as organisms retain the stain, the greater the decolorisation of the tissues in which they lie, the clearer will be the results. Gram's Method and its Modifications. — In the methods already described the tissues, and more especially the nuclei, usually retain some stain when decolorisation has reached the point to which it can safely go without the, bacteria themselves being affected. In the method of Gram, now to be detailed, this does not occur, for the stain can here be removed completely from the ordinary tissues, and left only in the bacteria. All kinds of bac- teria, however, do not retain the stain in this method, and therefore in the systematic description of any species it is customary to state whether it is, or is not, stained by Gram's method — by this is meant, as will be understood from what has been said, whether the particular organism retains the colour after the latter has been completely removed from the tissues. It must, however, be remarked that some tissue elements may retain the stain as firmly as any bacteria, e.g., keratinised epithelium, calcified particles, the granules of mast cells, and sometimes altered red blood corpuscles, etc. GRAM'S STAIN. 1 1 1 In Gram's method the essential feature is the treating of the tissue, after staining, with a solution of iodine. This solution is spoken of as Gram's solution, and has the following composition : — Iodine . . i part Potassium iodide . . 2 parts Distilled water . .300 parts The following is the method : — 1. Stain in aniline oil gentian-violet {vide p. 109) for about five minutes, and wash in water. 2. Treat the section or film with Gram's solution till its colour >. becomes a purplish black — generally about half a minute or a minute ■ is sufficient for the action to take place. 3. Decolorise with absolute alcohol or methylated spirit till the I colour has almost entirely disappeared, the tissues having only a faint violet tint. 4. Dehydrate completely, clear with xylol and mount. In the case of film preparations, the specimen is simply washed in water, dried and mounted. Before (4) a contrast stain is often used {vide infra). Gram's method, when carefully used, generally gives quite satisfactory results, but sometimes a precipitate of the gentian-violet is left in the tissues, and sometimes the specimen decolorises very slowly and the bacteria lose the stain in the process. We find that the result is more certain if in (1) the alcoholic gentian- violet be mixed with carbolic acid solution (1:20) instead of aniline oil (water, and if in (3) clove oil be used in decolorising after the specimen has been sufficiently dehydrated with alcohol or methylated spirit. The clove oil decolorises rapidly, and must then be washed thoroughly out with xylol, or, if a contrast stain is to be used, with alcohol. Nicolle has modified Gram's method by staining with carbol-gentian-violet as described, and, in (3) decolorising with a mixture of acetone one part and alcohol two parts. This also gives very good results. In Weigert's modifica- tion, aniline oil is used in (3) as the decolorising agent 112 MICROSCOPIC METHODS. instead of alcohol (p. 107). Other modifications have been introduced, of which only Kiihne's need be mentioned. Killings Modification of Grant's Method. 1 . Stain for five minutes in a solution made up of equal parts of saturated alcoholic solution of crystal-violet (" krystall-violet ") and 1 per cent solution of ammonium carbonate. 2. Wash in water. 3. Place for two to three minutes in Gram's iodine solution, or in the following modification by Kiihne : — Iodine 2 parts Potassium iodide .... 4 parts Distilled water . . . .100 parts For use, dilute with water to make a sherry-coloured solution. 4. Wash in water. 5. Decolorise in a saturated alcoholic solution of fluorescein (a saturated solution in methylated spirit does equally well). 6. Dehydrate, clear and mount. Contrast Stains. — With these methods, especially that of Gram's, it is often advantageous, after decolorisation, to co.unterstain the tissues with a dye different in colour from that retained by the bacteria. Lithia carmine or alum carmine may be used for contrast-staining in Gram's method. The sections here are stained first with the contrast, and then treated by Gram's method. (This is the most, satisfactory procedure.) . . In the case of the following stains the contrast-staining is not carried out till the tissues have been subjected to the bacterial stain and decolorised as far as necessary. . Safranin.— Stock solution is a 1 per cent solution of safranin dissolved in equal parts of methylated spirit and water. For use, dilute one part with five of water, and stain for thirty seconds. Bismarck-brown. — Stock solution — saturated solution in equal parts of alcohol and water. Stain for from two to three minutes. Both safranin and Bismarck-brown are excellent nuclear stains, and in cases where Gram's method has been used, colour most of the organisms which are left unstained by the violet. Eoun maybe used as a 1-1000 watery solution, and applied lor about a minute. It is a good ground stain, but does not bring out the nuclei. TUBERCLE STAINS. "3 Stain for Tubercle and Leprosy Bacilli. — These bacilli cannot be well stained with a simple watery solution of a basic aniline dye. This fact can easily be tested by attempting to stain a film of a tubercle culture with such a solution. They require a powerful stain containing a mordant, and must be exposed to the stain for a long time, or the action of the latter may be aided by a short application of heat. When once stained, however, they resist decolorising with very powerful reagents. The smegma bacillus also resists decolorising with strong acids (P- 255)- Any combination of gentian- violet or fuchsin with aniline oil or carbolic acid or other mordant will stain the bacilli named, but the following methods are most commonly used : — Ziehl-Neelsen Carbol-Fuchsin Stain. Basic fuchsin i part Absolute alcohol . ... . 10 parts Solution of carbolic acid (i : 20) . 100 parts 1. Place the specimen in this fluid, and having heated it till steam rises, allow it to remain there for five minutes, or allow it to remain in the cold stain for from twelve to twenty-four hours. (Films and paraffin sections are usually stained with hot stain, loose sections with cold ; in hot stain the latter shrink. ) • 2. Decolorise with 20 per cent solution of strong sulphuric acid, nitric acid, or hydrochloric acid, in water. In this the tissues become yellow. 3. Wash well with water. The tissues will regain a faint pink tint. If the colour is distinctly red, the decolorisation is insufficient, and the specimen must be returned to the acid. As a matter of prac- tice, it is best to remove the preparation from the acid every few seconds and wash in water, replacing the specimen in the acid and re-washing till the proper pale pink tint is obtained. 4. Contrast stain with a saturated watery solution of methylene- blue for half a minute, or with saturated Bismarck-brown for from two to three minutes. 5. Wash well with water. In the case of films, dry and mount. In the case of sections, dehydrate, clear and mount. n4 MICROSCOPIC METHODS. FraenkeVs modification of the Ziehl-Neehen Stain. Here the process is shortened by using a mixture containing both the decolorising agent and the contrast stain. The sections or films are stained with the carbol-fuchsin as above described, and then placed in the following solution : — They are treated with this till the red colour has quite disappeared and been replaced by blue. The subsequent stages are the same as in No. 5, supra. Leprosy bacilli are stained in the same way, but are rather more easily decolorised than tubercle bacilli, and it is better to use only 5 per cent sulphuric acid in j decolorising. In the case of specimens stained either by the original Ziehl-Neelsen method, or by Fraenkel's modification, the tubercle or leprosy bacilli ought to be bright-red, and the tissue blue or brown, according to the contrast stain used. Other bacteria which may be present are also coloured: with the contrast stain. The Staining of Spores.— If bacilli containing spores are stained with a watery solution of a basic aniline dye the spores remain unstained. The spores either take up the stain less readily than the protoplasm of the bacilli or they have a resisting envelope which prevents the stain penetrating to the protoplasm. Like the tubercle bacilli, when once stained they retain the colour with consider- able tenacity. The following is the method for staining spores : — 1 Stain cover-glass films as for tubercle bacilli. 2" Decolorise with 1 per cent sulphuric acid in water or with methylated spirit. This removes the stain from the bacilli. Distilled water ..... Absolute alcohol .... Nitric acid . Methylene-blue in crystals to saturation. 50 parts 30 parts 20 parts STAINING OF FI AG ELLA. "5 3. Wash in water. 4. Stain with saturated watery methylene-blue for half a minute. 5. Wash in water, dry, and mount in balsam. The result is that the spores are stained red, the protoplasm of the bacilli blue. The spores of some organisms lose the stain more readily than those of others, and for some, methylated spirit is a sufficiently strong de- colorising agent for use. If sulphuric acid stronger than 1 per cent is used the spores of many bacilli are readily decolorised. Moller recommends that before being stained, the films should be placed in chloroform for 2 minutes, and then in a 5 per cent solution of chromic acid for \-2 minutes. This procedure has an advantage in some cases, though in many it is unnecessary. The Staining of Capsules.— In many capsulated bacteria the capsule can be fixed by means of glacial acetic acid. Films are made and while wet are placed in this acid for a few seconds. The super- fluity is removed with filter-paper, and the preparation treated with gentian-violet in aniline oil water repeatedly till all the acetic acid is removed. It is then washed with a 1-2 per cent solution of sodium chloride and examined in the same solution. The Staining of Flagella. — The staining of the flagella of bacteria is the most difficult of all bacteriological procedures, and it requires considerable practice to ensure that good results shall be obtained. Many methods have been introduced, of which the three following are the most satisfactory. Preparation of Films. — In all the methods of staining flagella, young cultures on agar should be used, say a culture incubated for from twelve to eighteen hours at 370 C. A very small portion of the growth is taken on the point of a platinum needle and carefully mixed in a little water in a watch glass ; the amount should be such as to produce scarcely any turbidity in the water. A film is then made by placing a drop on a clean cover-glass and carefully spreading it out with the needle. It is allowed to dry in the air and is then passed twice or thrice through a flame, care being taken not to over-heat it. The cover- glasses used should always be cleaned in the mixture of sulphuric acid and potassium bichromate described on Page 95- n6 MICROSCOPIC METHODS. i. Pi (field's Method as modified by Richard Mnir. Prepare the following solutions : — A. The Mordant. Tannic acid, 10 per cent watery solution, filtered Corrosive sublimate, saturated watery solution Alum, saturated watery solution . Carbol-fuchsin {vide p. 113) 10 c.c. 5 c.c. 5 c.c. 5 c.c. Mix thoroughly. A precipitate forms, which must be allowed to deposit, either by centrifugalising or simply by allowing to stand. Remove the clear fluid with a pipette and transfer to a clean bottle. The mordant keeps well for one or two weeks. B. The Stain. Alum, saturated watery solution . Gentian-violet, saturated alcoholic solution 10 c.c. 2 c.c. The stain should not be more than two or three days old when used. It may be substituted in the mordant in place of the carbol-fuchsin. The film having been prepared as above described, pour over it as much of the mordant as the cover-glass will hold. Heat gently over a flame till steam begins to rise, allow to steam for about a minute, and then wash well in a stream of running water for about two minutes Then dry carefully over the flame, and when thoroughly dry pour on some of the stain. Heat as before, allowing to steam for about a minute, wash well in water, dry and mount in a drop ot xylol balsam. . , This method has yielded the best results in our hands. 2. Loffler's Method. Two solutions must be made up. Loffier gives the following ^TnfM^Tf^I of a 20 per cent solution of tann, \n water add as many drops of a saturated solution of ferrous sulphate n wa e as wU give'the whole fluid a dark-violet tint. To this add 7 of ablution made by boiling 1 gramme of logwood with 8 c c nf water (after boiling, filter and make up to 9 c.c to compensate for evapo a ion The mixture of the tannin solution with the bgvvood sEn appears of a dirty dark-violet colour. If too much logwood s added particles are precipitated which make the fluid useless as a is aaaea pa 1 mordant fresh on each ^ 0^5 c=. of «. carMic J STAINING OF FLAGELLA. 117 solution makes the fluid more permanent without impairing its pro- perties. B. The Stain. — To 100 c.c. of a filtered saturated solution of aniline oil in water add 1 c.c. of a 1 per cent solution of sodium hydrate. The aniline water is ordinarily neutral. The addition of the soda renders it slightly alkaline. To this solution add 4-5 grm. of solid methylene-violet, methylene-blue or fuchsin, and shake well. When a preparation is to be stained, filter a few drops on to the cover-glass. Make a film as above described, and holding the cover-glass in a pair of forceps, pour on as much of the mordant A as the cover-glass will hold. Heat it carefully above a flame till steam begins to rise and then move the preparation gently in and out of the hot-air column over the flame for about a minute. Wash well in distilled water till every trace of mordant appears to be gone. If necessary, wash with absolute alcohol till only the film itself appears tinted violet with the mordant. Filter a few drops of stain B on to the cover, again heat till steam rises and leave in the warm stain for one minute. Wash well in distilled water, dry, and mount in xylol balsam. 3. Van Ermengem's Method for Staining Flagella. The films are prepared as above described. Three solutions are here necessary : — Solution A. {Bain fixateur) — Osmic acid, 2 per cent solution . 1 part Tannin, 10-25 Per cent solution . . . .2 parts Place the films in this for one hour at room temperature, or heat over a flame till steam rises and keep in the hot stain for five minutes. Wash with distilled water, then with absolute alcohol for three to four minutes, and again in distilled water, and treat with Solution B. {Bain sensibilisateur) — . 5 per cent solution of nitrate of silver in distilled water. Allow films to be in this a few seconds. Then without washing transfer to Solut ion C. {Bam Teductenr et ixinfoycaleuv} Gallic acid ....... 5 gr. Tannin Fused potassium acetate Distilled water . Keep in this for a few seconds 3 gr- . 10 gr. • 35o c.c. Then treat again with Solution B till the preparation begins to turn black. Wash, dry, and mount. It is better, as Mervyn Gordon recommends, to leave the specimen u8 MICROSCOPIC METHODS. in B for two minutes and then to transfer to C for one and a half to two minutes, and not to transfer again to B, It will also be found an advantage to use a fresh supply of C for each preparation, a small quantity being sufficient. The beginner will find the typhoid bacillus or the B. coli communis very suitable organisms to stain by this method. Although the results obtained by this method are sometimes excellent, they vary considerably. Frequently both the organisms and fiagella appear of abnormal thickness. This is due to the fact that the process on which the method depends is a precipitation rather than a true staining. The pictures on the whole are less faithful than in the other two methods. The Testing of Agglutinative and Sedimenting Properties of Serum. By agglutination is meant the aggregation into clumps of uniformly disposed bacteria in a fluid, by sedimentation the formation of a deposit composed of such clumps when the fluid is allowed to stand. Sedimentation is thus the naked-eye evidence of agglutination. The blood serum may acquire this clumping power towards a particular organism under certain conditions j these being chiefly met with when the individual is suffering from the disease pro- duced by the organism, or has recovered from it, or when a certain degree of immunity has been produced artificially by injections of the organism. The nature of this property will be discussed later. Here we shall only give the technique by which the presence or absence of the property may be tested. There are two chief methods, a microscopic and a naked eye, corresponding to the effects mentioned above. In both, the essential process is the bringing of the diluted serum into contact with the bacteria uniformly disposed in a fluid. In the former this is done on a glass slide, and the result is watched under the microscope ; the occurrence of the phenomenon is shown by the aggregation of the bacteria into clumps, and if the organism is motile this change is preceded or accompanied by more or less complete loss of motility. In the latter method the mixture is placed in an upright thin glass tube ; TESTING OF PROPERTIES OF SERUM. 119 sedimentation is shown by the formation within a given time (say 12 or 24 hours) of a somewhat flocculent layer at the bottom, the fluid above being clear. Two points should be attended to. Controls should always be made with normal serum, and the serum to be tested should never be brought in the undiluted condition into con- tact with the bac- teria. The stages of procedure are the following ; — 1. Blood is con- veniently obtained by pricking the lobe of the ear, which should previously have been washed with a mixture of alcohol and ether and allowed to dry.' The blood is drawn up into the bulbous por- tion of a capillary pipette, such as in Fig. 41, a. (These pipettes can be readily made by drawing out quill glass tubing in a flame. It is con- venient always to have several ready for use. ) The pipette is kept in the upright position, one end being closed. For purposes of transit, break off the bulb at the constriction and seal the ends. After the serum has separated from the coagulum the bulb is broken through near its upper end and the serum removed by means of another capillary pipette. The serum is then to be diluted. I 0 d Fig. 41. — Tubes used in testing agglutinating and sedimenting properties of serum. 120 MICROSCOPIC METHODS. 2. The serum may be diluted (a) by means of a graduated pipette — either a leucocytometer pipette (Fig. 41, b) or some corresponding form. In this way successive dilutions of 1 : io, 1 : 20, 1 : ioo, etc., can be rapidly made. This is the best method. (/;) By means of a capillary pipette with a mark on the tube, the serum is drawn up to the mark and then blown out into a glass capsule ; equal quantities of bouillon are successively measured in the same way and added till the requisite dilution is obtained, (c) By means of a platinum needle with a loop at the end (Delepine's method). A loopful of serum is placed on a slide and the desired number of similar loopfuls of bouillon are separately placed around on the slide. The drops are then mixed. A very convenient and rapid method of combining the steps 1 and 2 is to draw a drop of blood up to the mark 1 or .5 on a leucocytometer pipette and draw the bouillon after it till the bulb is filled. A dilution of 10 or 20 times is thus obtained. Then blow the mixture into a U-shaped tube (Fig. 41, c) and centrifugalise or simply allow the red corpuscles to separate by standing. (In this method of course the dilution is really greater than if pure serum were used, and allowance must therefore be made in comparing results. ) The presence of red corpuscles is no drawback in the case of the microscopic method, but when sedimentation tubes are used the corpuscles should be separated first. 3. The bacteria to be tested should be taken from young cultures, preferably not more than twenty-four hours old, incubated at 370 C. They may be used either as a bouillon culture or as an emulsion made by adding a small portion of an agar culture to bouillon. In the latter case the mass of bacteria on a platinum loop should be gently broken down at the margin of the fluid in a watch-glass. When n thick turbidity is thus obtained, any remaining fragments should first be removed and then the organisms should be uniformly mixed with the rest of the fluid. The bacterial emulsion ought to have a faint but distinct turbidity. (When the exact degree of sedimenting power of a serum is to be tested — expressed as the highest dilution in which it produces complete sedimentation within twenty-four hours — a standard quantity (by weight) of bacteria must be added to a given quantity of bouillon. This is not necessary for clinical diagnosis.) 4. To test microscopically, mix equal quantities (measured by a marked capillary pipette) of the diluted serum and the bacterial emulsion on a glass slide, cover with a cover-glass and examine under the micro- scope. The form of glass slide used for hang-drop cultures (Fig. 26) will be found very suitable. The ultimate dilution of the serum will, of course, be double the original dilution. To observe sedimentation mix equal parts of diluted serum and of bacterial emulsion and place in a thin glass tube— a simple tube with closed end or a U-tube. Keep in upright position for twenty-four hours. One of Wright's sedimentation tubes is shown in Fig. 41, d. Diluted serum is drawn up to fill the space mn, a small quantity of air GENERAL BACTERIOLOGICAL DIAGNOSLS. 121 is sucked up after it to separate it from the bacterial emulsion, which is then drawn up in the same quantity ; the diluted serum will then occupy the position kl. The fluids are then drawn several times up into the bulb and returned to the capillary tube so as to mix, and finally blown carefully down close to the lower end, which is then sealed off. The sediment collects at the lower extremity. General Bacteriological Diagnosis. Under this heading we have to consider the general routine which is to be observed by the bacteriologist when any material is submitted to him for examination. The object of such examination may be to determine whether any organisms are present, and if so, what organisms ; or the bacteriologist may simply be asked whether a particular organism is or is not present. In any case his inquiry must consist (1) of a microscopic examination of the material submitted; (2) of an attempt to isolate the organisms present j and (3) of the identification of the organisms isolated. We must, however, before considering these points look at a matter often neglected by those who seek a bacteriological opinion, viz. : the proper methods of obtaining and transferring to the bacteriologist the material which he is to be asked to examine. The general principles here are (1) that every precaution must be adopted to prevent the material from being contaminated with ex- traneous organisms ; (2) that nothing be done which may kill any organisms which may be proper to the inquiry ; and (3) that the bacteriologist obtain the material as soon as possible after it has been removed from its natural surroundings. The sources of materials to be examined, even in pathological bacteriology alone, are of course so varied that we can but mention a few examples. It is, for instance, often necessary to examine the contents of an abscess! Here the skin must be carefully purified by the usual surgical methods ; the knife used forThe incision is prefer- ably to be sterilised by boiling, the first part of the pus which escapes allowed to flow away (as it might be spoiled 122 GENERAL BACTERIOLOGICAL DIAGNOSIS. by containing some of the antiseptics used in the purifica- tion) and a little of what subsequently escapes allowed to flow into a sterile test-tube. If test-tubes sterilised in a laboratory are not at hand, an ordinary test-tube may be a quarter filled with water, which is then well boiled over a spirit-lamp. The tube is then emptied and plugged with a plug of cotton wool, the outside of which has been singed in a flame. Small stoppered bottles may be sterilised and used in the same way. A discharge to be examined may be so small in quantity as to make the procedure described impracticable. It may be caught on a piece of sterile plain gauze, or of plain absorbent wool, which is then placed in a sterile vessel. Wool or gauze used for this purpose, or for swobbing out, say the throat, to obtain shreds of suspicious matter, must have no antiseptic impreg- nated in it, as the latter may kill the bacteria present and make the obtaining of cultures impossible. Fluids from the body cavities, urine, etc., may be secured with sterile pipettes. To make one of these, take nine inches of ordinary quill glass -tubing, draw out one end to a capillary diameter, and place a little plug of cotton wool in the other end. Insert this tube through the cotton plug of an ordinary test-tube and sterilise by heat. To use it, remove test-tube plug with the quill tube in its centre, suck up some of the fluid into the latter, and re- place in its former position in the test-tube (Fig. 42). Another method very convenient for transport is to make two constrictions on the glass tube at suitable distances, ac- cording to the amount of fluid to be taken. The fluid is then drawn up into the part between the constrictions, but so as not to fill it completely. The tube is then broken through at both constrictions and the thin ends are sealed by heating in a flame. Fig. 42. — Test- tube and pipette arranged for obtain- ing fluids contain- ing bacteria. ROUTINE EXAMINATION OF MATERIAL 123 Solid organs to be examined should, if possible, be obtained whole. They may be treated in one of two ways. 1. The surface over one part about an inch broad is seared with a cautery heated to dull red heat. All superficial organisms are thus killed. An incision is made in this seared zone with a sterile scalpel, and small quantities of the juice are removed by a platinum loop to make cover- glass preparations and plate or smear cultures. 2. An alternative method is as follows : — The surface is sterilised by soaking it well with 1 to 1000 corrosive sublimate for half an hour. It is then dried, and the capsule of the organ is cut through with a sterile knife, the incision being further deepened by tearing. In this way 'a perfectly uncon- taminated surface is obtained. Hints are often obtained from the clinical history of the case as to what the procedure ought to be in examination. Thus, as a matter of practice, cultures of tubercle and often of glanders bacilli can be easily obtained only by inoculation experiments. Typhoid bacilli need hardly be looked for in the fasces after the first ten days of the disease, and so on. Routine Procedure in Bacteriological Examination of Material. — In the case of a discharge regarding which nothing is known the following procedure should be adopted:— (r) Several cover-glass preparations should be made. One ought to be stained with saturated watery methylene-blue, one with a stain containing a mordant such as Ziehl-Neelsen carbol-fuchsin, one by Gram's method. (2) (a) Gelatine plates should be made and kept at room temperature, (b) a series of agar plates or successive strokes on^agar tubes (p. 66) should be made and incubated at 37 C. Method (6) of course gives results more quickly. If microscopic investigation reveals the presence of bacteria, it is well to keep the material in a cool place till next day when, if no growth has appeared in the incubated agar, some other culture medium (e.g., blood serum or agar smeared with blood) may be employed. If growth has taken place say in the agar plates, one with about 200 or fewer colonies should be made the chief basis for research. In such a 124 GENERAL BACTERIOLOGICAL DIAGNOSIS. plate the first question to be cleared up is : Do all the colonies present consist of the same bacterium ? The final settlement of this question depends on microscopic examination, but it is seldom necessary to examine all the colonies in this way ; for particular bacteria when growing in mass in a colony frequently present characteristic appearances, which may be recognised even by the naked eye or at least by a \ inch or i inch objective. The shape of the colony, its size, the appearance of the margin, the graining of the substance, its colour, etc. are all to be. noted. One precaution is necessary, viz. it must be noted whether the colony is on the surface of the medium or in its substance, as colonies of the same bacterium may exhibit differences according to their position. The arrange- ment of the bacteria in a surface colony may be still more minutely studied by means of impression preparations. A cover-glass is carefully cleaned and sterilised by passing quickly several times through a Bunsen flame. It is then placed on the surface of the medium and gently pressed down on the colony. The edge is then raised by a sterile needle, it is seized with forceps, dried high over the flame, and treated as an ordinary cover- glass preparation. In this way very characteristic appearances may sometimes be noted and preserved, as in the case of the anthrax bacillus. The most accurate method, however, of examining the arrangement of individual bacteria in a colony is by the prolonged examination of hanging -drop cultures. For applying such a method to an organism growing at 370 C. a special incubator surrounding the microscope stage has been devised. The colonies on a plate having been classified, a microscopic examination of each group may be made by means of cover-glass preparations, and tubes of gelatine and agar are inoculated from each representative colony. Each of the colonies used must be marked for future reference, preferably by drawing a circle round it on the under surface of the plate or capsule with one of Faber's pencils for marking on glass, a number or letter being added for easy reference. GROWTH CHARACTERISTICS. 125 The general lines along which observation is to be made in the case of a particular bacterium may be indicated as follows : — r. Microscopic Appearances. — For ordinary descriptive purposes young cultures, say of 24 hours' growth, on agar should be used, though appearances in older cultures, such as involution forms, etc., may also require attention. Note (1) the form, (2) the size, (3) the appearance of the protoplasmic contents, especially as regards uniformity or irregularity of staining, (4) the method of grouping, (5) the staining reactions. Has it a capsule ? Does the bacterium stain with simple watery solutions? Does it require the use of stains containing mordants ? How does it behave towards Gram's method ? It is important to investigate the first four points both when the organism is in the fluids or tissues of the body and when growing in artificial media, as slight variations occur. It must also be borne in mind that slight variations are observed according to the kind and consistence of the medium in which the organism is growing. (6) Is it motile and has it flagella ? If so, how are they arranged ? (7) Does it form spores, and if so, under what conditions as to temperature, etc. ? 2. Growth Characteristics. — Here the most important points on which information is to be asked are, What are the characters of growth and what are the relations of growth- (1) to temperature, (2) to oxygen? These can be answered from some of the following experiments : A. Growth on gelatine. (1) Stab culture. Note (a) rate of growth ; (b) form of growth, (a) on surface, (/?) in substance; (c) presence or absence of liquefaction; (d) colour; (e) presence or absence of gas formation and of characteristic smell; (/) relation to reaction of medium. (2) Streak culture. (3) Shake culture. (4) Plate cultures. Note appearances of colonies (a) superficial, (b) deep. (5) Growth in fluid gelatine at 370 C. B. Growth on agar at 370 C. (1) Stab. (2) Streak. Also on glycerine agar, blood agar, etc. Appearances of colonies in agar plates. 126 GENERAL BACTERIOLOGICAL DIAGNOSIS. G. Growth in bouillon, (a) character of growth, (/>) smell, (c) reaction. D. Growth on special media, (i) Solidified blood serum. (2) Potatoes. (3) Lactose and other sugar media. Does fermentation occur and is gas formed? (4) Milk. Is it curdled or turned sour? (5) Litmus media. Note changes in colour. (6) Peptone solution. Is indol formed ? E. What is viability of organism on artificial media ? 3. Results of inoculation experiments on animals. By attention to such points as these a considerable knowledge is attained regarding the bacterium, which will lead to its identification. In the case of many well-known organisms, however, a few of the above points taken together will often be sufficient for the recognition of the species, and experience teaches what are the essential points as regards any individual organism. In the course of the systematic description of the pathogenic organisms, it will be found that all the above points will be referred to, though not in every case. The methods by which the morphological and biological character- istics of any growth may be observed have already been fully described. It need only be pointed out here that in giving descriptions of bacteria the greatest care must be taken to state every detail of investigation. Thus in any description of microscopic appearances the age of the growth from which the preparation was made, the medium employed, the temperature at which development took place must be noted, along with the stain which was used ; and with regard to the latter it is always preferable to employ one of the well-known staining combina- tions, such as Loffler's methylene-blue. Especial care is necessary in stating the size of a bacterium. The apparent size often shows slight variations dependent on the stain used and the growth conditions of the culture. Accurate measurements of bacteria can only be made by preparing microphotographs of a definite magnification and measuring, the sizes on the negatives. From these the actual sizes can easily be calculated. In describing bacterial cultures it must be borne in mind that the appearances often vary with the age. It is suggested that in the case of cultures grown at from 360 to 370 C. the appearances between 24 and 48 hours should be made the basis of description, and in the case of cultures grown between 18° and 220 C. the appearances between 48 and 72 hours should be employed. The culture fluids INOCULATION OF ANIMALS. 127 used must be made up and neutralised by the precise methods already described. The investigator must give every detail of the methods he has employed in order that his observations may be capable of repetition. Inoculation of Animals.1 The animals generally chosen for inoculation are the mouse, the rat, the guinea-pig, the rabbit, and the pigeon. Great caution must be shown in drawing conclusions from isolated experiments on rabbits, as these animals often manifest exceptional symptoms, and are very easily killed. Dogs are, as a rule, rather insusceptible to microbic disease, and the larger animals are too expensive for ordinary labora- tory purposes. In the case of the mouse and rat the variety must be carefully noted, as there are differences in suscepti- bility between the wild and tame varieties, and between the white and brown varieties of the latter. In the case of the wild varieties, these must be kept in the laboratory for a week or two before use, as in captivity they are apt to die trom very slight causes, and, further, each individual should be kept in a separate cage, as they show great tendencies to cannibalism. Of all the ordinary animals the most suscep- tible to microbic disease is the guinea-pig. Practically all inoculations are performed by means of the hypodermic syringe. The best variety is made on the ordinary model with metal mountings, asbestos washers, and preferably furnished with platinum iridium needles. Before use the needle is mounted on the piston and the syringe sterilised by toiling for five minutes. The materials used for inocu- lation are cultures, animal exudations, or the juice of oreans If the bacteria already exist in a fluid there is no difficulty Ihe needle is most conveniently filled out of a shallow conical test glass which ought previously to have been covered with a cover of filter paper and sterilised. If an inoculation is to be made from organisms growing on the surface of a solid medium, either a little ought to be scraped 1 Experiments on animals, of course, cannot be oerfhr^Zi in >*7 country wuhout a license granted by the Home Secretary ^ 128 INOCULATION OF ANIMALS. off and shaken up in sterile distilled water or .75 per cent salt solution to make an emulsion, or a little sterile fluid is poured on the growth and the latter scraped off into it. This fluid is then filtered into the test glass through a plug of sterile glass wool. This is easily effected by taking a piece of § in. glass-tubing 3 in. long, drawing one end out to a fairly narrow point, plugging the tube with glass wool above the point where the narrowing commences, and sterilising by heat. By filtering an emulsion through such a pipette, flocculi which might block the needle are removed. If a solid organ or an old culture is used for inoculation it ought to be rubbed up in a sterile porcelain or metal crucible with a little sterile distilled water, by means of a sterile glass rod, and the emulsion filtered as in the last case. The methods of inoculation generally used are : (1) by scarification of the skin ; (2) by subcutaneous injection ; (3) by intraperitoneal injection ; (4) by intravenous injection j (5) by injections into special regions, such as the anterior chamber of the eye, the substance of the lung, etc. Of these (2) and (3) are most frequently used. When an anaesthetic is to be administered, this is conveniently done by placing the animal, along with a piece of cotton wool or sponge soaked in chloroform, under a bell-jar or inverted glass beaker of suitable size. 1. Scarification. — A few parallel scratches are made in the skin of the abdomen previously cleansed, just sufficiently deep to draw blood, and the infective material is rubbed in with a platinum eyelet. The disadvantage of this method is that the inoculation is easily contaminated. The method is only occasionally used. 2. Subcutaneous Injection. — A hypodermic syringe is filled with the substance to be inoculated. The part chosen for inoculation is either near the root of the tail, or between the scapula?, the advantage being that the animal cannot suck the point of inoculation in such situations. The hair is cut off the part, and the skin purified with 1 to 1000 cor- rosive sublimate. The skin is then pinched up and, the INTRAPERITONEAL INJECTION. 129 needle being inserted, the requisite dose is administered. The wound is then sealed with a little collodion. 3. Intraperitoneal Injection. — This is best performed by means of a special form of needle. The needle is curved, and has its opening not at the point, but in the side in the middle of the arch (Fig. 43). The hair over the lower part of the abdomen is cut, and the skin purified with an antiseptic. The whole thickness of the abdominal walls is then pinched up by an assistant, between the forefingers and thumbs of the two hands. The needle is then plunged through the fold thus formed. The result is that the hole in the side of the needle is within the abdominal cavity, and the inoculation can thus be made. Intraperitoneal inocu- lation can also be practised with an or- dinary needle. The mode of procedure , FlG- «— Ho!" is similar but after the needle is' plunged '"^eZl through the abdominal fold, it is partially (at a) for intra, withdrawn till the point is felt to be free peritoneal inocu" in the peritoneal cavity when the in- latl°'1S' jection is made. There is little risk of injuring the intestines by either method. _ 4- Intravenous Injection.- -The vein most usually chosen is one of the auricular veins. The part has the hair removed, the skin is purified, and the vein made prominent by ZITKT ^ hTVeeVhe P°int of inoculation and the heart The needle is then plunged into the vein and the fluid injected. That it has perforated the vessel will be tionT, I I eS?Pe °f " littlG bl00d i and that the injec- knnwn h ^ ^ ^ °f the Vessel will be known by the absence of the small swelling which occurs in subcutaneous injection, If preferred, therein may be firs" inttduced SmPPmg Skin °Ver lt ThG need1^ is then 5- Inoculation into the Anterior Chamber of the Eve — Local anesthesia is established by applying a few drops of •3° INOCULATION OF ANIMALS. 2 per cent solution of hydrochlorate of cocaine. The eye is fixed by pinching up the orbital conjunctiva with a pair of fine forceps, and the edge of the cornea being perforated by the hypodermic needle, the injection is easily accom- plished. Sometimes inoculations are made by planting small pieces of pathological tissues in the subcutaneous tissue. This is especially used in the case of glanders and tubercle. The skin over the back is purified, and the hair cut. A small incision is made with a sterile knife, and the skin being separated from the subjacent tissues by means of the ends of a blunt pair of forceps, a little pocket is formed into which a piece of the suspected tissue is inserted. The wound is then closed with a suture, and collodion is applied. In the case of guinea-pigs, the abdominal wall is to be preferred as the site of inoculation, as the skin over the back is extremely thick. Injections are sometimes made into other parts of the body, e.g., the pleurae and the cranium. It is unnecessary to describe these, as the application of the general principles employed above, together with those of modern aseptic surgery, will sufficiently guide the investigator as to the technique which is requisite. After inoculation, the animals ought to be kept in com- fortable cages, which must be capable subsequently of easy and thorough disinfection. For this purpose galvanised iron wire cages are the best. They can easily be sterilised by boiling them in the large fish-kettle which it is useful to have in a bacteriological laboratory for such a purpose. It is preferable to have the cages opening from above. Other- wise material which may be infective may be scratched out of the cage by the animal. The general condition of the animal is to be observed, how far it differs from the normal, whether there is increased rapidity of breathing, etc. The temperature is usually to be taken. This is generally done per rectum. The thermometer (the ordinary 5 min. clinical variety) is smeared with vaseline, and the bulb inserted just within the sphincter, where it is allowed A UTOPSIES ON ANIMALS. 1 3 1 to remain for a minute ; it is then pushed well into the rectum for five minutes. If this precaution be not adopted a reflex contraction of the vessels may take place, which is likely to vitiate the result by giving too low a reading Autopsies on Animals dead or killed after Inoculation —These should be made as soon as possible after death It is necessary to have some shallow troughs, constructed either of metal or of wood covered with metal, conveniently with sheet lead, and having a perforation at each corner to admit a tape or strong cord. The animal is tightly stretched out in the trough and tied in position. The size of the trough will, therefore, have to vary with the size of the outstretched body of the animal to be examined In certain cases it is well to soak the surface of the animal n carbolic acid solution (1 to 20), or in corrosive sublimate (1 to 1000) before it is tied out. This not only to a certain extent disinfects the skin but, what is more im- portant, prevents hairs which might be infected with patho- genic products from getting into the air of the laboratory The ins ruments necessary are scalpels (preferably with metal handles), dissecting forceps, and scissors. They are to be sterilised by boiling for five minutes. This is conveniently done in one of the small portable sterilisers used bv surgeon, Two sets at least ought to be used n I autopsy and they may be placed, a&fter boiling, on a tenle glass plate covered by a bell jar. It is also nece sa y to have a medium-sized hatchet-shaped cautery, or 0Ther similar piece of metal. It is well to have prepared a few freshly-drawn-out capillary tubes stored in a s er e cvhn dncal glass vessel, and also some larger sterile glass pipeSes' The hair of the abdomen of the animal is Amoved If some of the peritoneal fluid is wanted a band 1^ u cauterised down the linea alba from the s^ut to ^ pubes, and another at right angles to the upper Tnd of tht an incision should be made in the m.VMi P1f\ena ot thls> and the abdominal walls ZZ lolXZ O„?o ^ capillary tubes should then be filled lith,L fl / F "^ «» ™ "eing afiowfd tutS 1 32 INOCULATION OF ANIMALS. the point sealed off; or a larger quantity, if desired, is taken in a sterile pipette. If peritoneal fluid be not wanted, then an incision may be made from the episternum to the pubes, and the thorax and abdomen opened in the usual way. The organs ought to be removed with another set of instru- ments and it is convenient to place them pending examina- tion in deep Petri's capsules (sterile). It is generally advisable to make cultures and film preparations from the heart's blood. To do this, open the pericardium, sear the front of the right ventricle with a cautery, make an incision in the middle of the part seared, and remove some of the blood with a capillary tube for future examination or, intro- ducing a platinum eyelet, inoculate tubes and make cover- glass preparations at once. To examine any organ sew die surface with a cautery, cut into it, and inoculate tubes and make film preparations with a platinum loop, tor removing small parts of organs for making inoculations on ubes, a small platinum spud is very useful, as the ordinary wires are apt to become bent. Place pieces of the organs m some preservative fluid for microscopic examination. ?he oTgans ought not to be touched with the fingers. When 2 poft cartel is concluded the body should nave corrosive sublimate or carbolic acid solution poured over ^ and ^ forthwith burned. The dissecting trough and aU the ins u mentl ought to be boiled for half an hour The amount of ^caution to be taken will, of course, depend on the character of the bacterium under investigation, but as a general rule every care should be used. CHAPTER IV. NON-PATHOGENIC MICRO-ORGANISMS— FUNGI. It is quite outside the scope of the present volume to describe any bacteria other than those giving rise to disease processes. In the course of his work the bacterio- logist frequently meets with ordinary saprophytic organisms. These may occur in diseased organs in which putrefaction has already begun to take place, and they may therefore appear in cultures made from such organs. Their source in cultures may, further, be by contamination from the air, or from the use of insufficiently sterilised vessels or instru- ments. The positive characters of the pathogenic bacteria will be given, and from these other bacteria must be dis- tinguished by the application of the methods of diagnosis already detailed,, or by the special methods still to be described. There occur, however, from time to time as contaminations of bacterial cultures, organisms of a more complicated structure than the bacteria, namely fungi, and therefore we shall describe a few of the typical forms of these. The fungi have probably descended from the algae, or both have had a common ancestor. This is shown by the close resemblances in structure and development which the two groups present to each other. The chief differences centre round the degeneration of structure which the adop- tion of parasitism and saprophytism entails on the fungi. In the algae, reproduction takes place in both sexual and 134 NON- PATHOGENIC MICRO-ORGANISMS. non-sexual ways. In the former case, certain cells called gametes are set apart, and by the union of two of these — the embryonic male and female elements — a new cell called a zygospore is formed which after a period of rest grows into a new individual. Sometimes there is a more definite male element, the antherozoid, and a female ele- ment, the oosphere, and the coalescence of these forms an oospore which subsequently behaves like a zygospore. In the non-sexual reproduction there are formed certain cells called sporangia, the protoplasm of which, without being re- inforced from that of another cell, proceeds to break up into the elements of new individuals often called, when motile, swarm-spores. Both forms of reproduction are usually manifested by each species. The degradation of the fungi consists in the gradual loss of the faculty of sexual repro- duction, so that, in the most extreme species of the group, it does not appear at all and only asexual reproduction can be traced. We shall now describe a few of the typical forms of these lower fungi which are often met with in bacteriological work. Mucorinse : Mucor Mucedo.— This form occurs especi- ally in the putrefaction of horse dung and also in other putrefactions. To the naked eye it appears as a white or brownish-white mass of fine filaments, from which, here and there, rise special filaments often several inches long, having at their terminations spherical brown swellings, the reproductive elements. Microscopically, the plant consists of branching non-septate filaments. Such a structure is called a mycelium. The non-sexual is the commonest form of reproduction {vide Fig. 44 A 4). One of the fila- ments grows out, at its termination a septum forms, and a globular swelling (the sporangium) appears. This sporan- gium possesses a definite membrane. Within it from he septum grows a club-shaped mass of protoplasm called the columella, to which are attached the swarm-spores formed from the breaking up of the rest of the protoplasm. Vv hen ripe the brood cell bursts, the brown swarm-spores are cast off and from each a new individual arises. Under certain FUNGI. 135 Fic;. 44. — A. Mucor mucedo ; (1) (2) (3) stages in formation of a zygospore ; (4) a sporangium containing spores. B. Oidium lactis. C. Aspergillus glaucus (De Hary) ; (1) mycelium ; (2) and (5) gonidiophore bearing spores ; (3) (4) a perithecium (4 contains rudimentary asci) ; (6) piece of gonidiophore ; («) sterigma ; (/>) spore. D. Branched gonidio- phore of penicillium glaucum hearing spores. E, F. Saccharomyces cerevisia.-, cells are budding. G. Ditto, formation of endospores (after 136 NON-PATHOGENIC MICRO-ORGANISMS. circumstances sexual reproduction occurs {vide Fig. 44 A 1-3). Two filaments approach each other, and a small piece of the protoplasm of each being cut off by a septum, these parts coalesce. A zygospore is thus formed from which a new filamentous individual arises. Ascomycetse : Oidium Lactis (Fig. 44 B). — This is a common organism in sour milk and sour bread. It can easily be cultivated on gelatine where the colonies appear to consist of fine white filaments radiating from a centre. Microscopically here and there the filaments (which may be branched) are broken up, especially at the ends, into short rod-shaped or oval segments often referred to as the oidia. These behave like spores. Non-sexual reproduc- tion also takes place by the formation, within certain special sporangia in the filament called asci, of a definite number of spores to which the special name of ascospores is applied. Perisporiacese : (1) Aspergillus Niger {vide Fig. 44 C). — This, with other varieties of the same group is of fre- quent occurrence, especially in vegetable putrefactions. It grows readily in gelatine. It consists, to the naked eye, like the other fungi described, of a mass of felted filaments which microscopically are seen to form a septate branching mycelium. Though it is a matter of doubt whether sexual reproduction takes place, two forms of reproduction occur, the variety depending largely on the nutrition of the plant. The less common form is effected by means of the for- mation of structures known as perithecia, and it may perhaps be that the perithecia owe their formation to a sexual act. From a mycelial branch there arises a filament (or hypha) which becomes specially coiled and transversely septate at its end. From the base of the lowest coil of the spiral two or three hyphse grow up towards its apex. One of these being the first to reach the apex was regarded by De Bary as a male organ. The others by branching copiously produce a mass of closely woven hyphse which form a closed wall to this structure, which is the perithelium referred to. Within it numerous asci arise, as the ultimate YEASTS AND TORULAl. 137 ramifications of branches given off by the central coiled hypha. Inside each ascus eight ascospores are produced. Ultimately all the structures lying within the perithecium, save the spores, undergo disintegration, so that the mature perithecium consists of a small hollow sphere within which lie the loose spores. These latter are ultimately freed by the decay of the wall of the perithecium and develop into new individuals. The commonest method of reproduction is by the formation of spores (gonidia or conidia), which are clearly of non-sexual origin. These are formed exter- nally in the hypha? and not inside sporangia. A filament grows out, and at its termination a club-shaped swelling is formed on which a series of flask-shaped cells, called sterig- mata (vide Fig. 44 C 6), are perched. At the free end of each of these, an oval body, the spore or gonidium is formed, and this becoming free, can give rise to a new individual. (2) Penicillium Glaucum. — This is perhaps the most common of all the fungi met with in bacteriological work. It is the common green cheese mould, and its spores are practically omnipresent. The mycelium is like that of the aspergillus. Perithecium formation takes place, but the commonest mode of reproduction is by gonidia (vide Fig. 44 D). A filament (called a gonidiophore) grows out, and at its end breaks up into a number of finger-like branches. On the point of each of these a flask-shaped sterigma is developed. On the end of this a row of oval spores appears. These break off, and can give rise to new individuals. Yeasts and Torulse : Saccharomyces, Torula, Myco- derma. — These are of the greatest importance, of course, in brewing and baking. They only concern us as being of not uncommon occurrence in the air. They consist of round or oval cells usually many times larger than bacteria. They often reproduce themselves by budding (vide Fig. 44 E, F), a portion of the cell protruding, and finally being cut off to form a new individual. Endogenous spore formation also occurs (vide Fig. 44 G). Many of the 1 38 NON- PA THO GENIC MICRO- ORG A N1SMS. torulae, when growing in colonies, are brilliantly coloured. What their true morphological relationships are it is diffi- cult to say, but they present many analogies to the oidia of such forms as oidium lactis. A knowledge of the above type forms will enable the student to recognise the more common fungi as such, when they present themselves to him. For further information on this group he is referred to De Bary's book on The Fungi. Certain fungi closely related to the above are pathogenic agents. Some aspergilli have been found to grow in the animal tissues and to produce death, and to the fungi also belong the saprolegnia ferax (the cause of a disease of salmon), the tinea tonsurans, and the Achorion Schoenleinii. CHAPTER V. RELATIONS OF BACTERIA TO DISEASE— THE PRODUCTION OF TOXINES BY BACTERIA. Introductory. — It has already been stated that a strict division of micro-organisms into saprophytes and true parasites cannot be made. No doubt there are organisms such as the bacillus of leprosy and the spirillum of relaps- ing fever which as yet have not been cultivated outside the animal body, and others, such as the gonococcus, which are in natural conditions always parasites associated with disease. But these latter can lead a saprophytic existence in specially prepared conditions, and there are many of the disease-producing organisms, such as the organisms of typhoid and cholera, which can nourish readily outside the body, even in ordinary conditions. The conditions of growth are however of very great importance in the study of the modes of infection in the various diseases, though they do not form the basis of a scientific division. A similar statement applies to the terms pathogenic and saprophytic, and even to the terms pathogenic and non- pathogenic. By the term pathogenic is meant the power which an organism has of producing morbid changes or effects in the animal body, either under natural conditions or in conditions artificially arranged as in direct experi- ment. Now we know of no organisms which will in all circumstances produce disease in all animals, and, on the other hand, many bacteria described as harmless saprophytes RELATIONS OF BACTERIA TO DISEASE. will produce pathological changes if introduced in sufficient quantity. When, therefore, we speak of a pathogenic organism, the term is merely a relative one, and indicates that in certain circumstances the organism will produce disease, though in the science of human pathology it is often used for convenience as implying that the organism produces disease in man in natural conditions. Modifying Conditions. — In studying the pathogenic effects in any instance, both the micro-organisms and the animal affected must be considered, and not only the species of each, but also its exact condition at the time of infection. In other words, the resulting disease is the product of the sum total of the characters of the infecting agent, on the one hand, and of the subject of infection, on the other. We may, therefore, state some of the chief circumstances which modify each of these two factors involved and, con- sequently, the diseased condition produced. i. The Infecting Agent. — In the case of a particular species of bacterium its effect will depend chiefly upon (a) its virulence, and (&) the number introduced into the body. To these may be added (c) the path of infection. The virulence, i.e. the power of multiplying in the body and producing disease, varies greatly in different conditions, and the methods by which it can be diminished or in- creased will be afterwards described {vide Chapter XIX.). One important point is that when a bacterium has been enabled to invade and multiply in the tissues of an animal, its virulence for that species is often increased. This is well seen in the case of certain bacteria which are normally present on the skin or mucous surfaces. Thus it has been repeatedly proved that the bacillus coli cultivated from a septic peritonitis is much more virulent than when taken from the bowel of the same animal. The virulence may be still more increased by inoculating from one animal to another in series— the method of passage. Widely different effects are, of course, produced on the virulence being altered. For example, a streptococcus which produces merely a local inflammation or suppuration, may produce a CONDITIONS MODIFYING PATHOGENICITY. 141 rapidly fatal septicaemia when its virulence is raised. Virulence also has a relation to the animal employed, as sometimes on being increased for one species of animal it is diminished for another. For example, streptococci, on being inoculated in series through a number of mice, acquire increased virulence for these animals, but become less virulent for rabbits. (Knorr.) The number of the organisms introduced, i.e. the dose of the infecting agent, is another point of importance. The healthy tissues can usually resist a certain number of pathogenic organisms of given virulence, and it is only in a few instances that one or two organisms introduced will produce a fatal disease, e.g., the case of anthrax in white mice. The healthy peritoneum of a rabbit can resist and destroy a considerable number of pyogenic micrococci without becoming inflamed, but if a larger dose be intro- duced, inflammation or suppuration will follow. Again, a certain quantity of a particular organism injected subcutane- ously may produce only a local inflammatory change, but in the case of a larger dose the organisms may gain entrance to the blood stream and produce septicaemia. There is, therefore, for a particular animal, a minimum lethal dose which can be determined by experiment only. The path of infection may alter the result, serious effects often following a direct entrance into the blood stream. Staphy- lococci injected subcutaneously in a rabbit may produce only a local abscess, whilst on intravenous injection multiple abscesses in certain organs may result and death may follow. Local inflammatory reaction with subsequent destruction of the organisms may be restricted to the site of infection or may occur also in the lymphatic glands in relation. The latter therefore act as a second barrier of defence, or as a filtering mechanism which aids in protecting against blood infection. This is well illustrated in the case of " poisoned wounds." In some other cases, however, the organisms are very rapidly destroyed in the blood stream, and Klemperer has found that in the dog, subcutaneous 142 RELATIONS OF BACTERIA TO DISEASE. injection of the pneumococcus produces death more readily than intravenous injection. , 2. The Subject of Infection. — Amongst healthy individ- uals susceptibility and, in inverse ratio, resistance to a particular microbe may vary according to (a) species, (b) race and individual peculiarities, (c) age. Different species of the lower animals show the widest variation in this respect, some being extremely susceptible, others highly resistant. Then there are diseases, such as leprosy, gonor- rhoea, etc., which appear to be peculiar to the human subject and have not yet been transmitted to animals. And further, there are others, such as cholera and typhoid, which do not naturally affect animals, and the typical lesions of which cannot be experimentally reproduced in them, or appear only imperfectly, although . pathogenic effects follow inoculation with the organisms. In the case of the human subject, differences in susceptibility to a certain disease are found amongst different races and also amongst individuals of the same race, as is well seen in the case of tubercle and other diseases. Age also plays an important part, young subjects being more liable to certain diseases, e.g., to diphtheria. Further, at different periods of life certain parts of the body are more susceptible, for example, in early life, the bones and joints to tubercular and acute suppurative affections. In increasing the susceptibility of a given individual, conditions of local or general diminished vitality play the most important part. It has been experimentally proved that conditions such as exposure to cold, fatigue, starvation, etc., all diminish the natural resistance to bacterial infection. Rats naturally immune can be rendered susceptible to glanders by being fed with phloridzin, which produces a sort of diabetes, a large amount of sugar being excreted in the urine (Leo). Guinea-pigs may resist subcutaneous in- jection of a certain dose of the typhoid bacillus, but if at the same time a sterilised culture of the bacillus coli be injected into the peritoneum, they quickly die of a general infection. Also a local susceptibility may be produced by SUSCEPTIBILITY AND RESISTANCE. 143 injuring or diminishing the vitality of a part. If, for example, previously to an intravenous injection of staphy- lococci, the aortic cusps of a rabbit be injured, the organisms may settle there and set up an ulcerative endocarditis ; or if a bone be injured, they may produce suppuration at the part, whereas in ordinary circumstances these lesions would not take place. The action of one species of bacterium is also often aided by the simultaneous presence of other species. In this case the latter may act simply as additional irritants which lessen the vitality of the tissues, but in some cases their presence also favours the development of a higher degree of virulence of the former. These facts, established by experiment (and many others might be given), illustrate the important part which local or general conditions of diminished vitality may play in the production of disease in the human subject. This has long been known by clinical observation. In normal conditions the blood and tissues of the body, with the exception of the skin and certain of the mucous surfaces, are bacterium- free, and if a few organisms gain entrance, they are de- stroyed. But if the vitality becomes lowered their entrance becomes easier and the possibility of their multiplying and producing disease greatly increased. In this way the favouring part played by fatigue, cold, etc., in the produc- tion of diseases of which the direct cause is a bacterium, may be understood. It is important to keep in view in this connection 'that many of the inflammation-producing and pyogenic organisms are normally present on the skin and various mucous surfaces. The action of a certain organism may devitalise the tissues to such an extent as to pave the way for the entrance of other bacteria ; we may mention the liability of the occurrence of pneumonia, erysipelas, and various suppurative conditions in the course of or following infective fevers. In some cases the specific organism may produce lesions through which the other organisms gain entrance, e.g., in typhoid, diphtheria, etc. It is not uncommon to find in the bodies of those who have died from chronic wasting disease, collections of micro- i44 RELATIONS OF BACTERIA TO DISEASE. cocci or bacilli in the capillaries of various organs, which have entered in the later hours of life ; that is to say, the bacterium-free condition of the blood has been lost in the period of prostration preceding death. The methods by which the natural resistance may be increased belong to the subject of immunity, and are described in the chapter on that subject. Modes of Bacterial Action. — In the production of disease by micro-organisms there are two main factors in- volved, namely (a) the multiplication of the living organisms after they have entered the body, and (/>), the production by them of poisons which may act both upon the tissues around and upon the body generally. The former corresponds to infection, the latter is of the nature of intoxication or poisoning. In different diseases one of these is usually the more prominent feature, but both are always more or less concerned. i. Infection and Distribution of the Bacteria in the Body. After pathogenic bacteria have invaded the tissues, or in other words after infection by bacteria has taken place, their further behaviour varies greatly in different cases. In certain cases they may reach and multiply in the blood stream, producing a fatal septicaemia. In the lower animals this multiplication of the organisms in the blood throughout the body may be very extensive (for example, the septi- cemia produced by the pneumococcus in rabbits) ; but m septicemia in man, it very seldom, if ever, occurs to so great a degree, the organisms rarely remain in large numbers in the circulating blood, and their detection in it during life by microscopic examination, and even by culture methods, is rare. In such cases, however, the organisms may be found post mortem lying in large numbers within the capillaries of various organs, e.g., in cases of septicemia produced by streptococci. (Relapsing fever forms an exception, as in it numerous organisms may be seen in a drop of blood.) In the human subject more frequently one of two things happens. In the first place, the organ- isms may remain local, producing little reaction around MODES OF BACTERIAL ACTION. 145 them, as in tetanus, or a well-marked lesion, as in diph- theria, pneumonia, etc. Or in the second place, they may pass by the lymph or blood stream to other parts or organs in which they settle, multiply, and produce lesions, as in tubercle. 2. Production of Chemical Poisons. — In all these cases the growth of the organisms is accompanied by the forma- tion of chemical products, which act generally or locally in varying degree as toxic substances. The toxic substances become diffused throughout the system, and their effects are manifested chiefly by symptoms such as the occurrence of fever, disturbances of the circulatory, respiratory, and nervous systems, etc. In some cases corresponding changes in the tissues are found, for example, the changes in the nervous system in diphtheria, to be afterwards described. The general toxic effects may be so slight as to be of no importance, as in the case of a local suppuration, or they may be very intense as in tetanus, or again, less severe but producing cachexia by their long continuance, as in tuberculosis. The occurrence of local tissue changes or lesions produced in the neighbourhood of the bacteria, as already mentioned, is one of the most striking results of bacterial action, but these also must be traced to chemical substances formed in or around the bacteria, and either directly or through the medium of ferments. In this case it is more difficult to demonstrate the mode of action, for, in the tissues the chemical products are formed by the bacteria slowly, con- tinuously, and in a certain degree of concentration, and these conditions cannot be exactly reproduced by experi- ment. Further, it is very doubtful whether all the chemical substances formed by a certain bacillus growing in the tissues are also formed by it in cultures outside the body, rhe separated toxine of diphtheria, like various vegetable and animal toxines {vide infra), however, possesses a local toxic action of very intense character, often producing extensive necrotic change. The injection of large quantities of many different patho- 10 ,46 RELATIONS OF BACTERIA TO DISEASE. genie organisms in the dead conditions results in the production of a local inflammatory change which may be followed by suppuration, this effect being possibly brought about by certain substances in the bacterial protoplasm common to various species, or at least possessing a com- mon physiological action (Buchner and others). When dead tubercle bacilli, however, are introduced into the blood stream, nodules do result in certain parts which have a resemblance to ordinary tubercles. In this case the bodies of the bacilli evidently contain a highly resistan and slowly acting substance which gradually diffuses around and produces effects (vide Tuberculosis). ItPmay be here pointed out that there is, however no relation between the toxic effects of an organism , and h extent to which it invades the tissues. Some of the danisms which produce the most highly toxic effects have a comparatively localised sphere of ^^Vnt the and others multiply with great freedom th £ugh°ut the blood while their toxic effects in proportion to their number aw small. But there is, on the other hand, no Cwn examp" of a bacterium multiplying in the living " sues without producing distinct local or g very r^^SS^uhted by the position and ^o/yowTh of the or Ssms? can be accounted for only by the formation of definite chemical substances which act ^ ^^e may say ^ as disease-producers, as in fact hei power multiply in the living body depends upon ^ products formed directly or ind recti) y t^^^^T^ on the products. TISSUE CHANGES PRODUCED BY BACTERIA. 147 Effects of Bacterial Action. These may be for convenience arranged in a tabular form as follows : — Tissue Changes. (r) Local changes, i.e., changes produced in the neighbourhood of the bacteria. Position (a) At primary lesion. (b) At secondary foci. Character (a) Vascular changes and tissue] Acute reactions L or (b) Degeneration and necrosis J Chronic. (2) Produced at a distance from the bacteria by absorption of toxines. (a) In special tissues, e.g., nerve cells and fibres, secreting cells, vessel walls, etc. (b) General effects of malnutrition, etc. Symptoms. (a) Associated with known tissue changes. (b) Without known tissue changes. Tissue Changes produced by Bacteria.— The effects of bacterial action are so various as to include almost all known pathological changes. They may be classified as local effects or lesions produced in the neighbourhood of the bacteria, and general changes which are produced in various parts of the system by the circulation of the bacterial products. As already stated, both the local and the general effects are due to the products of the bacteria, but the substances which produce local disturbances may not be whltyo \ "T6 ^ m°re concentr^d form as those which act on distant parts of the system. In diphtheria mate™- 6 PrfUCtS WhiGh P1'0duce the lo-> inflam- matory reaction and necrosis are probably not the same as 148 RELATIONS OF BACTERIA TO DISEASE. those which act on the nerve fibres and cells. Further, it may be again stated that the action of the products circulating in the system is often manifested more by symptoms than by tissue changes, though our knowledge of the latter, especially in the nervous system, is gradually being extended. (i) Local Lesions.— By this is meant the changes pro- duced in the neighbourhood of the bacteria. These changes are, on the one hand, of the nature of inflam- matory reaction, from the most intense vascular changes in acute inflammations to the more or less chronic pro- liferative changes especially of connective tissue, and, on the other hand, of the nature of cell-poisoning, leading to degeneration or necrosis, especially of the more highly- developed elements. They may be roughly classified as acute and chronic changes. As already pointed out, the effects of a given organism vary in different animals, and further, where the lesion is approximately the same in different animals, differences in their minor characters may be found. Examples of this latter are furnished m the case of tubercle. Position of Lesions.-^ some diseases the lesion has a snecial site ; for example, the lesion of typhoid fever and' to? a less -tent, that of diphtheria. In other cases it depends entirely upon the point of entrance, e.g malignant puLle and the conditions known as wound infections. In others again, there is a special tendency for certain Parts to be affected, as the upper parts of the lungs m tubercle. In some cases the site has a mechanical ^me^oiganisms gain an entrance to the blood from a orimarv lesion, directly or by the lymphatic system, they Say become destroyed! or they may -ttlem certain c,gan and produce their characteristic effects. The organs speci auv hable to be affected in this way vary in different dJeases Pyogenic cocci show a special tendency to s de n the capillaries of the kidneys and produce rnihary abscesses, whilst these lesions rarely occur in the spleen. LOCAL LESIONS. 149 On the other hand, the nodules in disseminated tubercle or glanders are much more numerous in the spleen than in the kidneys, which in the latter disease are usually free from them. The important point is that the position of the disseminated lesions is not to be explained by a mechanical process, such as embolism, but depends upon a special relation between the organisms and the tissues, which may be spoken of either as a selective power on the part of the organisms or a special suscepti- bility of tissues, possibly in part due to their affording to the organisms more suitable conditions of nutriment. Even in the case of the lesions produced by dead tubercle bacilli, a certain selective action is found. Acute Local Lesions. — The local inflammatory reaction presents different characters in different conditions. It may be accompanied by abundant fibrinous exudation, or by great catarrh (in the case of an epithelial surface), or by haemorrhage, or by cedema ; it may be localised or spreading in character ; it may be followed by suppuration, or may lead to necrosis. A few examples may be given. A great many different organisms cause an abundant fibrinous exudation. This, along with necrosis of epi- thelium, is the action of the diphtheria bacillus on a mucous membrane, and also of streptococci in certain conditions ; it is produced in the alveoli of the lung in croupous pneumonia by the pneumococcus and probably by other organisms, whilst fibrinous inflammation in serous cavities is produced by a great many different bacteria. The last statement also applies to numerous suppurative and catar- rhal conditions. The inflammatory change in a Peyer's patch in typhoid fever, though fibrinous exudation is less marked, is followed by necrosis, while in the malignant pustule of man, necrotic change attended by considerable haemorrhage is one of the chief features. The great variety in local reaction is well illustrated in the case of skin lesions produced by bacteria. The necrotic or degenerative changes affecting especially the more highly developed elements are chiefly produced by the direct action of the bacterial 150 RELATIONS OF BACTERIA TO DISEASE. poisons, though aided by the disturbances of nutrition involved in the vascular phenomena. In many of the acute inflammatory conditions, if not attended by a fatal result, the disease comes to a natural termination after a certain time, e.g., in pneumonia, ery- sipelas, etc. This fact, the explanation of which is not yet fully understood, has an important relation to the subject of immunity, and will be discussed later. It may also be pointed out that a well-marked inflammatory re- action is often found in animals which occupy a medium position in the scale of susceptibility, and that an organism which causes a general infection in a certain animal may produce only a local inflammation when its virulence is lessened. Chronic Local Lesions. — In a considerable number of diseases produced by bacteria the local tissue reaction is a more chronic process than those described. In other words, the specific irritant is less intense, so that there is less vascular disturbance and a greater preponderance of the proliferative processes, leading to new formation of tissue. This formation may occur in foci here and there, so that nodules of greater or less consistence result, or it may be more diffuse. Such changes especially occur in the diseases often known as the infective granulomata, of which tubercle, leprosy, glanders, actinomycosis, syphilis, etc., are examples. A hard and fast line, however, cannot be 'drawn between such conditions and those described above as acute. In glanders, for example, especially in man, the lesion produced by the glanders bacillus often approaches very nearly to an acute suppurative change, and sometimes actually is of this nature. Whilst in these diseases the fundamental change is the same, viz., a re- action to an irritant of minor intensity, the . exact structural characters and arrangement vary in different diseases In some cases the disease may be identified by the histological changes alone, but on the other hand, this is often impossible. In tubercle, for example, m addition to the proliferative change, a cellular necrosis GENERAL LESIONS. leading to caseation is produced by the bacilli, whereas in leprosy the latter does not occur, though there may be a certain amount of degeneration and vacuolation of cells. In tubercle, giant-cells of somewhat characteristic appearance are found ; in leprosy, large rounded cells often called " lepra-cells " occur in large numbers ; in actinomycosis bovis, there is an extensive growth of spindle-celled granula- tion tissue which may form large masses, and so on. Infection of other parts from the primary lesion takes place chiefly by the blood vessels and lymphatics, though sometimes along natural tubes such as the bronchi, intes- tine, etc. The organs specially liable to be the site of secondary lesions vary in different diseases, as already explained. (2) General Lesions produced by Toxines. — In the various infective conditions produced by bacteria, changes com- monly occur in certain organs unassociated with the presence of the bacteria ; these are produced by the action of bacterial products circulating in the blood. Many such lesions can be produced experimentally. The secreting cells of various organs, especially the kidney and liver, are specially liable to change of this kind. Cloudy swelling, which may be followed by fatty change or by actual necrosis with granular disintegration, is common. Hyaline change in the walls of arterioles may occur, and in certain chronic conditions waxy change is brought about in a similar manner. The latter has been produced in animals by the repeated injection of the staphylococcus aureus. Capillary haemorrhages are not uncommon, and are in many cases due to an increased permeability of the vessel walls, aided by changes in the blood plasma, as evidenced sometimes by diminished coagulability. Similar haemorrhages may follow the injection of some bacterial toxines, e.g., of diphtheria, and also of vegetable poisons, e.g., ricin and abrin. Skin eruptions occurring in the ex- anthemata are probably produced in the same way, though in many of these diseases the causal organism has not yet been isolated. We have, however, the important fact that corre- 152 RELATIONS OF BACTERIA TO DISEASE. spending skin eruptions may be produced by poisoning with certain drugs. In the nervous system degenerative changes have been found in diphtheria, both in the spinal cord and in the peripheral nerves, and have been re- produced experimentally by the products of the diphtheria bacilli. There is also experimental evidence that the bacillus coli communis and the streptococcus pyogenes may, by means of their products, produce areas of softening in the spinal cord, and this may furnish an explanation of some of the lesions found clinically. It is also possible that some serous inflammations may be produced in the same way. General malnutrition and cachexia are, of course, of common occurrence, and it is a striking fact found by experiment that after injection of bacterial products, e.g., of the diphtheria bacillus, a marked loss of body weight often occurs which may be progressive, and ultimately lead to the death of the animal. Symptoms. — Many of the symptoms occurring in bacterial affections are produced by the histological changes mentioned, as can be readily understood ; whilst in the case of others, corresponding changes have not yet been discovered. Of the latter, fever, with its disturbances of metabolism and manifold affections of the various systems, is the most important. The nervous system is especially liable to be affected — convulsions, spasms, coma, paralysis, etc. being common. The secretory function of the glands of the alimentary canal, of the salivary glands, may be dis- turbed or practically stopped, — a striking analogy to what is found in the action of various drugs. These tissue changes and symptoms are given only as illustrative examples, and the list might easily be greatly amplified. The important fact, however, is that nearly all, if not quite all, the changes found throughout the organs (without the actual presence of bacterid), and also the symptoms occurring in infective diseases, can either be experi- mentally reproduced by the injection of bacterial poisons or have an analogy in the action of drugs. TOXINES PRODUCED BY BACTERIA Thr Toxines Produced by Bacteria. Early Work on Toxines.— We know that bacteria are capable of giving rise to poisonous bodies within the animal body and also in artificial media. As we shall see, we know comparatively little of the actual nature of such bodies, and therefore we apply to them as a class the general term toxines. The fact that in the case of many diseases, undoubtedly caused by bacteria, the latter are not distributed throughout the body, directed attention to the necessity for the existence of such toxines in order to explain the general pathogenic effects occurring in such circumstances. The first to systematically study the pro- duction of poisonous bodies by bacteria was Brieger. This observer, directing attention to general putrefactive processes as they occurred under natural conditions, e.g., in putrefying flesh, etc., isolated a series of crystalline nitrogen-containing bodies giving the reactions of alkaloids, and which he called ptomaines. Similar bodies occurring in the ordinary metabolic processes of the body had pre- viously been described and called leucom nines. Brieger further isolated ptomaines from media containing pure cultures of many of the pathogenic bacteria. These ptomaines, however, on being injected into animals sus- ceptible to the corresponding diseases, in no case except, perhaps, in tetanus, reproduced characteristic symptoms ■ and even in tetanus the effects produced only bore a dis- tant resemblance to those caused by the injection of the bacillus itself. Brieger's methods of obtaining these bodies were faulty. For instance, his use of hot mineral acids in the treatment of the crude material was shown to be sufficient to cause serious changes in the complex albumin- ous bodies present. His earlier results have therefore only a historic interest. The introduction of the principle of rendering fluid cultures bacteria-free by filtration through unglazed porce- lain and its application by Roux and Yersin to obtain m the case of the B. diphtheria, a solution containing a fS4 THE TOXINES PRODUCED BY BACTERIA. toxine which reproduced the symptoms of this disease (vide Chap. XV.), encouraged the further inquiry as to the nature of this toxine. The products of the B. diphtheria; were investigated again by Brieger, now in conjunction with C. Fraenkel. The filtrate was evaporated to a third of its bulk in vacuo, at a temperature not exceeding 30° C, and was precipitated by alcohol. The precipitate was redis- solved in water, reprecipitated by alcohol, and this opera- tion being repeated six to eight times a final product in the form of a white powder was obtained. The chemical procedure was thus in principle simple, and the toxine stood the two tests given by the original fluid, "namely (1) its specific toxicity to animals, and (2) destruc- tion of its toxic power by two hours' exposure to a tem- perature of 5 8° C. This substance, if it did not consist entirely of the diphtheria toxine, certainly contained the latter and from resemblances observed in it to serum albumin, was called by its discoverers a toxalbumin. Similar toxic bodies were obtained in the same way from the bacteria of tetanus, typhoid, and cholera, and also from the staphylococcus aureus. In the case of tetanus, in the one experiment recorded, where the toxalbumin was injected into a guinea-pig, death with spasms and paralysis resulted With the other toxalbumins, however, though death occurred from their injection, no characteristic symptoms or pathological effects were observed. These toxalbumins presented no special chemical reaction hough the authors considered them allied to serum albumin. They probably consisted largely of alburnoses,* and con- tained the toxic bodies in mixture with ^}^^A The Occurrence of Bacterial Toxmes.— The following 1 In the digestion of albumins by the gastric an d pane reati ^ the alburnoses are a group of bodies formed P^^*? ^Ir not of peptone Like the ^^^X^^ They differ being coagulated by heat, and in being sugnuj y 1 fronfthe peptones in being preci p. ate ^SJd sulphate of of much sodium chloride, and J° ^ n^ The first alburnoses SSft, ^oTJVeX—^ "hetero-aibumose, ,hich OCCURRENCE OF BACTERIAL TOXINES. 155 may be regarded as the chief facts regarding bacterial toxines which have been revealed by the study, partly of the bodily tissues of animals infected by the bacteria con- cerned, partly by what occurs in artificial cultures of these bacteria. The dead bodies of certain species have been found to be very toxic. ' When, for instance, tubercle bacilli are killed by heat and injected into a susceptible animal tubercular nodules are found to develop round the sites where they have lodged. From this it is inferred that they must have contained characteristic toxines, seeing that characteristic lesions result. The bodies of the cholera vibrio are likewise toxic. Such intracellular toxines, as they have been called, may appear in the fluids in which the bacteria are living (1) by excretion in an unaltered or altered condition, (2) by the disintegration of the bodies of the organisms which we know are always dying in any bacterial growth. Sometimes the media in which bacteria are growing become extremely toxic. This is much greater in some cases than in others. The two best examples of bacteria producing soluble toxines are the diphtheria and tetanus bacilli. In these and similar cases when bouillon cultures are filtered bacterium-free by means of a porcelain filter, highly toxic fluids are obtained, which on injection into animals reproduce the highly characteristic symptoms of the corresponding diseases. In the case of the B. anthracis, at any rate when growing in artificial media, such toxine production is much less marked, a filtered bouillon culture being relatively non-toxic. It is probable, how- ever, that this may not occur when the bacillus is growing in an animal body, for we have often here well-marked differ in the insolubility of the latter in hot and cold water (insolubility and coagulability are quite different properties). They have been called the primary albumoses. By further digestion both pass into the second- ary albumose, deutero - albumose, which differs slightly in chemical reactions from the parent bodies, e.g., it cannot be precipitated from watery solutions by saturated sodium chloride unless a trace of acetic acid be present. Dysalbumose is probably merely a temporary modification 01 netero-albumose. Further digestion of deutero-albumose results in the formation of peptone. 156 THE TOXJNES PRODUCED BY BACTERIA. evidence of pathogenic effects being produced at a distance from the actual focus of bacterial growth. This is further an instance of what we have strong reason to believe some- times occurs, namely that the toxines produced by bacteria, when these are growing in the animal body, differ somewhat from the toxines produced by the' same bacteria growing ' in artificial media. Poisons appearing in cultures have been called extracellular toxines, but, as we shall see, we cannot as yet say whether they are excreted by the bacteria or whether they are produced by the latter acting on the constituents of the media. We therefore cannot as yet draw a hard and fast line between intra- and extracellular toxines, but the terms are convenient. The extracellular toxines are the more easily obtainable in large quantities, and it is their nature and effects which are best known. One other fact may be noted, namely, that different kinds of toxines, e.g., as regards action, destructibility by heat, etc., may be produced by the same bacillus. Such occurs in the case of the B. diphtherial The Nature of Toxines.— The earlier investigations upon toxines suggested that analogies exist between the modes of bacterial action and what takes place in ordinary gastric digestion, and the idea has been worked out for certain pathogenic bacteria by Sidney Martin. This observer took, not solutions artificially made up with albumoses but the natural fluids of the body or definite solutions of albumins, and further, never subjected the results of the bacterial growth to heat above 4o° C, nor to any stronger agent than absolute alcohol. He showed that albumoses and some- times peptones were formed by the action of the pathogenic bacteria studied, and further, that these albumoses were toxia In certain cases the process of splitting up of the albumins went further than in peptic digestion, and organic bases or acids might be formed. The characteristic symptoms of the diseases could be explained by compound ac tions in , wh ich the albumoses were responsible for some of the effect thj other bodies for others. The precise effects produced in the cases studied by Martin will be taken up under the NATURE OF TOXIN ES. 157 diseases he investigated. A similar digestive action has been traced in the case of the tubercle bacillus by Kiihne. Further evidence that bacterial toxines are either albu- moses or bodies having a still smaller molecule is furnished by C. J. Martin. This worker, by filling the pores of a Chamberland bougie with gelatine, has obtained what is practically a strongly supported colloid membrane through which dialysis can be made to take place under the great pressure say of compressed oxygen. He finds that in such an apparatus toxines, at least the two kinds tried, will pass through just as an albumose will. Brieger and Boer, working with bouillon cultures of diphtheria and tetanus, have, by precipitation with certain metallic salts, especially zinc chloride, separated bodies which show characteristic toxic properties, but which have the reactions neither of peptone, albumose, nor albuminate, and the nature of which is unknown. It has also been found that the bacteria of tubercle, tetanus, diphtheria, and cholera can produce toxines when growing in proteid-free fluids. In the case of diphtheria when the toxine is produced in such a fluid a proteid reaction appears. Of course this need not necessarily be caused by the toxine. Further investigation is here required, for Uschyisky, applying Brieger and Boer's method to a toxine so produced, states that the toxine body is not precipitated by zinc salts, but remains free in the medium. If the toxines are really non-proteid they may, on the one hand, be the final product of a digestive action, or they may be the manifestation of a separate vital activity on the part of the bacteria. On the latter theory the toxicity of the toxalbumins of Brieger and Fraenkel, and of the toxic albumoses of Martin may be due to the precipitation of the true toxines along with these other bodies. From the chemical standpoint this is quite possible. Up to the present no bacterial toxine has been isolated in a pure condition, and therefore we are ignorant of the chemical nature of such bodies. It is possible that present chemical methods are inadequate for the solution of the problem. The toxines we know best are the extracellular. 158 THE TOXINES PR OD UCED BY BAC7 'ERIA . They are certainly all uncrystallisable. They are soluble in water and they are dialysable ; they are precipitated along with proteids by concentrated alcohol. If they are proteids they are either albumoses or allied to the albumoses. They are relatively unstable, having their toxicity diminished or de- stroyed by heat (the degree of heat which is destructive varies much in different cases), light, and by certain chemical agents. Regarding the toxines which are more intimately associated with the bacterial cell we know much less, but it is probable that their nature is similar, though some of them at least are not so easily injured by heat, e.g., in the case of the tubercle bacilli already mentioned. There is evidence in the case of all toxines that the fatal dose for an animal varies directly with the body weight. The comparison of the action of bacteria in the tissues in the production of these toxines to what takes place in the gastric digestion, has raised the question of the pos- sibility of the elaboration by these bacteria of ferments by which the process may be started. The problem of toxine formation is thus still further complicated. Martin has described toxic albumoses as occurring in all the diseases he investigated, viz. anthrax, ulcerative endocarditis, diphtheria, and. tetanus. In each of these cases, therefore, we would be led to suppose that ferments might be primarily produced. Martin carries the analogy further, and sug- gests that, just as by the secretion of ferments into the intestine, the non-soluble albumins of the food are trans- formed into the soluble albumoses and peptones which are easily absorbed by the intestinal cells, so it is likely that bacteria may excrete ferments which, acting on the albumins in which they are living, may make the latter more available for subsequent absorption as food. Looked at from the side of the animal in or on which the bacteria are living, these products of digestion are toxic, and it is evident that, given a diffusible ferment, we may look on it as the primary toxic agent which acts by producing secondary non-diastatic poisons. Hitherto all attempts at the isolation of such ferments in a pure condition have failed. PART PLAYED BY FERMENTS. 159 The two diseases in which there is most evidence of a ferment action are diphtheria and tetanus. Apart from the fact that a digestive action has occurred, which the presence of albumoses in the body of an animal dead of these diseases affords, the chief available evidence for the exist- ence of ferments lies in this, that the toxic products of the bacteria involved lose their toxicity by exposure to a temperature which puts an end to the diastatic activity of such an undoubted ferment as that of the gastric juice. If a bouillon containing diphtheria toxine be heated at 650 C. for one hour, it is found to have lost much of its toxic effect. There is evidence, however, that there remains a substance unaffected by the heat which, as we shall see later, is also toxic. In the case of B. tetani growing in artificial media similarly treated, all the toxicity is lost by exposure at 650 C. Both in this disease and in diphtheria there is a still further fact which is adduced in favour of a ferment being concerned in the toxic action, namely, the existence of a definite period of incubation between the injection of the toxic bodies and the appearance of symptoms. This may be interpreted as showing that after the introduction of say a filtered bouillon culture, further chemical changes have to be set up in the body before the actual toxic effect is produced. In the pharmacological action of some simple chemical bodies, however, there is observed a delay in the appearance of symptoms, and with some poisons presently to be mentioned which are closely allied to the bacterial toxines an incubation period may not exist. Thus the toxic action of bacteria may be a very complicated process, and the initial elaboration of a ferment may occur. Such a ferment injected into an animal might give rise, by a process of digestion, to toxic substances of a non-diastatic nature. The injection of the latter, derived from previous digestive action outside an animal's body, might also give rise to toxic effects. Our knowledge of the subject is at present, however, too scanty for a definite opinion to be expressed as to whether ferments play a part in the action of pathogenic bacteria or not. i6o THE TOXINES PRODUCED BY BACTERIA. Similar Vegetable and Animal Poisons. — Within recent years it has been found that the bacterial poisons belong to a group of toxic bodies all presenting very similar properties, other members of which occur widely in the vegetable and animal kingdoms. Among plants the best known examples are the ricin and abrin poisons obtained by making watery emulsions of the seeds of the Ricinus communis and the Abrus precalorius (jequirity) respectively. From the Robinia pseudacacia another poison — robin — belonging to the same group is obtained. The chemical reactions of ricin and abrin correspond to those of the bacterial toxines. They are soluble in water, they are precipitable by alcohol, but being less easily dialysable than the albumoses they have been called toxalbumins. Their toxicity is seriously impaired by boiling, and they also gradually become less toxic on being kept. Both are among the most powerful poisons known — ricin being the more fatal. When injected subcutaneously a period of twenty-four hours usually elapses — whatever be the dose — before symptoms set in. Both tend to produce great inflammation at the seat of inoculation, which in the case of ricin may end in an acute necrosis ; in fatal cases hemorrhagic enteritis and nephritis may be found. Both act as irritants to mucous membranes, abrin especially being capable of setting up most acute conjunctivitis. It is also certain that the poisons of scorpions and of poisonous snakes belong to the same group. The poisons derived from the latter are usually called venenes, and a very representative group of such venenes derived from different species has been studied. To speak generally there is derivable from the natural secretions of the poison glands a series of venenes which have all the reactions of the bodies previously considered. Like ricin and abrin, they are not so easily dialysable as bacterial toxines, and therefore have also been classed as toxalbumins. Their properties are also similar ; many of them are destroyed by heat, but the degree necessary here also varies much. There is also evidence that in a crude venene there may be several poisons differently sensitive to heat. All the venenes are very powerful poisons, but here there is practically no period of incubation— the effects are almost immediate. The toxicity of the venenes varies much with the animal employed, but chiefly with the species of snake from which it was derived. For instance, .47 milligrammes of crude venene from the Indian cobra will kill a rabbit in three to four hours. In the case of the American rattlesnake the dose would be 3.5 milligrammes, and in that of the Australian hoplocephalm variegatus 2. 5 milligrammes. The general effects of these vary with the dose, and slight variations also exist between the effects of venenes of different snakes. Thus cobra poison is said to produce rapid paralysis of the lips, tongue, larynx, and respiratory apparatus, from which death results. On the other hand the venene of the daboia of Ceylon is said to cause violent general convulsions, succeeded by paralysis, but with very little respiratory affection In the case of a dose not sufficient to cause immediate THE THEORY OF TOXIC ACTION. 161 death from its general effects, often the most acute and widespread neerosis may occur in a few hours round the site of inoculation. The Theory of Toxic Action. — While we know little of the chemical nature of any toxines we may, from our knowledge of their properties, group together the tetanus and diphtheria poisons, ricin, abrin, snake poisons, and scorpion poisons. Besides the points of agreement already noted, all possess the further property that, as will be after- wards described, when introduced into the bodies of sus- ceptible animals they stimulate the production of substances called antitoxines. The nature of the antagonism between toxine and antitoxine will be discussed later. Here to explain what follows it may be stated (i) that the molecule of toxine most probably forms a chemical combination with the molecule of antitoxine, and (2) that it has been shown that toxine molecules may lose much of their toxic power and still be capable of uniting with exactly the same propor- tion of antitoxine molecules. From these and other circum- stances Ehrlich has advanced the view that the toxine mole- cule has a very complicated structure, and contains two atom groups. One of these, the haptophorous {a-KTtiv, to bind to), is that by which combination takes place with the antitoxine molecule and also with presumably corresponding'molecules naturally existing in the tissues. The other atom group he calls the toxophorous, and it is to this that the ""toxic effects are due. .This atom group is bound to the cell elements, e.g., the nerve cells in tetanus, by the haptophor- ous group. Ehrlich holds that the toxophorous group is the more complicated and also the less stable. It is known that, for instance, a diphtheria toxine obtained by the filtra- tion of a bouillon culture loses its toxicity when subjected to such physical agencies as light and heat, and to certain chemical substances. Ehrlich explains this on the theory that the toxophorous group undergoes disintegration. And if we suppose that the haptophorous group remains un- affected we can then understand how a toxine may have its toxicity diminished and still require the same proportion of antitoxine molecules for its neutralisation. To the bodies 1 1 1 62 TOXINES PRODUCED BY BACTERIA. whose toxophorous atom groups have become degenerated, Ehrlich gives the name toxoids. He states that he has found evidence that similar bodies may be directly formed by the diphtheria bacillus and not as the result of sub- sequent degeneration, and these he calls toxones. Such observations are of importance, not only as throwing light on the constitution of the toxine molecule, but also as affording an explanation of how altered toxines (toxoids) can act as immunising agents by stimulating antitoxine for- mation. The theory may also afford an explanation of what has been suspected, namely, that in some instances toxines derived from different sources may be related to one another. For example, Ehrlich has pointed out that ricin produces in a susceptible animal body an antitoxine which corresponds almost completely with that produced by another vegetable poison, robin {vide supra), though nan and robin are certainly different. This may be due to the fact that robin is a toxoid of ricin, i.e., their haptophorous groups correspond, while their toxophorous differ. The evidence on which Ehrlich's deductions are based is of a very weighty character, and will be again referred to in the chapter on Immunity. CHAPTER VI. SUPPURATION AND ALLIED CONDITIONS. The subject of suppuration is an exceedingly wide one, and embraces a great many pathological conditions which in their general characters and results are widely different Thus bacteriological research has shown that the same organism may in one case produce a simple local abscess of trifling importance, in another case multiple spreading suppurations in various organs, or again, under different conditions, an ulcerative endocarditis. The study of the pus-producing or pyogenic organisms, their paths of entrance and their effects on the tissues, constitutes one of the most important subjects in pathology. Suppuration will first be treated of in general, and reference will afterwards be made to certain inflammatory or suppurative conditions of special importance from the clinical standpoint. Nature of Suppuration.— Suppuration is not a specific disease, but rather a pathological condition which follows inflammation under certain circumstances. The process is best studied in the subcutaneous tissue or in a solid organ such as the kidney, and it is found that the following factors are involved. Consequent on the inflammatory condition here occur in the part affected (a) a progressive immigra- tion and accumulation of leucocytes, chiefly of the finely granular polymorphonuclear variety (the so-called "multi nuceated" leucocytes); {b) degeneration followed by necrosis of the special cells of the part, those most highly i64 SUPPURATION AND ALLIED CONDITIONS. organised being affected first; and (c) a liquefaction or digestion of the supporting elements of the tissue. Any previously-formed fibrin is also softened and disappears. The result is that the solid tissue becomes replaced by the cream-like fluid called pus, a fluid which does not coagulate, and in which the chief cellular elements are polymorpho- nuclear leucocytes, along with the degenerated cells of the part Suppuration is therefore to be distinguished, on the one hand, from a severe inflammation, in which, however, the tissue is not destroyed, and on the other hand, from necrosis or death of the tissue en masse. When, however, suppuration is taking place in a very dense fibrous tissue, liquefaction may be incomplete, and a portion of dead tissue or slough may remain in the centre, as is the case m boils In the case of suppuration in a serous cavity the two chief factors are the progressive leucocytic accumulation and the disappearance of any fibrin which may be present. The liquefaction of the formed tissue elements in sup- puration is believed to depend chiefly upon a peptomsing action of the organisms or of ferments produced by them, and the progressive leucocytic aggregation is most probably the effect of microbic products which attract the leucocytes or in other words exert a positive chemwtaxis We might expect that any organisms which could flourish in the tissues and exert these actions would produce suppuration and as a matter of fact a considerable number have been found to P°S¥he T^Ztl* pyuria may be first explained as these will be frequently used. Septicaemia is applied to conations in which the organisms multiply within the blood and give rise to symptoms of ^p^son^ iSySWh of duced by absorption of their toxines. THE BACTERIA OF SUPPURATION. 165 impossible to detect any in the blood taken by puncture of the skin during life, though they may be seen in large numbers in the capillaries of the kidneys, liver, etc., post mortem. The best examples of extensive bacterial multi- plication in the circulating blood are afforded by certain infections of the lower animals, e.g., anthrax in guinea-pigs or pneumococcus septicaemia in rabbits. The essential fact in pyaemia, on the other hand, is the occurrence of multiple abscesses in internal organs and other parts of the body. In most of the cases of typical pyaemia, common in pre-antiseptic days, the starting-point of the disease was a septic wound with bacterial invasion of a vein leading to thrombosis and secondary embolism. Multiple foci of suppuration may be produced, however, in other ways, as will be described below (p. 182). If the term "pyaemia" be used to embrace all such conditions, their method of production should always be distinguished. The Bacteria of Suppuration. A considerable number of species of bacteria have been found in the pus of acute suppurations, and of these many have been proved to be the causes of the condition, whilst of some others the exact action has not yet been fully determined. Ogston, who was one of the first to study this question (in 1 881), found that the organisms most frequently present were micrococci, of which some were arranged irregularly in clusters (staphylococci), whilst others formed chains (streptococci). He found that the former were more common in circumscribed acute abscesses, the latter in spreading suppurative conditions. Rosenbach shortly afterwards (1884), by means of cultures, differentiated several varieties of micrococci, to which he gave the follow- ing special names: staphylococcus pyogenes aureus, staphy- lococcus pyogenes aldus, streptococcus pyogenes, micrococcus pyogenes tenuis. Other organisms have been met with in 166 SUPPURATION AND ALLIED CONDITIONS. suppuration, such as staphylococcus pyogenes citreus, staphy- lococcus cereus albus, staphylococcus cereus flavus, bacillus pyogenes fcetidus (Passct), bacillus colt communis, bacillus lactis ozrogenes, bacillus pyocyaneus, micrococcus tetragenus, pneumococcus, pneumobacillus, diplococcus intracellulars meningitidis, and others. In secondary suppurations following acute specific dis- eases the corresponding organisms have been found in some cases, such as gonococcus, pneumococcus of Fraenkel, pneumobacillus of Friedlander, and the typhoid bacillus. Suppuration is also produced by the actinomyces and the glanders bacillus, and sometimes chronic tubercular lesions have a suppurative character. Staphylococcus Pyogenes Aureus. — Microscopical Char- acters.— This organism is a spherical coccus about .9 /x in diameter, which grows irregularly in clusters or masses (Fig. 45). It stains readily with all the basic aniline dyes, and retains the colour in Gram's method. , Cultivatio?i. — It grows readily in all the ordinary media at the room temperature, though much more rapidly at the tempera- ture of the body. In Fig. 45. — Staphylococcus pyogenes aureus young culture on agar, showing clumps of stab Cultures On pep- tone pclatine a streak cocci. Stained with weak carbol-fuchsin. x 1000. of growth is visible on the day after inoculation, and on the second or third day liquefaction commences at the top. As liquefaction proceeds, the growth falls to the bottom as a flocculent deposit, which soon assumes a bright-yellow colour, while a yellowish film may form on the surface, the fluid portion still remaining STAPHYLOCOCCUS PYOGENES AUREUS. 167 I turbid. Ultimately liquefaction extends out to the wall of the tube (Fig. 46). In gelatine plates colonies may be seen with the low power of the microscope in twenty -four hours, as little balls some- what granular on the surface and of brownish colour. On the second day they are visible to the naked eye as whitish-yellow points, which afterwards become more distinctly yellow. Lique- faction occurs around these, and little cups are formed, at the bottom of which the colonies form little yellowish masses. On agar, a stroke culture forms a line of abundant opaque growth, with smooth, shining surface, well formed after twenty-four hours at 370 C. Later it becomes bright orange in colour, and resembles a streak of yellow oil paint. Single colonies on the .surface of agar are circular discs of similar appearance, which may reach 2 mm. or more in diameter. On potatoes it grows well' at ordinary tem- perature, forming a somewhat abundant layer of orange colour. In bouillon it produces a uniform turbidity, which afterwards settles to the bottom as an abundant layer which assumes a brownish-yellow tint. In the various media it renders the reaction acid, and it coagulates milk, in which it readily grows. The cultures have a somewhat sour odour. It has considerable tenacity of life outside the body, Fig. 46. — Two stab cultures of staphylococcus pyogenes aureus in gelatine, (a) 10 days old, (d) 3 weeks old. Showing liquefaction of the medium and characters of growth. Natural size. 1 68 SUPPURATION AND ALLIED CONDITIONS. cultures in gelatine often being alive after having been kept for several months. It also requires a rather higher temperature to kill it than most spore -free bacteria, viz. 8o° C. for half an hour (Lubbert). The Staphylococcus pyogenes albus is similar in char- acter with the exception that its growth on all the media is white. The colour of the staphylococcus aureus may become less distinctly yellow after being kept for some time in culture, but it never assumes the white colour of the staphylococcus albus, and it has been found impossible to transform the one organism into the other. Both organisms are common in air, dust, and especially on the surface of the skin. The staphylococcus pyogenes citreus, which is less frequently met with, differs in the colour of the cultures being a lemon yellow, and is less virulent than the other two. The staphylococcus cereus albus and staphylococcus cereus flavus are of much less importance. They produce a wax- like growth on gelatine without lique- faction, hence their name. Streptococcus pyo- genes.— This organism is a coccus of slightly larger size than the staphylococcus aureus, about i fju in diameter, I* and forms chains which C If" *~ may contain a large number of members, especially when it is growing in fluids (Fig. 47). The chains vary somewhat in length in different specimens of the streptococcus, and on this ground varieties have been distinguished, e.g., the streptococcus brev.s and strepto- Fig. 47.— Streptococcus pyogenes, young culture on agar, showing chains of cocci. Stained with weak carbol-tuchsin. x 1000. STREPTOCOCCUS PYOGENES. 169 coccus longus (vide infra). As division may take place in many of the cocci in a chain at the same time, the appearance of a chain of diplococci is often met with. In young cultures the cocci are fairly uniform in size, but after a time their size presents considerable variations, many swelling up to twice their normal diameter. These are to be regarded as involution forms. In its staining reactions the streptococcus resembles the staphylococci described, being readily coloured by Gram's method. Cultivation. — In cultures outside the body the streptococcus pyogenes grows much more slowly than the staphylococci, and also dies out more readily, being in every respect a more delicate organism. In pep/one-gelatine a stab culture shows, about the second day, a thin line which in its subsequent growth is seen to be formed of a row of minute rounded colonies of whitish colour, which are more clearly separate at the lower part of the puncture. They do not usually exceed the size of a small pin's head, this size being reached about the fifth or sixth day. The growth does not spread on the .surface and no liquefaction of the medium occurs. The colonies in gela- tine plates have a corresponding appearance, being minute spheri- cal points of whitish colour. On the agar media, growth takes place along the stroke as a collection of small circular discs of semi -translucent appearance, which show a great tendency to remain separate (Fig. 48). The separate colonies remain small and do not usually exceed 1 mm. in diameter. 3 ' . ,:W. -.:?.mC: Fig. 48. —Culture of the streptococcus pyogenes on an agar plate, showing numerous colonies — three successive strokes. Twenty-four hours' growth. Natural size. 170 SUPPURATION AND ALLIED CONDITIONS. Cultures on agar kept at the body temperature may often be found to be dead after ten days. On potato^ as a rule, no visible growth takes place. In bouillon^ growth forms numerous minute granules which after- wards fall to the bottom, the deposit, which is usually not very abundant, having a sandy appearance. The appearance in broth, however, presents variations which have been used as an aid to distinguish different species of streptococci. It has been found that those which form the longest chains grow most distinctly in the form of spherical granules, those forming short chains giving rise to a finer deposit. To a variety which forms distinct spherules of minute size the term streptococcus conglomerates has been given. The question as to the existence of varieties of streptococcus pyogenes will be discussed below. Bacillus Coli Communis. — The microscopic and cultural characters are described in the chapter on typhoid fever. The bacillus lactis cerogenes and the bacillus pyogenes fectidus closely resemble it ; they are either varieties or closely related species. The former is distinguished by producing more abundant gas formation, and by its growth on gelatine, etc., being thicker and whiter than that of the bacillus coli. Bacillus Pyocyaneus. — This organism occurs in the form of minute rods 1.5 to 2 in length and less than .5 ^ in thickness. Occasion- ally two or three are found attached end to end. They are actively motile, and do not form spores. They stain readily with the ordinary basic stains, but are decolorised by Gram's method. Cultivation. — It grows readily on all the ordinary media at the room temperature, the cultures being distinguished by the ' formation of a greenish pigment. In puncture cultures in peptone-gelatine a greyish line appears in twenty-four hours, and at its upper part a small cup of liquefaction forms within forty-eight hours. At this time a slightly greenish tint is seen in the superficial part of the gelatine. The liquefaction extends pretty rapidly, the fluid portion being turbid and showing masses of growth at its lower part. The green colour becomes more and more marked and diffuses through the gelatine. Ultimately liquefaction reaches the wall of the tube. In plate cultures the colonies appear as minute whitish points, those in the surface being the larger. Under a low power of the microscope they have a brownish-yellow colour and show a nodulated surface, the superficial colonies being thinner and larger. Liquefaction soon occurs, the colonies on the surface forming shallow cups with small irregular masses of growth at the bottom, the deep colonies small spheres of MICROCOCCUS TETRAGENUS. 171 liquefaction. Around the colonies a greenish tint appears. On agar the growth forms an abundant slimy greyish layer which afterwards becomes greenish, and a bright green colour diffuses through the whole substance of the medium. On potatoes the growth is an abundant reddish-brown layer resembling closely that of the glanders bacillus, and the potato sometimes shows a greenish discoloration. From the cultures there can be extracted by chloroform a coloured body pyocyanin, which belongs to the aromatic series, and crystallises in the form of long, delicate bluish-green needles. On the addition of a weak acid its colour changes to a red. This organism has distinct pathogenic action in certain animals. Subcutaneous injection of small doses in rabbits may produce a local suppuration, but if the dose be large, spreading hemorrhagic cedema results, which may be attended by a septicemia, the organism occurring throughout the body. Intravenous injection may produce, according to the dose, rapid septicaemia with nephritis, or sometimes a more chronic condition of wasting attended by albuminuria. Microccocus Tetragenus.— This organism, first described by Gaffky, is characterised by the fact that it divides in two planes at right angles to one another (Fig. 49), and is thus generally found in the tissues in groups of four or tetrads, which are ^ often seen to be surrounded ^ %L by a capsule which stains *9 . * f faintly or not at all. The a 4" * W cocci measure 1 fi in dia- * ♦ \ + * meter. They stain readily *\ * with all the ordinary ^ ^ « stains, and also retain j> Jf * the stain in Gram's method. ' «K* It grows readily on all ♦ the media at the room * * £ temperature. In a punc- * . ture culture on peptone- « gelatine a pretty thick whitish line forms along Fig. 49. -Micrococcus tetragenus; young the track of the needle, culture on agar, showing tetrads, whilst on the surface there Stained with weak carbol-fuchsin. x 1000. is a thick rounded disc of whitish-yellow colour. The gelatine is not liquefied. The colonies in gelatine plates are rounded yellowish-white points, which under a low power show a granular or slightly nodulated surface ; the super- ficial colonies appear as opaque round drops of yellowish-white colour On the surface of agar and of potato the growth is an abundant moist layer of the same colour. The growth on all the media has a peculiar 172 SUPPURATION AND ALLIED CONDITIONS. viscid or tenacious character, owing to the gelatinous character of the sheaths of the cocci. White mice are exceedingly susceptible to this organism. Sub- cutaneous injection is followed by a general septicaemia, the organism being found in large numbers in the blood throughout the body. Guinea-pigs are less susceptible ; sometimes only a local abscess with a good deal of necrotic change results, sometimes there is also septicaemia. Diplococcus Intracellularis Meningitidis. — This organism was first found by Weichselbaum in the purulent exudate in a number of cases of cerebro-spinal meningitis, and has since been found by other observers in some epidemics of the disease. In the recorded cases it has been described as occurring in large numbers in the pus in the form of a rounded or oval diplococcus (with the long axis lying trans- versely), chiefly in the interior of leucocytes. In fact, it closely resembles the gonococcus both in morphological characters and in arrangement. Like the latter also it loses the stain in Gram's method. Its conditions of growth outside the body are somewhat limited. It grows best on agar and glycerine agar, forming a number of transparent colonies which run together to form a thin layer. Growth occurs most rapidly at the temperature of the body, and entirely ceases at the ordinary room temperature. Individual cultures die out after six days, but growth can be maintained indefinitely in successive sub-cultures. Inoculation by ordinary methods shows that it has little virulence for guinea-pigs, rabbits, etc. A number of experiments have been per- formed by introducing pure cultures under the dura, and in some cases meningitis and encephalitis have resulted, but the disease as it affects the human subject is not fully reproduced. From the constancy with which it has been found in the various cases of some epidemics there can be little doubt that it is the causal agent in a certain proportion of cases of cerebro-spinal meningitis. It is of interest to note that in a considerable number of such cases it has been detected during the disease in the nasal secretion, whereas in normal individuals it is very rare. Experimental Inoculation; — We shall consider chiefly the staphylococcus pyogenes aureus and the streptococcus pyogenes, as these have been most fully studied. It may be stated at the outset that the occurrence of suppuration depends upon the number of organisms intro- duced into the tissues, the number necessary varying not only in different animals {e.g., suppuration being much more easily produced in rabbits than in dogs) but also in different parts of the same animal, a smaller number producing suppuration in the anterior chamber of the eye, for example,. EXPERIMENTAL INOCULATION. 173 than in the peritoneum. The virulence of the organism also may vary, and corresponding results may be produced. Especially is this.so in the case of the streptococcus pyogenes. The staphylococcus aureus, when injected subcutaneously in suitable numbers, produces an acute local inflammation which is followed by suppuration, in the manner described above. The spread of the suppuration goes pari passu with the growth of the cocci. Wherever the condition is spread- ing the cocci are present in the tissues at the margin, but after it has ceased to spread they are practically confined to the pus. In the latter case reaction occurs on the part of the connective tissues in the form of cellular proliferation and formation of new capillaries, which lead to the forma- tion of a granulation tissue barrier. After this has been well formed the cocci diminish gradually in numbers and finally disappear. If a large dose is injected, the cocci may enter the blood stream in sufficient numbers to cause secondary suppurative foci in internal organs {cf. intra- venous injection). Intravenous injection in rabbits, for example, produces interesting results which vary according to the quantity used. If a considerable quantity be injected, the animal may die in twenty-four hours of a general septicaemia, numerous cocci being present in the capillaries of the various organs, often forming plugs. If a smaller quantity be used, the cocci gradually disappear from the circu- lating blood ; some become destroyed, while others settle in the capillary walls in various parts and produce minute abscesses. These are most common in the kidneys, where they occur both in the cortex and medulla as minute yellowish areas surrounded by a zone of intense congestion, and often haemorrhage. If one of these areas be examined microscopically before actual suppuration has occurred, it will be found that many of the capillaries are filled with cocci, and the tissues immediately around are necrosed, apparently by the action of the products of the organisms. At the margin of the necrosed area there is a dense zone of leucocytes which gradually extend inwards ; ultimately 174 SUPPURATION AND ALLIED CONDITIONS. purulent softening of the area occurs. The cocci may reach the interior of the tubules, where they may be often seen mixed with leucocytes, and in this way they reach the bladder. Similar small abscesses may be produced in the heart wall, in the liver, under the periosteum, and in the interior of bones, and occasionally in the striped muscles. Very rarely indeed, in experimental injection, do the cocci settle on the healthy valves of the heart. If, however, when the organisms are injected into the blood, there be any traumatism of a valve or of any other part of the body, they show a special tendency to settle at these weakened points. Experiments on the human subject have also proved the pyogenic properties of those organisms. Garre inoculated scratches near the root of his finger-nail with a pure culture, a small cutaneous pustule resulting ; and by rubbing a culture over the skin of the forearm he caused a carbun- cular condition which healed only after some weeks. Con- firmatory experiments of this nature have been made by Bockhart, Bumm, and others. When tested experimentally the staphylococcus pyogenes albus has practically the same pathogenic effects as the staphylococcus aureus. The streptococcus pyogenes is an organism the virulence of which varies much according to the diseased condition from which it has been obtained, and one which also loses its viru- lence rapidly in cultures. Even highly virulent cultures, if grown under ordinary conditions, in the course of time lose practically all pathogenic power. By passage from animal to animal, however, the virulence may be much increased, and pari passu the effects of inoculation are correspondingly varied. Marmorek, for example, has found that the virulence of a streptococcus can be enormously increased by growing it alternately (a) in a mixture of human blood serum and bouillon {vide page 53), and (b) in the body of a rabbit ; ultimately, after several passages it possesses a supervirulent character, so that even an ex- tremely minute dose introduced into the tissues of a rabbit VARIETIES OE STREPTOCOCCI. 175 produces rapid septicaemia with death in a few hours, the organisms being found in large numbers in the internal organs. It has been proved by Marmorek's experiments and those of others that the same species of streptococcus may produce at one time merely a passing local redness, at another a local suppuration, at another a spreading erysipe- latous condition, or again a general septicemic infection, according as its virulence is artificially increased. Such experiments are of extreme importance as explaining to some extent the great diversity of lesions in the human subject with which streptococci are associated. Varieties of Streptococci. — Formerly the streptococcus pyogenes and the streptococcus erysipelatis were regarded as two distinct species, and various points of difference between them were given. Further study, and especially the results obtained by modifying the virulence, have shown that these distinctions cannot be maintained, and now nearly all authorities are agreed that the two organisms are one and the same, erysipelas being produced when the strepto- coccus pyogenes of a certain standard of virulence gains entrance to the lymphatics of the skin. Petruschky in a recent publication has shown conclusively that a streptococcus cultivated from pus may cause erysipelas in the human subject. He obtained a pure culture of a streptococcus from a case of purulent peritonitis secondary to parametritis, the patient never having suffered from erysipelas. By inoculations with this culture he produced typical erysipelas in two women suffering from cancer. More recently a distinction has been drawn between a streptococcus longus, which corresponds to the streptococcus pyogenes, as it usually forms long chains, and is pathogenic to rabbits or mice, and a strepto- coccus brevis, which occurs in the mouth in normal conditions and is without pathogenic properties when tested experimentally. The growth of the former in bouillon forms a somewhat granular deposit, that of the latter a more abundant and flocculent deposit. Marmorek has, however, found that the same streptococcus may at one time grow m short, at another in long chains, and Kolle has shown that a strepto- coccus, which originally grew in long chains, formed only short chains after being repeatedly passed through the body of the mouse, the 176 SUPPURATION AND ALLIED CONDITIONS. appearance of the growth in bouillon being correspondingly altered (p. 170). Further, Widal and Besancon found that a streptococcus cultivated from the mouth and which was non-pathogenic, became pathogenic when inoculated along with the bacillus coli communis, and thereafter its virulence could be enormously increased by passing it through a series of animals. These latter observers also found that streptococci cultivated from the mouth of a smallpox patient were non- virulent, whilst those cultivated from the blood of the same patient post mortem were highly virulent, the probability being that those in the blood had been derived from those in the throat. There does not therefore seem at present sufficient evidence for looking upon these two varieties as distinct species. It is sufficient to bear in mind that strepto- cocci in the normal mouth are usually non-virulent, and grow in short chains. On the other hand, in some cases of very virulent strepto- coccus infection in the human subject we have found the organism occurring only in very short chains. The streptococcus conglomerates, so called from the appearance of the growth in bouillon, is to be regarded merely as another variety, which forms very long chains and is usually possessed of a high degree of virulence, though its distinctive characters are not permanent. It has often been obtained from the fauces in scarlet fever. • We may accordingly conclude that, though it cannot be definitely stated that all the streptococci concerned in the production of disease in the human subject are of the same species, we have not the means of classifying them as distinct species. Bacillus coli communis.-— The virulence of this organism also varies much and can be increased by passage from animal to animal. Injection into the serous cavities of rabbits produces a fibrinous inflammation which becomes purulent if the animal lives sufficiently long. If, however, the virulence of the organism be of a high order, death takes place before suppuration is established, and there is a septicemic condition, the organisms occurring in large numbers in the blood. Intravenous injection of a few drops of a virulent bouillon culture usually produces a rapid septicemia with scattered hemorrhages in various organs. Other Effects. -It has been found by independent observers that in cases where rabbits recover after intravenous injectron of bacillus coli communis, a certain proportion suffer from paralysis and sometimes SUPPURATION WITHOUT BACTERIA. from atrophy of muscles, especially of the posterior limbs, these symptoms being due to lesions of the cells in the anterior cornua of the spinal cord. Somewhat similar results have been obtained by others after inoculations with staphylococci and streptococci, a certain propor- tion only of the animals showing paralytic symptoms and corresponding changes in the spinal cord. The lesions are believed to be due chiefly to the action of the products of the organisms on the highly-organised nervous elements. Much further research requires to be done before the importance of these results can be properly estimated, but it is not im- probable that they will throw light on the causation of nervous lesions which occur in the human subject, and the etiology of which at present is quite obscure. Some observers, chiefly of the French school, con- sider that paralysis associated with cystitis, in which the bacillus coli communis is often present, may have such a causation, and that paralytic conditions following acute infective fevers may be produced by the products of pyogenic cocci which frequently occur in these conditions. Can Suppuration occur apart from Bacteria ? After it had been conclusively proved that bacteria were the chief causes of suppuration, a great many experiments were per- formed to determine whether it could be produced by simple chemical substances, such as croton oil, nitrate of silver, mercury, etc. In these experiments various means have been employed to ensure the absence of bacteria. In some cases the chemical substance to be tested was placed in a closed glass capsule, which, after being sterilised, was inserted in the tissues and was not broken until the external wound had healed up ; in other cases the capsule was made with pointed ends, so that it could be moved in the body of the animal to another part, and there broken. The general conclusion obtained by independent observers is that in these conditions suppuration usually does not follow, but that in certain animals and with certain chemical substances it may occur, the pus which forms showing no organisms on bacteriological examination. Such suppura- tion, however, never produces secondary abscesses in other parts, and it is still questioned by some whether the pus produced really corresponds histologically and chemically with pus naturally produced. Buchner showed that sup- puration could be produced by injections of dead bacteria for example, sterilised cultures of bacillus pyocyaneus^ 12 178 SUPPURATION AND ALLIED CONDITIONS. tubercle bacillus, and various others. The question, how- ever, is now rather of scientific than practical interest, and the general statement may be made that practically all cases of acute suppuration met with clinically are produced by the action of living micro-organisms. Lesions in the Human Subject produced by Pyogenic Bacteria. The following statement may be made with regard to the occurrence of the chief organisms mentioned, in the various suppurative and inflammatory conditions in the human subject. The account is, however, by no means exhaustive, as clinical bacteriology has shown that practi- cally every part of the body may be the site of a lesion produced by the pyogenic bacteria. It may also be noted that acute catarrhal conditions of cavities or tubes are in many cases also to be ascribed to their presence. The staphylococci are the most common causal agents in localised abscesses, in pustules on the skin, in car- buncles, boils, etc., in acute suppurative periostitis, in catarrhs of mucous surfaces, in ulcerative endocarditis, and in various pycemic conditions. They may also be present in septicaemia. Streptococci are especially found in spreading inflamma- tion with or without suppuration ; in diffuse phlegmonous and erysipelatous conditions, suppurations in serous mem- branes and in joints (Fig. 50). They also occur in acute suppurative periostitis and ulcerative endocarditis. Second- ary abscesses in lymphatic glands and lymphangitis are also,- we believe, more frequently caused by streptococci than staphylococci. They also produce fibrinous exuda- tion on the mucous surfaces, leading to the formation of false membrane in many of the cases of non-diphtheritic inflammation of the throat, which are met with in scarla- tina 1 and other conditions, and they are also the organisms 1 True diphtheria may also occasionally be associated with this disease, usually as a sequel. LESIONS IN THE HUMAN SUBJECT. 179 most frequently present in acute catarrhal inflammations of this situation. In puerperal peritonitis they are frequently found in a condition of purity, and they also appear to be the most frequent cause of puerperal septi- caemia, in which con- dition they may be found after death in the capillaries of various organs, though examination of the blood during life usually gives a nega- tive result. In py- aemia they are fre- quently present, though in most cases associated with other pyogenic organisms. Some cases of enteritis in infants —streptococcic enteritis— are also apparently due to a streptococcus, which, however, presents in cultures certain points of difference from the streptococcus pyogenes. The bacillus coll communis is found in a great many inflammatory and suppurative conditions in connection with the alimentary tract— for example, in suppuration in the peritoneum or in the extraperitoneal tissue with or with- out perforation of the bowel, in the peritonitis following strangulation of the bowel, in appendicitis and the lesions following it, in suppuration around the bile ducts etc It may also occur in lesions in other parts of the body — endo carditis, pleurisy, etc., which in some cases are associ- ated with lesions of the intestine, though in others such cannot be found. It is also frequently present in inflam- Z Z i ° 1 Unnarl PaSSag6S' CyStitis' Pyelitis> abscesses in the kidneys, etc., these lesions being in fact most fre quently caused by this organism. Fig. 50. — Streptococci in acute suppuration. Corrosive film ; stained by Gram's method and safranin. x 1000. i8o SUPPURATION AND ALLIED CONDITIONS. In certain cases of enteritis it is probably the causal agent, though this is difficult of proof, as it is much increased in numbers in practically all abnormal conditions of the intestine. We may remark that it has been repeatedly proved that the bacillus coli cultivated from various lesions is more virulent than that in the intestine, its virulence having- been heightened by growth in the tissues. The micrococcus tetragenus is often found in suppurations in the region of the mouth or in the neck, and also occurs in various lesions of the respiratory tract, in phthisical cavities, abscesses in the lungs, etc. Sometimes it is present alone, and probably has a pyogenic action in the human subject under certain conditions. In other cases it is associated with other organisms. Recently one or two cases of pyaemia have been described in which this organism was found in a state of purity in the pus in various situations. In this latter condition the pus has been described as possessing an oily viscous character, and as being often blood-stained. The bacillus pyocyaneus is rarely found alone in pus, though it is not infrequent along with other organisms. We have met with it twice in cases of multiple abscesses, in association with the staphylococcus pyogenes aureus. Lately some diseases in children have been described in which the bacillus pyocyaneus has been found throughout the body ; in these cases the chief symptoms have been fever, gastro-intestinal irritation, pustular or petechial erup- tions' on the skin, and general marasmus. Suppurative conditions, associated with the organisms of special diseases, will be described in the respective chapters. ■ , _ Mode of Entrance and Spread.— Many of the organisms of suppuration have a wide distribution in nature, and many also are present or the skin and mucous membranes of healthy individuals. Staphylococci are commonly present on the skin and also occur in the throat and other parts, and streptococci have a similar distribution and can very often be cultivated from the secretions of the mouth in ENTRANCE AND SPREAD OF BACTERIA. 181 normal conditions. The pneumococcus of Fraenkel and the pneumobacillus of Friedlander have also been found in the mouth and in the nasal cavity in normal conditions, whilst the bacillus coli communis is a normal inhabitant of the intestinal tract. The entrance of these organisms into the deeper tissues when a surface lesion occurs can be Fig. 51. — Minute focus of commencing suppuration in brain — case of acute ulcerative endocarditis. In the centre a small hemorrhage ; to right side dark masses of staphylococci ; zone of leucocytes at periphery. Alum carmine and Gram. x 50. readily understood. Their action will, of course, be favoured by any depressed condition of vitality. Though in normal conditions the blood is bacterium-free, we must suppose that from time to time a certain number of such organisms gain entrance to it from trifling lesions of the skin or mucous surfaces, the possibilities of entrance i82 SUPPURATION AND ALLIED CONDITIONS. from the latter being especially numerous. In most cases they are killed by the action of the healthy serum or cells of the body, and no harm results. (It is to be noted in this connection that it has been proved experimentally that, even in the case of a pathogenic organism, a considerable number can be destroyed in the blood of a healthy animal.) If, however, there be a local weakness, they may settle in that part and produce suppuration, and from this other parts of the body may be infected. Such a supposition as this is necessary to explain many cases of suppuration met with clinically. Thus the liability of diseased heart valves to become infected by organisms and thence to assume an ulcerative character, is well known. Conditions such as suppurative periostitis and osteomyelitis, multiple suppura- tive arthritis, suppurative inflammations of several serous surfaces, and other similar conditions can be explained only in the same way. In some cases of multiple suppura- tions due to staphylococcus infection, which we have had the opportunity to examine, only an apparently unimport- ant surface lesion was present ; whilst in others no lesion could be found to explain the origin of the infection. The organs or parts of the body affected vary much in different cases, the distribution being explicable only by selective action on the part of the organisms. In some cases the lungs are especially affected, in others the kidne>s, in others the bones or joints, and so on (Figs. 51, 52). The term cryfitogenetic has been applied by some writers to such cases in which the original point of infection cannot be found, but its use is scarcely necessary. The paths of secondary infection may be conveniently summarised thus : First, by lymphatics. In this way the lymphatic glands may be infected, and also serous sacs in relation to the organs where the primary lesion exists. Second, by natural channels, such as the ureters and the bile ducts, the spread being generally associated with an inflammatory condition of the lining epithelium. In this way the kidneys and liver respectively may be infected. Third, by the blood-vessels : (a) by a few organisms gaining THE PATHS OF PYOGENIC INFECTION. 183 entrance to the blood from a local lesion, and settling in a favourable nidus or a damaged tissue, the original path of infection often being obscure ; (b) by a septic phlebitis with suppurative softening of the thrombus and resulting embol- ism ; and we may add (c) by a direct extension along a vein, producing a spreading thrombosis and suppuration Fig. 52. — Secondary infection of a glomerulus of kidney by the staphylococcus aureus,1 in a case of ulcerative endocarditis. The cocci (stained darkly) are seen plugging the capillaries and also lying free. The glomerulus is much swollen, infiltrated by leucocytes, and partly necrosed. Paraffin section; stained by Gram's method and Bismarck-brown, x 300. within the vein. In this way suppuration may spread along the portal vein to the liver from a lesion in the alimentary canal, the condition being known as pyelo-phlebitis sup- purativa. 1 This organism was obtained in pure culture from the kidney. 1 84 SUPPURATION AND ALLIED CONDITIONS. Some conditions produced by the pyogenic organisms demand special mention on account of their clinical import- ance, namely, ulcerative endocarditis, acute suppurative periostitis and osteomyelitis, and erysipelas. Ulcerative Endocarditis. — This condition has been proved to be a bacterial infection of the valves of the heart, and may be produced by various organisms, chiefly pyogenic. Of these the staphylococci and streptococci are most frequently found. In some cases of ulcera- tive endocarditis following pneumonia, the pneumococcus (Fraenkel's) is present ; in others pyogenic cocci, especially streptococci. Other organisms have been cultivated from different cases of the disease, and some of these have received special names ; for example, the diplococcus endocarditidis encapsulatus, bacillus endocarditidis griseus (Weichselbaum), and others. In some cases the bacillus coli communis has been found, and occasionally in endo- carditis following typhoid the typhoid bacillus has been described as the organism present, but further observations on this point are desirable. The gonococcus also has been shown to affect the heart valves (p. 198), though this is a very rare occurrence. Tubercle nodules on the heart valves have been found in a few cases of acute tuberculosis, though no vegetative or ulcerative condition is produced. In some cases, though we believe not often, the organ- isms may attack healthy valves, producing a primary ulcera- tive endocarditis, but more frequently the valves have been the seat of previous endocarditis, secondary ulcerative endo- carditis being thus produced. In both conditions the affection of the valves usually occurs in the course of sup- purative or inflammatory conditions elsewhere, e.g., in osteomyelitis, in septic inflammations of the urinary pas- sages, in pyaemia and septicaemia, in the course of or following infective fevers, and not very infrequently as a sequel to acute pneumonia. Not infrequently, especially when the valves have been previously diseased, the source of the infection is quite obscure. It is evident that as the vegetations are composed for the most part of unorganised UL CERA Tl VE ENDOCARDITIS. material, they do not offer the same resistance to the growth of bacteria, when a few reach them, as a healthy cellular tissue does. On microscopic examination of the diseased valves the organisms are usually to be found in enormous numbers, sometimes forming an almost continuous layer on the sur- m £ Fig. 53.— Section of a vegetation in ulcerative endocarditis, showing numerous staphylococci lying in the spaces. The lower portion is a frar ment in process of separation. s Stained by Gram's method and Bismarck-brown. x 600. face, or occurring in large masses or clusters in spaces in the vegetation (Fig. 53). By their action a certain amount of softening or breaking down of the vegetations occurs and the emboli thus produced act as the carriers of infec- tion to other organs, and give rise to secondary suppura- tions. The kidneys, heart-wall, brain, and spleen are the 1 86 SUPPURATION AND ALLIED CONDITIONS. parts most frequently infected in this way (vide Figs. 5h 52). Experimental. — Occasionally ulcerative endocarditis is produced by the simple intravenous injection of staphylococci and streptococci into the circulation, but this is a very rare occurrence. It often follows, however, when the valves have been previously injured. Oith and Wyssokowitsch at a comparatively early date produced the condition by damaging the aortic cusps by a glass rod introduced through the carotid, and afterwards injecting staphylococci into the circulation. Similar experiments have since been repeated with streptococci, pneumococci and other organisms, with like result. Ribbert found that if a potato culture of the staphylococcus aureus were rubbed down so as to form an emulsion in salt solution, and then injected into the circulation, some minute fragments became arrested at the attachment of the chords tendineas and produced an ulcerative endocarditis. Acute Suppurative Periostitis and Osteomyelitis.- Special mention is made of this condition on account of its comparative frequency and gravity. Becker in 1883 described a coccus which he believed to be the special organism concerned in this disease, but it has been since completely proved that this organism is simply the staphylo- coccus pyogenes aureus. The great majority of cases are caused by the pyogenic cocci, of which one or two varieties may be present, the staphylococcus, however, occurring most frequently. Pneumococci have been found alone in some cases, and in a few cases following typhoid fever, apparently well authenticated, the typhoid bacillus has been found alone. In others again the bacillus coli communis is present. The affection of the periosteum or interior of the bones by these organisms, which is especially common in young subjects, may take place in the course of other affections produced by these organisms or in the course of infective fevers, but in a great many cases the path of entrance is quite unknown. That the organisms enter frequently by a small surface lesion, and are carried by the blood stream to the part affected, there can be no doubt. In the course of this disease serious secondary infections are always very liable to follow, such as small abscesses in the kidneys, ERYSIPELAS. 187 heart-wall, lungs, liver, etc., suppurations in serous cavities, and ulcerative endocarditis ; in fact, some cases present the most typical examples of extreme general staphylococcus infection. The entrance of the organisms into the blood stream from the lesion of the bone is especially favoured by the arrangement of the veins in the bone and marrow. Experimental. — Multiple abscesses in the bones and under the periosteum may occur in simple intravenous injection of the pyogenic cocci into the blood, and are especially liable to be formed when young animals are used. These abscesses are of small size, and do not spread in the same way as in the natural disease in the human subject. In experiments on healthy animals, however, the conditions are not analogous to those of the natural disease. We must presume that in the latter there is some local weakness or susceptibility which enables the few organisms which have reached the part by the blood. to settle and multiply. If, however, a bone be injured, e.g., by actual fracture or by stripping off the periosteum, before the organisms are injected, then a much more extensive suppuration occurs at the injured part. Erysipelas. — A spreading inflammatory condition of the skin maybe produced byavarietyof organisms, but the disease in the human subject in its characteristic form is almost invariably due to a streptococcus, as was shown by Fehleisen in 1884. He obtained pure cultures of the organism, and gave it the name of streptococcus erysipelatis ; and, further, by inoculations on the human subject as a therapeutic measure in malignant disease, he was able to reproduce ery- sipelas. As stated above, however, one after another of the supposed points of- difference between the streptococcus of erysipelas and that of suppuration has broken down, and it is now generally held that erysipelas is produced by the streptococcus pyogenes of a certain degree of virulence. It must be noted, however, that erysipelas passes from patient to patient as erysipelas, and purulent conditions due to streptococci do not appear liable to be followed by erysipe- las. On the other hand, the connection between erysipelas and puerperal septicaemia is well established clinically. The conditions which produce the special degree of virulence in the streptococcus for the occurrence of erysipelas are not yet fully known. 1 88 SUPPURATION AND ALLIED CONDITIONS. In a case of erysipelas the streptococci are found in large numbers in the lymphatics of the cutis and underlying tissues, just beyond the swollen margin of the inflammatory area. As the inflammation advances they gradually die out, and after a time their extension at the periphery comes to an end. In the affected area there are the usual changes found in inflammation, — great leucocytic emigration and serous exudation with formation of fibrin at places,— but there is no suppurative liquefaction of the tissues. The streptococci may extend to serous and synovial cavities and set up inflammatory or suppurative change, — peritonitis, meningitis, and synovitis may thus be produced. Methods of Examination. — These are usually of a com- paratively simple nature, and include (i) microscopic examination, (2) the making of cultures. (1) The pus or other fluids should be examined micro- scopically, first of all by means of film preparations in order to determine the characters of the organisms present. The films should be stained (a) by one of the ordinary solutions, such as carbol-thionin blue (p. 109), or a saturated watery solution of methylene -blue ; and (b) by Gram's method. The use of the latter is of course of high importance as an aid in the recognition. (2) As most of the pyogenic organisms grow readily on the gelatine media at ordinary temperatures, pure cultures can be readily obtained by the ordinary plate methods. But in many cases the separation can much more rapidly be effected by the method of successive streaks on agar tubes which are then incubated at 370 C. When the presence of pneumococci is suspected this method ought always to be used, and it is also to be preferred in the case of strepto- cocci. Inoculation experiments may be carried out as occasion arises. CHAPTER VII. GONORRHOEA, SOFT SORE, SYPHILIS. GONORRHCEA. Introductory. — The micrococcus now known to be the cause of gonorrhoea, and often spoken of as the gonococcus, was first described by Neisser, who in 1879 gave an account of its microscopical characters as seen in the pus of gonor- rhceal affections, both of the urethra and of the con- junctiva. He considered that this organism was peculiar to the disease, and that its characters were distinctive. The earlier announcements regarding pure cultures obtained on peptone -gelatine and other media, on which it does not really grow, are now known to be erroneous, but later it was successfully isolated and cultivated on solidified blood serum by Bumm and others. Its characters have since been minutely studied, and by inoculations of cultures on the human subject its causal relationship to the disease has been conclusively established. The Gonococcus. — Microscopical Characters. The organism of gonorrhoea is a small micrococcus which very often occurs in the diplococcus form, the adjacent margins of the two cocci being flattened, or even slightly concave, so that between them there is a small oval interval which does not stain. An appearance is thus presented which has been compared to that of two beans placed side by side {vide Fig. 54). When division takes place in the two iqo G0N0RRHCEA, SOFT SORE, SYPHILIS. I members of a diplococcus, a tetrad is formed, which, how- ever, soon separates into two sets of diplococci — that is to say, arrangement as diplococci is much commoner than as tetrads. Cocci in process of degenera- tion are seen as spherical elements of various size, some being considerably swollen ; they lie singly or in small groups. These organisms are found in large numbers in the pus of acute gonorrhoea, both in the male and female, and for the most part are contained within the leucocytes. In the earliest stage, when the secretion is glairy, a considerable number are lying free, or are adhering to the surface of desquamated epithelial cells, but when it becomes purulent the large proportion within leucocytes is a very striking feature. In the leucocytes they lie within the protoplasm, especially superficially, and are often so numerous that the leucocytes appear to be filled with them, and their nuclei are obscured. As the disease becomes more chronic, the gonococci gradually become diminished in number, though even in long-standing cases they may still be found in con- siderable numbers. They are also present in the purulent secretion of gonorrhceal conjunctivitis, also in various parts of the female genital organs when these parts are the seat of true gonorrhceal infection, and they have been found in some cases in the secondary infections of the joints in the disease, as will be described below. Fig. 54. — Portion of film of gonorrhceal pus, showing the characteristic arrangement of the gonococci within leucocytes. Stained with fuchsin. x 1000. Staining.- -The gonococcus stains readily and deeply CULTIVATION OF GONOCOCCUS. 191 with a watery solution of any of the basic aniline dyes — methylene-blue, fuchsin, etc. It is, however, easily decolor- ised, and it completely loses the stain by Gram's method — an important point in the microscopical examination. Cultivation of the Gonococcus. — This is attended with some difficulty, as the suitable media and conditions of growth are somewhat restricted. The most suitable media are solidified blood serum (especially human serum and rabbit's serum), "blood agar," and Wertheim's medium, which consists of one part of fluid serum, added to two parts of liquefied agar at a temperature of 400 C, and then allowed to solidify by cooling. The serum may be obtained from the blood of the human placenta ; pleuritic or other effusion may also be used. Growth takes place best at the temperature of the body, and ceases altogether at 250 C. Cultures are obtained by taking some pus . on the loop of the platinum needle and inoculating one of the media mentioned by leaving minute quantities here and there on the surface. The medium may be used either as ordinary " sloped tubes " or as a thin layer in a Petri's capsule. The young colonies are visible within forty-eight hours, and often within twenty-four hours. They appear around the points of inoculation as small semi-transparent discs of irregularly rounded shape, the margin being undulated and sometimes showing small processes. The colonies vary somewhat in size and tend to remain more or less separate. They generally reach their maximum size on the fourth or fifth day, and are usually found to be dead on the ninth day, sometimes earlier. On the medium of Wertheim the period of active growth and the duration of life are somewhat longer. Even if impurities are present, pure sub-cultures can generally be obtained by the above method from colonies of the gonococcus which may be lying separate. In the early stage of the disease the organism is present in the male urethra in practically pure condition, and if the meatus of the urethra be sterilised by wash- ing with weak solution of corrosive sublimate and then with absolute alcohol, and the material for inoculation be 192 GONORRHOEA, SOFT SORE, SYPHILIS. expressed from the deeper part of the urethra, cultures may often be obtained which are pure from the first. By successive sub-cultures at short intervals, growth may be maintained indefinitely, and the organism gradually flourishes more luxuri- antly. In culture the organisms have similar microscopic characters to those described (Fig- 55)> but show a remarkable tendency to undergo degenera- tion, becoming swollen and of various sizes, and staining very irregularly. Degener^ ated forms are seen even on the second _ .", day, whilst in a culture Fig. 55. — Gonococci, from a pure culture on blood agar of twenty-four hours' growth, four or five days old Some already are beginning to show the comparatively few nor- swollen appearance common in older cultures. ^ j be found Stained with carbol-thionm-blue. x 1000. < The less suitable the medium the more rapidly does degeneration take place. On ordinary agar and on glycerine agar growth does not take place, or is so slight that these media are quite unsuitable for purposes of culture. The organism does not grow on gelatine,1 potato, etc. Plate-Cultures. — The following ingenious method of plate-culture was introduced by Wertheim for the culture of the gonococcus. The medium of culture is a mixture of human blood serum and of ordinary agar (2 per cent) in equal parts. The serum, in a fluid and sterile condition, is put in suitable quantities into two or three test tubes and brought to a temperature of 400 C. These are then successively 1 Turro has announced that he has cultivated the gonococcus on acid gelatine, i.e., ordinary peptone-gelatine which has not been neutralised. We have failed to obtain any growth of the gonococcus on this medium, even when inoculation was made from a vigorous growth on blood agar. RELATIONS TO THE DISEASE. 193 inoculated with the pus or other material in the same manner as gelatine tubes for ordinary plates {vide p. 62). To each tube is added an equal part of ordinary agar which has been thoroughly liquefied by heating and allowed to cool also to 400 C. The mixture is then thoroughly shaken up and quickly poured out on a plate or Petri's dish and allowed to solidify, the plates being then incubated at a temperature of 37 C. The colonies of the gonococcus are just visible in twenty-four hours, and are seen both in the substance of the medium and on the surface. The deep colonies when examined with a lens are minute and slightly nodulated spheres, sometimes showing little processes, whilst those on the surface are thin discs of larger diameter with wavy margin and rather darker centre. In this way the gonococcus may be separated from fluids which are contaminated with a considerable number of other organisms. Relations to the Disease— The gonococcus is invari- ably present in the urethral discharge in gonorrhoea, and also in other parts of the genital tract when these are the seat of true gonorrheal infection. Its presence in these difterent positions has been demonstrated not only by microscopic examination but also by culture From the description of the conditions of growth in culture, it will be seen that a life outside the body in natural conditions is practically impossible— a statement which corresponds with the clinical fact that the disease is always transmitted directly by contagion. Inoculations of pure cultures on the urethra of lower animals, and even of apes, is followed by no effect, but a similar statement can be made with regard to inoculations of gonorrhceal pus itself. In fact hitherto it has been found impossible to reproduce the disease by any means in the lower animals. On a consider- able number of occasions inoculations of pure cultures have been made on the human urethra, both of the male and female and the disease, with all its characteristic symptoms, has resulted. (Such experiments have b en performed independently by Bumm, Steinschneider, Wer theim, and others.) The causal relationship of the SsLf tHe>diSeaSe ^ thGref0re been --Pletely of ^ : 7t ^ u ter6Sting t0 n°te h0W the editions of growth and the pathogenic effects of the organism aeree with the characters of the natural disease. g *3 194 G ONORRH (HA , SOFT SORE, SYPHILIS. Intraperitoneal injections of pure cultures of the gono- coccus in white mice produce a localised peritonitis with a small amount of suppuration, the organisms being found in large numbers in the leucocytes (Wertheim). They also penetrate the peritoneal lining and are found in the sub- endothelial connective tissue, but they appear to have little power of proliferation, they soon disappear, and the inflammatory condition does not spread. Injection of pure cultures into the joints of rabbits, dogs, and guinea-pigs causes an acute inflammation, which, however, soon sub- sides, whilst the gonococci rapidly die out ; a practically similar result is obtained when dead cultures are used. These experiments show that while the organism, when present in large numbers, can produce a certain amount of inflammatory change in these animals, it has little or no power of multiplying and spreading in their tissues. Toxine of the Gonococcus. — De Christmas has cultivated the gonococcus in a mixture of one part of ascitic fluid and three parts of bouillon, and has found that the fluid after twelve days' growth has toxic properties. At this period all the organisms are dead and the toxic substances are apparently derived from the breaking down of the bodies of the organisms. Such a fluid constitutes the " toxine." 1 he toxic substances are precipitated along with the proteids by alcohol, and the precipitate after being desiccated possesses the toxic action. In youno- rabbits injection of the toxine produces suppuration: this is well seen in the anterior chamber of the eye, where hypopyon results. The most interesting point, however, is with regard to its action on mucous surfaces ; for, while in the case of animals it produces no effect, its introduction into the human urethra causes acute catarrh, attended with purulent discharge. He found that no tolerance to the toxines resulted after five successive injections at intervals. Distribution in the Tissues.— The gonococcus having been thus shown to be the direct cause of the disease, some additional facts may be given regarding its presence both in the primary and secondary lesions. In the human urethra the gonococci penetrate the mucous membrane, passing chiefly between the epithelial cells, causing a loosening and desquamation of many of the latter and inflammatory reaction in the tissues below, attended witB DISTRIBUTION OF GONOCOCCUS. 195 great increase of secretion. There occurs also a gradually increasing emigration of leucocytes which take up a large number of the organisms. It is to be noted, however, that though there is such an abundant phagocytosis, the cocci within the leucocytes are usually quite healthy in appear- ance, and the establishment of the phagocytosis is not followed by a rapid cure of the disease. The organisms also penetrate the subjacent connective tissue, and are especially found along with extensive leucocytic emigration around the lacunae. Here also many are contained within leucocytes. They are constantly being carried to the surface by leucocytes and discharged, but by multiplication they are able to maintain their footing till such a time as the disease comes naturally to an end. Even, however, when the gonococci have disappeared from the urethral discharge, they may still be present in the deeper part of the mucous membrane of the urethra, possibly also in the prostate, and may thus be capable of producing infection. The prostatic secretion may sometimes be examined by making pressure on the prostate from the rectum when the patient has almost emptied his bladder, the secretion being afterwards discharged along with the remaining urine. (Foulerton). In acute gonorrhoea there is often a con- siderable degree of inflammatory affection of the prostate and vesiculae seminales, but whether these conditions are due to the presence of gonococci in the affected parts we have not at present the data for determining. A similar statement also applies to the occurrence of orchitis and also of cystitis in the early stage of gonorrhoea. During the more chronic stages other organisms appear in the urethra aid in maintaining the irritation, and may produce some of the secondary results. The bacillus coli, the pyogenic cocci, etc., are often present, and may extend along the urethra to the bladder and set up cystitis, though in this they may be aided by the passage of a catheter. It is then also that buboes usually occur, often associated with the presence of a small ulcer in the urethra. Though the bacteriology of these cannot yet be said to be fully worked 196 GONORRHCEA, SOFT SORE, SYPHILIS. out, they are certainly sometimes produced by the ordinary pyogenic organisms and by some varieties of diplococci which are often present in the urethra in abnormal conditions. It may be mentioned here that Wertheim cultivated the gonococcus from a case of chronic gonorrhoea of two years' standing, and by inoculation on the human subject proved it to be still virulent. In the disease in the female, gonococci are almost in- variably present in the urethra, the situation affected next in frequency being the cervix uteri. They do not appear to infect the lining epithelium of the vagina of the adult unless some other abnormal condition be present, but they do so in the gonorrhceal vulvo-vaginitis of young subjects. They have also been found in suppurations in connection with Bartholini's glands, and sometimes produce an inflam- matory condition of the mucous membrane of the body of the uterus. They may also pass along the Fallopian tubes and produce inflammation of the mucous membrane there. From the pus in cases of pyosalpinx they have been culti- vated in a considerable number of cases. According to the results of various observers they are present in one out of four or five cases of this condition, usually unassociated with other organisms. Further, in a large proportion of the cases in which the gonococcus has not been found no organisms of any kind have been obtained from the pus, and in these cases the gonococci may have been once present and have subsequently died out. Lastly, they may pass to the peritoneum and produce peritonitis, which is usually of a local character. It is chiefly to the methods of culture supplied by Wertheim that we owe our extended knowledge of such conditions. In gonorrheal conjunctivitis the mode in which the gonococci spread through the epithelium to the subjacent connective tissue is closely analogous to what obtains in the case of the urethra. Their relation to the leucocytes in the purulent secretion is also the same. Microscopic examination of the secretion alone in acute cases often gives positive evidence and pure cultures may be readily GONOCOCCUS IN JOINT AFFECTIONS, ETC. 197 obtained on blood-agar. As the condition becomes more chronic the gonococci are less numerous and a greater proportion of other organisms may be present. Relatiotis to Joint Affections, etc. — The relations of the gonococcus to the sequels of gonorrhoea form a subject of great interest and importance, but one on which further knowledge is still required. The reason of this is that till within the last few years the cultivation of the gonococcus had been a matter of considerable difficulty, and without cultures it is not possible to be absolutely certain of the identity of the organism, especially when present only in small numbers, there being other species of diplococci, some of which have been cultivated from the urethra in normal and diseased conditions, and which resemble the gonococcus not only in microscopical characters, but also in staining reaction. At present, however, the following statements may be made. First, in a certain number of cases of arthritis following gonorrhoea the gonococcus has been found microscopically, and pure cultures have been obtained, e.g., by Neisser, Lang, Bordoni-Uffreduzzi, and others. A similar statement applies to inflammation of the sheaths of tendons following gonorrhoea. Secondly, in a large proportion of cases no organisms have been found Thirdly, in some cases, especially in those associated with extensive suppuration, occasionally of a pysemic nature various pyogenic cocci have been found to be present It must therefore be" considered that a secondary infection of the joints by the gonococcus, evidently by way of the blood stream, can occur, and it remains to be determined in what proportion of cases it does so. In the instances in which the gonococcus has been found in the joints, the fluid present has been described as being usually of a whitish- yellow tint, somewhat turbid, and containing shreds of hbnn-hke material, sometimes purulent in appearance. It has also been noted in one or two cases, where the surface of the synovial membrane has been carefully examined, hat the gonococci have been much more numerous there than in the fluid— a circumstance which may explain some 198 GONORRHOEA, SOFT SORE, SYPHILIS. of the negative results when the fluid alone is examined. In one case Bordoni-Uffreduzzi cultivated the gonococcus from a joint-affection, and afterwards produced gonorrhoea in the human subject by inoculating with the culturrs obtained. In another case in which pleurisy was present along with arthritis the gonococcus was cultivated from the fluid in the pleural cavity. The existence of a gonorrheal endocarditis has been established by recent observations. Cases apparently of this nature occurring in the course of gonorrhoea have been described by Leyden, Michaelis, Thayer and Blumer, and others. In these there were present in the vegetations micrococci which, in their posi- tion within leucocytes, in their microscopical characters, and in their staining reactions, corresponded to gonococci. Cultures of the gonococcus were not obtained, but no other organisms grew on the media used, a circumstance which is in favour of the view that the organisms present were really gonococci. Quite recently, however, Rendu has cultivated the organisms from a diseased heart-valve. Thayer and Blumer, moreover, obtained from the blood during life colonies of an organism in every respect re- sembling the gonococcus. After six days the growths were found to have died and subcultures were not obtained. In this' case about 2 c.c. of blood were taken by venesection and added to melted agar, as in Wertheim's method. So far as we know it is the only record of successful culture from the blood. Methods of Diagnosis. — For microscopical examination dried films of the suspected pus, etc., may be stained by any of the simple solutions of the basic aniline stains. We prefer methylene- or thionin-blue, as they do not over- stain, and the films do not need to be decolorised. Stain- ing for one minute is sufficient. It is also advisable to stain by Gram's method, and it is a good plan to put at one margin of the cover-glass a small quantity of culture of staphylococcus aureus if available, in order to have a standard by which to be certain that the supposed gonococci are really decolorised. Regarding the value of microscopic SOFT SORE. 199 examination alone, we may say that the presence of a large number of micrococci in a urethral discharge having the characters, position, and staining reactions described above, is practically conclusive that the case is one of gonorrhoea. There is no other condition in which the sum total of the microscopical characters is present. We con- sider that it is sufficient for purposes of clinical diagnosis, and therefore of great value; in the acute stage a diagnosis can thus be made earlier than by any other method. The mistake of confusing gonorrhoea with such conditions as a urethral chancre with urethritis, will also be avoided. Even in chronic cases the typical picture is often well maintained, and microscopic examination alone gives a definite positive result. When other organisms are present, and especially when the gonococci are few in number, it is difficult, and in some cases impossible, to give a definite opinion, as a few gonococci mixed with other organisms cannot be recognised with certainty. This is often the condition in chronic gonorrhoea in the female. Microscopic examina- tion, therefore, though often giving positive results, will sometimes be inconclusive. Cultures alone supply the absolute test, and when the organism is present in an apparent, condition of purity, Wertheim's medium or blood- agar should be used. If other organisms are present, we are practically restricted to Wertheim's plate method. Soft Sore. Within recent years a considerable amount of attention has been directed to the bacteriology of this condition, owing to the discovery of a somewhat characteristic bacillus in the affected parts. This organism was first described by Ducrey in 1889, who found it in the purulent discharge from the ulcerated surface; and later, in 1892, TJnna described its appearance and distribution as seen in sections through the sores. The statements of these observers regarding the presence and characters of this organism have been fully confirmed by other observers. 200 GONORRH(EA% SOFT SORE, SYPHILIS. Microscopical Characters. — This organism occurs in the form of minute oval rods measuring about 1.5 //, in length, and .5 fi in thickness. It is found mixed with other organisms in the purulent discharge from the surface, and is chiefly arranged in small groups or in short chains. When studied in sections through the ulcer it is found in the superficial part of the floor, but more deeply situated than other organisms, and may be present in a state of purity amongst the leucocytic infiltration. In this position it is usually arranged in chains which may be of consider- able length, and which are often seen lying in parallel rows between the cells. Both in the tissues and in the secretions the bacilli chiefly occur in the free condition, but occasionally a few may be contained within leucocytes. This bacillus takes up the basic aniline stains fairly readily, but loses the colour very rapidly when a decolor- ising agent is applied. Accordingly, in film preparations when dehydration is not required, it can be readily stained by most of the ordinary combinations, though Loffler's or Kuhne's methylene-blue solutions are preferable, as they do not overstain. In sections, however, great care must be taken in the process of dehydration, and the aniline-oil method (vide p. 107) should be used for this purpose, as alcohol decolorises the organism very readily. A little of the methylene-blue or other stain may be with advantage added to the aniline oil used for dehydrating. This organism has not yet been successfully cultivated outside the body, though practically every medium has been tried for this purpose. Ducrey, however, succeeded in separating it from other organisms by the following method. He produced a series of pustules by successive inoculations in the human subject on the skin, which had been previously sterilised, the pustules being afterwards protected from contamination by watch-glasses fixed in position. He found that in this method the other organisms gradually died off, while the characteristic bacilli persisted and at about the fifth or sixth inoculation might be present alone. Further, the pus containing the bacilli in a pure SOFT SORE. 20 1 condition still produced the typical lesion on inoculation. Even when the organisms were thus separated he failed to obtain any growth on the numerous media which he employed. The evidence that this organism is the causal agent in the affection accordingly rests on the facts well established that the organism is apparently always present in the discharge from the sore, and in its tissues ; that it has been observed hitherto in no other form of ulceration, and that it is sharply marked off from saprophytic organisms by the fact that is has not been obtained in cultures outside the body. Regarding the presence of this organism in the buboes associated with soft sore there is some uncertainty. A considerable number of observers have failed to find it, and have also failed to produce a characteristic soft sore by inoculation with pus withdrawn from a bubo under aseptic precautions. When a chancroid condition follows in a bubo which has been opened, they accordingly con- sider that it has been secondarily inoculated with the bacillus. On the other hand one or two observers have found the bacillus in unopened buboes. Audry, for example, in a bubo before suppuration had occurred, found it lying in little groups of two or three within leucocytes in the lymph channels ; and in this case inoculation with the material from the bubo produced the typical lesion. Krefting also found it in buboes in some cases. It is therefore possible that the buboes associated with soft sore are caused by the same organisms, but that as suppuration occurs they in great part die off. It seems certain at least, from the results of various workers, that in many cases the ordinary pyogenic organisms are not present in the suppurating buboes. In connection with the two diseases, gonorrhoea and soft sore, it is of special interest to note in the case of the former how restricted are the conditions of growth outside the body of the organism which produces the disease, and in the case of the latter that attempts to cultivate the 202 CON OR A' II QiA, SOFT SORE, SYPHILIS. supposed causal organism outside the body have entirely failed. Syphilis. Regarding the relation of bacteria to this disease, we cannot be said at present to possess much definite know- ledge. Most interest, however, is attached to the observa- tions of Lustgarten, who in 1884 described a characteristic bacillus both in the primary sore and in the lesions in in- ternal organs. He found it in all of sixteen cases which he examined. This bacillus somewhat resembles the tubercle bacillus in shape and size. It occurs in the form of slender rods, straight or slightly bent, about 3 to 4 n in length, often forming little clusters either within cells or lying free in the lymphatic spaces. Like the tubercle bacillus it takes up the basic aniline stains with difficulty, but it is much more easily decolorised by mineral acids. Lustgarten stained the tissues for twenty-four to forty- eight hours in aniline-water solution of gentian violet ; and then, after washing them in alcohol, placed them for ten seconds in a 1.5 per cent solution of permanganate of potassium. They were then treated with sulphurous acid, which removes the brown precipitate formed, and decolorises the sections. They were then washed in water, dehydrated, and mounted. The observations of other workers have given contradictory results. De Michele and Radice, for example, found Lustgarten's bacilli in the tissues in forty-five out of sixty-four cases examined, while, on the other hand, other observers have failed to find them. Apart, however, from negative results obtained by many, criticism has been made in other ways. It has been alleged by some that Lustgarten's bacilli is merely the smegma bacillus which has penetrated the affected tissues. This bacillus, which was first described by Alvarez and Tavel, occurs in the smegma prceputiale, and morphologically resembles somewhat the tubercle bacillus, but is more easily decolorised. The above explanation, however, SYPHILIS. 203 would not account for the presence of the bacilli in the internal organs, where they were observed by Lustgarten and others. And further, there are minor points of differ- ence between this smegma bacillus and Lustgarten's bacillus. It has also been suggested by some that the organisms described by Lustgarten are merely tubercle bacilli which have been accidentally present in the affected tissues. Those, however, who have found the former organism in the tissues agree that it can be readily distinguished from the tubercle bacillus, as it does not resist decolorising with strong acids. This explanation of the presence of these bacilli in the tissues is really without definite support. The organism has not been cultivated outside the body, though, in view of what we know with regard to some other diseases, this fact in itself does not form a grave objection. In the absence, however, of definite evidence as to its invariable presence in the lesions, its relations to the disease are still highly problematical. It may also be noticed that this organism has been found in the tertiary lesions, which are usually believed to be non-infectious. Other organisms have been described as present in syphilitic lesions, notably one quite recently by Van Niessen. This organism is a pleomorphous bacillus belonging to the higher bacteria. He claims not only to have demonstrated it, both in the tissues and in the blood, but to have obtained it in pure culture from a number of cases. Until confirmation of his results has been obtained it is unnecessary to give details. CHAPTER VIII. ACUTE PNEUMONIA. Introductory.- — Pneumonia, as a clinical term, is applied to several conditions which present differences in pathological anatomy and in origin. There is, first of all, the acute croupous or lobar pneumonia, in which an inflammatory process attended by abundant fibrinous exudation affects, by continuity, the entire tissue of a lobe or of a large portion of the lung. This type is, both in its clinical symptoms and pathological effects, familiar. Secondly, there is the acute catarrhal or lobular pneumonia, where the inflammatory process spreads from the capillary bronchi to the air vesicles, and leads to a catarrhal con- solidation of patches of the lung tissue. Up till 1889 acute catarrhal pneumonia was comparatively rare except in children. In adults it was chiefly found as a secondary complication to some condition such as diphtheria, typhoid fever, etc. Since the first recent great epidemic of in- fluenza in the year named, however, it has been of much more frequent occurrence in adults, has assumed a very fatal tendency, and has presented the formerly quite un- usual feature of being sometimes the precursor of gangrene of the lung. Moreover, not only has the prevalent type of pneumonia (the term being used in its widest sense) changed through the occurrence of a greater proportion of catarrhal cases, but it appears to be now more common to find cases which microscopically present a mixed type, i.e., in TYPES OF PNEUMONIA. 205 which both an acute croupous condition and an acute catarrh occur in the same lung. Besides these two clinical types of pneumonia there is another group of cases which are somewhat loosely denominated septic pneumonias, and which may arise in two ways : (1) by the entrance into the trachea and bronchi of discharges, blood, etc., which form a nidus for the growth of septic organisms, and thus infect the tissue of the lung ; (2) from secondary pyogenic infection by means of the blood stream from suppurative foci in other parts of the body. (See Chapter on Suppuration, etc.) We shall see that bacteria have been found associated with all these types of pneumonia. Special importance is attached to acute croupous pneumonia on account of its course and characters, but reference will also be made to the other forms. Historical. — Acute lobar pneumonia for long, both popularly and medically, had been supposed to be an effect of exposure to cold ; but there were not wanting those who' were dissatisfied with this view of its etiology. Not only did many cases occur where no such exposure could be traced, but it had been observed that the disease sometimes occurred epidemically, and was occasionally contracted by hospital patients lying in beds adjacent to those occupied by pneumonia cases. Further, the sudden onset and definite course of the disease conformed to the type of an acute infective fever. It was thus suspected by some that it might in reality be due to a specific infection. The first con- tributor to the modern view of its etiology was Friedlander, whose results (published in 1882-83) were briefly as follows. In the bronchial contents and in sections of pneumonic lungs, there were cocci, adherent usually in pairs, and possessed of a definitely con- toured capsule which was faintly but distinctly stained. These cocci could be isolated and grown on gelatine, and on inoculation in mice they produced definite pathogenic effects. Instead of developing pneumonia, however, the animals died of a kind of septicemia with inflammation of the serous membranes. The blood and the exudation in serous cavities contained numerous capsulated diplococci. Though of course this was not proof that the cocci were the cause of the disease in man, Friedlander brought forward the growing tendency to regard pneumonia as an infectious disease, the alleged universal occurrence of his cocci in the lungs of persons dead of the disease, and the patho- genic capacities of these cocci in animals, as indications that an etio- logical factor had been discovered. Various criticisms of Friedlander's 206 ACUTE PNEUMONIA. views soon appeared, and there is little doubt that many of the organ- isms seen by T'riedlander were really Fraenkel's pneumococcus to be presently described. By many observers it was found that the sputum of healthy men, when injected into animals, sometimes caused death, with the same symptoms as in the case of the injection of Friedlander's coccus ; and in the blood and serous exudations of such animals capsulaled diplo- cocci were found. A. Fraenkel investigated this subject, and found that the sputum of pneumonic patients was much more fatal and more constant in its effects than that of healthy individuals. The cocci which were found in animals dead of this "sputum septicaemia" as it was called, differed from Friedlander's cocci in several respects to be presently studied. Fraenkel further investigated a few cases of pneu- monia, and isolated from them cocci identical in microscopic appear- ances, cultures, and pathogenic effects, with those isolated in sputum septicaemia. The most extensive investigations on the whole question were those of Weichselbaum, published in 1886. This author examined 129 cases of the disease, and included in his survey not only acute croupous pneumonia, but lobular and septic pneumonias. From them he isolated four groups of organisms. ( 1 ) Diplococcus pneumonia. This he described as an oval or lancet-formed coccus, corresponding in appearance and growth characters to Fraenkel's coccus. (2) Strepto- coccus pneumonia. This was less common than the last, was rounder, and formed longer and more twisted chains, but on the whole pre- sented similar characters. It was more vigorous in its growth, and could grow below 20° C, though it preferred a temperature of 370 C. (3) Staphylococcus pyogenes aureus. (4) Bacillus pneumonia. This was a short rod-shaped organism, which must be classed among the bacilli. Weichselbaum, however, was of opinion that it was identical with Friedlander's pneumococcus. Of these organisms the diplococcus pneumoniae was by far the most frequent, being observed in 94 cases of the 129 examined, and isolated by cultures in 54. It also occurred in all forms of pneumonia. Next in frequency was the streptococcus pneumoniae, and lastly the bacillus pneumoniae. Inoculation experiments were also performed by Weichsel- baum with each of the three characteristic cocci he isolated. The diplococcus pneumoniae and the streptococcus pneumoniae both gave pathogenic effects of a similar kind in certain animals. The general result of these earlier observations was to establish the occurrence in connection with pneumonia of two species of organisms, each having its distinctive characters, viz. : — 1. Fraenkel's pneumococcus, which is recognised to be identical with the coccus of "sputum septicaemia," with CHARACTERS OF BACTERIA OF PNEUMONIA. 207 Weichselbaum's diplococcus pneumonias, and probably also with his streptococcus pneumonias. 2. Friedlander 's pneumococcus (now known as Fried- lander's pneumobacillus), which is almost certainly the same as the bacillus pneumoniae of Weichselbaum. We shall use the terms " FraenkePs pneumococcus " and " Friedlander's pneumobacillus," as these are now usually applied to the two organisms. Microscopic Characters of the Bacteria of Pneumonia. — Methods. — The organisms present in acute pneumonia can best be examined in film preparations made from pneu- monic lung (preferably from a part in a stage of acute con- gestion or early hepatization) or from the gelatinous parts of pneumonic sputum (here again preferably when such sputum is either rusty or occurs early in the disease), or in sections of pneumonic lung. Such preparations may be stained by any of the ordinary weak stains, such as a watery solution of methylene-blue, but Gram's method is to be preferred, with safranin or Bismarck-brown as a contrast stain. Ziehl- Neelsen carbol-fuchsin is also very suitable ; ft is best either to stain with it for only a few seconds, or to overstain and then decolorise with alcohol till the ground of the prepara- tion is just tinted. In such preparations as the above, and even in specimens taken from the lungs immediately after death (as may be quite well done by means of a hypodermic syringe), putrefactive and other bacteria may be present, but those to be looked for are capsulated organisms which may be of either or both of the varieties mentioned. (1) Fraenkel's Pneumococcus. — This organism occurs in the form of small oval cocci, about 1 ^ in longest diameter, arranged generally in pairs (diplococci), but also in chains of four to ten (Fig. 56). The free ends are often pointed like a lancet, hence the term diplococcus lanceolatus has also been applied to it. These cocci have round them a capsule, which usually appears as an unstained halo, but is some- times stained more deeply than the ground of the prepara- tion. This difference in staining depends, in part at least, on the amount of decolorisation to which the pre- 208 A C U'l 'E PNE UA/ON/. I . paration has been subjected. The capsule is rather broader than the body of the coccus, and has a sharply defined external margin. This organism takes up the basic aniline stains with great readiness, and also retains the stain in Gram's method. It is the organism of by far the most fre- quent occurrence in true croupous pneu- monia, and in fact may be said to be rarely absent. (2) Fried/tinder's Pneumobacillus. — As seen in the sputum and tissues, this or- ganism both in its ap- pearance and arrangement, as also in the presence of a capsule, somewhat resembles Fraenkel's pneumococcus, and it was at first described as the "pneumococcus." The form, however, is more of a short rod-shape, and it has blunt rounded ends; it is also rather broader than Fraenkel's pneumococcus. It is now usually classed amongst the bacilli, especially in view of the fact that in cultures elongated rod forms may occur. The capsule has the same general characters as that of Fraenkel's organism. Friedlander's pneumobacillus stains readily with the basic aniline stains, but loses the stain in Grain's method, and is accordingly coloured with the contrast stain, — safranin or Bismarck-brown, as above recommended. A valuable means is thus afforded of distinguishing it from Fraenkel's pneumococcus in microscopic preparations. Friedlander's organism is much less frequently present in pneumonia than Fraenkel's ; sometimes it is associated with the latter, very rarely it occurs alone. Fig. 56. — Film preparation of pneumonic sputum, showing numerous pneumococci (Fraenkel's) with capsules; some are ar- ranged in short chains. Stained with carbol-fuchsin. x 1000. CULTIVATION OF PNEUMOCOCCUS. 209 In sputum preparations the capsule of both pneumo- cocci may not be recognisable, and the same is sometimes true of lung preparations. This is probably due to changes which occur in the capsule as the result of changes in the vitality of the organisms. Sometimes the difficulty of recognising the capsule when it is present, is due to the refractive index of the fluid in which the specimen is mounted being almost identical with that of the capsule. This difficulty can always be overcome by having the ground-work of the preparation tinted. The Cultivation of Fraenkel's Pneumococcus. It is usually difficult, and sometimes impossible, to isolate this coccus directly from pneumonic sputum. On culture media it has not a vigorous growth, and when mixed with other bacteria it is apt to be overgrown by the latter. To get a pure culture it is best to insert a small piece of the sputum beneath the skin of a rabbit or a mouse. In about forty- eight hours the animal will die, with numerous capsulated pneumococci throughout its blood. From the heart-blood . cultures can be easily obtained. Cultures can also be got pst mortem from the lungs of pneumonic patients by streaking a number of agar or blood-agar tubes with a scraping taken from the area of acute congestion or com- mencing red hepatization, and incubating them at 37 0 C. The colonies of the pneumococcus appear as almost trans- parent small discs which have been compared to drops of dew (Fig. 57). This method is also sometimes successful in the case of sputum. The appearances presented in cultures by different varieties of the pneumococcus vary somewhat. It always grows best on blood serum or on Pfeiffer's blood agar It usually grows well on ordinary agar or in bouillon, but not so well on glycerine agar. In a stroke culture on blood serum grovyth appears as an almost transparent pellicle along the track, with isolated colonies at the margin. On amr media it is more manifest, but otherwise has similar characters. The appearances are similar to those of a culture of streptococcus pyogenes, but the growth is less x4 2IO ACUTE PNEUMONIA. vigorous, and is more delicate in appearance. A similar statement also applies to cultures in gelatine at 22° C, growth in a stab culture appearing as a row of minute points which remain of small size ; there is, of course, no liquefaction of the medium. On agar plates colonies are almost invisible to the naked eye, but under a low power of the microscope appear to have a compact finely granular centre and a pale trans- parent periphery. In bouillon, growth forms a slight turbidity, which settles to the bottom of the vessel as a slight dust-like deposit. On pota- toes, as a rule, no growth appears. Cultures on such media may be maintained for one or two months, if fresh sub-cultures are made every four or five days, but they tend ul- timately to die out. They also rapidly lose their virulence, so that four or five days after isolation from an animal's body their pathogenic action is already diminished. Eyre and Washbourne, however, have succeeded in maintaining cultures in a condition of constant virulence for at least three months by growing the organisms on agar smeared with rabbits' blood. The agar must be prepared with Witte's peptone, must not be heated over ioo° C, and after neutralisation (rosolic acid being used as the indicator) must have .5 per cent of normal sodium hydrate added. The tubes when inoculated are to be kept at 37.50 C. and sealed to prevent evaporation. In none of the ordinary artificial media do pneumococci develop a capsule. They usually appear as diplococci, but in preparations made from the surface of agar or from bouillon, shorter or longer chains may be observed (Fig. 58). After a few days' growth Fig. 57. — Stroke culture of Fraenkel's pneumococcus on blood agar. The colonies are unusually large and distinct. 24 hours' growth at 37° C. Natural size. CULTIVATION OF PNEUMOBACILLUS. 211 they lose their regular shape and size, and involution forms appear. Usually the pneumococcus does not grow below 2 2° C, but forms in which the virulence has disappeared often grow well at 20° C. Its optimum • \ temperature is 370 ** J * C.j its maximum 42 0 ^* f\* s C. It is preferably / # \ % an aerobe, but can *» -•*/ exist without oxy- } \** j V, gen. It prefers a \ « 1 slightly alkaline •. medium to a neutral, \ » * and does not grow v. ^ on an acid medium. ^ % \ These facts show that I when growing out- X** side the body on •* artificial media, the Fig. 58.— Fraenkel's pneumococcus from a pneumocOCCUS is . a Pureculture on blood agar of twenty-four hours' pnmnnr,t;,,0l„ A ^ • Srowth' some in Pairs, some in short chains. Comparatively dell- Stained with weak carbol-fuchsin. x 1000. cate organism. The Cultivation of Friedlander's Pneumobacillus. This organism, when present in sputum or in a pneumonic lung, can be readily separated by making ordinary gelatine plate cultures, or a series of successive strokes on agar tubes. The surface colonies always appear as white discs which become raised from the surface so as to appear like little knobs of ivory. From these, pure cultures can be readily obtained. The appearance of a stab culture in gelatine growth is very characteristic. At the site of the puncture there is on the surface a white growth heaped up, it may be fully one -eighth of an inch above the level of the gelatine ; along the needle track there is a white granular appearance, so that the whole resembles a white" round- headed nail driven into the gelatine (Fig. 59). Hence the name « nail-like " which has been applied. Occasionally bubbles of gas develop along the line of growth. There is 212 ACUTE PNEUMONIA. no liquefaction of the medium. On sloped agar it forms a \ **■ » Fig. 6o. — Friedlander's pneumobacillus,1 from a young culture on agar ; showing some rod-shaped forms. Stained with thionin-blue. x iooo. very white growth with a shiny lustre, which, when touched with a platinum needle, is found to be of a viscous consistence. In cultures much longer rods are formed than in the tissues of the body (Fig. 6o). On the sur- face of potatoes it forms an abundant moist white .layer. Friedlander's bacillus has active fermenting powers on sugars, though varieties isolated by different observers vary in the degree in which such powers are possessed. It always seems capable of acting on dextrose, lactose, maltose, dextrin, and mannite, and sometimes also Fig. 59. — Stab cul- ture of Friedlander's pneumobacillus in peptone gelatine, show- ing the nail -like ap- pearance ; ten clays' growth. Natural size. 1 The apparent size of this organism, on account of the nature of ite sheath, varies much according to the stain used. If stamed with a strong Sin, e.g., carbol-fuchsin, its thickness appears nearly twice as great as ,s shown in the figure. DISTRIBUTION OF PNEUMOBA CTERIA. 213 on glycerine. The substances produced by the fermenta- tion vary with the sugar fermented, but include ethylic alcohol, acetic acid, lsevolactic acid, succinic acid, along with hydrogen and carbonic acid gas. The amount of acid produced from lactose seems only exceptionally sufficient to cause coagulation of milk. The Occurrence of the Pneumobacteria in Pneumonia and other Conditions. — The pathological anatomy of pneumonia is so fully dealt with in all text -books on pathology, that it is unnecessary for us to do more than emphasise its strictly bacteriological features. Capsulated organisms have been found in every variety of the disease — in acute croupous pneumonia, in broncho-pneumonia, in septic pneumonia. In the great majority of these it is Fraenkel's pneumococcus which both microscopically and culturally has been found to be present. Friedlander's pneumobacillus occurs in only about 5 per cent of the cases. It may be present alone or associated with Fraenkel's organism. In a case of croupous pneumonia the pneumococci are found all through the affected area in the lung, especially in the exudation in the air-cells. They also occur in the pleural exudation and effusion, and in the lymphatics of the lung. The greatest number are found in the parts where the inflammatory process is youngest, e.g., in an area of acute congestion in a case of croupous pneumonia, and therefore such parts are prefer- ably to be selected for microscopic examination, and as the source of cultures. Sometimes there occur in pneu- monic consolidation areas of suppurative softening which may spread diffusely. In such areas the pneumococci occur with or without ordinary pyogenic organisms streptococci being the commonest concomitants. In other cases, especially when the condition is secondary to influenza, gangrene may supervene and lead to destruc- tion of large portions of the lung. In these a great variety of bacteria, both aerobes and anaerobes, are to be found In ordinary broncho-pneumonias also Fraenkel's pneumo- coccus is usually present, sometimes along with pyogenic 214 ACUTE PNEUMONIA. cocci ; in the broncho-pneumonias secondary to diphtheria it may be accompanied by the diphtheria bacillus, and also by pyogenic cocci ; in typhoid pneumonias the typhoid bacillus or the B. coli may be alone present or be accom- panied by the pneumococcus, and in influenza pneumonias the influenza bacillus may occur. In septic pneumonias the pyogenic cocci in many cases are the only organisms discoverable, but the pneumococcus may also be present. Especially important, as we shall see, from the point of view of the etiology of the disease, is the occurrence in other parts of the body of pathological conditions associ- ated with the presence of the pneumococcus. By direct extension to neighbouring parts empyema, pericarditis, and lymphatic enlargements in the mediastinum and neck may take place ; in the first the pneumococcus may occur either alone or with pyogenic cocci. But distant parts may be affected, and the pneumococcus may be found in suppurations and inflammations in various parts of the body (subcutaneous tissue, joints, kidneys, liver, etc.), in otitis media, ulcerative endocarditis (p. 184), and menin- gitis. These conditions may take place either as complica- tions of pneumonia, or they may constitute the primary disease. The occurrence of meningitis is of special im- portance, for next to the lungs the meninges appear to be the parts most liable to attack by the pneumococcus. A large number of cases have been investigated by Netter, who gives the following tables of the relative frequency of the primary infections by the pneumococcus in man : — (1) In adults — Pneumonia . . 65.95 per cent Broncho-pneumonia \ Capillary bronchitis / Meningitis . Empyema . Otitis . Endocarditis Liver abscess (2) In children 46 cases were investigated. In 29 the primary affection was otitis media, in 12 broncho-pneumonia, in 2 meningitis, in 1 pneumonia, in 1 pleurisy, in 1 pericarditis. 15.85 13.00 8-53 2.44 1.22 1.22 EXPERIMENTAL INOCULATION. 215 Thus in children the primary source of infection is in a great many cases an otitis media, and Netter concludes that infection takes place in such conditions from the nasal cavities. Experimental Inoculation. — The pneiimococcus of Fraenkel is pathogenic to various animals. The sus- ceptibility of different species, as Gamaleia has shown, varies to a considerable extent. The rabbit, and especially the mouse, are very susceptible; the guinea-pig, the rat, the dog, and the sheep occupy an intermediate position ; the pigeon is quite immune. In the more susceptible animals the general type of the disease produced is not pneumonia, but a general septicemia. Thus, if a rabbit or a mouse be injected subcutaneously with pneumonic sputum, or with a scraping from a pneumonic lung, death occurs in from twenty-four to forty-eight hours. There is some fibrinous infiltration at the point of inoculation, the spleen is often enlarged and firm, and the blood contains capsulated pneumococci in large numbers (Fig. 61). If the seat of inoculation be in the lung, there generally results pleuritic effusion on both sides, and in the lung there may be a process somewhat resembling the early stage of acute croupous pneumonia in man. There are often also pericarditis and enlargement of spleen. We have already stated that cultures of the pneumococci on artificial media in a few days begin to lose their virulence. Now, if such a partly attenuated culture be injected sub- cutaneously into a rabbit, there is greater local reaction ; pneumonia, with exudation of lymph on the surface of the pleura, and a similar condition in the peritoneum, may occur. In sheep greater immunity is marked by the occurrence, after subcutaneous inoculation, of an enormous local sero-fibrinous exudation, and by the fact that few pneumococci are found in the blood stream. Intra-pul- monary injection in sheep is followed by a typical pneu- monia, which is generally fatal. The dog is still more immune; in it also intra-pulmonary injection is followed by a fibrinous pneumonia, which is only sometimes fatal. 2 I 6 ACUTE PNEUMONIA. Inoculation by inhalation appears only to have been per- formed in the susceptible mouse and rabbit; here also septicemia resulted. The general conclusion to be drawn from these experi- ments thus is that in highly susceptible animals virulent pneumococci produce a general septicaemia; whereas in more FlG. 6i. — Capsulated pneumococci in blood taken from the heart of a rabbit, dead after inoculation with pneumonic sputum. Dried film, fixed with corrosive sublimate. Stained with carbol-fuchsin and partly decolorised. x iooo. immune species there is an acute local reaction at the point of inoculation, and if the latter be in the lung, then there may result pneumonia, which, of course, is merely a local acute inflammation occurring in a special tissue, but identical in essential pathology with an inflammatory reaction in any other part of the body. When a dose of pneumococci EXPERIMENTAL INOCULATION. 217 sufficient to kill a rabbit is injected subcutaneously in the human subject, it gives rise to a local inflammatory swelling with redness and slight rise of temperature, all of which pass off in a few days. It is therefore justifiable to suppose that man occupies an intermediate place in the scale of susceptibility, probably between the dog and the sheep, and that when the pneumococcus gains an entrance to his lungs, the local reaction in the form of pneumonia occurs. Analogies to the facts just stated are afforded in the case of other diseases caused by bacteria. Thus, for example, the anthrax bacillus produces in the human subject more marked inflammatory reaction, and is more restricted to the local lesions, than in the much more susceptible guinea- pig, in which it produces a rapidly fatal septicaemia. An analogous result is also obtained when, instead of taking animals of different susceptibility, the same species of animal is used, but the virulence of the organism is altered ; for example, a streptococcus, as already 'stated, producing at one time an erysipelatous condition, causes an acute septicaemia when its virulence is increased. The occurrence in the lung of inflammatory conditions due to other causes does not make it less likely that the great majority of cases of acute pneumonia which occur under natural conditions have as the causal agent the pneumococcus. For in the latter we have an organism with certain very definite microscopic and biological characters, which is certainly present in the great majority of, if not in all, cases of the disease. Its action as a pro- ducer of general septicaemia in animals, we have seen, finds a perfectly rational explanation in the different degrees of susceptibility which exists towards it in different species In this connection the occurrence of manifestations of general infection associated with pneumonia in man is of the highest importance. We have seen that meningitis and other inflammations are not very rare complication? of he disease, and such cases form a link connecting the local disease in the human subject with the general septicemic processes which may be produced artificially 2l8 ACUTE PNEUMONIA. in the more susceptible representatives of the lower animals. A fact which has, in the minds of some, rather mili- tated against the pneumococcus being the cause of pneu- monia is the discovery of this organism in the saliva of healthy men. This fact was early pointed out by Pasteur, and also by Fraenkel, and the observation has been con- firmed by many other observers. It can certainly be isolated from the mouths of a considerable proportion of normal men, from their nasal cavities, etc., being probably in any particular individual more numerous at some times than at others, and sometimes being entirely absent. This can be proved, of course, by inoculation of susceptible animals. Such a fact, however, does not necessarily imply that the pneumococcus is not the cause of pneumonia. It only im- plies the importance of predisposing causes in the etiology of the disease, and it is further to be observed that we have corresponding facts in the case of the diseases caused by pyogenic staphylococci, streptococci, the bacillus . coli, etc. It is probable that by various causes the vitality and power of resistance of the lung are diminished, and that then the, pneumococcus gains an entrance. In relation to this possi- bility we have the very striking facts that in the irregular forms of pneumonia, secondary to such conditions as typhoid and diphtheria, the pneumococcus is very frequently present, alone or with other organisms. Apparently the toxines produced by such bacteria as the B. typhosus and the B. diphtheria can devitalise the lung to such an extent that secondary infection by. the pneumococcus is more likely to occur and set up pneumonia. We can therefore understand how much less definite devitalising agents such as cold, alcoholic excess, etc., can play an important part in the causation of pneumonia. In this way also other abnormal conditions of the respiratory tract, a slight bronchitis, etc., may play a similar part. It is more difficult to explain why sometimes the pneumo- coccus is associated with a spreading inflammation, as in croupous pneumonia, whilst at other times it is localised ETIOLOGICAL RELATLONS TO PNEUMONIA. 219 to the catarrhal patches in broncho -pneumonia. It is quite likely that in the former condition the organism is possessed of a higher order of virulence, though of this we have no direct proof. We have, however, a closely analogous fact in the case of erysipelas, which, we have stated reasons for believing, is produced by a streptococcus which, when less virulent, causes only local inflammatory and suppurative conditions. Summary. — We may accordingly summarise the facts regarding the relation of Fraenkel's pneumococcus to the disease by saying that it can be isolated from nearly all cases of acute croupous pneumonia, and also from a con- siderable proportion of other forms of pneumonia. When injected into the lungs of moderately insusceptible animals it gives rise to pneumonia. If, in default of the crucial experiment of intra-pulmonary injection in the human subject, we take into account the facts we have discussed, we are justified in holding that it is the chief factor in causing croupous pneumonia, and also plays an important part in other forms. Pneumonia, in the widest sense of the term, is, however, not a specific affection, and various inflammatory conditions in the lungs can be set up by the different pyogenic organisms, by the bacilli of diphtheria, of influenza, etc. The possibility of Friedlander's pneumobacillus having an etiological relationship to pneumonia has been much disputed. Its discoverer found that it was pathogenic towards mice and guinea-pigs, and to a less extent towards dogs. Rabbits appeared to be immune. The type of the disease was of the nature of a septicaemia. No extended experiments, such as those performed by Gamaleia with Fraenkel's coccus, have been done, and therefore we cannot say whether any similar pneumonic effects are produced by it in partly susceptible animals. The organism appears to be present alone in a small number of cases of pneumonia, and the fact that it also appears to have been the only organism present in certain septicemic complications of pneumonia, such as empyema and meningitis, render it 220 ACUTE PNEUMONIA. possible that it may be the causal agent in a few cases of the disease. The Toxines of Fraenkel's Pneumococcus. — Pneumonia is a disease which presents in many respects the characters of an acute poisoning. In very few cases does death take place from the functions of the lungs being interfered with to such an extent as to cause asphyxia. It is from cardiac failure, from grave interference with the heat -regulating mechanism, and from a general nervous depression that death usually results. These considerations, taken in connection with the fact that in man the pneumococci are usually confined to the lung, suggest that they may produce their general effects by means of toxines. The subject has been investigated by Emmerich and Fowitsky and by G. and F. Klemperer. The latter isolated from recent bouillon cultures, by the methods of Brieger and Fraenkel (p. 154) bodies having the reactions of the toxalbumins obtained in the case of other bacteria. When injected, these tox- albumins (which they called " pneumotoxin ") produced symptoms in rabbits, and when they were derived not from, bouillon cultures but from the blood of animals dead of the disease, they could produce fatal effects. We have seen that ordinarily the pneumococci rapidly lose their virulence in artificial media, and therefore instead of letting bouillon cultures go on for a month, as in the case of diphtheria, the Klemperers had to be content with two days' growth to obtain the maximum effect. We can say little of the true nature of these toxines. Their activity is interfered with by an hour's exposure at 6o° C, but, as in the case of other toxines, whether they are really proteids, or non- ' proteid bodies carried down with the latter in the methods of precipitation used, we do not know. Immunisation against the Pneumococcus. — Animals can be immunised against the pneumococcus either by inocula- tion with attenuated cultures or by the injection of toxic bodies derived from cultures. The former can be effected by cultures which have become attenuated by growth on artificial media, or by the naturally attenuated cocci which IMMUNISATION AGAINST PNEUMOCOCCUS. 221 occur in the sputum after the crisis of the disease. Netter effected immunisation by injecting an emulsion of the dried spleen of an animal dead of pneumococcus septicaemia. Here the cocci were attenuated by the drying. Virulent cultures killed by heating at 620 C. have also been used, immunisation being here accomplished by the intracellular toxines. The Klemperers found that injection of rusty sputum kept at 6o° C. for one to two hours and then filtered, and of toxine similarly treated, had a like result. In all cases one or two injections of the modified bacteria or toxine were sufficient for immunisation. It was three days in the case of intravenous injection, and fourteen days in the case of subcutaneous injection, before immunity was established, and the latter lasted a month or more. The immunity was accompanied by the development in the blood of antitoxic substances which had no effect either outside or inside the body in killing the pneumococci, but merely neutralised their toxines. Such antitoxines not only protected a rabbit against subsequent inoculation with pneumococci, but if injected within twenty-four hours after inoculation, prevented death. These results have been generally confirmed by later observers. In this connection an interesting fact observed by Mennes may be noted, namely, that normal leucocytes only become phagocytic towards pneumococci when they are lying in the serum of an animal immunised against this bacterium. The production of antitoxines may shed new light on what occurs in man in the case of recovery from pneumonia. The view has been advanced that the crisis so character- istic of a non-fatal case of the disease takes place when the balance of antitoxine against toxine is in favour of the former. The pneumococci after the crisis, as has been proved both culturally and by inoculation experiments, are still vital and virulent, though not so virulent as when the fever is at its height. On them directly the antitoxine has no effect, but any toxine now elaborated by them is neutralised and has no longer either local or general pathogenic effects' 222 ACUTE PNEUMONIA. A fact interesting as corroborating the view that the pneumococcus is really the cause of acute lobar pneumonia, is that the serum of patients who have recovered from pneumonia has in a certain proportion of cases a protective effect against the pneumococcus in rabbits. So far as our knowledge goes, such a protective serum is specific, or in other words, protects only against the organism by the action of which its protective properties have been produced, and therefore it must be against the pneumococcus that the human subject requires protection in pneumonia. The Klemperers treated a certain number of cases of human pneumonia by serum derived from immune animals, and apparently with a certain measure of success. The most exact work on this subject is that of Washbourn, who, as already described, has succeeded in obtaining pneumo- coccus cultures of constant virulence. This observer immunised a pony by using (i) broth cultures killed by one hour's exposure to 6o° C; (2) living agar cultures; (3) living broth cultures. From this animal a serum of high protective power was obtained. It protected susceptible animals against many times an otherwise fatal dose, and it also had a curative action, only, however, when injected very soon after inoculation. It has also been used in human pneumonias. Although it apparently causes the temperature to fall, experience of its use is so limited that as yet no definite conclusion as to its value can be drawn. A similar remark applies to anti-pneumonia serum prepared by other observers. Methods of Examination.— These have been already described, but may be summarised thus: (1) Microscopic. Stain films from the densest part of the sputum or from the area of spreading inflammation in the lung by Gram's method and by carbol-fuchsin, etc. (p. 207), in the latter case without decolorising the ground-work of the prepara- tion. . , : (2) By cultures. (a) FraenkePs pneumococcus. Witj similar material make successive strokes on agar, blood agar or blood serum. The most certain method, however, METHODS OF EXAMINATION. 223 is to inject some of the material containing the suspected cocci into a rabbit. If the pneumococcus be present the animal will die, usually within forty-eight hours, with numerous capsulated pneumococci in its heart blood. With the latter inoculate tubes of the above media and observe the growth. (6) Friedlander's pneumobacillus can be readily isolated either by ordinary gelatine plates or by successive strokes on agar media. CHAPTER IX. TUBERCULOSIS. The cause of tubercle was proved by Koch in 1882 to be the organism now universally known as the tubercle bacillus. Probably no other single discovery has had a more im- j portant effect on medical science and pathology than this. It has not only shown what is the real cause of the disease, but has also supplied infallible methods for determining what are tubercular lesions and what are not, and has also given the means of studying the modes and paths of in- fection. A definite answer has in this way been supplied to many questions which were previously the subject of endless discussion. Historical.— Klencke in 1843 made the statement that he had pro- duced tuberculosis in rabbits by intravenous injection of tubercular material, but he only concluded from these experiments that the cells of tubercles could multiply and reproduce the disease, and he appears to have placed little importance on the discovery. Villemin has the honour of having been the first to investigate the infectious character of tubercle by systematic experiments, and to demonstrate the regularity with which tuberculosis can be transmitted by inoculation with tubercular material. His first observations were published in 1865. He produced tuberculosis in animals not only by tubercular material from the human subject, but also by portions of what were known as the ferlsucht nodules in cattle, and came to the conclusion that perlsucht was due to the same virus as tubercle. He concluded that this virus was comparable in its mode of action with that of other infectious diseases. . . , n -a These views, however, aroused a storm of opposition from all sines. The opposition was at first chiefly on theoretical grounds, but later HISTORY OF RESEARCH ON TUBERCLE. 225 also from experimental results. Investigators who repeated Villemin's experiments obtained similar results so far as the production of tuberculosis by tubercular material was concerned, but many found that tuberculosis also followed inoculation with non-tubercular material (such as pus from pyemic abscesses, portions of decomposed tissue, etc.), and even by the mere introduction of setons. The general opinion came to be strongly against the existence in tubercle of an infective agent of specific nature, and along with this there prevailed great confusion as to the distinction between tubercular and non- tubercular lesions. Armanni, in 1873, by scarification of the cornea and inoculation with tubercular material, produced in that situation a small tubercular ulcer, which was afterwards followed by general tuberculosis. Such a result he found never followed inoculation with non-tubercular material. But it was the work of Cohnheim and Salomonsen along similar lines which was chiefly instrumental in altering the prevailing opinion as to the nature of tubercle. By inoculation of the anterior chamber of the eye of rabbits with tubercular material they found that in many cases the results of irritation soon disappeared, but that after a period of incubation, usually about twenty-five days, small tubercular nodules appeared in the iris; afterwards the disease gradually spread, leading to a tubercular disorganisation of the globe of the eye. Later, the lymphatic glands became involved, and finally the animal died of acute tuberculosis. The question remained as to the nature of the virus the specific character of which was thus established, and this question was answered by the work of Koch. The announcement of the discovery of the tubercle bacillus was made by Koch in March 1882, and a full account of his researches appeared m 1884 (Mitth. a. d. k. Gsndhtsamte., Berlin). Koch's work on this subject will remain as a classical master-piece of bacteriological research, both on account of the great difficulties which he successfully overcame and the completeness with which he demonstrated the relations of the organism to the disease. The two chief difficulties were, first, the demonstration of the bacilli in the tissues and secondly, the cultivation of the organism outside the body For with regard to the first, the tubercle bacillus cannot be demonstrated' by a simple watery solution of a basic aniline dye, and it was only after prolonged staining for twenty-four hours with a solution of methylene blue with caustic potash added, that he was able to reveal the presence of the organism. Then, in the second place, all attempts to cultivate it on the ordinary media failed, and he only succeeded in obtaining growth on solidified blood serum, the method of preparing which he himself devised, inoculations being made on this medium from the organs of animals artificially rendered tubercular. The fact that growth did not appear till the tenth day at the earliest, might easily have led to the hasty conclusion that no growth took place. All difficulties T5 226 TUBERCULOSIS. were, however, successfully overcome. He cultivated the organism by the above method from a great variety of sources, and by a large series of inoculation experiments on various animals, performed by different methods, he conclusively proved that bacilli from these different sources produced the same tubercular lesions and were really of the same species. His work was the means of showing conclusively that such conditions as lupus, " white swelling" of joints, scrofulous disease of glands, etc., are really tubercular in nature. Tuberculosis in Animals. — Tuberculosis is not only the most widely spread of all diseases affecting the human subject, and produces a mortality greater than any other, but there is probably no other disease which affects the domestic animals so widely. We need not here describe in detail the various tubercular lesions in the human subject, but some facts regarding the disease in the lower animals may be given, as this subject is of great importance in rela- tion to the infection of the human subject. Amongst the domestic animals the disease is com- monest in cattle (bovine tuberculosis), and in them the lesions are very various, both in their character and dis- tribution. In most cases the lungs are affected, and contain numerous rounded nodules, many being of con- siderable size; these may be softened in the centre, but are usually of pretty firm consistence and may be calcified. There may be in addition caseous pneu- monia, and also small tubercular granulations. Along with these changes in the lungs, the pleurae are also often affected, and show numerous nodules, some of which may be of large size, firm and pedunculated, the condition being known in Germany as Perhucht, in France as pommehcrc. Lesions similar to the last may be chiefly confined to the peritoneum and pleurse. In other cases, again, the abdo- minal organs are principally involved. The udder becomes affected in a certain proportion of cases of tuberculosis in cows_in 3 per cent according to Bang— but primary anec- tion of this gland is very rare. Tuberculosis is also a com- paratively common disease in pigs, in which animals it m many cases affects the abdominal organs, in other cases produces a sort of caseous pneumonia, and sometimes THE TUBERCLE BACILLUS. 227 is met with as a chronic disease of the lymphatic glands, the so-called " scrofula " of pigs. Tubercular lesions in the muscles are less rare in pigs than in most other animals. In the horse the abdominal organs are usually the primary scat of the disease, the spleen being often enormously enlarged and crowded with nodules of various shapes and sizes ; sometimes, however, the primary lesions are pulmonary. In sheep and goats tuberculosis is a rare occurrence, especially in the . former animals. It also occurs spon- taneously in dogs, cats, and in the large carnivora. It is also sometimes met with in monkeys in confinement, and leads to a very rapid and widespread affection in these animals, the nodules having a special tendency to soften and break down into a pus-like fluid. Tuberculosis in fowls (avian tuberculosis) is a common and very infectious disease, nearly all the birds in the poultry-yard being sometimes affected. The relation of avian to mammalian tuberculosis is discussed below. From these statements it will be seen that the disease in animals presents great variations in character, and may differ in many respects from that met with in the human subject. The tubercle nodules may be of so large a size, e.g., in the horse and ox, as to be described as sarcoma-like ; they may be tough and firm, with little or no caseation, or they may be softened in the centre, more resembling abscesses, or again there may be an eruption of very minute granulations. However different their naked- eye appearances may be, they are built up histologically on the same plan, and of greater importance still is the fact that they are all produced by the tubercle bacillus. An account of the lesions experimentally produced will be given later. & Tubercle Bacillus — Microscopical Characters. — Tu- bercle bacilli are minute rods which usually measure 2.5 to 3.5 m length, and. 3 p in thickness, i.e. in proportion to their length they are comparatively thin organisms (Figs 62 and 63). Sometimes, however, longer forms, up to c a or more in length, are met with, both in cultures and in 228 TUBERCULOSIS. the tissues. They are straight or slightly curved, and are of uniform thickness, or may show slight swelling at their extremities. When stained they appear uniformly coloured, Fig. 62.— Tubercle bacilli, from a pure ganism it is extremely of the unstained points. Accordingly, we find that some consider these to be spores, while others find that it is impossible to stain them by any means whatever, and consider that they are really of the nature of vacuoles. Against their being spores is also the fact that many occur in one bacillus. Others again hold that some of the con- densed and highly-stained particles are spores. It is impossible to speak dogmatically on the question at present. We can only say that the younger bacilli stain uniformly, and that in the older forms inequality in staining is met with, but it has not been definitely proved that this always indicates spore formation. The bacilli in the tissues occur scattered irregularly or in little masses. They are usually single, or two are attached end to end and often form in such a case an obtuse angle. True chains are not formed, but occasionally short filaments are met with. In cultures the bacilli form masses in which the rods are closely applied to one another or may present small uncoloured spots along their course, with darkly-stained parts between. In the case of the tubercle bacillus, as of many other or- ganisms, a consider- able amount of discussion has taken place as to the oc- currence of spores. In such a minute or- culture on glycerine agar. Stained with carbol-fuchsin. x 1000. difficult to recognise the exact characters ABERRANT FORMS 229 and arranged in a more or less parallel manner. Tubercle bacilli are quite devoid of motility. Aberrant Forms. — Though such are the characters of the organism as usually met with, other appearances are sometimes found. In old cultures, for example, very much \ t is often phtMsical Sputum ; the^ are lo»ger &™ Film preparation, stained with carbol-fuchsin and methylene -blue. X IOOO. " larger elements may occur. These may be in the form of long filaments, which may be swollen or clubbed at their extremities, may be irregularly beaded, and may even show the appearance of branching. Such forms have been studied by Metchnikoff, Maffucci, Klein, and others. Their significance has been variously interpreted, for while some look upon them as degenerated or involution forms, others 230 TUBERCULOSIS. regard them as indicating a special phase in the life history of the organism, allying it with the higher bacteria. This latter view, however, has many facts against it, especially the circumstance that these aberrant forms are chiefly met . with when the organisms are undergoing retrogressive change. The question, however, is one which at present is not definitely settled. Staining Reactions. — The tubercle bacillus takes up the ordinary stains with great slowness, and very faintly, and for successful staining one of the most powerful solutions ought to be employed, e.g., gentian-violet or fuchsin, along with aniline oil water or solution of carbolic acid. Further, such staining solutions require to be applied for a long time, or the staining must be accelerated by heat, the solution being warmed till steam arises and the specimen allowed to remain in the hot stain for two or three minutes. This resistance to staining Koch attributes to the presence in the bacterial protoplasm of two monatomic fatty acids. As stated above, Koch at first used a solution of methylene- blue with caustic potash added, but even this method stains somewhat faintly, and he afterwards abandoned it in favour of the combination of aniline oil with gentian-violet, intro- duced by Ehrlich. One of the best and most convenient methods is the Ziehl-Neelsen method (see p. 113)- The bacilli present this further peculiarity, however, that after staining has taken place they resist decolorising by solutions which readily remove the colour from the tissues and from other organisms which may be present. Such decolorising agents are sulphuric or nitric acid in 20 per cent solution. Preparations can thus be obtained in which the tubercle bacilli alone are coloured by the stain first used, and the tissues can then be coloured by a contrast stain. Leprosy bacilli, however, retain the stain in the same way, though not so firmly, as tubercle bacilli, and thus constitute an exception to this reaction being peculiar to the latter. rI he smegma bacillus also may retain the colour in the above method of staining (vide p. 255). The spores of many bacilli become decolorised more readily than tubercle CULTIVATION OF TUBERCLE BACILLUS. 231 bacilli, though some retain the colour with equal tenacity. Tubercle bacilli also stain by Gram's method, but the results are inferior to those obtained with carbolic fuchsin. Cultivation. — The medium first used by Koch was inspissated blood serum {vide p. 50). If inoculations are made on this medium with tubercular material free from other organisms, there appear from the tenth to fourteenth day minute points of growth of dull whitish colour, rather ir- regular, and slightly raised above the surface. In such cultures they usually reach only a com- paratively small size and remain separate, becoming confluent only when many occur close together. Koch compared the appearance of these to that of small. dry scales. In sub-cul- tures, however, growth is more luxuriant and may come to form a dull wrinkled film of whitish colour, which may cover the greater part of the surface of the serum and at the bottom of the tube may grow over the surface of the condensation water on to the glass (Fig. 64, A). The growth is always of a dull appearance and has a considerable degree of consistence, it being difficult to dissociate a portion thoroughly in a drop on B C Fig. 64. — Cultures of tubercle bacilli glycerine agar. A and B. Mammalian tubercle bacilli ; A is an old culture, B one of a few weeks' growth. C. Avian tubercle bacilli. The growth is whiter and smoother on the surface than the others. 232 TUBERCULOSIS. of water. In older cultures the growth may acquire a slightly brownish or buff colour. When the small colonies are examined under a low power of the microscope they are seen to be extending at the periphery in the form of wavy or sinuous streaks which radiate outward and which have been compared to the flourishes of a pen. The central part shows similar markings closely interwoven. These streaks are composed of masses of the bacilli arranged in a more or less parallel manner. On glycerine agar, which was first introduced by Nocard and Roux as a medium for the culture of the tubercle bacillus, growth takes place in sub-cultures at an earlier date and progresses more rapidly than on serum, but, strangely enough, this medium is not suitable for obtaining cultures from the tissues, inoculations with tubercular material usually yielding a negative result. The growth has practically the same characters as on serum, but is more luxuriant. It, however, tends to lose its virulence more rapidly than when grown on serum. In glycerine broth, especially when the layer is not deep, tubercle bacilli grow readily in the form of little white masses which fall to the bottom and form a powdery layer. If, however, the growth be started on the surface it spreads superficially as a dull whitish, wrinkled pellicle which may reach the walls of the flask ; this mode of growth is specially suitable for the production of tuber- culin {vide infra). The culture has a peculiar fruity and not unpleasant odour. On ordinary agar and on gelatine media no growth takes place. It was at one time believed that the tubercle bacillus would only grow on media containing animal fluids, but of late years it has been found that growth takes place also on a purely vegetable medium, as was first shown by Pawlowsky in the case of potatoes. Sander has shown that the bacillus grows readily on potato, carrot, macaroni, and on infusion of these substances, especially when glycerine is added. He also found that cultures from tubercular lesions could be more easily made on potato than on glycerine agar. Glycerinated potato can be prepared by covering the slices POWERS OF RESISTANCE. 233 in Roux's tubes with 5 per cent glycerine in water during the first period of sterilisation. In the case of the decoc- tions used by Sander, as in glycerine broth, the growth forms a wrinkled membrane on the surface. The optimum temperature for growth is 370 to 380 C. Growth ceases above 420 and usually below 2 8°, but on long-continued cultivation outside the body and in special circumstances, growth may take place at a lower tempera- ture, e.g., Sander found that growth took place in potato broth even at 220 to 230 C. Powers of Resistance.— Tubercle bacilli have consider- able powers of resistance to external influences, and can retain their vitality for a long time outside the body in various conditions. In this respect they resemble bacilli which are known to possess spores, and this is really the chief argument in favour of the presence of spores in tubercle bacilli, though their resisting power is considerably less than that of most spore -containing bacilli. Dried phthisical sputum has been found to contain still virulent bacilli or their spores after two months, and similar results are obtained when the bacilli are kept in distilled water for several weeks. So also they resist for a long time the action of putrefaction, which is rapidly fatal to many pathogenic organisms. Sputum has been found to contain living tubercle bacilli even after being allowed to putrefy for several weeks (Fraenkel, Baumgarten), and the bacilli have been found to be alive in tubercular organs which have been buried in the ground for a similar period. They are not killed by being exposed to the action of the gastric juice for six hours, or to a temperature of -30 C. for three hours, even when this is repeated several times. It has been found that when completely dried they can resist a tempera- ture of ioo° C. for an hour, but, on the other hand, exposure in the moist condition to 7oc C. for the same time is usually fatal. It may be stated that raising the temperature to 100 C. kills the bacilli in fluids and in tissues, but in the case of large masses of tissue care must be taken that this temperature is reached throughout. They are killed in less 234 TUBERCULOSIS. than a minute by exposure to 5 per cent carbolic acid, and both Koch and Straus found that they are rapidly killed by being exposed to the action of direct sunlight. Action on the Tissues. — The local lesion produced by the tubercle bacillus is the well-known tubercle nodule, but though the typical structure is often described as consisting of a central giant cell surrounded by a zone of compara- tively large and somewhat spindle-shaped cells (epithelioid cells), and again by an outer zone of lymphocytes or small uninucleated leucocytes, the structure varies in different situations and according to the intensity of the action of the bacilli. A considerable discussion has taken place as to the exact origin of the elements composing the tubercle follicle. In the case of the iris its formation was fully studied by Baum- garten, and his views we consider to be correct regarding the ordinary mode of formation. Before describing the exact changes which occur in the tissues, it may be stated that the action of the bacillus is twofold. On the one hand, by its irritation it induces tissue reaction in the form of proliferative changes and leucocytic infiltration, and on the other hand, it causes degenerative changes in the cells around, which afterwards result in their death. After the bacilli gain entrance to a connective tissue such as that of the iris, their first action appears to be on the connective tissue cells, which become somewhat swollen and undergo mitotic division, the resulting cells being distinguishable by their large size and pale nuclei. These constitute the so-called epithelioid cells. These prolifera- tive changes may be well seen on the fifth day after inocula- tion or even earlier. A small focus of proliferated cells is thus formed in the neighbourhood of the bacilli and about the same time numbers of leucocytes— chiefly lymphocytes —begin to appear at the periphery and gradually become more numerous. Soon, however, the action of the bacilli as cell-poisons comes into prominence, the changes first occurring in the centre of the focus. The epithelioid cells become swollen ACTION ON THE TISSUES. 235 and somewhat hyaline, their outlines become indistinct, whilst their nucleus stains faintly, and ultimately loses the power of staining. The cells in the centre, thus altered, gradually become fused into a homogeneous substance and this afterwards becomes somewhat granular in appearance. If the central necrosis does not take place very quickly, then giant-cell formation may occur in the centre, this con- stituting one of the characteristic features of the tubercular lesion. The giant cells in tubercle are large, rounded, or oval protoplasmic masses, often with numerous processes, and containing a varying number of oval nuclei somewhat poor in chromatin, which are often arranged in a ring towards the periphery, sometimes collected in a clump towards one end, and sometimes lying irregularly. The centre of a giant cell often shows signs of degeneration, such as hyaline change and vacuolation, or it may be more granular than the rest of the cell. Though there has been a considerable amount of dis- cussion as to the mode of origin of the giant cells, we think there can be little doubt that in most cases they result from enlargement of single epithelioid cells, the nucleus of which undergoes proliferation without- the protoplasm dividing. Sometimes cells a little larger than epithelioid cells may be seen, which contain only two or three nuclei ; these may be young giant cells. Some consider that the giant cells result from a fusion of the epithelioid cells ; but though there are occasionally appearances which suggest such a mode of formation, it cannot be regarded as of common occurrence. In some cases of acute tuberculosis, when the bacilli become lodged in a capillary the endothelial cells of its wall may proliferate, and thus a ring of nuclei be formed round a small central thrombus. Such an occurrence gives rise to an appearance closely resembling a typical giant cell. Giant cells are found especially when the caseous change is relatively not very active— that is, in circumstances where the formative processes have time to come into play. If the centre of the nodule becomes caseous, giant cells may 236 TUBERCULOSIS. be formed later in the cellular tissue at the periphery. According to the view here stated, both the epithelioid and the giant cells are of connective tissue origin ; and we can see no sufficient evidence for the view held by some observers, chiefly of the French school, that they are formed from leucocytes which have emigrated from the capillaries. Such are the usual changes which occur on the intro- duction of the bacilli into connective tissue ; but the tubercle nodule has not always the same mode of formation and structure. In very acute tuberculosis of the spleen, for example, a group of bacilli may often be seen to have caused cellular necrosis around them before any tissue proliferation has taken place, and it may be only at the margin of the larger and older follicles that epithelioid cells are well seen. In acute cases, also, the commencement of the tubercle nodule may sometimes be traced to a clump of leucocytes surrounding bacilli in a capillary ; such an appearance may sometimes be met with in the liver. The great varieties in the appearance of tubercular lesions depend upon the number of the bacilli and their manner of spread, and accordingly on the proportion in which the proliferative and degenerative changes occur. We thus find that cellular proliferation is especially marked when the bacilli are few in number. There can be no doubt, we think, from a careful study of the tubercular lesions, that the cell necrosis and ulti- mate caseation depend upon the products of the bacilli, and are not due to the fact that the tubercle nodule is non- vascular. This non-vascularity itself is to be explained by the circumstance that young capillaries cannot grow into a part where tubercle bacilli are active, and that the already existing capillaries become thrombosed, owing to the action of the bacillary products on their walls, and ultimately disappear. At the periphery of tubercular lesions there may be considerable vascularity and new formation of capillaries. The general symptoms of tuberculosis — pyrexia, perspira- DISTRIBUTION OF BACILLI. 237 tion, wasting, etc., are to be ascribed to the absorption and distribution throughout the system of the toxic products of the bacilli. The occurrence of waxy change in the organs is believed by some to be chiefly due to the products of other, especially pyogenic, organisms secondarily present in the tubercular lesions, e.g., phthisical cavities. This matter, however, requires further elucidation. Presence and Distribution of the Bacilli. — A few facts may be stated regarding the presence of bacilli, and the numbers in which they are likely to be found in tubercular lesions. On the one hand, they may be very few in number and difficult to find, and on the other hand, they may be present in very large numbers, sometimes forming masses which are easily visible under the low power of the microscope. They are usually very few in number in chronic lesions, whether the latter are tubercle nodules with much con- nective tissue formation or old caseous collections. In caseous material one can sometimes see a few bacilli faintly stained, along with very minute unequally stained granular points, some of which may possibly be spores of the bacilli. Whether they are spores or not, the important fact has been established that tubercular material in which no bacilli can be found microscopically, may be proved, on experi- mental inoculation into animals, to be still virulent. In such cases the bacilli may be present in numbers so small as to escape observation, or it may be that their spores only are present. In subacute lesions, with well-formed tubercle follicles and little caseation, the bacilli are generally scanty. They are most numerous in acute tubercular lesions, especi- ally where caseation is rapidly spreading, for example, in such conditions as caseous catarrhal pneumonia (Fig. 65), acute tuberculosis of the spleen in children, which is often attended with a good deal of rapid caseous change, etc. In acute miliary tuberculosis a few bacilli can generally be found in the centre of the follicles ; but here they are often much more scanty than one would expect. The tubercle bacillus is one which not only has comparatively slow growth, 238 TUBERCULOSIS. but retains its form and staining power for a much longer period than most organisms. This is true of the bacilli both in cultures and also in the tissues. As regards their position in the tissues, the bacilli are usually scattered irregularly or in small groups amongst the cells or granular material. Most of the bacilli lie free, and their occurrence within the cells is relatively un- common, there being in this respect a contrast to what is seen in the lesions in leprosy. Occasionally we find them within the giant cells, in which they may be arranged in a somewhat radiate manner at the periphery, occasionally also DISTRIBUTION OF BACILLI. 239 in epithelioid cells and in leucocytes ; but these are by no means frequent sites. The above statements, however, apply only to tuber- culosis in the human subject, and even in this case there are exceptions. In the ox, on the other hand, the presence of tubercle bacilli within giant cells is a very common Fig. 66. — Tubercle bacilli in giant cells, showing the radiate arrange- ment at the periphery of the cells. Section of tubercular udder of cow. Stained with carbol-fuchsin and Bismarck-brown. x 1000. occurrence ; and it is also common to find them in con- siderable numbers scattered irregularly throughout the cellular connective tissue of the lesions, even when there is little or no caseation present (Fig. 66). In tuberculosis in the horse and in avian tuberculosis the numbers of bacilli may be enormous, even in lesions which are not specially acute ; and considerable variation 240 TUBERCULOSIS. both in their number and in their site is met with in tuber- culosis of other animals. Cellular necrosis and caseation ' occur in proportion to the numbers of the bacilli present, much more readily in some animals than in others, prob- ably owing to different degrees of susceptibility of their tissues. In discharges from tubercular lesions which are breaking down, tubercle bacilli are usually to be found. In the sputum of phthisical patients their presence can be demon- strated almost invariably at some period, and sometimes their numbers are very large (for method of staining see p. 113). Several examinations may, however, require to be made ; this should always be done before any conclusion as to the non-tubercular nature of a case is come to. In cases of genito-urinary tuberculosis they are often present in the urine ; but as they are much diluted it is difficult to find them unless a very complete forma- tion of deposit is al- lowed to take place. This deposit is ex- amined in the same way as the sputum. It is, however, much easier to obtain their separation by means of the centrifuge. If this method is em- ployed, bacilli can usually be detected, though sometimes their number may be very small; here, especially, repeated examinations may be necessary. The bacilli often occur in little clumps, as shown in Fig. 67. In tubercular ulceration of the intestine their presence in the faeces may be demonstrated, Fig. 67. — Tubercle bacilli in urine ; show- ing one of the characteristic clumps, in which they often occur. Stained with carbol-fuchsin and methylene- blue. x 1000. EXPERIMENTAL INOCULATION. 241 as was first shown by Koch ; but in this case their dis- covery is usually of little importance, as the intestinal lesions, as a rule, occur "only in advanced stages when diagnosis is no longer a matter of doubt. Experimental Inoculation. — -Tuberculosis can be arti- ficially produced in animals by infection in a great many different ways — by injection of the bacilli into the sub- cutaneous tissue, into the peritoneum, into the anterior chamber of the eye, into the veins ; by feeding the animals with the bacilli ; and, lastly, by making them inhale the bacilli suspended in the air. The exact result, of course, varies in different animals and according to the method of inoculation, but we may state generally that when introduced locally into the tissues of a susceptible animal, the bacilli usually produce the lesions above described, terminating finally in a local caseation ; that there then occurs a tubercular affection of the neigh- bouring lymphatic glands, and that lastly there may be a rapid extension of the bacilli to other organs by the blood stream and the production of general tuberculosis. Of the animals used for the purpose, the guinea-pig is most susceptible. When a guinea-pig is inoculated subcutaneously with tubercle bacilli from a culture, or with material containing them, such as phthisical sputum, a local swelling gradually forms which is usually well marked about the tenth day. This swelling becomes softened and caseous, and breaks down, leading to the formation of an irregularly ulcerated area with caseous lining. The lymphatic glands in relation to the parts can generally be found to be enlarged, and of somewhat firm consistence, about the end of the second or third week. Later, in them also caseous change occurs, and a similar condition may spread to other groups of glands b turn, passing also to those on the other side of the body. During the occurrence of these changes, the animal loses weight, shows general disturbance of nutrition, gradually becomes cachectic, and dies, death occurring sometimes within six weeks, sometimes not for two or three months 16 242 TUBERCULOSIS. Post mortem, in addition to the local and glandular changes, an acute tuberculosis is usually present, the spleen being specially affected. This organ is swollen, and is studded throughout by numerous tubercle nodules, which may be minute and grey, or larger and of a yellowish tint. If death has been long delayed, calcification may have occurred in some of the nodules. Tubercle nodules, though rather less numerous, are also present in the liver and in the lungs, the nodules in the latter organs being usually of smaller size though occasionally in large numbers. The extent of the general infection varies ; sometimes the chronic glandular changes constitute the outstanding feature. Intraperitoneal injection of pure cultures produces a local lesion in the form of an extensive tubercular infiltra- tion and thickening of the omentum, sometimes attended with acute tubercles all over the peritoneum. There is a caseous enlargement of the retroperitoneal and other lymphatic glands, and later there may be a general tuber- culosis. Intravenous injection produces a typical acute tuberculosis, the nodules being usually more numerous and of smaller size, while death follows more rapidly the larger the numbers of bacilli injected. Guinea-pigs, when fed with tubercle bacilli, or with sputum or portions of tissue containing them, readily contract an intestinal form of tuberculosis, lesions being present in the lymphoid tissue of the intestines, in the mesenteric glands, and later in the; internal organs. Whatever be the path of infection when a guinea-pig has died after inoculation, attempts to cultivate the bacilli may fail, though they are present in an apparently normal condition in the lesions. The bacilli have, in such a case, probably died in the struggle of the tissues against them, but the intracellular toxines which still remain have been absorbed and have caused the death of the animal. Rabbits are less susceptible than guinea-pigs, and in them the effects of subcutaneous inoculation are very variable ; sometimes the lesions remain local, sometimes a general tuberculosis is set up. Otherwise the reactions are much on AVIAN TUBERCULOSIS. 243 the same nature. Dogs are much more highly resistant, but tuberculosis can be produced in them by intraperitoneal injection of pure cultures (Koch), or by intravenous injection (Maffucci). In the latter case there results an extensive eruption of minute miliary tubercles. Tuberculosis can also be easily produced in susceptible animals by making them inhale the bacilli. Koch, for this purpose, used pure cultures which were mixed with dis- tilled water, and then distributed in the air by means of a spray. Rabbits, guinea-pigs, and mice were exposed by this means to inhalations for half an hour on three successive days, and were afterwards kept in healthy conditions. Some of the rabbits and guinea-pigs died within four weeks, the others were killed at the end of that time, and all showed pulmonary lesions which, in the case of the rabbits and the guinea-pigs, were in the form of patches of caseating catarrhal pneumonia. To obtain pure cultures of the tubercle bacillus, the acute lesions in the organs of an animal recently killed should be selected, e.g., the spleen of a guinea-pig with early acute tuber- culosis. If the lesions are subacute or chronic with much caseation, attempts at cultivation often fail. It would appear as if a considerable number of bacilli required to be present to start the growth, or it may be that many of the bacilli found microscopically are actually dead. The portions of tissue must, of course, be taken with aseptic precautions, the knives, scissors, etc., to be used being carefully sterilised, and the inoculations should be made on solidified blood serum. Avian Tuberculosis. — There can be no doubt that the bacilli present in tuberculosis of the various mammals mentioned are of the same variety, though differences in virulence may be occasionally noticed. There has, how- ever, of late years been considerable discussion as regards the identity of the bacilli in avian and mammalian tuber- culosis. In the tubercular lesions in birds there are found bacilli which correspond in their staining reactions and in their morphological characters with those in mammals, but 244 TUBERCULOSIS. differences are observed in cultures, and also on experi- mental inoculation. These differences were first described by Maffucci and by Rivolta, but special attention was drawn to the subject by a paper read by Koch at the Inter- national Medical Congress in 1890. Koch stated that he had failed to change the one variety of tubercle bacillus into the other, though he did not conclude therefrom that they were quite distinct species. On glycerine agar and on serum, the growth of tubercle bacilli from birds is more luxuriant, has a moister appear- ance (Fig. 64, C), and, moreover, takes place at a higher temperature, 43. 50 C, than is the case with ordinary tubercle bacilli. Experimental inoculation brings out even more distinct differences. Tubercle bacilli derived from the human subject, for example, when injected into birds, usually fail to produce tuberculosis, whilst those of avian origin very readily do so. Birds are also very susceptible to the disease when fed with portions of the organs of birds containing tubercle bacilli, but they can consume enormous quantities of phthisical sputum without becoming tubercular (Straus, Wurtz, Nocard). No doubt, on the other hand, there are cases on record in which the source of infection of a poultry - yard has apparently been the sputum of phthisical patients. Again, tubercle bacilli cultivated from birds have not the same effect on inocula- tion of mammals, as ordinary tubercle bacilli. When guinea-pigs are inoculated subcutaneously they usually resist infection, though occasionally a fatal result follows. In the latter case, usually no tubercles visible to the naked eye are found, but numerous bacilli may be present in internal organs, especially in the spleen, which is much swollen. Further, intravenous injection even of large quantities of avian tubercle bacilli, in the case of dogs, leads to no effect, whereas ordinary tubercle bacilli produce acute tuberculosis. [The rabbit, on the other hand, is comparatively susceptible to avian tuberculosis (Nocard).] There is, therefore, abundant evidence that the bacilli derived from the two classes of animals show important AVIAN TUBERCULOSIS. 245 differences, and, reasoning from analogy, we might infer that probably the human subject also would be little susceptible to infection from avian tuberculosis. The question remains, are these differences of a permanent character ? Now it has been found that occasionally the inoculation of fowls with tubercle bacilli from the human subject produces tuberculosis, and that, when this occurs, the disease can be readily transmitted to other fowls. Also in some cases, inoculation with avian tubercle bacilli produces ordinary tubercle nodules in guinea-pigs and rabbits (Courmont and Dor), and, in other cases, these lesions are found after the bacilli have been passed through the tissues of a number of guinea-pigs. The matter seems permanently settled by the experiments of Nocard, in which mammalian tubercle bacilli have been made to acquire all the characters of those of avian origin. The method adopted was to place bacilli from human tuberculosis in small collodion sacs containing bouillon and then to insert each sac in the peritoneal cavity of a fowl. The sacs were left in situ for periods of four to eight months. They were then removed, cultures were made from their contents, fresh sacs were inoculated and introduced into other fowls. In these conditions the bacilli are subjected only to the tissue juices, the wall of the sac being impervious both to bacilli and to leucocytes, etc. After one sojourn of this kind, and still more so after two, the bacilli are found to have acquired some of the characters of avian tubercle bacilli, but are still non-virulent to fowls. After the third sojourn, however, they have acquired this property and pro- duce in fowls the same lesion as bacilli derived from avian tuberculosis. It therefore appears that the bacilli of avian tuberculosis are not a distinct and permanent species, but a variety which has been modified by growth in the tissues of the bird. Evidently also there are degrees of this modification, according to the period of time during which the bacilli have passed from bird to bird. We may add that tuberculin prepared from avian tubercle bacilli has the same action as the ordinary tuberculin. 246 TUBERCULOSIS. Action of dead Tubercle Bacilli. — The remarkable fact has been established by independent investigators that tubercle bacilli in the dead condition, when introduced into the tissues in sufficient numbers, can produce tubercle- like nodules. Prudden and Hodenpyl, by intravenous injection in rabbits of cultures sterilised by heat, produced in the lungs small nodules in which giant cells were occa- sionally present, but no caseation, and which were charac- terised by more growth of fibrous tissue than in ordinary tubercle. The subject has been very fully investigated with confirmatory results by Straus and Gamaleia, who find that, if the number of bacilli introduced into the circula- tion is large, there result very numerous tubercle nodules with well-formed giant cells, and occasionally traces of caseation. The bacilli can be well recognised in the nodules by the ordinary staining method. In these experi- ments the bacilli were killed by exposure to a temperature of 1 1 50 C. for ten minutes before being injected. Similar nodules can be produced by intraperitoneal injection. Subcutaneous injection, on the other hand, produces a local abscess, but in this case no secondary tubercles are found in the internal organs. Further, in many of the animals inoculated by the various methods a condition of marasmus sets in and gradually leads to a fatal result, there being great emaciation before death. These experi- ments, which have been confirmed by other observers, show that even after the bacilli are dead they preserve their staining reactions in the tissues for a long time, and also that there are apparently contained in the bodies of the dead bacilli certain substances which act locally, producing proliferative and, to a less extent, degenerative changes, and which also markedly affect the general nutrition. The long period during which the tubercle bacillus, as com- pared with other organisms, retains even when dead its morphological and staining characters, is a very striking feature. Stockman has recently found that an animal in- oculated with large numbers of dead tubercle bacilli after- wards gives the tuberculin reaction. SOURCES OF HUMAN TUBERCULOSIS. 247 Practical Conclusions. — From the facts above stated with regard to the conditions of growth of the tubercle bacilli, their powers of resistance, and the paths by which they can enter the body and produce disease (as shown by experiment), the manner by which tuberculosis is naturally transmitted can be readily understood. Though the ex- periments of Sander show that tubercle bacilli can multiply on vegetable media to a certain extent at warm summer temperature, it is doubtful whether all the conditions necessary for growth are provided to any extent in nature. At any rate, the great multiplying ground of tubercle bacilli is the animal body, and tubercular tissues and secretions containing the bacilli are the chief, if not the only, means by which the disease is spread. The tubercle bacilli leave the body in large numbers in the sputum of phthisical patients, and when the sputum becomes dried and pulverised they are set free in the air. Their powers of resistance in this condition have already been stated. As examples of the extent to which this takes place, it may be said that their presence in the air of rooms containing phthisical patients has been repeatedly demonstrated. Williams placed glass plates covered with glycerine in the ventilat- ing shaft of the Brompton Hospital, and after five days found, by microscopic examination, tubercle bacilli on the surface, whilst Klein found that guinea-pigs kept in the ventilating shaft became tubercular. Cornet produced tuberculosis in rabbits by inoculating them with dust collected from the walls of a consumptive ward. Tubercle bacilli are also discharged in considerable quantities in the urine in tubercular disease of the urinary tract, and also by the bowel when there is tubercular ulceration, but, so far as the human subject is concerned, the great means of disseminating the bacilli in the outer world is dried phthisi- cal sputum, and the source of danger from this means can scarcely be over-estimated. Every phthisical patient ought to be looked upon as a fruitful source of infection to those around, and the sputum ought in every case to be collected in special receptacles and thoroughly sterilised either by 248 TUBERCULOSIS. boiling or by the addition of a 5 per cent solution of carbolic acid. Another great source of infection is the milk of cows affected with tuberculosis of the udder. In such cases the presence of tubercle bacilli in the milk can usually be readily detected by centrifugalising it, and then examining the deposit microscopically, or by inoculating an animal with it. As pointed out by Woodhead and others, the milk from cows thus affected is probably the great source of tabes mesenterica, which is so common in young sub- jects. In these cases there may be tubercular ulceration of the intestine, or it may be absent. Woodhead found that out of 127 cases of tuberculosis in children, the mesenteric glands showed tubercular affection in 100, and that there was ulceration of the intestine in 43. It is especially in children that this mode of infection occurs, as in the adult ulceration of the intestine is rare as a primary affection, though it is common in phthisical patients as the result of infection by the bacilli in the sputum which has been swallowed. There is less risk of infection by means of the flesh of tubercular animals, for, as stated by the recent Tuberculosis Commission, in the first place, tuber- culosis of the muscles of oxen being very rare, there is little chance of the bacilli being present in the flesh unless the surface has been smeared with the juice of the tuber- cular organs, as in the process of cutting up the parts ; and in the second place, even when present they will be destroyed if the meat is thoroughly cooked. We may state, therefore, that the two great modes of infection are by inhalation and by ingestion, of tubercle bacilli. By the former method the tubercle bacilli will in most cases be derived from the human subject ; in the latter, probably from tubercular cows, though contamina- tion of food by tubercular material from the human subject may also occur. It is quite probable that bacilli from these two sources may be of somewhat different virulence towards the human subject, but at present we have npt the means of speaking definitely on this point. Both in inhalation KOCH'S TUBERCULIN. 249 and in ingestion, tubercle bacilli may lodge about the pharynx 'and thus come to infect the pharyngeal lymphoid tissue, tonsils, etc., tubercular lesions of these parts being much more frequent than was formerly supposed. Thence the cervical lymphatic glands may become infected, and afterwards other groups of glands, bones, or joints, and internal organs. Koch's Tuberculin.— We have seen that the pathology of tuberculosis indicates that the tubercle bacillus can act on tissues with which it is not immediately in contact, and therefore it is natural to ask whether it, like other organisms, produces definite toxic bodies. What knowledge we have of the latter is secondary to the bringing forward by Koch in 1890-91 of a substance called " tuberculin," which he introduced as a curative agent for tubercular affections. He had observed that if in a guinea-pig suffering from the initial local induration occurring after subcutaneous inocu- lation with tubercle bacilli, a second subcutaneous inocula- tion of tubercle bacilli, or of dead cultures of the same, was practised in another part of the body, superficial ulceration occurred in the primary tubercular nodule, the wound healed, and the animal did not succumb to tuber- culosis. This reaction was further studied by means of the above mentioned tuberculin, which consisted of a glycerine bouillon culture of tubercle in which the bacilli had been killed by heat, and which had then been concentrated by evaporation. It thus contains the dead and often macerated bacilli, the substances indestructible by boiling which existed in these bacilli, non- volatile products formed by them from the food material when alive and the concentrated remains of the bouillon and glycerine. The injection of .25 c.c. of tuberculin into a healthy man causes, in three to four hours, malaise, tend- ency to cough, laboured breathing, and moderate pyrexia • all of which pass off in twenty-four hours. The injection (the site of the injection being quite unimportant), how- ever of .01 c.c. into a tubercular person gives rise to similar symptoms, but in a much more aggravated form 250 TUBERCULOSIS. and in addition there occurs around any tubercular focus | great inflammatory reaction, resulting in necrosis and a j casting off of the tubercular mass, when this is possible. These appearances can be well seen in such a superficial ] tuberculosis as lupus. The bacilli are, it was shown, not I killed in the process. Koch's theory of the action of the I substance was that the tubercle bacillus ordinarily secretes a body having a necrotic action on the tissues. When this is injected into a tubercular patient, the proportion | present round a tubercular focus is suddenly increased, 1 inflammatory reaction takes place around, and necrosis of the spreading margin occurs very rapidly, the material con- ! taining the living or dead bacilli being thrown off en masse instead of being disintegrated piecemeal. The hopes which the introduction of tuberculin raised! that a curative agent against tuberculosis had been dis-J covered were soon seen not to be justified. It was very difficult to see how the necrosed material which it produced and which contained the still living bacilli could be got ridl of either naturally, as would be necessary in the case of a small tubercular deposit in a lung or a lymphatic gland, or artificially, as in a complicated joint-cavity where surgical interference could be undertaken. Not only so, but the ulceration which might be the sequel of the necrosis appeared to open a path for fresh infection. Soon facts were reported which justified these criticisms. Cases where rapid acute tubercular conditions ensued on the use of tuberculin were reported, and in a few months the treat- ment was practically abandoned. The conditions in guinea-pigs on which the discovery was based have since been found not to be of universal occurrence. Recent results appear to show that the tuberculin reaction (i.e., fever and local necrosis round tubercular deposits, following injection of tuberculin) is not yet fully understood ; for on the one hand other substances besides products of the tubercle bacillus may g»e rise to similar effects in tubercular animals, and, on the other a similar reaction can take place in other diseases where there J locally in the body a deposit of new tissue. Matthes has fo. instance ound that albumoses and peptones isolated from the ordinary peptic TOXINES OF THE TUBERCLE BACILLUS. 251 digestion of various albumins give the same reaction in tubercular guinea-pigs. The injection of milk, lactic acid, ricin all give a similar result, before the discovery of tuberculin, Gamaleia had found that tubercular animals were very susceptible to the toxines of the vibrio Metchnikovi, and later Metchnikoff found that a similar susceptibility existed towards the toxines of the bacillus of fowl cholera. Buchner found that a group of albuminous bodies which he called proteines, and which he extracted from the bodies of the B. anthracis, B. mallei, and B. prodigiosus, produced the tuberculin reaction, and he considered that the active body in tuberculin was probably of the same nature, and had a similar source. There is, however, no evidence that the sub- stances so derived from different bacteria are identical. While .the tuberculin reaction has thus been obtained with other bodies besides tuberculin, a similar reaction has taken place when tuberculin has been injected into persons suffering from diseases other than tubercle, e.g., cancer, sarcoma, syphilis. Further investigations on this subject are thus required. The Toxines of the Tubercle Bacillus. — Koch's work on tuberculin showed that there could be separated from tubercle cultures substances the necrotic action of which on certain tissues was capable of explaining a great patho- logical feature of tuberculosis. The inquiries which this fact stimulated were first directed towards finding out what the substance was to which tuberculin owes its action. Hunter stated that tuberculin consists chiefly of (1) albumoses, chiefly proto- and deutero-albumose, with small quantities of hetero-albumose and a trace of dysalbumose ; (2) alkaloidal substances, two of which can be obtained in the form of platinum compounds of their hydrochlorate salts; (3) extractives, mucin, inorganic salts, etc. He prepared two modifications of tuberculin, one of which con- tained all that could be precipitated by 70 per cent alcohol, and the other all that was left. The former, which of" course contained a larger proportion of albumoses, produced less fever than the latter. From this fact it appeared that the necrotic action on the tissues and the fever-producing effects were not necessarily caused by the same body in tuberculin. The most complete analyses of tuberculin were carried out by Kiihne, who generally confirmed Hunter's results, except that he found peptone also present. He 252 TUBERCULOSIS. also found an albumose not previously described, and which he named acro-albumose. He observed, however, that the same constituents were present when uninoculated glycerine bouillon was incubated. The relative proportions present in cultures varied, but both in this experiment and in otln rs performed with solutions containing the higher albumoses in a pure condition, Kiihne found that after the tubercle bacillus had been growing, there appeared a larger propor- tion of the lower albumoses, i.e., those formed just prelimin- ary to the production of peptone. This indicates that the bacillus has a digestive action on albumin. Whether the albumoses thus formed are the toxic bodies in tuberculin is doubtful. Kiihne found that all the varieties he isolated gave a tuberculin reaction to tubercular guinea-pigs, so that they might all simply be carriers of the real toxine. This view he corroborated by a further experiment. Tubercle bacilli were grown in a glycerine medium which contained leucin, tyrosin, asparagin, etc., instead of peptone. Thus no proteid matter was present. The fluid after culture was analysed as before and no albumoses or peptones were found to be present, but only an albuminate. It neverthe- less had the same effect on tubercular animals as tuberculin. The toxines of tubercle are thus possibly not of the nature of albumoses. Of their real nature we are still ignorant. From what is known, it is possible that they do not to any great extent diffuse out into the culture media. It has been found that if tubercle cultures are filtered germ-free the filtrate does not give such a marked tuberculin reaction as 1 the unfiltered fluid. Maragliano has found that such a fluid, however, causes in animals lowering of temperature and sweating, and further that if it is heated at ioo° C. it now gives a much more marked tuberculin reaction. From this he infers that there is diffused out into the culture fluid a body allied to the toxalbumins of Brieger and Fraenkel, which is destroyed by heat, and which has a temperature- lowering action. When this body is destroyed in a tubercle filtrate, any intracellular poison which may be present from the maceration of the bodies of the dead bacilli always ANTITUBERCULAR SERUM. 253 present in a growing culture, is unantagonised and now gives the usual reaction. It is thus probable that more than one toxic body may be formed by the tubercle bacillus. The Use of Tuberculin in the Diagnosis of Tuberculosis in Cattle. — This is now the chief use to which tuberculin is put. In cattle, tuberculosis may be present without giving rise to apparent symptoms. It is thus important from the point of view of human infection that an early diagnosis should be made. The method is applied as follows. The animals are kept twenty-four hours in their byres and the temperature is taken every three hours, from four hours before the injection till twenty-four after. The average temperature in cattle is 102.2° F. ; 30 to 40 centi- grammes of tuberculin are injected, and if the animal be tubercular the temperature rises 20 or 30 F. in eight to twelve hours and continues elevated for ten to twelve hours. Bang, who has worked most at the subject, lays down the principle that the more nearly the temperature approaches 1040 F. the more reason for suspicion is there. He gives a record of 280 cases where the value of the method was tested by subsequent post-mortem examination. He found that with proper precautions the error was only 3.3 per cent. The method is largely practised on the Continent, and ought to be more widely applied. Immunisation against the Tubercle Bacillus : Anti- tubercular Serum.— Tuberculosis differs from other diseases against which animals can be immunised in that there is no evidence that one attack protects against a second, further, we have no means of obtaining truly attenuated tubercle bacilli. Many attempts at immunisation have however, been made. It has been thought by some thai the tubercle bacilli from so-called scrofulous glands are less virulent than those say from phthisis, but apparently here sufficient attention has not been paid to the difference of the numbers of bacilli injected in each case, and this appears to be a very important point. Experiments have also been brought forward which appear to show that the injection of bacilli from avian tuberculosis could protect the 254 TUBERCULOSIS. dog against bacilli derived from man. But these ;uc not yet conclusive. Further, many attempts have been made at immunisation against the tubercle bacillus by the employ- ment of its toxic products. The most successful have been those of Maragliano. We have seen that this author dis- tinguishes between the toxic bodies contained in the bodies of the bacilli (which withstand, unchanged, a temperature of ioo° C.) and those secreted into the culture fluid (which are destroyed by heat). The substance used by him for immunising his animals consists of three parts of the former and one of the latter. Commencing with 2 mgrms. of the mixture he increases the dose by 1 mgrm. daily, till a dose of 40 to 50 mgrms. is reached. This latter quantity is in- jected daily for six months, by which time a high degree of immunity has been reached. - The animals employed are the dog, the ass, the horse. The serum obtained from these is capable of protecting healthy animals against an otherwise fatal dose of tuberculin. Maragliano does not appear to have studied the effects of this serum on tuber- cular animals, but it has been tried in a great number of cases of human tuberculosis, 2 c.c. being injected sub- cutaneously every two days. Improvement is said to have taken place in a certain proportion, especially of mild non- febrile cases. Active Immunisation by Intracellular Toxines. — Koch in 1897 published the results of further researches on tuberculosis. These consisted ( 1 ) of an attempt to immunise animals against the tubercle bacillus by employing its intracellular toxines ; (2) of trying to utilise such an im- munisation to aid the tissues of an animal already attacked with tubercle the better to combat the effects of the bacilli. To obtain the intracellular toxines is difficult, as they appear to be intimately connected in the protoplasm with several very insoluble fatty acids. The method was as follows. Bacilli from young virulent cultures were dried in vacuo, and then well rubbed up with an agate pestle and mortar, treated with distilled water and centrifugalised. The clear fluid was decanted, and is called by Koch " tuberculin 0. ' ACTIVE IMMUNISATION AGAINST TUBERCLE. 255 The remaining deposit was again dried, bruised, treated with water and centrifugal ised, the clear fluid being again decanted. This process was repeated with successive residues till, on centrifugalisation, at last no residue remained. All the fluids were then put together, and these form what Koch calls " tuberculin R." He states that it differs from tuberculin O, and also from tuberculin as originally made, in that it contains the substances present in the bacilli, which are insoluble in glycerine. Tuberculin O produces the tuberculin reaction like the original glycerine extract, but tuberculin R only does so in large doses. Koch states that the latter when injected into animals in repeated and increasing doses, ^ mgrm. being the initial dose, produces immunity against the original extract, against tuberculin O, and against living and virulent tubercle bacilli. As supplied commercially, each c.c. corresponds to 10 mgrms. of dried bacilli. He also treated guinea-pigs already infected, and stated that there was a tendency to improvement in the tubercular lesions. Baumgarten, however, in a series of experiments found that this did not occur. Cases of early phthisis in man and of lupus have been treated with "tuberculin R," no dose being given which raises the temperature more than .5° F. Though cases of lupus have been recorded in which improvement has taken place little success has attended the use of this substance as a remedial agent. Smegma Bacillus. -Tins organism is of importance, as in form and staining reaction it somewhat resembles tubercle bacillus and may be mistaken for it It occurs often in large numbers in the smegma pne- putiale and m the region of the external genitals, especially where there is an accummulation of fatty matter from the secretions. Morphologi- cally it is a slender slightly curved organism, like the tubercle bacillus but usually distinctly shorter (Fig. 68). Like the tubercle bacillus it stains with some difficulty and resists decolorisation with strong minera acids Most observers ascribe the latter fact to the fatty matter with Z otr rUrr°Unded' finTd 'hat if the is" treated 2 alcoho the organism is easily decolorised. Czaplewski, however who chums t0 have cultivated it on various media, finds hat £ cu ture it shows resistance to decolorisation both with alcohol and With acids, and considers, therefore, that the TtjTi* £ t?£ 256 TUBERCULOSIS. the surrounding fatty medium. We have found that in smegma it can be readily decolorised by a minute's exposure to alcohol after the usual treatment with sulphuric «-^_ acid, and thus can be ^■^L.. readily distinguished from the tubercle bacillus. We, moreover, believe that minor points of dif- ference in the microscopic appearances of the two organisms are quite sufficient to make the , w , , experienced observer t'j suspicious if he should meet with the smegma bacillus in urine, and lead him to apply the decolorising test. Diffi- culty will only occur when a few scattered bacilli retaining the Film prepara- fuchsin occur. Its cultivation is attended with much diffi- culty. In fact, Czap- lewski alone appears to have succeeded. On serum it grows in the form of yellowish-grey irregularly rounded colonies about 1 mm. in diameter, sometimes becoming confluent to form a comparatively thick layer. He found that it also grew on glycerine agar and in bouillon. It is non-pathogenic to various animals which have been tested. Methods of Examination.— (1) Microscopic examination. Tuberculosis is one of the comparatively few diseases in which a diagnosis can usually be definitely made by micro- scopic examination alone. In the case of sputum one of the yellowish fragments which are often present ought to be selected ; dried films are then prepared in the usual way and stained by the Ziehl-Neelsen method (p. 113). ±n ™e case of urine or other fluids a deposit should first be obtained by centrifugalising a quantity in a test-tube or by allowing the fluids to stand in a tall glass vessel (an ordinary burette is very convenient). Film preparations are then made with the deposit and treated as before. If a negatn e result is obtained in a suspected case, repeated examination t t Fig. 68.- — Smegma bacilli tion of smegma. Ziehl-Neelsen stain. x 1000. METHODS OF EXAMINATION. 257 should be undertaken. To avoid risk of contamination with the smegma bacillus the meatus of the urethra should be cleansed and the urine first passed should be rejected, or the urine may be drawn off with a sterile catheter. As stated above it is only exceptionally that difficulty will arise to the experienced observer from this cause. (For points to be attended to, vide p. 255.) (2) Inoculation. — The guinea-pig is the most suitable animal. If the material to be tested is a fluid it is injected subcutaneously ; if solid or semi -solid it is placed in a small pocket in the skin or it may be thoroughly broken up in sterile water or other fluid and the emulsion injected. By this method, material in which no tubercle bacilli can be found microscopically may sometimes be shown to be tubercular. (3) Cultivation. — Owing to the difficulties this is usually quite impracticable as a means of diagnosis, and it is also unnecessary. The best method to obtain pure cultures is to produce tuberculosis in a guinea-pig by inoculation with tubercular material, and then, killing the animal after five or six weeks, to inoculate tubes of solidified blood serum, under strict aseptic precautions, with portions of a tuber- cular organ, e.g., the spleen. >7 CHAPTER X. LEPROSY. Leprosy is a disease of great interest, alike in its clinical and pathological aspects ; whilst from the bacteriological point of view also, it presents some striking peculiarities. The invariable association of large numbers of character- istic bacilli with all leprous lesions is a well-established fact, and yet, so far, attempts to cultivate the bacilli outside the body, or to produce the disease experimentally in animals, have been attended with failure. Leprosy, so far as is known, is a disease which is confined to the human subject, but it has a very wide geographical distribution. It occurs in certain parts of Europe — Norway, Russia, Greece, etc., but is commonest in Asia, occurring in Syria, Persia, etc. It is prevalent in Africa, being especially found along the coast, in the Pacific Islands, in the warmer parts of North and South America, and also to a small extent in the northern part of North America. In all these various regions the disease presents the same general features, and the study of its pathological and bacteriological characters, wherever such has been carried on, has yielded similar results. Pathological Changes. — Leprosy is characteristically a chronic disease, in which there is a great amount of tissue change, with comparatively little necessary impairment of the general health. In other words, the local irritative effects of the bacilli are well marked, often extreme, whilst the toxic phenomena are proportionately at a minimum. PA THOLO GICAL CHA NGES. 259 There are two chief forms of leprosy. The one, usually called the tubercular form — lepra tuberosa or tuberculosa — is characterised by the growth of granulation tissue in a nodular form or as a diffuse infiltration in the skin, in mucous membranes, etc., great disfigurement often result- ing. In the other form, the anaesthetic, — maculo - an- 0 Ai r.'.-V. vl%^v Fig. 69.— Section through leprous skin, showing the masses of cellular granulation tissue in the cutis ; the dark points are clumps of bacilli deeply stained. 3 Paraffin section ; Ziehl-Neelsen stain. x 80. aesthetic of Hansen and Looft — the outstanding changes are in the nerves, with consequent anaesthesia, paralysis of muscles, and trophic disturbances. In the tubercular form the disease usually starts with the appearance of erythematous patches attended by a small amount of fever, and these are followed by the development of small nodular thickenings in the skin, especially of the 26o LEPROSY. face, of the backs of hands and feet, and of the extensor aspects of arms and legs. These nodules enlarge and pro- duce great distortion of the surface, so that, in the case of the face, an appearance is produced which has been described as " leonine." The thickenings occur chiefly in the cutis, to a less extent in the subcutaneous tissue (Fig. 69). The epithelium often becomes stretched over them, and an oozing surface becomes developed, or actual ulcera- tion may occur. The cornea and other parts of the eye, the mucous membrane of the mouth, larynx, and pharynx may be the seat of similar nodular deposits. Internal organs, especially the spleen, liver, and testicles, may become secondarily affected. In all situations the change is of the same nature, — a sort of chronic inflammatory condition attended by abundant formation of granulation tissue which may be of a nodular character, or more diffuse in its arrangement. In this tissue a large proportion of the cells are of rounded or oval shape, like hyaline leucocytes ; a number of these may be of comparatively large size, and may show vacuolation of their protoplasm and a vesicular type of nucleus. These are often known as "lepra cells." Amongst the cellular elements there is a varying amount of stroma, which in the earlier lesions is scanty and delicate, but in the older lesions may be very dense. Periarteritis is a common change, and very fre- quently the superficial nerves become involved in the nodules and undergo atrophy. The tissue in the leprous lesions is comparatively vascular, at least when young, and, unlike tubercular lesions, never shows caseation. Some of the lepra cells may contain several nuclei, but we do not meet with cells resembling in their appearance tubercle giant cells, nor does an arrangement like that in tubercle follicles occur. . In the anasthetic form the lesion of the nerves is the outstanding feature. These are the seat of diffuse infiltra- tions which lead to the destruction of the nerve fibres. In the earlier stages, in which the chief symptoms are pams along the nerves, there occur patches on the skin, often ot BA CILL US OF LEPR OS Y. considerable size, the margins of which show a somewhat livid congestion. Later, these patches become pale in the central parts, and the periphery becomes pigmented. There then follow remarkable series of trophic disturbances in which the skin, muscles, and bones are especially involved. The skin often becomes atrophied, parchment-like, and anaesthetic ; frequently pemphigoid bullae or other skin eruptions occur. The bones become atrophied, and, owing to the irregular affection of the muscles, great dis- tortion of the extremities may result. Partly owing to injury to which the feet and arms are liable from their anaesthetic condition, and partly owing to trophic disturb- ances, necrosis and separation of parts are liable to occur. In this way great distortion results. The lesions in the nerves are of the same nature as those described above, that is, they are the result of a chronic inflammatory process, but the granulation tissue is less in amount, and has a greater tendency to undergo cicatricial contraction. This is to be associated with the fact that the bacilli are present in fewer numbers. Bacillus of Leprosy. — This bacillus was first observed in leprous tissues by Hansen in 187 1, and was the subject of several communications by him in 1874 and later. Further researches, first by Neisser in 1879, and afterwards by observers in various parts of the world, agreed in their main results, and confirmed the accuracy of Hansen's observations. The bacilli as seen in scrapings of ulcerated leprous nodules, or in sections, have the following characters. They are thin rods of practically the same size as tubercle bacilli, which they also resemble both in appearance and in staining reaction. They are straight or slightly curved, and usually occur singly, or two may be attached end to end; but they do not form chains. When stained they may have a uniform appearance, or the protoplasm may be fragmented, so that they appear like short rows of cocci. They often appear tapered at one or both extremities • occasionally there is slight club-like swelling. Degenerated and partially broken down forms are also seen. They take 262 LEPROSY. up the basic aniline stains rather more readily than tubercle bacilli, but in order to stain them deeply a powerful stain, such as carbol-fuchsin, is necessary. When stained, they strongly resist decolorising, though they are more easily decolorised than tubercle bacilli. The best method is to stain with carbol-fuchsin as for tubercle bacilli, but to use a Fig 70 —Superficial part of leprous skin; the cells of the granulation tissue appear as dark patches, owing to the deeply-stained bacilli in their interior. In the upper part a process of epithelium is seen. Paraffin section; stained with carbol-fuchsin and Bismarck-brown, x 500. weaker solution of sulphuric acid, say 5 per cent in de- colorising; in the case of films and thin sections, decolor- ising with such a solution for fifteen seconds is usually sufficient. Thereafter the tissues are coloured by a contrast stain, such as a watery solution of methylene -blue M p. it 3). The bacilli are also readily stained by Gram. POSITION OF THE BACILLI. 263 method. Regarding the presence of spores practically nothing is known, though some of the unstained or stained points may be of this nature. We have, however, no means of testing their powers of resistance. Leprosy bacilli are non-motile. Position of the Bacilli. — They occur in enormous num- Fig. 71. — High-power view of portion of leprous nodule showing the arrangement of the bacilli within the cells of the granulation tissue. Paraffin section ; stained with carbol-fuchsin and methylene-blue. x 11 00. bers in the leprous lesions, especially in the tubercular form. In fact, so numerous are they that the granulation tissue in sections, properly stained as above, presents quite a red colour under a low power of the microscope. The bacilli occur for the most part within the protoplasm of the round cells of the granulation tissue, and are often so 264 LEPROSY. numerous that the structure of the cells is quite obscured (Fig. 70). They are often arranged in bundles which con- tain several bacilli lying parallel to one another, though the bundles lie in various directions (Fig. 71). The appearance thus presented by the cells filled with bacilli is very character- istic. Bacilli are also found free in the lymphatic spaces, but the greater number are undoubtedly contained within the cells. They are also found in spindle-shaped con- nective tissue cells, in endothelial cells, and in the walls of blood vessels. They are for the most part confined to the connective tissue, but a few may be seen in the hair follicles and glands of the skin. Occasionally one or two may be found in the surface epithelium, where they probably have been carried by leucocytes, but this position is, on the whole, exceptional. They also occur in large numbers in the lymphatic glands associated with the affected parts. In the internal organs — liver, spleen, etc., when leprous lesions are present, the bacilli are also found though in relatively fewer numbers. In the nerves in the anaesthetic form they are comparatively few, and in the sclerosed parts it may be impossible to find any. There are few also in the skin patches referred to above as occurring in this form of the disease. Their spread is chiefly by the lymphatics, though distri- bution by the blood stream also occurs. They have been said to be found in the blood during the presence of fever and the eruption of fresh nodules, and they have also been observed in the blood vessels post mortem, being chiefly contained within leucocytes. Recent observations (e.g., those of Doutrelepont and Wolters) show that the bacilli may be more widely spread throughout the body than was formerly supposed. A few may be detected in some cases in various organs which show no structural change, especially in their capillaries. The brain and spinal cord are practically exempt. Relations to the Disease. — Attempts to cultivate the leprosy bacilli outside the body have so far been unsuccess- ful. From time to time announcements of successful culti- RELATIONS OF BACILLI TO THE DISEASE. 265 vations have been made, but one after another has proved to be erroneous. A similar statement may be made with regard to experiments on animals. If a piece of leprous tissue be introduced subcutaneously in an animal, such as the rabbit, a certain amount of induration may take place around it, and the bacilli may be found unchanged in appear- ance weeks or even months afterwards, but no multiplica- tion of the organisms occurs. The only exception to this statement is afforded by the experiments of Melcher and Orthmann, who inoculated the anterior chamber of the eye of rabbits with leprous material, the inoculation being followed by an extensive growth of nodules in the lungs and internal organs, which they affirmed contained leprosy bacilli. It has been questioned, however, by several authorities whether the organisms in the nodules were really leprosy bacilli, and up to the present we cannot say that there is any satisfactory proof that the disease can be transmitted to any of the lower animals. It would also appear that the disease is not readily inoculable in the human subject. In a well-known case described by Arning, a criminal in the Sandwich Islands was inoculated in several parts of the body with leprosy tissue. Two or three years later, well-marked tubercular leprosy appeared and led to a fatal result. This experiment, however, is open to objections, as the individual before inoculation had been exposed to infection in a natural way, having been frequently in contact with lepers. In other cases inoculation experiments on healthy subjects, and inoculations in other parts of leprous individuals have given negative results. It has been supposed by some that the failure to obtain cultures and to reproduce the disease experimentally may be partly due to the bacilli in the tissues being dead. That many of the leprous bacilli are in a dead condition is quite possible, in view of the long period during which dead tubercle bacilli introduced into the tissues of animals retain their form and staining reaction I here is also the fact that from time to time in leprous subjects there occur attacks of a certain amount of fever 266 LEPROSY. which are followed by a fresh outbreak of nodules, and it would appear that especially at these times multiplication of the bacilli takes place more actively. The facts stated with regard to cultivation and inocu- lation experiments go to distinguish the leprosy bacillus all the more strongly from other organisms. Some have supposed that leprosy is a form of tubercle, or tubercle modified in some way, but for this there appears to us to be no evidence. Both from the pathological and from the bacteriological point of view the diseases are distinct. It should also be mentioned that tubercle is a not uncommon complication in leprous subjects, in which case it presents the ordinary characters. The mode by which leprosy is transmitted has been the subject of great controversy, and is one on which authorities still hold opposite opinions. Some consider that it is a hereditary disease, or at least that it is transmitted from a parent to the offspring ; others again that it is transmitted by direct contact. There appears to be no doubt, however, that on the one hand leprous subjects may bear children free from leprosy, and that on the other hand, healthy individuals entering a leprous district may contract the disease, though this rarely occurs. Of the latter occurrence there is the well-known instance of Father Damien, who contracted leprosy after going to the Sandwich Islands. In view of the fact that we must regard the bacillus as the cause of the disease, it is highly probable that in certain conditions it may be transmitted by direct contact, though its contagiousness is not of a high order. In leprosy, therefore, there is an organism which is invariably present in the disease, and has a special relation to the changes in the tissues. This organism can be distinguished from all other known organisms, and is lound in no other condition. Further, all the tissue changes in leprosy can be readily explained by the presence of a low form of irritation, such as is afforded by this organism] The evidence stated must be accepted as to its being the cause of the disease, though absolute proof is still wanting METHODS OF DIAGNOSIS. 267 owing to failure to cultivate the organism outside the body. Methods of Diagnosis. — Film preparations should be made with the discharge from any ulcerated nodule which may be present, or from the scraping of a portion of excised tissue, and should be stained as above described. The presence of large numbers of bacilli situated within the cells and giving the staining reaction of leprosy bacilli, is conclusive. It is more satisfactory, however, to make microscopic sections through a portion of the excised tissue, when the structure of the nodule and the arrangement of the bacilli can be readily studied. The points of difference between leprosy and tubercle have already been stated, and in most cases there is really no difficulty in distinguish- ing the two conditions. CHAPTER XI. GLANDERS AND RHINOSCLEROMA . Glanders. The bacillus of glanders (bacillus mallei ; Fr., bacille de la morve; Ger.,. Rotzbacillus) was discovered by Loffler and Schutz, the announcement of this discovery being made towards the end of 1882. They not only obtained pure cultures of this organism from the tissues in the disease, but by experiments on horses and other animals conclu- sively established its causal relationship. These have been fully confirmed. The same organism has also been culti- vated from the disease in the human subject, first by Weichselbaum in 1885, who obtained it from the pustules in a case of acute glanders in a woman, and by inoculation of animals obtained results similar to those of Loffler and Schutz. Within recent years a substance, maliein, has been obtained from the cultures of the glanders bacillus by a method similar to that by which tuberculin was prepared, and has been found to produce corresponding effects in animals suffering from glanders to those produced by tuber- culin in tuberculous animals. The Natural Disease.— Glanders chiefly affects the equine species— horses, mules, and asses. Horned cattle, on the other hand, are quite immune, whilst goats and sheep occupy an intermediate position, the former being rather NATURAL OCCURRENCE OF GLANDERS. 269 more susceptible and occasionally suffering from the natural disease. It also occurs in some of the carnivora — cats, lions, and tigers in menageries, which animals are infected from the carcases of animals affected with the disease. Many of the small rodents are highly susceptible to inoculation {vide infra). Glanders is also found in man as the result of direct inoculation on some wound of the skin or other part by means of the discharges or diseased tissues of an animal affected, and hence is commonest amongst grooms and others whose work brings them into contact with horses. In horses the lesions are of two types, to which the names " glanders " proper and " farcy " have been given, though both may exist together. In glanders proper the septum nasi and adjacent parts are chiefly affected, there occurring in the mucous membrane nodules at first firm and of somewhat translucent grey appearance. The growth of these is attended usually by inflammatory swelling and pro- fuse catarrhal discharge. Afterwards the nodules soften in the centre, break down, and give rise to irregular ulcera- tions. Similar lesions, though in less degree, may be found in the respiratory passages. Associated with these lesions there is usually implication of the lymphatic glands in the neck, mediastinum, etc. ; and there may be in the lungs, spleen, liver, etc., nodules of the size of a pea or larger, of greyish or yellow tint, firm or somewhat softened in the centre, and often surrounded with a congested zone. The term " farcy " is applied to the affection of the super- ficial lymphatic vessels and glands, which is specially seen where infection takes place through an abrasion of the skin, such as is often produced by the rubbing of the harness. The lymphatic vessels become irregularly thickened, so as to appear like knotted cords, and the lymphatic glands associated become enlarged and firm, though suppurative softening usually follows, and there may be ulceration. These thickenings are often spoken of as "farcy buds" and "farcy pipes." In farcy also, secondary 270 GLANDERS. nodules may occur in internal organs and the nasal mucous membrane. In the ass the disease runs a more acute course than in the horse. In man the disease is met with in two forms, an acute and a chronic ; though intermediate forms also occur, and chronic cases may take on the characters of the acute disease. The site of inoculation is usually on the hand or arm, by means of some scratch or abrasion, or possibly along a hair follicle, sometimes on the face, and occasionally on the mucous membrane of the mouth, nose, or eye. In the acute form there appears at the site of inoculation inflammatory swelling, attended often with spreading redness, and the lymphatics in relation to the part also become inflamed, the appearances being those of a, " poisoned wound." These local changes are soon followed by marked constitutional disturbance, and by an eruption on the surface of the body, at first papular and afterwards pustular, and later there may form in the subcutaneous tissue and muscles larger masses which soften and suppurate, the pus being often mixed with blood ; suppuration may occur also in the joints. In some cases the nasal mucous membrane may be secondarily infected, and thence inflam- matory swelling may spread to the tissues of the face ; in others it remains free. The patient usually dies in two or three weeks, sometimes sooner, with the symptoms of rapid pyaemia. In addition to the lesions mentioned there may be foci, usually suppurative, in the lungs (attended often with pneumonic consolidation), in the spleen, liver, bone- marrow, salivary glands, etc. In the chronic form the local lesion results in the formation of an irregular ulcer with thickened margins and sanious, often foul, discharge. The ulceration spreads deeply as well as superficially, and the thickened lymphatics have a great tendency to ulcerate also, though the lymphatic system is not so prominently affected as in the horse. Deposits form also in the sub- cutaneous tissue and muscles, and the mucous membrane may become affected. The disease may run a very chronic course, lasting for months, and recovery may occur, though, THE GLANDERS BACILLUS. 271 on the other hand, the disease may take on a more acute character and rapidly become fatal. The Glanders Bacillus — Microscopical Characters. — The glanders bacilli are minute rods, straight or slightly curved, with rounded ends, and about the same length as tubercle bacilli, but distinctly M thicker (Fig. 72). They show, however, M considerable varia- M tions in size and in appearance, and their protoplasm is often broken up into a number of deeply- stained portions with unstained intervals between. These riG. 72. — (jlanders bacilli amongst broken- Characters are seen down cells. Film preparation from a glanders both in the tissues nodu,e >n a- guinea-pig. and in cultures, but, Stained with weak carbol-fuchsin. x 1000. as in the case of many organisms, irregularities in form and size are more pronounced in cultures (Fig. 73), short filamentous forms 8 to 12 p in length being sometimes met with, but these are on the whole rare. The organism is non-motile. In the tissues they usually occur irregularly scattered amongst the cellular elements ; a few may be contained within leucocytes and connective tissue corpuscles, but the position of most is extracellular. They are most abundant in the acute lesions, in which they may be found in con- siderable numbers ; but in the chronic nodules, especially when softening has taken place, they are few in number, and it may be impossible to find any in sections. They have less powers of persistence, and disappear in the tissues much more quickly than tubercle bacilli. There has been dispute as to whether or not they 272 GLANDERS. pure contain spores. Some consider certain of the unstained portions to be of that nature, and it has been claimed that these can be stained by the method for staining spores (Rosenthal). But it is very doubtful that such is the case ; the appearances cor- respond rather with mere breaks in the protoplasm, such as are met with in many other bacilli which do not contain spores, and the compara- tively low powers of resistance of glanders bacilli containing these so-called spores is strongly against their being of that nature. The powers of resistance is after all the important practical point. Staining. — The glanders bacillus differs widely from the tubercle bacillus in its staining reactions. It stains with simple watery solutions of the basic stains, but somewhat faintly (better when an alkali or a mordant, such as carbolic acid, is added), and even when deeply stained it readily loses the colour when a decolorising agent such as alcohol is applied. Loffler and Schutz recommended staining of sections in an alkaline solution of methylene-blue for five minutes and then decolorising for a few seconds in water, 10 c.c, to which were added ten drops of a concentrated solution of sulphurous acid and one drop of a 5 per cent solution of oxalic acid. We have, however, obtained the best results by carbol-thionin-blue (p. 109), and we prefer to dehydrate by the aniline-oil method. In film preparations of fresh glanders nodules the bacilli can be ready found by staining with any of the ordinary combinations, e.g., carbol- FiG. 73. — Glanders bacilli, from a culture on glycerine agar. Stained with carbol - fuchsin and partially decolorised to show segmentation of protoplasm. x 1000. CULTIVATION OF GLANDERS BACILLUS. 273 thionin-blue or weak carbol-fuchsin. By using a stain of suitable strength no decolorising agent is necessary, the film being simply washed in water, dried, and mounted. Gram's method is quite inapplicable, the glanders bacilli rapidly losing the stain in the process. Cultivation. — (For the methods of separation vide infra.) The glanders bacillus grows readily on most of the ordinary media, but a somewhat high temperature is necessary, growth taking place most rapidly at 35 0 to 370 C. Though a certain amount of growth occurs down to 210 C, a temperature above 250 C. is always desirable. On agar and glycerine agar in stroke cultures growth appears along the line as a uniform streak of greyish-white colour and somewhat transparent appearance, with moist- looking surface, and when touched with a needle is found to be of rather slimy consistence. Later it spreads laterally for some distance, and the layer becomes of slightly brownish tint. On serum the growth is somewhat similar but more transparent, the separate colonies being in the form of round and almost clear drops. In subcultures on these media at the body temperature growth is visible within twenty-four hours, but when fresh cultures are made from the tissues it is not visible till the second day. Serum, however, is much more suitable for cultivating from the tissues than the agar media ; on the latter it is sometimes difficult to obtain growth. In broth, growth. forms at first a uniform turbidity, but soon settles to the bottom, and after a few days forms a pretty thick flocculent deposit of slimy and somewhat tenacious consistence. On potato the glanders bacillus flourishes well and produces a characteristic appearance, incubation at a high temperature, however, being necessary. If inoculation is made to potato from another medium, growth proceeds rapidly, and on the third day has usually formed a trans- parent layer of slightly yellowish tint, like clear honey in appearance. On subsequent days, the growth still extends and becomes darker in colour and more opaque, till about 18 274 GLANDERS. the eighth day it has a reddish-brown or chocolate colour, while the potato at the margin of the growth often shows a greenish-yellow staining. The characters of the growth on potato along with the microscopical .appearances are quite sufficient to distinguish the glanders bacillus from every other known organism (sometimes the cholera organism and the B. pyocyaneus produce a somewhat similar appearance, but they can be readily distinguished by their other characters). The potato is also a suitable medium for starting cultures from the tissues ; in this case minute transparent colonies become visible on the third day and afterwards present the appearances just described. Powers of Resistance. — The glanders bacillus is not killed at once by drying, but usually loses its vitality after fourteen days in the dry condition, though sometimes it lives longer. It is not quickly destroyed by putrefaction, having been found to be still active after remaining two or three weeks in putrefying fluids. In cultures the bacilli retain their vitality for three or four months, if, after growth has taken place, they be kept at the temperature of the room ; on the other hand, they are often found to be dead at the end of a month when kept constantly at the body temperature. They have comparatively feeble resistance to heat and antiseptics. Loffler found that they were killed in ten minutes in a fluid kept at 550 C, and in two to three minutes by a 5 per cent solution of carbolic acid. Boiling water and the ordinarily used antiseptics are very rapid and efficient disinfectants. We may summarise the characters of the glanders bacillus by saying that in its morphological characters it resembles somewhat the tubercle bacillus, but is thicker, and differs widely from it in its staining reactions. For its cultivation the higher temperatures are necessary, and thl growth on potato presents most characteristic features. Experimental Inoculation. — In horses subcutaneous injection of the glanders bacillus in pure culture reproduces all the important features of the disease. This fact was established at a comparatively early date by Loffler and EXPERIMENTAL INOCULA TION. 275 Schutz, who, after one doubtful experiment, successfully inoculated two horses in this way, the cultures used having been grown for several generations outside the body. In a few days swellings formed at the sites of inoculation, and later broke down into unhealthy-looking ulcers. There was the usual involvement of the lymphatic vessels and glands, and symptoms due to affection of the nasal mucous membrane also appeared after some time, there being the characteristic discharge. One of the animals died ; after a few weeks the other, showing symptoms of cachexia, was killed. In both animals, in addition to ulcerations on the surface with involvement of the lymphatics, there were found post mortem, nodules in the lungs, softened deposits in the muscles, and also affection of the nasal mucous membrane, nodules, and irregular ulcerations. The ass is even more susceptible than the horse, the disease in the former running a more rapid course, but with similar lesions. The ass can be readily infected by simple scarification and inoculation with glanders secretion, etc. (Nocard). Of small animals, field-mice and guinea-pigs are the most susceptible. Strangely enough, house-mice and white mice enjoy an almost complete immunity. In field-mice subcutaneous inoculation is followed by a very rapid disease, usually leading to death within eight days, the organisms becoming generalised and producing numerous minute nodules, especially in the spleen, lungs, and liver. In the guinea-pig'the disease is less acute, though secondary nodules in internal organs are usually present in consider- able numbers. At the site of inoculation an inflammatory swelling forms, which soon softens and breaks down, leading to the formation of an irregular crateriform ulcer with indurated margins. The lymphatic vessels become in- filtrated, and the corresponding lymphatic glands become enlarged to the size of peas or small nuts, softened, and semi-purulent. The animal sometimes dies in two or three weeks, sometimes not for several weeks. Secondary nodules, in varying numbers in different cases, may be 276 GLANDERS. found in the spleen, lungs, bones, nasal mucous membrane, testicles, ovaries, etc. ; in some cases a few nodules are found in the spleen alone. Intraperitoneal injection in the male guinea-pig is followed, as pointed out by Straus, by a very rapid and semi-purulent affection of the tunica vaginalis, shown during life by a great swelling and redness of the testicles, which may be noticeable in two or three days. By this method there occur also numerous small nodules on the surface of the peritoneum. Rabbits are less susceptible than guinea-pigs, and the effect of sub- cutaneous inoculation is somewhat uncertain. Accidental inoculation of the human subject with pure cultures of the bacillus has in more than one instance been followed by the acute form of the disease and a fatal result. Action on the Tissues. — From the above facts it will be seen that in many respects glanders presents an analogy to tubercle as regards the general characters of the lesions and the mode of spread. In the guinea-pig, for example, there are in both diseases a local swelling, an implication of lymphatics in connection with the part, and, lastly, a spread to internal organs and other parts by means of the blood vessels. When the tissue changes in the two diseases are compared, certain differences are found. The glanders bacillus causes a more rapid and more marked inflammatory reaction. There is more leucocytic infiltra- tion and less proliferative change which might lead to the formation of epithelioid cells. Thus the centre of an early glanders nodule -shows a dense aggregation of leucocytes, many of which are polymorpho-nuclear, and have recently emigrated from the vessels, whilst the tissue elements between may be more or less degenerat- ing, or may show proliferative changes. And further, the inflammatory change may be followed by suppurative softening of the tissue, especially in certain situations, such as the subcutaneous tissue and lymphatic glands. The nodules, therefore, in glanders, as Baumgarten puts it, occupy an intermediate position between miliary ab- scesses and tubercles. The diffuse coagulative necrosis MODE OF SPREAD. 277 and caseation which are so common in tubercle do not occur to the same degree in glanders, and typical giant cells are not formed. The nodules in the lungs show leucocytic infiltration and thickening of the alveolar walls, whilst the vesicles are filled with catarrhal cells ; i.e., there is reaction both on the part of the connective tissue, and of the endothelium of the air vesicles, whilst at the periphery of the nodules connective tissue growth is present in proportion to their age. The tendency to spread by the lymphatics is always a well-marked feature, and when the bacilli gain entrance to the blood-stream, they soon settle in the various tissues and organs. Accordingly, even in acute cases it is usually quite impossible to detect the bacilli in the circulating blood, though sometimes they have been found. It is an interesting fact, shown by observations of the disease both in the human subject and in the horse, as well as by experiments on guinea-pigs, that the mucous membrane of the nose may become infected by means of the blood — another example of the tendency of organisms to settle in special sites. Mode of Spread. — Glanders usually spreads from a diseased animal by direct contagion with the discharge from the nose or from the sores, etc. So far as infection of the human subject goes, no other mode is known. There is no evidence that the disease is produced in man by inhalation of the bacilli in the dried condition. Some authorities consider that pulmonary glanders may be pro- duced in this way in the horse, whilst others maintain that in all cases there is first a lesion of the nasal mucous membrane or of the skin .surface, and that the lung is affected secondarily. Babes, however, found that the disease could be readily produced in susceptible animals by exposing them to an atmosphere in which pulverised cultures of the bacillus had been. He also found that inunction of the skin with vaseline containing the bacilli might produce the disease, the bacilli in this case entering along the hair follicles. 278 GLANDERS. Agglutination of Glanders Bacilli. — Shortly after the discovery of agglutination in typhoid fever, M'Fadyean showed that the serum of glandered horses possessed the power of agglutinating glanders bacilli. His later observations show that in the great majority of cases of glanders a I in 50 dilution of the serum produces marked agglutina- tion in a few minutes, whilst in the great majority of non-glandered animals no effect is produced under these conditions. The test performed in the ordinary way is, however, not absolutely reliable, as exceptions occasionally occur in both directions, i.e., negative results by glandered animals and positive results by non-glandered animals. He finds that a more delicate and reliable method is to grow the bacillus in bouillon containing a small proportion of the serum to be tested. In this way he has obtained a distinct sedi- menting reaction with a serum which did not agglutinate at all distinctly in the ordinary method. Further observations are still required to determine the precise value of the test. Mallein and its Preparation.— Mall ein is obtained from cultures of the glanders bacillus grown for a suitable length of time, and, like tuberculin, is really a mixture comprising (1) substances in the bodies of the bacilli and (2) their soluble products, not destroyed by heat, along with substances derived from the medium of growth. It was at first obtained from cultures on solid media by extracting with glycerine or water, but is now usually prepared from cultures m glycerine bouillon. Such a culture, after being allowed to grow for three or four weeks, is sterilised by heat either in the autoclave at 1 15 C. or by steaming at 100° C. on successive days. It is then faltered through a Chamberland filter. The filtrate constitutes fluid mallein. Usually a little carbolic acid (.5 per cent) is added to prevent it from decomposing. Of such mallein 1 c.c. is usually the dose for a horse ( M 'Fadyean) . Foth has prepared a dry form of mallein by th owing he filtrate of a broth culture, evaporated to one-tenth of its bulk, into twenty-five or thirty times its volume of alcohol. A white precipitate is formed, which is dried over calcium chloride and then under an air- pump. A dose of this dry mallein is .05 to .07 grm. P The use of Mallein as a means of Diagnosis.-!* .using mallein for the diagnosis of glanders, the temperature of the animal ought to , be observed for some hours beforehand, and, after subcutaneous injection of a suitable dose, it is taken at definite intervals, -.according to M^adyean at the sixth, tenth, fourteenth and eighteenth .hours . after wards, and on the next day. Here both the local reaction _ and [ the temperature are of importance. In a glandered animal a the site ot Sedation there is a fomewhat painful local «^*^££j diameter of five inches at least, the maximum size not being ; attainea until twenty-four hours afterwards. The temperature rises I 5 to a CJ ormoT:, tL maximum generally occurring ! If the temperature never rises as much as \\ , the leaction is cons, doubtful " In the negative reaction given by an animal free fiom METHODS OF EXAMINATION. 279 glanders, the temperature often rises 1°, the local swelling reaches the diameter of three inches at most, and has much diminished at the end of twenty-four hours. In the case of dry mallein, local reaction is less marked. Veterinary authorities are practically unanimous as to the great value of the mallein test as a means of diagnosis. We cannot as yet speak as to its applicability to diagnosis of the disease in the human subject. Methods of Examination. — Microscopic examination in a case of suspected glanders will at most reveal the presence of bacilli corresponding in their characters to the glanders bacillus. An absolute diagnosis cannot be made by this method. Cultures may be obtained by making successive strokes on blood serum or on glycerine agar (preferably the former), and incubating at 370 C. The colonies of the glanders bacillus do not appear till two days after. This method often fails unless a considerable proportion of the glanders bacilli are present. Another method is to dilute the secretion or pus with sterile water, to varying degrees, and then to smear the surface of potato with the mixture, the potatoes being incubated at the above temperature. The colonies on potatoes may not appear till the third day. The most certain method, however, is by inoculation of a guinea-pig, either by subcutaneous or intraperitoneal in- jection. By the latter method, as above described, lesions are much more rapidly produced, and are more character- istic. If, however, there have been other organisms present, the animal may die of a septic peritonitis, though even in such a case the glanders bacilli will be found to be more numerous in the tunica vaginalis, and may be cultivated" from this situation. In the case of horses, etc., a diagnosis will, of course, be much more easily and rapidly effected by means of mallein. Rhinoscleroma. This disease is considered here as, from the anatomical changes, it also belongs to the group of infective granulo- mata. It is characterised by the occurrence of chronic nodular thickenings in the skin or mucous membrane 2SO R II I NO S CLE R OMA . of the nose, or in the mucous memhrane of the pharynx, larynx, or upper part of the trachea. It is scarcely ever met with in this country, hut is of not very uncommon occurrence on the Continent, especially in Austria. In the granulation tissue of the nodules there are to be found numerous round and rather large cells, which have peculiar characters and are often known as the cells of Mikulicz. Their protoplasm contains a collection of somewhat gelatin- ous material which may fill the cell and push the nucleus to the side. Within these cells there is present a character- istic bacillus, occurring in little clumps or masses lying chiefly in the gelatinous material. A few bacilli also lie free in the lymphatic spaces around. This organism was first observed by Frisch, and is now known as the bacillus of rhinoscleroma. The bacilli have the form of short oval rods, which, when lying separately, can be seen to possess a distinct capsule, and which ' in all their microscopical characters correspond closely with Friedlander's pneumo- bacillus. They are usually present in the lesions in a state of purity. It was at first stated that they could be stained by Gram's method, but more recent observa- tions show that like Friedlander's organism they lose the stain. From the affected tissues this bacillus can be easily cultivated by the ordinary methods. In the characters of its growth in the various culture media it presents a close similarity to that of the pneumobacillus, as it also does in its fermentative action in milk and sugar-containing fluids. The nail-like appearance of the growth on gelatine is said to be less distinct, and the growth on potatoes is more transparent and may show small bubbles of gas ; otherwise it resembles the pneumobacillus. It is doubtful whether any distinct line of difference can be drawn between the two organisms so far as their microscopical and cultural characters are concerned. The evidence that the organisms described are the cause of this disease consists in their constant presence and their special relation to the affected tissues, as already described. RI1IN0SCLER0MA. From these facts alone it is highly probable that they are the active agents in the production of the lesions. Experi- mental inoculation has thrown little light on the subject, though one observer has described the production of nodules on the conjunctivae of guinea-pigs. The relation of the rhinoscleroma organism to that of Friedlander is, however, still a matter of doubt, and the matter has been further complicated by the fact that a bacillus possessing closely similar characters has been found to be very fre- quently present in ozcena, and is often known as the bacillus ozaitice. The last-mentioned organism is said to have more active fermentative powers. From what has been stated it will be seen that a number of organisms closely allied in their morphological characters, have been found in the nasal cavity in healthy or diseased conditions. From what we know, however, of other diseases, it is not improbable that though presenting these close resemblances, they may be distinct species, and may cause distinct patho- logical conditions in man. The subject is one on which more light is still required. CHAPTER XII. ACTINOMYCOSIS. Actinomycosis is a disease of special interest, inasmuch as it is the most important example of a microbic affection in which the parasite belongs to a higher order, and presents greater complexity of form, than the ordinary bacteria show. It is related, by the characters of the pathological changes, to the diseases which have been described. The disease affects man in common with certain of the domestic animals, though it is more frequent in the latter, especially in oxen, swine, and horses. The parasite was first discovered in the ox by Bollinger, and described by him in 1877, the name actinomyces or ray fungus being from its appearance applied to it by the botanist Harz. In 1878 Israel described the parasite in the human subject, and in the following year Ponfick identified it as being the same as that found in the ox. Since that time a large number of cases have been observed in the human subject, the result of investigation being to show that it affects man much more frequently than was formerly supposed. The disease in man is characterised by chronic suppurative processes, which often extend to internal organs, producing a sort of chronic pyaemia ; the disease in the pig is of somewhat similar nature. In the ox, on the other hand, and also in the horse, the lesions are characterised by an abundant formation of granulation tissue, often resulting in tumour- like masses of considerable size. CHARACTERS OF THE ACTINOMYCES. 283 Naked-eye Characters of the Parasite. — The actino- myces grows in the tissues in the form of little round masses or colonies, which, when fully developed, are easily visible to the naked eye, the largest being about the size of a small pin's head, whilst all sizes below this may be found. When suppuration is present, they lie free in the pus ; when there is no suppuration, they are embedded in the granulation tissue, but are usually surrounded by a zone of softer tissue. They may be transparent or jelly-like, or they may be opaque and of various colours — white, yellow, greenish, or almost black. The appearance depends upon their age and also upon their structure, the younger colonies being more or less transparent, the older ones being generally opaque. Their colour is modified by the presence of pig- ment and by degenerative change, which is usually accom- panied by a yellowish coloration. They are generally of soft, sometimes tallow -like, consistence, though sometimes in the ox they are gritty, owing to the presence of calcareous deposit. They may be readily found in the pus by spreading it out in a thin layer on a glass slide and holding it up to the light. They are sometimes described as being always of a distinctly yellow colour, but this is only occasion- ally the case ; in fact, in the human subject they occur much more frequently as small specks of semi-translucent appear- ance, and of greenish-grey tint. Microscopical Characters. — Whilst there is still dispute as to the exact ■ botanical position of the actinomyces, most authorities regard it as a pleomorphous bacterium belonging to the streptothrix group (p. 20). This view appears to us to be the correct one. In the colonies, as they grow in the tissues, three morphological elements may be described, namely, filaments, coccus -like bodies, and clubs. 1. The filaments are comparatively thin, measuring about .5 fx in diameter, but they are often of great length. They are composed of a central protoplasm enclosed by a sheath. The latter, which is most easily made out in the older filaments with granular protoplasm, occasionally contains 284 ACTINOMYCOSIS. granules of dark pigment. In the centre of the colony the filaments interlace with one another, and form an irregular network which may be loose or dense ; at the periphery they are often arranged in a somewhat radiating manner, and run outwards in a wavy or even spiral course. They also show branching, a character which at once distinguishes Fig. 74.— Actinomycosis ot human liver, showing a colony of the parasite composed of a felted mass of filaments surrounded by pus. Paraffin section ; stained by Gram's method and with safranin. x 500. them from the ordinary bacteria. Between the filaments there is a finely granular or homogeneous ground substance. Most of the colonies at an early stage are chiefly constituted by filaments loosely arranged ; but later, part of the growth may become so dense that its structure cannot be made out. This dense part, starting excentrically, may grow round the colony to form a hollow sphere, from the outer surface CHARACTERS OF THE ACTINOMYCES. 285 of which filaments radiate for a short distance (Fig. 74). The filaments usually stain uniformly in the younger colonies, but some, especially in the older colonies, may be segmented so as to give the appearance of a chain of bacilli or of cocci, though the sheath enclosing them may generally be distinguished. Rod-shaped and spherical forms may also be seen lying free. 2. Coccus-like Bodies. — The formation of these from filaments has already been described, but it is doubtful if all are of the same nature. Like other species of strepto- thrix the actinomyces when growing on a culture medium shows on its surface filaments growing upwards in the air, the protoplasm of which becomes segmented into rounded spores or gonidia. In natural conditions outside the body these gonidia become free and act as new centres by growing out into filaments. They have higher powers of resistance than the filaments though less than the spores of most of the lower bacteria. It is probable that some of the spherical bodies formed within filaments when growing in the tissues have the same significance, i.e. are gonidia, whilst others may be merely the result of degenerative change. Some observers have described young colonies largely composed of spherical forms as if these multiplied by division, but this latter point is still doubtful. Both the filaments and the spherical bodies are readily stained by Gram's method. 3. Clubs. — These are elongated pear-shaped bodies which are seen at the periphery of the colony, and are formed by a sort of hyaline swelling of the sheath around the free extremity of a filament (Fig. 75). They are usually homogeneous and structureless in appearance. In the human subject the clubs are often comparatively fragile structures which are easily broken down, and may some- times be dissolved in water. Sometimes they are well seen when examined in the fresh condition, but in hardened specimens are no longer distinguishable. In specimens stained by Gram's method they are not coloured by the violet, but take readily a contrast stain, such as picric acid, 286 ACTINOMYCOSIS. rubin, etc.; sometimes a darkly -stained filament can be seen running for a distance in the centre, and may have a knob-like extremity. In many of the colonies in the human subject the clubs are absent. In the ox, on the other hand, where there are much older colonies, the clubs constitute the most prominent feature, whilst in most Pig 7S. -Actinomyces in human kidney, showing clubs radially arranged and surrounded by pus. The filaments had practically dts "'''Baffin section ; stained with hematoxylin and rubin. x 500. colonies the filaments are more or les Regenerated and i rnav sometimes be impossible to find any. They often "dense fringe ^^^^^^1 by Gram's method retain the violet s am They have J fact undergone some further chemical change which prd duces the altered staining reaction. Clubs showing mter- TISSUE LESIONS. 287 mediate staining reaction have been described byM'Fadyean. The view that the clubs are organs of fructification has been abandoned by most authorities, and there appears to us little evidence in support of it. Tissue Lesions. — In the human subject the parasite produces by its growth a chronic inflammatory change, usually ending in a suppuration which slowly spreads. In some cases there is a comparatively large production of granulation tissue, with only a little softening in the centre, so that the mass feels solid. This condition is sometimes found in the subcutaneous tissue, especially when the disease has not advanced far, and also in dense fibrous tissue. In most cases, however, and especially in internal organs, suppuration is the outstanding feature. This is to be associated with abundant growth of the parasite in the filamentous form. In an organ such as the liver, multiple foci of suppuration are seen at the spreading margin of the disease, presenting a honeycomb appearance which is some- what characteristic, whilst the colonies of the parasite may be seen in the pus with the naked eye. In the older parts the abscesses have become confluent, and formed large areas of suppuration. The pus is usually of greenish- yellow colour, and of somewhat slimy character. In cattle the tissue reaction is more of a formative type, there being abundant growth of granulation tissue, which may result in large tumour-like masses, usually of more or less nodulated character. The cells immediately around the colonies are usually irregularly rounded, or may even be somewhat columnar in shape, whilst farther out they become spindle-shaped and concentrically arranged. It is not uncommon to find leucocytes or granulation tissue invading the substance of the colonies, and portions of the parasite, etc., may be contained within leucocytes or within small giant cells which are sometimes present. A similar invasion of old colonies by leucocytes is sometimes seen in human actinomycosis. Origin and Distribution of Lesions.~The lesions in the human subject may occur in almost any part of the body, 288 ACTINOMYCOSIS. the paths of entrance being very various. In many cases the entrance takes place in the region of the mouth — probably around a decayed tooth — by the crypts of the tonsil, or by some abrasion. Swelling and suppuration may then follow in the vicinity and may spread in various directions. The periosteum of the jaw or the vertebra may thus become affected, caries or necrosis resulting, or the pus may spread deeply in the tissues of the neck, and may even pass into the mediastinum. Occasionally the parasite may enter the tissues from the cesophagus, and in a considerable number of cases the primary lesion is in some part of the intestine, generally of the large intestine. The parasite penetrates the wall of the bowel, and may be found deeply between the coats, surrounded by puru- lent material. Ulceration, and sometimes a considerable amount of necrosis may follow. Thence it may spread to the peritoneum or to the extraperitoneal tissue, the retro- ceecal connective tissue and that around the rectum being not uncommonly seats of suppuration produced in this way. A peculiar affection of the intestine has been described, in which slightly raised plaques are found both in the large and small intestines, these plaques being com- posed almost exclusively of masses of the actinomyces along with epithelial cells. This, however, is a very rare condition. The path of entrance may also be by the respiratory passages, the primary lesion being pulmonary or peribronchial ; extensive suppuration in the lungs may result. Infection may also occur by the skin surface, and lastly, by the female genital tract, as in a case recorded by j Grainger Stewart and Muir, in which both ovaries and both Fallopian tubes were affected. When the parasite has invaded the tissues by any of these channels, secondary or " metastatic " abscesses may occur in internal organs. The liver is the organ most frequently affected, though abscesses may occur in the lungs, brain, kidneys, etc. In such cases the spread takes place by the blood stream, and it is possible that leucocytes may be the carriers of the infection, as it is not uncommon CULTIVATION OF ACTINOMYCES. 289 to find leucocytes in the neighbourhood of a colony con- taining small portions of the filaments in their interior. In the ox, on the other hand, the disease usually remains quite local, or spreads by continuity. It may produce tumour-like masses in the region of the jaw or neck, or it may specially affect the palate or tongue, in the latterproducing enlargement and in- duration, with nodular thickening on the sur- face— the condition known as " woody tongue." Source of the Para- site.— There is a con- siderable amount of evidence to show that outside the body the parasite grows on grain, especially on barley. Both in the ox and in the pig the parasite has been found growing around fragments of grain embedded in the tissues. There are besides, in the case nf tho, K„™„ u , 7 '~ Cultures of the actinomyces on Of the human sub- glycerine agar, of about three weeks' growth ; ject, a certain number showing the appearances which occur. The Of cases in which gr°.Th in ,A iS at pIaces somewhat corrugated , , La on the surface. Natural size. there was a history of penetration of a mucous surface by a portion of grain and in a considerable proportion of cases the patient has been exposed to infection from this source. The position of the lesions in cattle is also in favour of such a view Ihe conditions of growth outside the body in a natural *9 ago ACTINOMYCOSIS. condition are, however, not known, nor has the parasite been cultivated from any source outside the body. Cultivation (for methods of isolation see later). — The actinomyces grows on a variety of media, though on all its rate of growth is somewhat slow. Growth takes place at the ordinary room temperature, but very slowly, the tem- perature of the body being much more suitable. On agar or glycerine agar at 37° C, growth is generally visible on the third or fourth day in the form of little .-Actinomyces, from a culture on a tOUgh consistence, surface of the agar. Older growths often show on the surface a sort of corrugated aspect, and may sometimes present the appearance of having been dusted with a brownish-yellow powder (Fig. 76). The organism grows well in the anaerobic condition on agar, and for this pur- pose unopened eggs also, either in the fresh or boiled condition, have been used, inoculation being effected by drilling in the shell a small hole which is afterwards closed. The growth on potatoes is somewhat similar to that on agar. On gelatine the same tendency to grow in little spherical transparent drops which gradually en- large and form rounded projections of a reddish -yellow tint and somewhat transparent appear- ance, like drops of amber. The growths tend to remain sepa- rate, and even when they become con- fluent, the nodular character is main- tained. They have VARIETIES OF ACTINOMYCES. 2gi masses is seen, and the medium becomes very slowly liquefied. When this occurs the liquefied portion has a brownish colour and somewhat syrupy consistence, and the growths may be seen at the bottom, as little balls, from the surface of which filaments radiate. In the cultures at an early stage the growth is composed of branching filaments, which stain uniformly (Fig. 77), but later some of the superficial filaments may show segmenta- tion into gonidia. True clubs are not formed in cultures, though slight bulbous thickenings may be seen at the end of some of the filaments. Varieties of Actinomyces and Allied Forms.— It is possible that in the cases of the disease described in the human subject there may be more than one variety or species of parasite belonging to the same group. Gasperini has described several varieties of actinomyces bovis according to the colour of the growths, and a similar condition may obtain in the case of the human subject. Wolff and Israel cultivated from two cases of "actinomycosis" in man a streptothrix which differs in so many important points from the actinomyces that it is now regarded as a distinct species. Eppinger also obtained from a brain abscess another species of streptothrix, and there is also the strepto- thrix madura? presently to be described. In diseases of the lower animals several other forms have been found. For example, a streptothrix has been shown by Nocard to be the cause of a disease of the ox,— "farcin du boeuf,"— a disease in which also there occur tumour-like masses of granulation tissue. The so- called diphtheria of calves and " bacillary necrosis " in the ox are probably both produced by another streptothrix which grows diffusely in the tissues in the form of fine felted filaments. Further investiea tion may show that some of these or other species may occur in the human subject in conditions which are not yet differentiated. Experimental Inoculation.— Some observers (eg Bos- trom) have obtained negative results by inoculation on animals, but Israel and Wolff succeeded in the case of guinea-pigs and rabbits. Intraperitoneal injection of the parasite in the bacillary or filamentous form was followed in a month by the production of nodules in the peritoneum from the size of a pea to that of a plum. The nodules were composed of granulation tissue, vascular on the surface and containing fatty pus in which colonies of typical 292 ACTINOMYCOSIS. structure lay. These colonies showed clubs, though clubs were not present in the culture injected. The disease can also be reproduced by direct inoculation from an animal affected. Methods of Examination and Diagnosis. — As actino- mycosis cannot be diagnosed with certainty apart from the discovery of the parasite, a careful examination of the pus in obscure cases of suppuration should always be undertaken. As already stated, the colonies can be recog- nised with the naked eye, especially when some of the pus is spread out on a piece of glass. If one of these is washed in salt solution and examined unstained, the clubs, if present, are at once seen on microscopic examination. Or the colony may be stained with a simple reagent such as picrocarmine, and mounted in glycerine or Farrant's solution. To study the filaments, a colony should be broken down on a cover glass, dried, and stained with a simple solution of any of the basic aniline dyes, such as gentian-violet, though better results are obtained by carbol- thionin-blue, or by carbol-fuchsin diluted with five parts of water. If the specimen be over-stained, it may be decolorised by weak acetic acid. Cover-glass preparations of this kind and also of cultures, are readily stained by these methods, but in the case of sections of the tissues Gram's method, or a modification of it, should be used to show the filaments, etc., a watery solution of rubin being afterwards used to stain the clubs. By this method, very striking preparations may be obtained. To obtain cultures, tubes of glycerine agar should be inoculated with portions of the colonies and incubated at ^7° C Owing to the slow growth of the actinomyces, however, the obtaining of pure cultures is difficult, unless the pus is free from contamination with other organisms. Madura Disease. Madura disease or mycetoma in many respects closely resembles actinomycosis, and is produced by a somewhat MADURA DISEASE. 293 similar parasite, though it is now pretty certain that the organisms in the two conditions are of different species. This disease is comparatively common in India and in various other parts of the tropics. It most frequently affects the foot ; hence the disease is often spoken of as "Madura foot." The hand is rarely affected. In the parts affected there is a slow growth of granulation tissue which has an irregularly nodular character, and in the centre of the nodules there occurs purulent softening which is often followed by the formation of fistulous openings and ulcers. There occur great enlargement and distortion of the part and frequently caries and necrosis of the bones. Within the softened cavities and also in the spaces between the fibrous tissue, small rounded bodies or granules, bearing a certain resemblance to the actinomyces, are present. These may have a yellowish or pinkish colour, compared from their appearance to fish roe, or they may be black like grains of gunpowder, and may by their conglomeration form nodules of considerable size. Hence a yellow or pale, and a black variety of the disease have been distinguished. In both varieties the granules mentioned reach a rather larger size than in actinomycosis. When the roe-like granules are examined microscopically, they are found, like the actinomyces, to show in their interior an abundant mass of branching filaments with mycelial arrangement. There are also present at the peri- phery, structures which have a resemblance to the clubs in actinomyces. These structures often have an elongated wedge shape, forming an outer zone to the colony, and in some cases the filaments can be found to be connected with them. In the black variety, in many cases, the pigment is so abundant that all internal structure is obscured. In some cases, however, filaments may be found as in the yellow variety. As a rule, however, the parasite appears to be in a more or less degenerated condition, and the tissue round about the black masses is usually fibrous in character, their presence not being associated with much softening. 294 A CTINOM YCOSIS. Regarding the exact relations of this organism there is still doubt. Kanthack considered that it had all the important characters of the actinomyces and belonged to the same family, though without cultures he could not state definitely that the two species were identical. He also considered that the parasite was of the same nature in the pale and black varieties, and that probably the latter was a degenerated form of the former. Boyce and Surveyor, on the other hand, described the parasite as being composed of long non-branching septate filaments, and regarded it as belonging to the hyphomycetes or moulds. Vincent, how- ever, has obtained cultures from a case of the disease occurring in Tunis, and the organism obtained, though resembling the actinomyces in many respects, is a distinct species. Vincent gives to it the name streptothrix Madurce. The chief points of difference are the following : the strepto- thrix Madura does not liquefy gelatine ; its cultures on the agar media have a reddish colour ; it flourishes readily on certain vegetable infusions on which the actinomyces does not grow. Unlike the actinomyces again, it does not grow under anaerobic conditions, and so far its inoculation on animals has not been followed by any pathogenic effects. The results of Vincent would, therefore, show that the two organisms belong to the same genus, but are distinct species. It would, however, still require to be shown that the disease in the case studied by him was identical with that common in India, and also that in these conditions the parasite is always the same. It may also be mentioned that Madura disease differs from actinomyces, not only in its geographical distribution, but also in its clinical characters. Its course, for example, is of an extremely chronic nature, and though the local disease is incurable except by opera- tion, the parasite never produces secondary lesions in internal organs. Vincent also found that iodide of potas sium, which has a high value as a therapeutic agent mmany cases of actinomycosis, had no effect in the case studied by ■ CHAPTER XIII. ANTHRAX.1 OTHER NAMES. SPLENIC FEVER, MALIGNANT PUSTULE, WOOLSORTER'S DISEASE. GERMAN, MILZBRAND ; FRENCH, CHARBON.2 Introductory. — Anthrax is a disease occurring epidemically among the herbivora, especially sheep and oxen, in which animals it has the characters of a rapidly fatal form of septicaemia with splenic enlargement, attended by an extensive multiplication of characteristic bacilli through- out the blood. The disease does not occur as a natural affection in man, but may be communicated to him directly or indirectly from animals, and it may then appear in certainly two and possibly three forms. In the first there is infection through the skin, in which a local lesion, the I malignant pustule," occurs. In the second form infection takes place through the respiratory tract. Here very aggravated symptoms centred in the thorax, with rapidly fatal termination, follow. Thirdly, an infection may probably take place through the intestinal tract, which is 1 [n even recent works on surgery the term "anthrax" may be found applied to any form of carbuncle. Before its true pathology was known the local variety of the disease which occurs in man and which is now called "malignant pustule" was known as " malignant carbuncle." _ 2 This must be distinguished from " charbon symptomatique," which is quite a different disease. 296 ANTHRAX. now the first part to give rise to symptoms. In all these forms of the affection in the human subject, the bacilli arc in their distribution much more restricted to the local lesions than is the case in the ox, their growth and spread being attended by inflammatory oedema and often by haemorrhages. Historical Summary. — Historical researches leave little doubt that from the earliest times anthrax has occurred among cattle. For a long time its pathology was not understood, and it went by many names. During the early part of the present century much attention was paid to it, and, with a view to finding out its nature and means of spread, various conditions attaching to its occurrence, such as those of soil and weather, were exhaustively studied. Pollender in 1849 pointed out that the blood of anthrax animals contained numerous rod-shaped bodies which he conjectured had some causal connection with the disease. In 1863 Davaine announced that they were bacteria, and originated the name bacillus anthracis. He stated that unless blood used in inoculation experiments on animals contained them, death did not ensue. Though this conclusion was disputed, still by 'the work of Davaine and others the causal relationship of the bacilli to the disease had been nearly established when the work of Koch appeared in 1876. This constituted that observer's first contribu- tion to bacteriology, and did much to clear up the whole subject. Koch confirmed Davaine's view that the bodies were bacteria. He observed in the blood of anthrax animals the appearance of division, and from this deduced that multiplication took place in the tissues. He observed them under the microscope dividing outside the body, and noticed spore formation taking place. He also isolated the bacilli in pure culture outside the body, and by inoculating animals with them, produced the disease artificially. In his earlier experiments he failed to produce death by feeding susceptible animals both with bacilli and spores, and as the intestinal tract was, in his view, the natural path of infection, he considered as incomplete the proof of this method of the spontaneous occurrence of anthrax in herds of animals. Koch's ob- servations were, shortly afterwards, confirmed in the mam by Pasteur, though controversy arose between them on certain minor points. Moreover, further research showed that the disease could be produced in animals by feeding them with spores, and thus the way in which the disease might spread naturally was explained. The Bacillus Anthracis.— Anthrax as a disease in man is of comparative rarity. Not only, however, is the bacillus anthracis easy of growth and recognition, but in its growth it illustrates many of the general morphological. BACILLUS ANTHRA CIS. 297 characters of the whole group of bacilli, and is therefore of the greatest use to the student. Further, its behaviour when inoculated in animals illustrates many of the points raised in connection with such difficult questions as the general pathogenic effects of bacteria, immunity, etc. Hence an enormous amount of work has been done in investigating it in all its aspects. If a drop of blood is taken immediately after death from an auricular vein of a cow, for example, which has died from anthrax, and examined microscopically, it will be found to contain a great number of large non-motile bacilli. On making a cover-glass preparation from the same source, and staining with watery methylene-blue, the characters of the bacilli can be better made out. They are about 1.2 /j, thick or a little thicker, and 6 to 8 /x, long, though both shorter and longer forms also occur. The ends are sharply cut across, or may be slightly dimpled so as to resemble somewhat the proximal end of a phalanx. Their protoplasm is very finely granular, and sometimes appears surrounded by a thin unstained capsule. When several bacilli lie end to end in a thread, the capsule seems common to the whole thread (Fig. 82). They stain well with all the basic aniline dyes and are not decolorised by Gram's method. Plate Cultures. — From a source such as that indicated, it is easy to isolate the bacilli by making gelatine or agar plates. If, after . twelve hours' incubation at 37° C, the latter be examined under a low objective, colonies will be observed. They are to be recognised by beautiful wavy wreaths like locks of hair, radiating from the centre and apparently terminating in a point which, however, on examination with a higher power is observed to be a fila- ment which turns upon itself (Fig. 78). The whole colony is, in fact, probably one long thread. Such colonies are very suitable for making impression preparations {vide p. 124) which preserve permanently the appearances described On examining such with a high power, the wreaths are seen to be made up of bundles of long filaments lying 298 ANTHRAX. parallel with one another, each filament consisting of a chain of bacilli lying end to end, and similar to those observed in the blood (Fig. 79). On gelatine plates, after from twenty-four to thirty-six hours at 200 C, the same ap- pearances manifest themselves, and later they are accompanied by liquefaction of the gelatine. In gelatine plates, however, instead Fig. 78.— -Surface colony of the anthrax of the characteristically bacillus on an agar plate, showing the wreathed appearance at characteristic appearances. x 30. the margin, the colonies sometimes give off radiating spikelets ir- regularly nodulated, which produce a star- like form. These spikelets are com- posed of spirally twisted threads. From such plates the bacilli can be easily isolated, and the appearances of pure cultures on various media studied. Appearances of Cultures. — In bouil- lon, after twenty-four hours' incubation at 370 C. there is usually the appearance of irregularly spiral pIG_ 79. —Anthrax bacilli, arranged in chains, from a twenty-four hours' culture on agar at 37 C. , , . Stained with fuchsin. x 1000. 1 APPEARANCES OF CULTURES. 299 threads suspended in the liquid. These, on being examined, are seen to be made up of bundles of parallel chains of bacilli. Later, growth is more abundant, and forms a floc- culent mass at the bottom of the fluid. In gelatine stab cultures, the charac- teristic appearance can be best observed when a low proportion, say 7 J percent, of gelatine is present, and when the tube is directly inoculated from anthrax blood. In about two days there radiate out into the medium from the needle track numberless very fine spikelets which enable the cultures to be easily recognised. These spikelets are longest at the upper part of the needle track (Fig. 80). Not much spread takes place on the surface of the gelatine, but here liquefaction commences, and gradually spreads down the stab and out into the medium, till the whole of the gelatine may be liquefied. Gelatine slope cultures exhibit a thick , felted growth, the edges of which show the wreathed appearance seen in plate cul- tures. Liquefaction here soon ploughs a trough in the surface of the medium. Sometimes "spiking" does not take place in gelatine stab cultures, only Fig. So. —Stab little round particles of growth occurring culture of the anthrax down the needle track, followed by liquefaction. As has been shown by Richard Muir, this property of spiking can be restored by growing the bacillus for twenty-four hours on blood agar at 37° C. Agar sipped cultures have the appearance of similar cultures in gelatine, though, of course no liquefaction takes place. Blood serum sloped cultures present the same appear bacillus 111 peptone- gelatine ; seven days' growth. It shows the " spiking " and also, at the surface, com- mencing liquefaction. Natural size. 300 ANTHRAX. ances as those on agar. The margin of the surface growth on any of the solid media shows the characteristic wreathing seen in plate colonies. On potatoes there occurs a thick felted white mass of bacilli showing no special characters. Such a growth, however, is useful for studying sporulation. The anthrax bacillus will thus grow readily on any of the ordinary media. It can usually be sufficiently recog- nised by its microscopic appearance, by its growth on agar or gelatine plates, and by its growth in gelatine stab cul- tures. The growth on plates is specially characteristic, and is simulated by no other pathogenic organism. Among the non-pathogenic bacteria the only organism which has ; similar colonies is the bacillus figurans, and the resemblance is only a distant one. The Biology of the B. Anthracis. — Koch found that the bacillus anthracis grows best at a temperature of 35°oC-| Growth, i.e., multiplication, does not take place below 120 C. or above 450 C. In the spore-free condition the bacilli have comparatively low powers of resistance. They do not j stand long exposure to 6o° C, and if kept at ordinary tem- perature in the dry condition they are usually found to be dead after a few days. The action of the gastric juice is rapidly fatal to them, and they are accordingly destroyed in the stomachs of healthy animals. They are also soon killed in the process of putrefaction. They can, however, be cooled below the freezing-point without dying. The bacillus can grow without oxygen, but some of its vital functions are best carried on in the presence of this gas. Thus in anthrax cultures the liquefaction of gelatine always commences at the surface and spreads downwards. Growth is more rapid in the presence of oxygen, and spore forma- tion does not occur in its absence. The organism may be classed as a facultative anaerobe. Sporulation.— Under certain circumstances sporulation occurs in anthrax bacilli. The morphological appearances are of the ordinary kind. A little highly-refractile speck appears in the protoplasm about the centre of the bacillus; SPOKULATION. 301 body the from this gradually increases in size until it forms an oval about the same thickness as the bacillus lying bacillary protoplasm (Fig. 81). The latter gradually loses its staining capacities and finally disappears. The spore thus lies free as an oval highly- refractile body which does not stain by ordinary methods, but which can be easily stained by the special methods described for such a purpose (p. 114). When the spore is again about to as- sume the bacillary form the capsule is apparently absorbed, and the protoplasm within grows out, taking on the ordinary rod-shaped form. According to most observers sporulation never occurs within the body of an animal suffering from anthrax. Koch attributes this, probably rightly, to the absence of free oxygen. The latter gas he found necessary to the occur- rence of spores in cultures outside the body. Many, how- ever, are inclined to assign as the cause of sporulation the absence of the optimum pabulum, which in the case of anthrax is afforded by the animal tissues. Besides these conditions there is another factor necessary to sporulation, viz. a suitable temperature. The optimum temperature for spore production is 320 C. Koch found that spore-forma- tion did not occur below 180 C. Above 420 C. not only does sporulation cease, but Pasteur found that if bacilli were kept at this temperature for eight days they did not regain the capacity when again grown at a lower tempera- Fig. 81. — Anthrax bacilli spores (the darkly coloured bodies) ; a three days' culture on agar at 370 C. Stained with carbol-fuchsin and methylene- blue. x 1000. 3°2 ANTHRAX. ture. In order to make them again capable of sporing it is necessary to adopt special measures, such as passage through the bodies of a series of susceptible animals. Anthrax spores have extremely high powers of resistance. In a dry condition they will remain viable for a year or more. Koch found they resisted boiling for five minutes ; and dry heat at 1400 C. must be applied for several hours to kill them with certainty. Unlike the bacilli, they can resist the action of the gastric juice for a long period of time. They are often used as test objects by which the action of germicides is judged. For this purpose an emulsion is made by scrap- ing off a surface culture and rubbing it up in a little sterile water. Into this sterile silk threads are dipped, which, after being dried over strong sulphuric acid in a dessicator, can be kept for long periods of time in an unchanged condition. For use they are placed in the germicidal solution for the desired time, then washed with water to remove the last traces of the reagent and laid on the surface of agar or placed in bouillon, in order that if death has not occurred growth may be observed. Anthrax in Animals. — Anthrax occurs from time to time epidemically in sheep, cattle, and, more rarely, in horses and deer. These epidemics are found in various parts of the world, although they are naturally most far- reaching where legal precautions to prevent the spread of infection are non-existent. All the countries of Europe are from time to time visited by the disease, but in some it is much more common than in others. In Britain the death- rate is small, but in France the annual mortality among sheep was probably 10 per cent of the total number in the country, and among cattle 5 per cent. These figures, how- ever, have been largely modified by the system of preven- tive treatment which will be presently described. In sheep and cattle the disease is specially virulent. An animal may suddenly drop down, with symptoms of collapse, quickening of pulse and respiration, and dyspnoea, and death may occur in a few minutes. In less acute cases the animal is appai- ently out of sorts, and does not feed ; its pulse and respira- NATURAL ANTHRAX IN ANIMALS. 303 tion are quickened ; rigors occur, succeeded by high temperature ; there is a sanguineous discharge from the bowels, and bloody mucus may be observed about the mouth and nose. There may be convulsive movements, there is progressive weakness, with cyanosis, death occurring in from twelve to forty-eight hours. In the more prolonged cases widespread oedema and extensive enlargement of lymphatic glands are marked features ; and in the glands, especially about the neck, actual necrosis with ulceration may occur, constituting the so-called anthrax carbuncles. Such subacute conditions are especially found among horses, which are by nature not so susceptible to the disease as cattle and sheep. On post-mortem examination of an ox dead of anthrax, the most noticeable feature — one which has given the name " splenic fever " to the disease — is the enlargement of the spleen, which may be two or three times its natural size. It is of dark-red colour, and on section the pulp is very soft and friable, sometimes almost diffluent. A cover-glass preparation may be made from the spleen and stained with watery methylene-blue. On examination it will be found to contain enormous numbers of bacilli mixed with red corpuscles and leucocytes, chiefly lymphocytes and the large uninucleated variety (Fig. 82). Pieces of the organ may be hardened in absolute alcohol, and sections cut in paraffin. These are best stained by Gram's method. Micro- scopic examination of such shows that the structure of the pulp is considerably disintegrated, whilst the bacilli swarm throughout the organ, lying irregularly amongst the cellular elements. The liver is enlarged and congested, and may be in a state of acute cloudy swelling. The bacilli are present in the capillaries throughout the organ, but are not so numerous as in the spleen. The kidney is in a similar condition, and here the bacilli are chiefly found in the capillaries of the glomeruli, which often appear as if injected with them The lungs are congested and may show catarrh, whilst bacilli are present in large numbers throughout the capillaries, and may also be found in the air cells, probably 3°4 ANTHRAX. as the result of rupture of the capillaries. The blood throughout the body is usually fluid and of dark colour. The lymphatic system generally is much affected. The glands, especially the mediastinal, mesenteric, and cervical glands, are enlarged and surrounded by cedematous tissue, the lymphatic vessels are swollen, and both glands and PlG 82 —Scraping from spleen of guinea-pig dead of anthrax, showing the bacilli mixed with leucocytes, etc. (Same appearance as in the ox.) 1 ' Corrosive film " stained with carbol-thionm-blue. x 1000. vessels may contain numberless bacilli. The heart may be in a state of cloudy swelling, and the blood in its cavities contains bacilli, though in smaller numbers than that in the capillaries. The intestines are enormously congested, the epithelium more or less desquamated, and the lumen filled with a bloody fluid. From all the organs the bacilli can be easily isolated by stroke cultures on agar. EXPERIMENTAL ANTHRAX. 305 It is important to note the existence of great differences in susceptibility to anthrax in different species of animals. Thus the ox, sheep (except those of Algeria, which only succumb to enormous doses of the bacilli), guinea-pig, and mouse are all very susceptible, the rabbit slightly less so. The last three are of course most used for experimental inoculation. We have no data to determine whether the disease occurs among these in the wild state. Less sus- ceptible than this group are the horse, deer, goat, in which the disease occurs from time to time in nature, as it also does, though rarely, in the pig. The human subject may be placed next in order of susceptibility, man thus occupy- ing a medium position between the highly susceptible and the relatively immune animals. The white rat is highly immune to the disease, while the brown rat is susceptible. Adult carnivora are also very immune, and the birds and amphibia are in the same position. With these differences in susceptibility there are also great variations in the pathological effects produced in the natural or artificial disease. This is especially the case when we consider the distribution of the bacilli in the bodies of the less susceptible animals. Instead of the widespread occurrence described above, they may be con- fined to the point where they first gained access to the body and the lymphatic system in relation to it, or may be only very sparsely scattered in organs such as the spleen (which is often not enlarged), the lungs, or kidneys. Never- theless the cellular structure of the organs even in such a case may show changes, a fact which is important when we consider the essential pathology of the disease. Experimental Inoculation.— Of the animals commonly used m laboratory work, white mice and guinea-pigs are the most susceptible to anthrax, and are generally used for test inoculations. If a small quantity of anthrax bacilli t>e injected into the subcutaneous tissue of a guinea-pig a fatal result follows, usually within two days. Post mortem around the site of inoculation the tissues, owing to intense inflammatory oedema, are swollen and gelatinous in appear- 20 3o6 ANTHRAX. ance, small haemorrhages are often present, and on micro- scopic examination numerous bacilli are seen. The internal organs show congestion and cloudy swelling, with some- times small haemorrhages, and their capillaries contain enormous numbers of bacilli, as has already been described k f+.-A- FlG 8. - Portion of kidney of a guinea-pig dead of anthrax, showing the bacilli in the capillaries, especially of die glomerulus. Paraffin section ; stained by Gram's method and Bismarck - browd x 300. in the case of the ox (Fig. 83) ; the spleen also shows a corresponding condition. Highly susceptible animals may be infected by being made to inhale the bacilli or ther spores and also by being fed with spores, a general infec tion rapidly occurring by both methods. Anthrax in the Human Subject.-As we have noted man occupies a middle position in the scale of suscept ANTHRAX IN MAN. 307 bility to anthrax. It is always communicated to him from animals, and usually is seen among those whose trade leads them to handle the carcases or skins of animals which have died of the disease. It occurs in two principal forms, the main difference between which is the site of entrance of the organism into the body. In one, the path of entrance is through cuts or abrasions in the skin, or through the hair follicles. A local condition called a " malignant pustule " develops, which may lead to a general infection. This variety occurs chiefly among butchers and those who work among hides (foreign ones especially). In Britain the workers of the latter class chiefly liable are the hide-porters and hide-workers in south-eastern London. In the other variety of the disease, the site of infection is the trachea and bronchi, and here a fatal result almost always follows. The cause is the inhalation of dust or threads from wool which has been taken from sheep dead of the disease, and which has been contaminated with blood or secretions con- taining the bacilli, these having afterwards formed spores. From the fact that this variety occurs in the centres of the wool-stapling trade (in England, chiefly in Yorkshire), it is called " woolsorter's disease." (1) Malignant Pustule.— -This usually occurs on the exposed surfaces— the face, hands, forearms, and back, the last being a common site among hide-porters. One to three days after inoculation a small red painful pimple appears, soon becoming a vesicle, which may contain clear or blood- stained fluid, and is rapidly surrounded by an area of intense congestion. Central necrosis occurs and leads to the malignant pustule proper, which in its typical form appears as a black eschar often surrounded by a ring of vesicles, these in turn being surrounded by a congested area. l«rom this pustule as a centre, subcutaneous oedema spreads especially in the direction of the lymphatics ; the neighbour- ing glands are enlarged. There is fever with general malaise. On microscopic section of the typical pustule the central eschar is noticed to be composed of necrosed tissue and degenerating blood cells; the vesicles are 3o8 ANTHRAX. formed by the raising of the stratum corneum from the rete Malpighi. Beneath them and in their neighbourhood the cells of the latter are swollen and cedematous, the papillae being enlarged and flattened out and infiltrated with inflammatory exudation, which also extends beneath the centre of the pustule. In the tissue next the eschar necrosis is commencing. The subcutaneous tissue is also cedematous, and often infiltrated with leucocytes. The bacilli exist in the periphery of the eschar and in the neighbouring lym- phatics, and, to a certain extent, in the vesicles. It is very important to note that widespread oedema of a limb, en- largement of neighbouring glands, and fever may occur while the bacilli are still confined to the immediate neigh- bourhood of the pustule. Sometimes the pathological process goes no further, the eschar becomes a scab, the inflammation subsides, and recovery takes place. In the majority of cases, however, if the pustule be not excised, the oedema spreads, invasion of the blood stream may occur, and the patient dies with, in a modified degree, the patho- logical changes detailed with regard to the acute disease in cattle. In man the spleen is usually not much enlarged,, and the organs generally contain few bacilli I he actua cause of death is therefore the absorption of toxines It may here be said that early excision of an anthrax pustule, especially when it is situated in the extremities, is followed, in a large proportion of cases, by recovery. (2 wllsorteSs Disease.-^ pathology of this affection was worked out in this country especial y by Greenfield The local lesion is usually situated in the lower part of he uachea or in the large bronchi, and is in the form patches in the mucous membrane often with ^°^af into then, The tissues are cedematous, and the eel uto elements are separated, but there is usual 1 tt le or no necrosis There is enormous enlargement of the mediastinal "nchial glands, and hemorrhagic mfiltration of the cellular tissue in the region. There are pleural and P «I cardial effusions, and hemorrhagic spots occur beneath ti| e cus membranes. The lungs show collapse and edema. TOXINES OF THE BACILLUS ANTHRA CIS. 309 There may be cutaneous cedema over the chest and neck, with enlargement of glands, and the patient rapidly dies with symptoms of pulmonary embarrassment, and with a varying degree of pyrexia. It is to be noted that in such cases, though numerous bacilli are present in the bronchial lesions, in the lymphatic glands, and affected tissues in the thorax, comparatively few may be present in the various organs, such as the kidney, spleen, etc., and sometimes it may be impossible to find any. (3) It is probable that infection occasionally takes place through the intestine ; but this condition is rare. In such cases there is a local lesion in the intestinal mucous mem- brane, of similar nature to that in the bronchial form, with a corresponding affection of the mesenteric glands. The Toxines of the Bacillus Anthracis. — Various theories were formerly held as to the mode in which the anthrax bacillus produces its effects. One of the earliest was the mechanical, according to which it was supposed that the serious results were produced by extensive block- ing of the capillaries in the various organs by the bacilli. According to another, it was supposed that the bacilli used up the oxygen of the blood, thus leading to starvation of the tissues. Though such modes of action may occur to a small extent, we now know that in anthrax, as in other diseases, the important local and general effects are pro- duced by specific poisons formed by the bacilli. We have therefore to consider the nature of these toxic bodies. During the years 1889-90 several papers were published dealing with the toxines of the bacillus anthracis. Hankin, investigating the means of conferring immunity against the disease, isolated from cultures in a bouillon made from Liebig's meat juice an albumose which he considered to be the toxine. His reason for thinking so was that, while the injection of very small doses of this substance (one five millionth to one ten millionth of the weight of an animal) lengthened the incubation period of the disease, and might even ward off a fatal attack, the injection of larger doses hastened the death of the animal. Very full researches on ;io ANTHRAX. the subject were carried out by Sidney Martin. This observer used alkali-albumin on which to grow the bacillus, this medium approaching most closely to the environ- ment of the latter when growing in the animal body. From cultures in this medium, concentrated by evapora- tion either at ioo° C. or in vacuo at 35° to 450 C, there were isolated proto-albumose, deutero - albumose, and traces of peptone. The albumoses differed from those which occur in ordinary digestion, in being strongly alkaline in their reaction. This alkalinity, Martin held, was due to traces of an alkaloidal body of which the albumoses were the precursors, and which were formed when the process of digestion of the alkali - albumin by the bacillus was allowed to go on further. By the albu- moses and the alkaloid pathogenic effects were pro- duced in animals, closely similar to those produced by the bacilli themselves. Martin adduced evidence to show that of the symptoms of the disease, the fever was mostly due' to the albumoses, while the oedema and congestion were mostly due to the alkaloid which acted as a local irritant He showed that prolonged boiling destroyed the activity of the albumoses, but not that of the alkaloid. Further, from the body fluids of animals dead of anthrax he isolated poisonous bodies identical with those produced bv the bacilli growing in this artificial medium. Hankin, in a later research with Wesbrook, arrived at the conclusion that the bacillus anthracis produces a ferment which dit- fusing out into the culture fluid, elaborates albumoses from the proteids present in it. The bacilli also produce albumoses directly without the intervention of a ferment. The albumoses produced in the latter way, when injected in small doses, cause in susceptible animals immunity against subsequent inoculation with virulent bacilli, but are only toxic to animals not very susceptible to the disease. Marnier, after cultivating the B. anthracis m peptone solu- tion containing certain salts, removed all the albumos from the resultant liquid, and from them, either by dialysis or extraction with glycerine, isolated a body, which gave no MODE OF SPREAD IN NATURE. reactions of albuminoid matter, peptone, propeptone, or alkaloid. This he considers the toxine. It killed animals susceptible to anthrax by a sort of cachexia, and in suitably small doses could be used to immunise them against sub- sequent inoculation with virulent bacilli. It was chiefly retained within the bacilli when these were growing in the most favourable conditions. Unlike the toxines of tetanus and diphtheria, and unlike ferments, it was not destroyed by heating to no0 C. The toxine produced by the B. anthracis growing in a fluid medium remains intimately associated with the bacterial protoplasm, as such cultures when filtered are relatively non-toxic. From this account of the researches into the toxines of the B. anthracis, it will be seen that our knowledge is far from complete. It is difficult to say what interpretation is to be put on the results of Hankin and Wesbrook. The researches of Marmier rather indicate that, as is the case with the toxines of other bacilli, the toxine of anthrax may belong to a group of non-proteid bodies of whose chemical nature we are in complete ignorance. Be this as it may, the results detailed open up a way for our arriving at an idea of the true pathology of the disease. The bacilli in all parts of the body, whether directly or intermediately by ferments, produce bodies toxic to tissue cells. Further, bacilli confined locally produce by this means effects on distant tissues. This explains how in certain cases, while the bacilli are still locally confined, there may occur oedema spreading from the pustule, and pyrexia. The Spread of the Disease in Nature. — We have seen that the B. anthracis rarely, if ever, forms spores in the body, and if the bacilli could be confined to the blood and tissues of the carcase, it is certain that anthrax in an epidemic form would rarely occur. For it has been shown by many observers that in the course of the putrefaction of such a carcase the anthrax bacilli rapidly die out, and that after ten days or a fortnight very few remain. But it must be remembered that while still alive, an animal is shedding into the air by the bloody excretions from the mouth, nose 312 ANTHRAX. and bowel, myriads of bacilli which may rapidly spore, and thus arrive at a very resistant stage. These lie on the surface of the ground and are washed off by surface water. At certain seasons of the year the temperature is, however, sufficiently high to permit of their germination, and also of their multiplication, as they can undoubtedly grow on the organic matter which occurs in nature. They can again form spores. It is in the condition of spores that they are dangerous to susceptible animals. In the bacillary stage, if swallowed, they will be killed by the acid gastric con- tents ; but as spores they can pass uninjured through the stomach, and, gaining an entrance into the intestine, infect its wall, and ultimately reach, and multiply in, the blood. It is known that in the great majority of cases of the disease in sheep and oxen, infection takes place thus from the intestine. It was thought by Pasteur that worms were active agents in the natural spread of the disease by bring- ing to the surface anthrax spores. Koch made direct experiments on this point, and could get no evidence that this was the case. He thinks it much more probable that the recrudescence of epidemics in fields where anthrax carcases have been buried, is due to persistence of spores on the surface which has been infected by the cattle when alive. The Disposal of the Carcases of Animals dead of Anthrax. -It is extremely important that anthrax carcases should be disposed of in such a way as to prevent their becoming future sources of infection. I f anthrax be suspected as the cause of death no post-mortem examination should be made, but only a small quantity of blood be removed from an auricular vein for bacteriological investigation. If such a carcase be now buried in a deep pit surrounded by quicklime, little danger of infection will be run. The bacilli being confined within the body will not spore, and will die during the process of putrefaction. The danger of sporu ation taking place is, of course, much greater when an animal has died of an unknown disease which on post-mortem examination has proved to be anthrax, but similar measures for burial must fad here adopted. In some countries anthrax carcases are burned and thTs! if practicable, is of course the best means of treating hem The chief source of clanger to cattle subsequently, however, proceeds Lm the infection of fiefds, yards, and byres with the offd and he discharge from the mouths of anthrax animals. All material that can 1. 1 [MUNIS A TION A GAINS T ANTHRA X. 313 be recognised as such should be burned along with the straw in which the animals have lain. The stalls or buildings in which the anthrax cases have been must be limewashed. Needless to say, the greatest care must be taken in the case of men who handle the animal or its carcase that they have no wounds on their persons, and that they thoroughly disinfect themselves by washing the hands, etc., in 1 to 1000 solution of corrosive sublimate, and that all clothes soiled with blood, etc., from anthrax animals be thoroughly boiled or steamed for half an hour before being washed. The Immunising of Animals against Anthrax. — Having ascertained that there was ground for believing that in cattle one attack of anthrax protected against a second, Pasteur (in the years 1880-82) elaborated a method by which a mild form of the disease could be given to animals, which rendered harmless a subsequent inoculation with virulent bacilli. He found that the continued growth of anthrax bacilli at 42 0 to 430 C. caused them to lose their capacity of producing spores, and also gradually to lose their virul- ence, so that after twenty-four days they could no longer kill either guinea-pigs, rabbits, or sheep. Such cultures constituted his pre?nier vaccin, and protected against the subsequent inoculation with bacilli which had been grown for twelve days at the same temperature, and the attenua- tion of which had therefore not been carried so far. The latter constituted the deuxihne vaccin. It was further found that sheep thus twice vaccinated now resisted inocu- lation with a culture which usually would be fatal. The method was to inoculate a sheep on the inner side of the thigh by the subcutaneous injection, from a hypodermic syringe, of about five drops of the premier vaccin ; twelve days later to again inoculate with the deuxihne vaccin; fourteen days later an ordinary virulent culture was injected without any ill result. This method was applicable also to cattle and horses, about double the dose of each vaccine being here necessary. Extended experiments in France generally confirmed earlier results, and the method was, before long, used to mitigate the disease, which in many departments was endemic and a very great scourge. Since that time the method has been regularly in use. It is 3i4 ANTHRAX. difficult to arrive at a certain conclusion as to its merits. Undoubtedly a certain number of animals die of anthrax cither after the first or second vaccination, or during the year following vaccination. At the end of a year the immunity is lost in about 40 per cent of the animals vac- cinated ; and thus to be permanently efficacious the pro would have to be repeated every year. Further, the immunity is much higher in degree if, after the first and second vaccinations, an inoculation with virulent anthrax is performed. Everything being taken into account, however, there is no doubt that the mortality from natural anthrax is much diminished by this system. Statistics are available for the twelve years 1882-93. During that time 3,296,815 sheep were vaccinated, with a mortality, either after the first or second vaccination or during the subsequent twelve months, of .94 per cent, as contrasted with the ordinary mortality in all the flocks of the districts, of 10 per cent. During the same time 438,824 cattle were vaccinated, with a mortality of . 34 per cent, as contrasted with a probable mortality of 5 per cent if they had been unprotected. Other means of immunising animals against anthrax have been elaborated, but these have a more strictly scientific interest. In dealing with the toxines of anthrax we have already referred to the work of Hankin and: Wesbrook on this point. We have also seen that Marmier succeeded in immunising animals by using a toxine isolated by him. Even, however, as a method of immunising animals for scientific observations Pasteur's method still obtains. Serum Anticharbo-nneux.— The properties of the serum of animals vaccinated against anthrax have been investigated by Marchoux. The animals were immunised in the usual way The serum of sheep and especially of rabbits was found to afford a certain degree of protection to susceptible animals against subsequent inoculation with virulent baculfl It also exhibited a small degree of curative action. Wnefl it was injected immediately after inoculation with the bacdli a certain number of the animals survived, but m propor- ME THODS OF EXAM1NA TION. 315 tion as the symptoms of the disease (oedema, fever, etc.) were established, so was the curative effect diminished, even though large doses of the serum were employed. These results have been in the main confirmed by other observers. Methods of Examination. — These include (a) micro- scopic examination ; (l?) the making of cultures ; and (c) test inoculations. (a) Microscopic Examination. — In a case of suspected malignant pustule, film preparations should be made from the fluid in the vesicles or from a scraping of the incised or excised pustule, and stained with a watery solution of methylene -blue and also by Gram's method. By this method practically conclusive evidence may be obtained ; but sometimes the result is doubtful, as the bacilli may be very few in number. In all cases confirmatory evidence should be obtained by culture. Occasionally they are so scanty that both film preparations made from different parts and even cultures may give negative results, and yet a few bacilli may be found when a section of the pustule is examined. It should be noted that the greatest care ought to be taken in handling the part, as otherwise the diffusion of the bacilli into the surrounding tissues may be aided and the condition greatly aggravated. The examina- tion of the blood in cases of anthrax in man usually gives negative results, with the exception of very severe cases, when a few bacilli may be found in the blood shortly before death, though even then they may be absent. (b) Cultivation. — A small quantity of the material used for microscopic examination should be taken on a platinum needle, and successive strokes made on agar tubes, which are then incubated at 370 C. At the end of twenty-four hours anthrax colonies will appear, and can be readily recognised from their wavy margins, by means of a hand lens. They should also be examined microscopically .by means of film preparations. (c) Test Inoculations. — A little of the suspected material should be mixed with some sterile bouillon or water, and 3i6 ANTHRAX. injected subeutaneously into a guinea-pig, or it may be introduced into the subcutaneous tissue by means of a seton. If anthrax bacilli are present, the animal usually dies within two days, with the changes in internal organs already described. CHAPTER XIV. TYPHOID FEVER— BACILLI ALLIED TO THE TYPHOID BACILLUS. OTHER NAMES. ENTERIC FEVER: GASTRIC FEVER. GER- MAN, TYPHUS ABDOMINAL1S : ABDOMINALTYPHUS I UNTERLEIBSTYPHUS. FRENCH, LA FIEVRE TYPHOIDE. Historical Summary. — During the early part of the bacteriological era many observers had described various micrococci and bacilli, and even higher forms, as occurring in the neighbourhood of the typhoid ulcers and in the tissues generally, but the first definite descriptions of what is now known as the bacillus typhosus appeared about 1880-8 1, when the papers of Eberth, Koch, and Klebs were published. On account of priority of publication and of the general confirmation of his observations by later observers, the credit of the discovery is generally assigned to Eberth, and the bacillus typhosus is often called Eberth's bacillus. This worker investigated in all forty cases of the disease, and in twenty-two he found microscopically what he considered to be characteristic bacilli in the intestinal ulcers, in the spleen, and in the lymphatic glands. These are now identified with the bacillus typhosus. He, however, made no attempts to grow them outside the body. This important step was taken by Gaffky (1884), who confirmed Eberth's observations on the occurrence of the bacilli in 3«8 TYPHOID / / / / A\ the organs of typhoid cases, and succeeded in obtaining from the spleen pure cultures in gelatine. He further described very fully the morphological character of the bacilli both in cultures and when occurring in the body. He held that the bacilli were not putrefactive, as they did not produce putrefactive effects on artificial media ; but all his attempts to reproduce by their means the disease in different species of animals (including monkeys) were unsuccessful. The position, therefore, was that in the great majority of cases of typhoid fever, characteristic bacilli could be found and isolated in pure culture, but that these did not give rise to the disease in animals. During the years succeeding the publication of the work of Eberth, Koch, and Gaffky, the results of these investigators were confirmed so far as they went, but ljttle further advance was made. In 1885, Escherich, working on the first appearance of organisms in the bowel of the new-born infant, described a bacillus which he named the bacillus coll communis (often subsequently named the bacterium coli commune and also Escherich's bacillus). This also was shown to be identical with the bacillus neapolitanus which Emmerich found in the intestines of the victims of a cholera epidemic at Naples. Weisser, who worked at the subject, pointed out that the B. coli was a normal inhabitant of the human intestine ; and, further, comparing the growth characters of this bacillus with those of the typhoid bacillus, noted the similarities which exist between the two microbes. Doubt was thus cast on the causal relationship of Eberth's bacillus to typhoid fever. From this time forward, the question of the morphological relation- ships of the two organisms has played an important part in the bacterio- logical investigation of the subject. There has been much controversy as to whether they are varieties of the same species, and also as to whether, in view of the fact that the B. coli is a normal inhabitant of the human intestine, the B. typhosus may not originate de novo from it in every case. The result, however, is a growing conviction that for an unknown time, at any rate, the two have been distinct species. The Bacillus Typhosus. — Microscopic Appearances. Most observers will agree with Gaffky in attributing any failure to find typhoid bacilli in the organs of a typhoid patient to the difficulties of the search. Numerous sections of different parts of a spleen, for example, may be examined before a characteristic group is found. The best tissues for BACILLUS TYPHOSUS. 319 examination are a Peyer's patch where ulceration has not yet commenced or where it is just commencing, the spleen, the liver, or a mesenteric gland. The spleen and liver are better than the other tissues named, as in the latter the presence of the B. coli is more frequent. From scrapings of such solid organs dried films may be prepared and stained for a few minutes in the cold by any of the strong staining solutions, - I; with carbol- thion in-blue, or with Ziehl-Neelsen's car- bol-fuchsin diluted with five parts of distilled water. As a rule, decolorising f or is not necessary. For the proper obser- vation of the arrange- ment of the bacilli in the tissues, paraffin sections should be prepared and stained in carbol - thionin- blue for a few min- utes, or in Loffler's Fig. 84. — A specially large clump of ty- phoid bacilli in a spleen. The individual bacilli are only seen at the periphery of the mass. (In this spleen, enormous numbers of typhoid bacilli were shown by cultures to be present in a practically pure condition.) Paraffin section ; stained with carbol-thionin- blue. x 500. methylene -blue for one or two hours. The bacilli take up the stain somewhat slowly, and as they are also easily decolorised, the aniline oil method of dehydration may be used with advantage {vide p. 1 07). In such preparations the characteristic appearance to be looked for is the occurrence' of groups of bacilli lying between the cells of the tissue (Fig. 84). The individual bacilli are 2 ,x to 4 p. long, with somewhat oval ends, and 5 a in thickness. Sometimes filaments 8 fi to 10 ^ long may be observed, though they are less common than in cultures It is evident that one of the short oval forms may frequently 320 TYPHOID FEVER. in a section be viewed endwise, in which case the appear- ance will be circular. This appearance accounts for some, at least, of the coccus-like forms which have been described. The bacilli are decolorised by ('.rani's method. Isolation and Appearances of Cultures. — To grow the organism artificially it is best to isolate it from the spleen, as it exists there in greater' numbers than in the other solid organs, and may be the sole organism present even some time after death. The spleen is removed whole, and a portion of its capsule is seared with a cautery to destroy all superficial contami- nating organisms. A small incision is made into the organ with a sterile knife, a little of the pulp removed by a platinum needle, and agar or gelatine plates are prepared, or successive strokes made on agar tubes. On the agar media the growths are visible after twenty- four hours' incuba- tion at 370 C. On agar plates the superficial colonies appear as circular spots, dull white by reflected light, bluish-gray by transmitted light. Colonies in the substance of the agar are small, and appear as minute round points. When viewed under a low objective, the surface colonies are found to be very transparent (requiring a small diaphragm for their definition), finely granular in appearance, and with a very coarsely crenated and well-defined margin. The deep colonies are usually spherical, sometimes lenticular in shape, and are smooth or finely granular on the surface, and more opaque Fig. 85. — Typhoid bacilli; from a young culture on agar, showing some filamentous forms. Stained with weak carbol-fuchsin. x 1000. MORPHOLOGICAL CHARACTERS. 321 than the superficial colonies. On making cover-glass preparations, the bacilli are found to present the same microscopic appearances as are observed in preparations from solid organs, except that there may be a greater number of the longer forms which may almost be called filaments (Fig. 85). The same is true of films made from young gelatine colonies. Sometimes the diversity in the length of the bacilli is such as to throw doubts on the purity of the culture. Its purity of course can be readily tested by preparing plates from it in the usual way. As a general rule in a young (twenty-four to forty-eight hours old) colony, grown at a uniform tempera- ture, the bacilli are plump, and the protoplasm stains uniformly. In old cultures or in cultures which • have been exposed to change of temperature, the protoplasm stains only in parts ; there may be an appearance of vacuolation either at the centre or at the ends of the bacilli, or a bacillus may resemble a string of irregular coccus-like bodies. It is these appearances which have led some to believe that the typhoid bacillus forms spores. Gaffky described the latter as highly refractile bodies occurring at the ends of the bacillus ; and others have thought that the coccus-like bodies are spores. Cultures containing either have, however, been found to be not more highly resistant than those containing ordinary bacilli ; further, the staining reactions of such bodies are not those of spores, so that now it is generally believed that spore-formation does not occur in the typhoid bacillus. Motility ---In hanging-drop preparations the bacilli are found to be actively motile. The smaller forms have a darting or rolling motion, passing quickly across the field whilst some show rapid rotatory motion. The filamentous forms have an undulating or serpentine motion, and move more slowly. Hanging-drop preparations ought to be made from agar or broth cultures not more than twenty- four hours old. In older -cultures the movements are less Flagdla.—On being stained by the appropriate methods 322 TYPHOID FEVER. {vide p. 1 1 6) the bacilli are seen to possess many long wavy flagella which are attached all along the sides and to the ends (Fig. 86). They are more numerous, longer, and more wavy than those of the B. coli. Characters of Cultures. — Stab cultures in peptone-gelatine give a somewhat characteristic appearance. On the surface FlG. 86. —Typhoid bacilli, from a young culture on agar, showin flagella. " Stained by Van Ermengem s method. x iooo. of the medium, growth spreads outwards from the puncture as a thin film or pellicle, with irregularly wavy margin (Fig 87 A). It is semi-transparent, and of bluish-wnite colour ' Ultimately this surface growth may reach the wall of the tube. Not infrequently, however, the surface growtn is not well marked. Along the stab there is an opaque whitish line of growth, of finely nodose appearance. 1 here APPEARANCES OE CULTURES. 323 is no liquefaction of the medium, and no formation of gas. In stroke cultures there is a thin bluish-white film, but it does not spread to such an extent as in the case of the surface growth of a stab culture (Fig. 87, B). In gelatine plates also the superficial and deep colonies present corre- Fig. 87. • A. Stab culture of the typhoid bacillus in gelatine, five days' growth B. Stroke culture of the typhoid bacillus on gelatine, six days' growth. S?fb,cuIture °f the bacillus coh in gelatine, nine days' growth ; the eelatine is split in its lower part owing to the formation of gas. ' gelatlne ls sponding differences. The former are delicate semi-trans- parent films, with wavy margin, and are much larger than the colonies in the substance, which appear as small round points. These appearances, which are well seen on the third or fourth day, resemble those seen in agar plates, as already described in the method of isolation ; but on gela- 324 TYPHOID FEVER. tine the surface colonies are rather more transparent than those on agar. Their characters, as seen under a low power of the microscope, also correspond. In stroke cultures on agar there is a bluish-grey film of growth, with fairly regular margins, but without any char- acteristic features. This film is loosely attached to the surface, and can be easily scraped off. The growth on potatoes is most important. For several days (at ordinary temperature) after inoculation there is apparently no growth. If looked at obliquely, the surface appears wet, and if the surface is scraped with the platinum loop a glistening track is left ; a cover-glass preparation shows numerous bacilli. Later, however, a slight pellicle with a dull, somewhat velvety surface, may appear, and this may even assume a brown appearance. These characterise appearances are only seen when a fresh potato with an aci reaction has been used. In bouillon incubated at 37 C. for twenty-four hours, there is simply a uniform turbidity. Cover-glass prepara- tions made from such sometimes show filamentous forms of considerable length, without apparent segmentation. Conditions of Growth, «*.-The optimum temperature of the typhoid bacillus is about 37° C, though it alsol flourishes well at the room temperature. It will not grow bdow o° C. or above 4*° C. Growth takes place in aerobic as well as in aerobic conditions. Its powers ^resistance correspond with those of most non-spor,ng bacteria. It is killed by exposure for half jnto-J C or for two or three minutes at 1 00 C. 1 yphoid bacilli kept in distilled or in ordinary, tap water have usually been found to be dead after three weeks (Frankland). f°UTdhe Bacillus Coli Communis. -This bacillus , » ; the chj nrcnnism present in the small intestine in normal conditions, and 4Tmany other bacteria, it also inhabits the large tnte'stm During typhoid fever, and other patholog.cd conditions affecting the intestines, it is relatively and absi u^renormously increased in the latter situate, where may sometimes be almost the only bacillus present. Its BACILLUS CO LI COMMUNIS. 325 relations to various suppurative and inflammatory conditions are described in the chapter on Suppuration (p. 179). Microscopically it has the same appearances and staining reaction as the typhoid bacillus, and like the latter also presents variations in size, though it is usually somewhat shorter (Fig. 88). It is motile, and possesses lateral flagella, which, however, are fewer in number and some- what shorter than those of the typhoid bacillus. It is easily isolated from the stools of men and animals by any of the ordinary methods. After, e.g., twenty- • four hours' incubation at 37° C. on agar, there are large super- ficial colonies and small deep colonies in the plates. To the naked eye they are denser and more glistening than those of typhoid when viewed by transmitted Film Fig. 88. — Bacillus coli communis, light, and rather of PreParation from a young culture on agar. „ l • , , . , Stained with weak carbol-fuchsin. x 10 a brownish-white colour. Under a low objective the colonies again appear denser than those of the typhoid bacillus and more granular. On ordinary gelatine and agar media the appearances are similar to those of the typhoid bacillus, but the growth is whiter, thicker, and more opaque, and gives the impres- sion of having greater vigour. In the case of gelatine stab cultures, a few gas bubbles sometimes develop in the medium (Fig. 87, C.) On potatoes in forty-eight hours there is a distinct film of growth of brownish tint and moist-lookmg surface, which rapidly spreads and becomes thicker. I his contrasts very markedly with the colourless film of the B. typhosus. 326 TYPHOID FEVER. The Comparative Culture Reactions of the B. typhosus and the B. coli. — The importance of the relationships between the B. typhosus and the B. coli has caused great j attention to be paid to their biological characters, in order | to facilitate the distinction of the one from the other. Some of these we have already noted. Of the morpho- j logical characters the growth on potatoes is the most important. As has been pointed out by Wathelet, and also by Klein, differences exist in the growth of the two bacilli in melted gelatine. A gelatine tube is inoculated, and, instead of being kept at the room temperature, is placed in the incubator at 370 C, at which temperature it is of course fluid. In such cultures, in the case of the j B. typhosus, there is a general turbidity of the gelatine, I while with the B. coli there are large flocculi developed which float on the surface. It is, however, to physiological differences between the bacilli, rather than to morphologi- cal, that importance is to be attached. Several important points are to be studied hereon. (1) The Fermentation of Sugars.— Chantemesse and Widal were the first to show that the B. coli produced an acid fermentation in lactose (milk sugar). Their method was as follows. To tubes of 2 per cent lactose bouillon about 1 gramme of sterilised calcium carbonate was added in each case, and the tubes were then sterilised. On inoculating such a tube with B. coli, the acid produced by the fermen- tation (chiefly lactic acid) acts on the calcium carbonate, setting free bubbles of carbon dioxide which collect on the surface of the liquid. The production of acid ir 1 lactose gelatine by the B. coli can also be observed by adding to tubes sufficient blue litmus to make the whole distinctly blue If a stab culture be made in such a tube, a reo colour diffuses out in the gelatine from the line of growth, and bubbles of gas also form. Later the medium .becomes decolorised by reduction of the litmus The ^ntf lactose or other sugars to a simple solution of peptone, however, gives more accurate results (p. 86). REACTIONS OF B. TYPHOSUS AND B. CO LI. 327 The fermentation of lactose by the B. coli may also be demonstrated by means of Petruschky's lil/nits-zohey. The preparation of this medium, which is somewhat difficult, is as follows. Fresh milk is slightly warmed, and sufficient very dilute hydrochloric acid is added to cause precipitation of the casein, which is now filtered off. Dilute sodium hydrate solution is added up to, but not beyond, the point of neutralisation, and the fluid steamed for one to two hours, by which procedure any casein which has been converted into acid albumin by the hydrochloric acid, is precipitated. This is filtered off, and a clear, colourless, perfectly neutral fluid should result. Its chief constituent, of course, will be lactose. To this, 5 per cent of a saturated alcoholic solution of litmus is added, the medium is put into tubes and then sterilised. After growth has taken place, the amount of acid found can be estimated by dropping in standardised soda solution, till the tint of an uninoculated tube is reached. The fermentation of sugars is a very important effect of the growth of the B. coli. In a culture on a medium equally rich in lactose, for example, and peptone, the former will be broken up and the latter be left practically un- affected. According to the first results of Chantemesse and Widal, the B. typhosus did not ferment lactose. Petruschky, however, states that it can do so in litmus whey. Much seems to depend upon what other constituents are present in the medium. Pere has confirmed the earlier view, but finds that the typhoid bacillus, though it cannot ferment cane sugar or lactose, can originate such a change in arabinose, galactose, levulose, and glucose. The fer- mentative power of the typhoid bacillus is thus, though existent, much less. active than that of the B. coli; and as a matter of practical experience the formation of bubbles of gas in Chantemesse and Widal's lactose medium is rarely observed. The test? may, therefore, be taken in conjunction with others, as of use in diagnosing the identity of the bacillus. Curdling of Milk by the B. Coll.— This probably depends on the fermentation of the lactose of the milk, and the throwing down of the casein by the resulting lactic acid ; but the action may be a more complicated one, as milk can be curdled by organisms which do not possess acid- forming properties. 32« TYPHOID FEVER. Formation of Acids in Ordinary Media. — If ordinary litmus bouillon or gelatine be inoculated with the B, typhosus or the B. coli, a production of acid will be j observed during the early period of growth, but the acid reaction is more quickly produced by the B. coli. With such media Pere found that in the case of both microbes there I was for forty-eight hours a production of acid. At the end of five days, however, typhoid cultures were alkaline, and in cultures of B. coli the acidity, though present, was diminished. Ordinary media contain sugars derived from the meat from which they are made, and the acidity might proceed from the fermentation of these. In media made with pure syntonin or peptone, though there was an initial slight acid formation, especially with the B. coli, still in the case of both organisms at the end of four days the reaction was alkaline. The reaction is, therefore, probably a double one, but the resulting acidity in ordinary cases may be due to fermentative changes in carbohydrates. * Here again the acid-forming capacities of the B. typhosus are inferior I to those of the B. coli. (2) Production of Gas by the B. coli. — The production 1 of gas in various media by the B. coli can be demonstrated by any of the methods described (p. 86). Shake cultures I are usually employed. According to Klein the gas pro- duced is methane. We have found, however, that in a shake culture in peptone solution with 10 per cent gelatine added the B. coli produces no gas, but bubbles rapidly form if the medium has added to it a trace of lactose. No such development of gas occurs in a shake culture of typhoid in any of these media. (3) Formation of IndoL— Among the bacteria capable of forming indol is to be classed the B. coli. Indol can be recognised in bouillon cultures of the B. coli three to four days old by the usual tests {vide p. 87). As there is no evidence that it can produce nitrites, a small quantity of the latter must be added. The typhoid bacillus never gives this reaction when growing in ordinary conditions, but on the other hand, it appears that some varieties of the B coli fail to produce it also. Peckham, however, has shown that if the typhoid bacillus be grown in peptone solution, after a few generations of three days each it may RE A C 7 'IONS OF B. TYPHOSUS AND B. COLL 329 acquire the property of producing indol. The formation of indol by an organism after the first transference to peptone solution from one of the ordinary media may, however, be accepted as evidence in favour of the organism not being the typhoid bacillus. It is to be noted here that the presence of lactose in a medium prevents the production of indol by the B. coli. The indol reaction thus ought to be sought for in a sugar-free medium. (4) Growth on Phenolated Gelatine It was at one time thought that gelatine with .2 per cent carbolic acid added inhibited the growth of all bacteria but the typhoid bacillus. It has been found, however, that the growth of the B. coli is also unaffected by such a medium, though it prevents the growth of most putrefactive organisms which liquefy gelatine. (5) The Application of the Agglutination Test in Dis- tinguishing B. typhosus from B. coli.— The scope of the application of this test will be discussed later {see Im- munity). Here we may say that a negative result obtained with a suspected B. typhosus culture is of greater value than a positive result obtained with a suspected B. coli culture. It is only to be taken in conjunction with the other means of differentiating the two organisms, and is not strictly a crucial test. It will thus be seen that the diagnosis between the B typhosus and the B. coli is a matter of no small difficulty I he points to be attended to in making such a diagnosis are given in the accompanying table. There is no evidence that the one organism ever passes into the other. Klein nas found that both after prolonged sojourn in distilled and tap water, and also after passage through the bodies of a series of animals, each organism still preserves its original characters Statements as to their identity usually rest on theoretical considerations, or on purely negative evidence. U-ea difficulties sometimes arise in consequence of a bacillus being found which, while giving a number of the characteristics of either one or the other, fails to give some o he characteristic tests, or only gives them very slowly J his is especially true of organisms related to the B coli 330 I V I'll 01 D FEVER. It has consequently become common to speak of the typhoid group and the coli group in order that such varieties may be included. In the coli group cases may be met with which do not give an indol reaction, which do not curdle milk, or which do not produce gas, and Gordon even includes varieties producing alkali, or slowly liquefying gelatine. Two of the most important varieties, the bacillus enteritidis (Gaertner) and the bacillus of psitta- cosis, are described below. Methods of the Diagnosis of the Typhoid Bacillus. The Differences between the B. typhosus and the B. coli: — B. Typhosus. Flagella more numerous, longer, and more wavy. In artificial media growth gener- ally slower and not so vigorous. Growth on fresh acid potatoes, a nearly transparent film. Very slight acid production in ordinary media, followed sometimes by production of alkali. Fermentation of lactose very slight if any. Milk not coagulated. Gelatine shake cultures— no gas formation. Production of indol in ordinary bouillon — nil. Agglutination. Bacilli become 'clumped together and motion- less in the serum of a typhoid patient. (A similar reaction is given by the blood serum of an animal immunised against the typhoid bacillus.) B. Colt. Flagella fewer and shorter. Growth faster and more vigorous. Growth on potatoes, a brownish pellicle. Well-marked acid production. Fermentation pronounced. Milk coagulated. Abundant gas formation round colonies. Well-marked indol production. In most cases the bacilli remain actively motile, but sometimes clumping occurs. Bacillus Enteritidis (Gaertner). -In 1888 Gaertner in invest* number of cases of illness resulting from eatmg the fle.h of a gating a ALLIED BACILLI. 33i diseased cow, isolated, not only from the meat but from the spleen of a man who died, a bacillus which presents all the characteristics of the B. typhosus except that it ferments lactose and is very pathogenic to animals. In the latter, whatever the method of introduction, there is an intense hemorrhagic enteritis with swelling of the lymph follicles. The distribution of the bacilli varies in different cases, but usually they are present in the solid organs. In man also the symptoms are centred in the intestine, and hence the name given to the bacillus. During recovery a very characteristic point is the occurrence of desquamation. Since it was described by Gaertner others have isolated the bacillus under similar circumstances. Its toxic products have been found to be very pathogenic to animals, and in man cases of illness have occurred when broth made from the diseased flesh has been partaken of. When there is an infection by the bacillus itself being present, symptoms usually begin after twenty-four hours. Many cases, however, of an earlier illness have occurred, no doubt due to the action of toxines already existing in the meat. During the last few years, in some epidemics of meat- poisoning, similar bacilli differing slightly from Gaertner's bacillus have been isolated, e.g. by Durham, and it is probable that here also we have to do not with one variety but with a group of bacilli. The Psittacosis Bacillus. — When parrots are imported from the tropics in large numbers many die of a septicemic condition in which an enteritis, it may be hemorrhagic, is a marked feature. There is intense congestion of all the organs and peritoneal ecchymoses. From the spleen, bone marrow, and blood there has been isolated a short actively motile bacillus with rounded ends which does not stain by Gram's method. It grows on all ordinary media, and on potato resembles B. coli. It does not liquefy gelatine, does not ferment lactose, does not curdle milk and gives no indol reaction. The parrot is most susceptible to its action, but it also causes a fatal hemorrhagic septicemia in guinea-pigs, rabbits, mice, pigeons and fowls, the bacilli after death being chiefly in the solid organs. From affected parrots the disease appears to be readily communicable to man, chiefly, it is probable, from the feathers being soiled by infective excrement. Several small epidemics have been recognised and investigated in Pans. After about ten days' incubation, headache, fever, anorexia occur, followed by great restlessness, delirium, vomiting, often diarrhoea, and albuminuria. Frequently broncho -pneumonia supervenes and a fatal result has followed in about a third of the cases observed. The organism has been isolated from the blood of the heart. The psittacosis bacillus is evidently one of the typhoid group, a fact which is further borne out by the observation that it is clumped by a typhoid serum— 1 ■ 10 (normal serum having no result). The clumping is, however, said' to be incomplete, as the bacilli between the clumps may retain their motility. It differs from the typhoid bacillus in its growth on potatoes and in its pathogenicity. 332 TYPHOID FEVER. Pathological Changes in Typhoid Fever. — As these are sufficiently described in ordinary pathological text- books, we can confine our attention solely to their bacterio- logical aspects. It is generally recognised that the inflam- mation and ulceration in the Peyers patches and solitary glands of the intestine are the central features of the disease. In the early stage these have a swollen and slightly pinkish appearance, and microscopically it is seen that there has been produced an acute inflammatory condition, attended with extensive leucocytic emigration ; sometimes small haemorrhages may be observed. It is at this period that the typhoid bacilli are most numerous in the patches, groups being easily found between the cells. There follows a necrosis of the cells which may involve the whole tissue of the patch, and a slough forms which on being cast off leaves an ulcer. This necrosis is evidently in chief part the result of the action of the toxic products of the bacilli, which now gradually disappear from their former positions, though they may still be found in the deeper tissues and at the spreading margin of the necrosed area. They also occur in the lymphatic spaces of the muscular coat. It' is important to note that the ulcers in a fatal case of typhoid may vary much in numbers. The whole lower third of the small intestine may be ulcerated, or only two or three ulcers may be present even in a case where death has occurred by the severity of the fever. Further, small ulcers may also occur in the lymphoid follicles of the large intestine. The condition of the mesenteric glands in typhoid is important. Those corresponding to the affected part of the intestine are usually enlarged, sometimes to a very great extent, the whole mesentery being filled with glandular masses. In such glands there may be acute inflammation attended with haemorrhages, and even necrosis in patches may occur, but this is never the outstanding feature as m the case of the Peyer's patches. Sometimes on section the glands are of a pale -yellowish colour, the contents being diffluent and consisting largely of leucocytes. Typhoid PA THOLOGTCAL CHANGES. 333 bacilli may be isolated both from the glands and the lymphatics connected with them, but the B. coli is in addition often present. The spleen is enlarged, — on section usually of a fairly firm consistence, of a reddish-pink colour, and in a state of congestion. Of all the solid organs it usually contains the bacilli in greatest numbers. They can be seen in sections, occurring in clumps between the cells, there being no evidence of local reaction round them. Similar clumps may occur in the liver in any situation, and without any local reaction. In this organ, however, there are often small foci of leucocytic infiltration, in which, so far as our experience goes, bacilli cannot be demonstrated. Clumps of bacilli may also occur in the kidney. _ In addition to these local changes in the solid organs there are also widespread cellular degenerations in the form of cloudy swelling of the specialised cells of the liver and kidney, or of the muscular fibres of the heart. A granular disintegration of such cells may occur. As they may exist altogether apart from local presence of the bacilli, these changes suggest the circulation in the blood of soluble poisons. In the lungs there may be bronchitis, patches of congestion and of acute broncho-pneumonia. In these, typhoid bacilli may some- times be observed, but evidence of a toxic action depressing the powers of resistance of the lung tissue is found in the fact that the pneumococcus is frequently found in such complications of tvphoid fever. ' r The nervous system shows little change, though meningitis associated either w.th the typhoid bacillus, with the B. coli, or with the strepto- coccus pyogenes has been observed. The typhoid bacilli probably travel by the blood stream, but they have not been frequently isolated from the blood. Whether they have ever been found in the roseolar spots which occur in typhoid fever is a subject of dispute. The fact that the typhoid bacilli are usually con- fined to certain organs and tissues, shows that they must have a selective action on certain tissues. To sum up the pathology of typhoid fever we have in it a disease, the centre of which lies in the lymphoid tissue in and connected with the intestine. In this situation we must have an irritant, against which the inflammatory re- action is set up, and which in the intestine is sufficiently 334 TYPHOID FEVER. powerful to cause necrosis. The affections of the other organs of the body suggest the circulation in the blood of poisonous substances capable of depressing cellular vitality, and producing histological changes. Suppurations occurring in connection with Typhoid Fever. — The relation of the typhoid bacillus to such conditions has been the subject of much discussion, and it must be observed at the outset that statements as to its isolation from pus, etc., can be accepted only when all the points available for the diagnosis of the organism have been attended to. On this understanding the following summary may be given. In a small proportion of the cases examined the typhoid bacillus has been the only organism found. This has been the case in subcutaneous abscesses, in suppura- tive periostitis, suppuration in the parotid, abscesses in the kidneys, etc., and probably also in one or two cases of ulcerative endocarditis. But in the majority of cases, other organisms, especially the B. coli and the pyogenic micro- cocci, have been obtained, the typhoid bacillus having been searched for in vain. It has, moreover, been experimentally shown, notably by Dmochowski and Janowski, that suppura- tion can be experimentally produced by injection in animals, especially in rabbits, of pure cultures of the typhoid bacillus, the occurrence of suppuration being favoured by conditions of depressed vitality, etc. These observers also found that when typhoid bacilli were injected along with pyogenic staphylococci, they died out in the pus more quickly than the latter. So that in clinical cases where the typhoid bacillus is present alone, it is improbable that other or- ganisms were present at an earlier date. Pathogenic Effects produced in Animals by the Typhoid Bacillus —There is no disease known to veterinary science which can be said to be identical with typhoid, nor is there any evidence of the occurrence of the typhoid bacillus under ordinary pathological conditions in the bodies of animals Even before any bacteriological investigation, unsuccessful attempts had been made to communicate the disease to animals by feeding them on typhoid dejecta, and we have PATHOGENIC EFFECTS. 335 seen that Gaffky did not succeed in communicating the disease to animals by feeding them with bacilli, though many different species were inoculated. Nevertheless pathogenic effects caused by feeding have been observed. Typhoid bacilli are killed by a very short exposure to the gastric juice, and Beumer and Peiper, taking this into account, neutralised the gastric juice with soda before the introduction of the bacteria, and slowed the intestinal peristalsis with opium, as Koch did in the case of cholera. By this method they caused death in rabbits and guinea- pigs in from twelve hours to four or five days. Post mortem there were swelling of the spleen, congestion of the liver and kidneys, hyperemia especially of the upper part of the intestine, but the typical typhoid lesions were not reproduced. Remlinger has obtained more important results by feeding rabbits on vegetables soaked in water containing typhoid bacilli. In a certain proportion of animals symptoms appeared about the sixth day and the contamination of the food was then stopped. The illness which followed was characterised by general weakness, diarrhoea, and pyrexia (the temperature curve being of the nature of that seen in human typhoid), and Widal's reaction (vide infra) was obtained. In some cases recovery took place after 8 to 12 days' illness; sometimes death after 12 to 18 days. Post mortem there was observed conges- tion of the small intestine, especially of the last part, and of Peyer's patches, enlargement of mesenteric glands and spleen, and in the latter typhoid bacilli were present. The blood was sterile. As the earlier feeding experiments were not convincing attention was directed to the pathogenic effects produced by introducing the bacilli into the blood or lymph streams Here Beumer and Peiper obtained results similar to those which they produced by feeding. Sirotinin, however, showed that dead cultures produced the same effects, and Wys- sokowitch observed that living bacilli injected into the peri- toneum rapidly decreased in numbers. Many, therefore, held that there was no evidence that the bacilli multiplied in 336 TYPHOID FEVER. the blood, and considered that the effects were due to toxic bodies injected along with the bacilli. Other observers did not confirm Sirotinin's results with the injection of dead cultures, and Pfeiffer is probably correct in holding that the diverse results obtained were due to differences in the virulence of the cultures used. Ordinary laboratory cultures of B. typhosus are usually non-pathogenic. They can, however, be made virulent in various ways. This Chantemesse and Widal effected by injecting along with it the sterilised products of the streptococcus pyogenes, and Sanarelli used for the same purpose sterilised cultures of the B. coli. The method of the latter was as follows. .5 c.c. of a bouillon typhoid culture twenty-four hours old was in- jected subcutaneously into a guinea-pig, and at the same time 10 to 12 c.c. of sterilised old culture of B. coli were introduced into the perito- neal cavity. The animal died in from twelve to fourteen hours with typhoid bacilli in the peritoneum and a few in the blood and organs. From the former situation bouillon cultures were made and used for the subcutaneous injection of a second animal, which also received intraperitoneally some sterilised B. coli culture, a less quantity of the latter being now found sufficient to cause death in the same time. In a series of animals thus inoculated, each from the previous member, less and less B. coli culture was found sufficient until this could be dispensed with altogether, the typhoid bacilli alone being sufficient After about thirty such passages a culture of a typhoid bacillus of exalted virulence was obtained. Sidney Martin has obtained virulent cultures by pass- ing bacilli, derived directly from the spleen of a person dead of typhoid fever, through the peritoneal cavities of a series of guinea-pigs. _ ( „ Sanarelli found that the intraperitoneal injection of a few drops of a culture of highly-exalted virulence, or the subcutaneous injection of 3 to 4 c.c, caused in guinea- nies and rabbits illness and death in from twelve to twenty- four hours. After injection the temperature first rose and then gradually sank till death, and there were flatulence and abdominal tenderness. Post mortem the spleen was enlarged and hemorrhagic, the liver enlarged and fatty, the kidneys congested, whilst the intestine showed con- TOXINES OF B. TYPHOSUS. 337 gestion, with excess of mucous secretion and desquamation of the intestinal epithelium. The Peyer's patches and solitary glands were enormously infiltrated, sometimes almost purulent, and they contained typhoid bacilli which were also found in the mesenteric lymphatics and glands, and in the spleen. Sanarelli states that by whatever path the bacilli were introduced into the body the brunt of the pathological effects always fell on the intestine and ab- dominal organs ; and with regard to the bacilli themselves, though they might be found in the blood, their usual site was in the solid organs, especially the spleen. Pfeiffer could not confirm Sanarelli's observations with regard to a special affection of the Peyer's patches, but the cultures used by him did not possess so exalted a virulence as those of Sanarelh. The latter's observations, at any rate, leave no doubt that, provided the cultures be sufficiently virulent, the typhojd bacillus can multiply m the body of an animal and rapidly produce a fatal result. The Toxic Products of the Typhoid Bacillus — We must next look at what has been done in investigating the oxic bodies, which the pathological anatomy of the disease leads us to suspect are elaborated by the bacillus typhosus Bneger, in his earlier work on the ptomaines, stated thai bouillon cultures of the typhoid bacillus eight weeks old contained a base (typhotoxin) having the formula CH NO This observation has only a historic interest, and Brieger H hedbv F Tl\ UP'JZ 'ater (l89°>' in the P*P- ished by Fraenkel and himself he describes among other ovalbumins one formed by the typhoid bacillu"g The authors obtained it-by making bouillon cultures germ-f ee by filtering through a Cumberland's filter after concentra on £ one^third of the original volume by e£pSS£ 30 L. A precipitate was then obtained by adding ten volumes of alcohol acidified with acetic acid. The pre £ red^solved in water and reprecipitated wkh ~ rw- hdiruthat ;n rer and again »ated b" .rtung with sulphate of ammonium in nowdpr rrui Prec,p,.ate was a third time dissolved in ZJ^L^ 338 TYPHOID FEVER. What remained in the dialyser was the toxic body. It gave the reactions of the group of toxalbumins, and was ener- getic in pathogenic effects. These, however, still did not reproduce the appearances seen either in the natural or artificial disease. In view of the uncertain results thus obtained in the search for the toxines of typhoid in a pure condition, later observers have been content to work with fluids containing these toxines in mixture with other bodies. Sanarelli, who has also investigated the toxic action of the typhoid bacillus, thinks that various bodies may be concerned. He pre- pared the toxine by growing the virulent bacillus on 2 per cent glycerine bouillon for one month at 370 C. and eight months at the room temperature. It was then kept for some days at 6o° C. to kill and macerate the bacilli. A clear fluid could be decanted off which contained the toxic substances, many being no doubt derived from the bacterial bodies. When injected subcutaneously into guinea- pigs in the proportion of 1.5 c.c. per 100 grms. of body-weight, it caused death in twenty hours. There was progressive fall of temperature, abdominal pains and distension, and bloody stools. Post mortem there was peritoneal exudation rich in leucocytes and an enlarged spleen. The intestine was congested, especially the small intestine, and the lym- phatic patches were infiltrated and congested. The other organs were normal. Sidney Martin has found that cultures (especially virulent cultures) in broth, when filtered germ free, contain toxines causing lowering 01 temperature, loss of weight and diarrhoea ^ the only changes were fatty degeneration of the heart. 1 ne bodies of the bacilli killed by chloroform vapour were much more toxic, similar effects, however, being pro- duced, and here Ihe toxicity could be further increased by heating the bodies of the bacilli for a few minutes t| about 60" C. The latter result may be caused .1 the poisons being liberated by the breaking up of the bodies* Ihe bacilli. No change in the Peyenan patches , as ever found, though diarrhoea was a constant symptom. IMMUNISATION AGAINST B. TYPHOSUS. 339 animals appear, however, either to have died sooner or survived much longer than in Sanarelli's experiments Martin found that if the B. coli and B. enteritidis had L BTvohT CXalted KSUltS Similar t0 th0Se seen -th he B typhosus were obtained. He further points out hat not only the poisons of these three related bS£ bu those of nan and abrin produce similar results The general conclusions to be drawn from all these observations is that there exist in the bodies of typho d bacilli toxic bodies, that in artificial cultures these do not pass to any great degree out into the surrounding medium ^LtltfZPWdUCe 6ffeCtS °n the "^estin'e ^hteTs evidence that these are not peculiar to the toxines of the B typhosus. As to the nature of the typhoid tox nes we c"th;n? h*%t ^ h0W6Ver' "ound in the case of the typhoid bacillus there is very little digestive action, such as occurs with the bacilli J diphthenflnd Bacmus^r^T 0' Animals against Typhoid Bacillus.-In considering this question we must noteM immunisation against the living bacilli • /o\ > ' against their toxines - and ITfu (a) lmmumsation & cu uuxines, and (3) the relations between th*** wo conditions. Earlier observers had been TccessfS n accustoming mice -to the typhoid bacillus by the succS ive Wassermann in ,h„ 1 Br,eSer> Kitasato, and to ~uV o:;,orf ,rarches,on sufficiently nutritive ,n ' , gh ",e medium ™s APplyi„gythrsUt™Vceip e° tope™"/t multiplication. culture very virulentt Sice fofth^Z 'o? *7 * bouil on, and then tili^ tv. • ys 011 such a « ^ C. for ^ * h^ 'njected into each of a series of m ™ '/ then ^ days ,ater , was fou~S nt^S 34o TYPHOID FEVER. an otherwise fatal dose of the original living virulent cul- ture The experiments were repeated on guinea-pigs with a similar result, and it was also found that the serum of a guinea-pig thus immunised could, if transferred to another guinea-pig, protect the latter from the subsequent injection of a dose of typhoid bacilli to which it would naturally suc- cumb Chantemesse and Widal, Sanarelli, and also Pfeiffer, succeeded in immunising guinea-pigs against the subsequent intraperitoneal injection of virulent living typhoid bacilli, by repeated and gradually increasing intraperitoneal or sub- cutaneous doses of typhoid cultures in bouillon, in which the bacilli had been killed by heat or chloroform vapour Experiments performed with serum derived from typhoid patients and convalescents have been ^duced as bearing on the matter. Many observers had noticed that the serum of men convalescent from typhoid had an inimical effect on typhoid bacilli; and these results have been con- firmed by Pfeiffer, whose technique was less open to objection than that of most previous workers. He found Sat the serum of healthy men had such an action but in a mSch less degree. The method was to mix the serum and Z bacUU in a little bouillon, and inject the whole intr* iio guinea-pig, He found that when the htter did not die, the bacilli became motionless and ap, pi ntly dead, and that plate cultures made after a tinie Cm the exudation containing them, remained sterile. The SL^^ptfta* has, therefore, antimicrohc powers, but the e is no evidence that it contains any antitoxic pigs, death took place as 11 d h t whllJ received the toxines alone. Sanaiein aiso urn , Pfeiffer found that by using the serum of immunised g PATHOGENIC ACTION OF B. COIL 341 he could, to a certain extent, protect other animals against the subsequent injection of virulent living typhoid bacilli On trying to use the agent in a curative way, i.e., injecting it only after the bacilli had begun to produce their effects, he got little or no result. There is thus evidence that the serum of persons who have recovered from typhoid fever, and the serum of animals artificially immunised against virulent typhoid bacilli, protect from these bacilli. There is no evidence that the serum has much power in neutralising the products of these bacilli. We have thus this curious fact. Animals are immunised by injections of the toxines of a bacillus • , their serum, however, has no effect in neutralising its toxines, but only aids in the destruction of the bacilli which produce the toxines. Similar results have been obtained in the case of cholera. Typ^ol/tS^V0/ hthe Bf *? aHd ^ Relation t0 that of U» rewnSihW 5 already, seen that the B. coli is probably phok ? -ef thrt rrence of rr of the abscesses ^ virulent B ^a^^^S/T^^ ** by ««* Sanarelli holds that they ^ f 1 uT t hTf " °f ^ * *Pho"* nature of a septicemia S 1 mer partake more of the while in the S ter the dhS. J ^ P™ditis' "d peritonitis ; 342 7YPII0ID FEVER. ward as confirmatory of the fact of their being distinct species. Thus Sanarelli accustomed the intestinal mucous membrane of guinea-pigs to toxines derived from an old culture of the B. coli, by introducing clay by clay small quantities of the latter into the stomach. When a relatively large dose could be tolerated, it was found that the introduc- tion in the same way of a small quantity of typhoid toxine was followed by fatal result. Pfeiffer also found that while the serum of conval- escents from typhoid paralysed the typhoid bacilli, it had no more effect on similar numbers of B. coli than the serum of healthy men. General View of the Kelationship of the B. typhosus to Typhoid Fever. — i. We have in typhoid fever a disease having its centre in and about the intestine, and acting secondarily on many other parts of the body. In the parts most affected there is always a bacillus present, microscopically resembling other bacilli, especially the B. coli, which is a normal inhabitant of the animal intestine. This bacillus can be isolated from the characteristic lesions of the disease and from other parts of the body as de- scribed, and further, it is found by culture reactions to differ from the B. coli. The whole series of culture reactions, however, must be investigated before a particular bacillus is identified as the B. typhosus, and no weight must be attached to any observations made on the subject^ when this has not been done. Here the important point, however, is that a bacillus giving all the reactions of the typhoid bacillus has never been isolated except from cases of typhoid fever, or under circumstances that make it possible for the bacillus in question to have been derived from a case of typhoid fever. There is no evidence that the B coli can be transformed into the typhoid bacillus, or the typhoid bacillus into the B. coli, though of course this does not preclude the possibility of the one having been * originally derived from the other. All practically are now agreed that two separate bacilli exist, the B. coh and the B" T Against the etiological relationship of the latter to the disease several facts may be adduced Fnst, there J the comparative difficulty of the isolation of the B. typhosus from the stools of typhoid patients. We have pointed out, RELATION OF B. TYPHOSUS TO TYPHOID. 343 however, that the latter can be isolated during the first ten days of the disease, and that the extraordinary multiplica- tion of the B. coli, which takes place in any pathological condition of the intestine, sufficiently explains the failures in the later stages. The second and great difficulty in the way of accepting the etiological relationship of the B. typhosus lies in the comparative failure to cause the disease in animals. We have noted, however, that in nature animals do not suffer from typhoid fever. The experiments of Sanarelli ought to have considerable weight in this connection. No other observer has exalted to such a degree the virulence of the typhoid bacillus, which is certainly the rational procedure when dealing with a re- fractory animal. In a way this is unfortunate, for at present Sanarelli's results can be neither confirmed nor denied. We must for the present provisionally accept his statements that both the bacilli of exalted virulence, and what is even more important, the toxines derived from them, give rise to selective pathological changes in Peyer's patches and the mesenteric glands. 3. The observations of Pfeiffer and others on the pro- tective power against typhoid bacilli shown, on testing in animals, to belong to the serum of typhoid patients and convalescents, and the peculiar action of such serum in im- mobilising and causing clumping of the bacilli {vide infra) are also of great importance. These very important facts may thus be accepted as indirect evidence of the patho- genic relationships of the typhoid bacillus to the disease. According to our present results we must thus hold that the bacillus typhosus constitutes a distinct species of bacterium, and that there is strong reason for accepting it as the cause of typhoid fever. The Serum Diagnosis of Typhoid Fever.— This method of diagnosis is based on the fact that living and actively motile typhoid bacilli if placed in the diluted serum of a patient suffering from typhoid fever, within a very short time lose their motility and become aggregated into clumps. The researches which led up to the discovery will 344 TVrilOID FEVER . be described in the chapter on Immunity. We shall find that in many diseases the serum has this property of causing agglutination of cultures of the causal bacterium. The principles on which the possession of the faculty depends, and also its significance, are obscure, and even in the case of the typhoid bacillus, where an enormous amount of work has been done, we do not know the true interpreta- tion of some of the facts which have been observed. The methods by which the test can be applied have already been described (p. 118). (1) It will be there seen that the loss of motility and clumping may be observed microscopically. If a prepara- tion be made by the method detailed (typhoid serum in a dilution of, say, 1 130 having been employed), and examined at once under the microscope, the bacilli will usually be found actively motile, darting about in all directions. In a short time, however, these movements gradually become slower, the bacilli begin to adhere to one another, and ultimately become completely immobile and form clumps by their aggregation, so that no longer are any free bacilli noticeable in the preparation. When this occurs the reaction is said to be complete. If the clumps be watched still longer a swelling up of the bacilli will be observed, with a granulation of the protoplasm, so that their forms can with difficulty be recognised. In a preparation similarly made with non-typhoid serum the individual bacilli can be observed separate and actively motile for many hours. (2) A corresponding reaction visible to the naked eye is obtained by the " sedimentation test," the method of apply- ing which has also been described (p. 1 20). Here at the end of twenty-four hours the bacilli form a mass like a precipi- tate at the bottom of the mixture of bacterial emulsion and diluted typhoid serum, while the upper part remains clear. A similar preparation made with normal serum gives a diffuse turbidity at the end of twenty-four hours. The test in this form has the disadvantage of taking longer time than the microscopic method, but it is useful as a control ; in nature it is similar. SERUM DIAGNOSIS. 345 Such is what occurs in the case of a typical reaction. I here are several details, however, which require attention, and on which the value of the method as a means of diagnosis Largely depends. The race of typhoid bacillus employed is important. All races do not give uniformly the same results, though it is not known on what this difference of susceptibility depends. The bacteriologist must, therefore, apply a process of selection to the races at his disposal, with a view to obtaining one which gives the best result in the greatest number of undoubted cases of typhoid fever, and which gives as little reaction as possible with normal sera or sera derived from other diseases. This latter point is important, as some races react very readily to non-typhoid sera. Again, care must be taken as to the state of the culture used. The suitability of a culture may be impaired by varying the conditions of its growth Con- tinued growth of a race in surroundings very favourable to vegetative activity makes it less suitable for use ZZ test as the bacilli tend naturally to adhere in clumps, which may be mistaken for those produced by the reaction. Wyatt Johnson recommends that the stock culture should be kept rarTubcnltagar a7°0m temPerature ^ maintained by agar sub-cultures made once a month. For use in anolvina he test bouillon sub-cultures are made and ncubS fof twenty-four hours at 3f C. As the reaction of the medium has also an important effect on the sensitiveness of a culture he recommends that such bouillon should first be made neutral to phenol-phthalein, and then have added to t Zee or four per cent of normal hydrochloric acid. When these precautions are taken a growth occurs which only gives a thetcilliUm " th;Kb°UiIl0n Wkh0Ut of P^&^L^^ °f ^ dUUti°n °fm Tf i r_ Lue occurrence of clumping is most imnortnnr It has been found that if the degree of dilut.o" 'be t00 dTIi ™n-t>'Ph°'d may cause dumping If p$ .^tt tshrId ahrays be ™de 346 TYPHOID FEVER. a complete reaction. Some typhoid sera have, however, very powerful agglutinating properties, and may in a com- paratively short time produce a reaction when diluted many hundreds of times. The conditions giving rise to such sera are not known, and the cases from which they are derived are not necessarily of a severe type. With highly diluted sera not only may the reaction be delayed but it may be incomplete. Here, what is usually seen is that the clumps formed are small, many bacilli being left free. These latter may either have been rendered motionless or they may still be motile. No diagnosis is conclusive which is founded on the occurrence of such an in- complete clumping alone. Seeing that low dilutions sometimes give a reaction with non-typhoid sera, great discussion has taken place as to what is the minimum dilution at which, when complete clumping occurs, it may safely be said that the reaction is due to the specific action of a typhoid serum. The general consensus of opinion, with which our own experience agrees, is that when a serum in a dilution of i : 30 causes complete clumping in half an hour, it may safely be said that it has been derived from a case of typhoid fever. Suspicion should be enter- tained as to the diagnosis if a lower dilution is required, or if a longer time is required. The reaction given by the serum in typhoid fever usually begins to be observed about the seventh day of the disease, though occasionally it has been found as early as the fifth day, and sometimes it does not appear till the third week or later. Usually it gradually becomes more marked as the disease advances, and it is still given by the blood of convalescents from typhoid, but cases occur in which it may permanently disappear before convalescence sets in. How long it lasts after the end of the disea ha not yet been fully determined, but in many cases t has been found after several months at least. As a rule tl region is more marked where the fever is of a pronounced character In the milder cases it is less pronounced J sometases which from the clinical symptoms were almost SERUM DIAGNOSIS. 347 certainly typhoid, the reaction has apparently been found to be absent. It has been found that the reaction is not only obtained with living bacilli, but in certain circumstances also with bacilli that have been killed. This last may be effected by keeping the bacilli at 6o° C. for an hour. If a higher temperature be used, sensitiveness to agglutination is im- paired. The capacity is also still retained if a germicide be employed. Here Widal recommends the addition of one drop of formalin to 1 50 drops of culture. The reaction, however, tends to be less complete. It may be remarked that while clumping is taking place where dead cultures are used, active Brownian movement among the free bacteria may be noticed, which may lead the observer to doubt whether the bacilli are really dead. Besides the blood serum it has been found that the reaction is given in cases of typhoid fever by pericardial and pleural effusions, by the bile and by the milk, and also to a slight degree by the urine. The blood of a foetus may have little agglutinating effect though that of its mother may have given a well marked reaction. It may here also be mentioned that a serum will stand exposure for an hour at 58 C. without having its agglutinating power much diminished.. Higher temperatures, however, cause the property to be lost. T/le Agglutination of Organisms other than the B Typhosus by Typhoid Serum.- -It was at first thought that re' action in typhoid fever would afford a reliable method of dis- tinguishmg the typhoid bacillus from the B. coli Thoueh many races of the latter give no reaction with a typhoid serum, there are others which react positively. Usually however, a lower dilution and a longer time are required for a result to be obtained, and the reaction is often Tn complete. It has also been found that other organ sms te^h^f group' **; Gaertner's baci"us -d perhapS the bacillus of psittacosis, react in a similar way Ihe reaction as a method of distinguishing between these forms is thus not reliable, but in certain Jse may 348 TYPHOID FEVER. be of value in giving confirmation to other tests. Then is a point in this connection regarding which further light is required. Many races of B. coli in use have been isolated from typhoid cases, and we as yet do not know what effect a sojourn in such circumstances may have on its subsequent sensitiveness to agglutination by typhoid serum. The discovery that the exhibition of Widal's reaction is not confined to the B. typhosus has caused great attention to be paid to the sensitiveness to different sera shown by it and by other allied organisms. It has been found that not only typhoid sera but the sera of healthy persons, and of those suffering from diseases other than typhoid fever, may occasionally clump typhoid bacilli even when con- siderably diluted. It has not, however, been sufficiently noted that, as Christophers has pointed out, a large pro- portion of similar sera will clump the B. coli in dilutions of from i : 20 to 1 : 200, and no doubt many of the reactions shown by typhoid sera towards B. coli are due to the pre- existence in the individuals of an agglutinative property towards the bacillus. It has been shown that both the B. coli and the B. typhosus are clumped by the normal serum of the horse, the ass, and the rabbit, and it has been found that the serum of an animal immunised against either of these bacilli sometimes clumps both, and sometimes also in addition the B. enteritidis, though usually the dilutions necessary differ. It may also be remarked that in such immunised animals the best agglutinating result is not always obtained with subcultures of the race by which im- munisation was effected. It is evident that these results are of great interest, and may ultimately throw light on the true nature of agglutination. With regard to the value of the serum reaction there is little doubt. In nearly 95 per cent of cases of typhoid it can be obtained in such a form that no difficulty is experienced if the precautions detailed above are observed. The causes of possible error may be summarised as follows . the serum of the person may naturally have the capacity 01 clumping typhoid bacilli ; there may have been an attacK VACCINATION AGAINST TYPHOID 349 of typhoid fever previously with persistence of agglutinative capacity; the case may be one of disease caused by an allied bacillus ; the disease may have a quite different cause, and yet the serum may react with typhoid bacilli ; the disease may be typhoid fever and yet no reaction may occur. The most important of these sources of error is that with which diseases caused by allied organisms are concerned, as it is probable that all the forms which these take in man have not been recognised. The very wide application of the Widal reaction has elicited the fact that it is given in many cases of slight, transient and ill-defined febricuke, which occur especially when typhoid fever is prevalent! Our knowledge of these is still insufficient to justify our setting all of them down as cases of aborted typhoid There is no doubt that, taking all the facts into account, the cases where the reaction gives undoubtedly correct information so far outnumber those in which an error may be made that it must be looked on as a most valuable aid to diagnosis. In concluding we may point out here that the fact of a typhoid serum clumping allied bacilli in no way, so far as our present knowledge goes, justifies doubt being cast on the specific relation of the typhoid bacillus to typhoid fever. Vaccination against Typhoid.— The principles of the immunisation of animals against typhoid bacilli have been applied by Wright and Semple to man in the following way. Typhoid bacilli are obtained of such virulence that a quarter of a twenty-four hours' old agar culture when administered hypodermically will kill a guinea-pig of from 35° to 4°o grammes. Such a culture is emulsified in bouillon and killed by being raised to 6o° C. and kept at that for five minutes. The vaccination is accomplished by the hypodermic injection of from one-twentieth to one-fourth of such a dead culture. The effects of such an injection are some local tenderness, and it may be swelling, and a general feeling of restlessness for some hours with occa- sionally a slight rise of temperature. The possibility of this vaccination preventing the development of typhoid 35o TYPHOID FEVER. fever after exposure to natural infection must rest on experience, but as most of those who have been subjected to it are medical officers in the British services, who are likely to be exposed to such infection abroad, this ex- perience may soon be gained. It has been found in cases where the method has been practised that in the course of a fortnight a positive Widal's reaction was obtained, and such an occurrence is probably evidence of the acquisition of a certain degree of immunity. Anti-typhoid Serum. — Bokenham grew virulent typhoid bacilli for three weeks on bouillon containing ten per cent of alkali albumin, and filtering the cultures through porcelain obtained a filtrate which, though non-toxic to guinea-pigs, probably had immunising properties. Starting with this and afterwards using it alternately with killed cultures, he immunised a horse and found that the serum had neutral- ising power for typhoid bacilli when the latter mixed with it were injected into guinea-pigs. When injection of the serum was followed by injection of bacilli, the pathogenic action of the latter was to a certain extent prevented, and there was also evidence of the serum possessing curative properties. Methods of Examination. — The methods of microscopic! examination, and of isolation of typhoid bacilli from the spleen post mortem, have already been described. They may' be isolated from the Peyer's patches, lymphatic glands, etc by a similar method. During life, typhoid bacilli may be obtained in culture in the following ways : — (a) From the Spleen. — This is the most certain method of obtaining the typhoid bacillus during the continuance of a case. The skin over the spleen is purified and, a sterile hypodermic syringe being plunged into the organ, there is withdrawn from the splenic pulp a droplet of fluid, from which plates are made. In a large proportion of cases of typhoid the bacillus may be thus obtained, failure only occurring when the needle does not happen to touch a bacillus. Numerous observations have shown that pro- vided the needle be not too large, the procedure is quit* safe. Its use, however, is scarcely called for. METHODS OF EXAMINATION. 35i (b) From the Urine. — Typhoid bacilli are present in the urine in about twenty-five per cent of cases, especially late in the disease, probably chiefly when there are groups in the kidney substance. For methods of examining sus- pected urine, see p. 78. (c) From the Stools. — During the first ten days of a case of typhoid fever, the bacilli can be isolated from the stools by the ordinary plate methods — preferably in phenolated gelatine. After that period, though the continued in- fectiveness of the disease indicates that they are still present, their isolation is practically hopeless. We have seen that after ulceration is fairly established by the slough- ing of the necrosed tissue, the numbers present in the patches are much diminished and therefore there are fewer cast-off into the intestinal lumen, and that in addition there is a correspondingly great increase of the B. coli, which thus causes any typhoid bacilli in a plate to be quite outgrown. From the fact that the ulcers in a case of typhoid may be very few in number, it is evident that there may be at no time very many typhoid bacilli in the intestine. We may add that the microscopic examination of the stools is useless as a means of diagnosing the presence of the typhoid bacillus. Isolation from. Water Supplies.— A great deal of work has been done on this subject. It is evident that if it is difficult to isolate the bacilli from the stools it must a fortiori much more difficult to do so when the latter are enormously diluted by water. Some have held that the typhoid bacillus has never been isolated from suspected water, and have adduced this as an argument against its etiological relationship to the disease. The considerations just advanced, however, militate against such a view The B typhosus has been isolated from water during epidemics I his was done by Klein in the outbreaks in recent years at Worthing and Rotherham. The B. coli is, as might be expected, the organism most commonly isolated in such circumstances. In the case of both bacteria, the whole series of culture reactions must be gone through before 352 TYPHOID FEVER. any particular organism isolated is identified as the one or the other ; probably there are saprophytes existing in nature which only differ from them in one or two reactions. In the examination of water, the addition of .2 per cent carbolic acid to the medium inhibits to a certain extent the growth of other bacteria, while the B. typhosus and the B. coli are unaffected. In examining waters, the ordinary plate methods are generally used. Klein, however, niters a large quantity through a Berkefeld filter and, brushing off the bacteria retained on the porcelain, makes cultures. A much greater concentration of the bacteria is thus obtained. CHAPTER XV. DIPHTHERIA. There is no oetter example of the valuable contributions of bacteriology to scientific medicine than that afforded in the case of diphtheria. Not only has research supplied as ,n the case of tubercle, a means of distinguishing true diphtheria from conditions which resemble it, but the study of the toxines of the bacillus has explained the manner by which the pathological changes and characteristic symptoms of the disease are brought about, and has led to the dis- ™,l V r « °;emb™»<=. but made no cultivations. It was first cidti K puunsnea in 1864, and to him we owe the first arm.m( „f characters in cultures and of some of its pathogenic effects on a, il ? The organism is for these reasons known as th* Klebs Sffler WHl •He s„„i« of „,t,oh inquiry, Sltl^S: 354 DIPHTHERIA. said to have been definitely established by the brilliant researches of Roux and Yersin, who made an extensive study of its characters and life history, and showed that the most important features of the disease could be produced by means of the separated toxines of the organism. Their experiments were published in 1888-90. A considerable amount of further light has been thrown on the subject by the work of Sidney Martin, who has found that there can be separated from the organs in cases of diphtheria substances which act as nerve poisons, and also produce other phenomena met with in diphtheria. General Facts. — Without giving a description of the pathological changes in diphtheria, it will be well to men- tion the outstanding features which ought to be considered in connection with its bacteriology. In addition to the formation of false membrane, which may prove fatal by mechanical effects, the chief clinical phenomena are the symptoms of general poisoning, great muscular weakness, tendency to syncope, and albuminuria ; also the striking paralyses which occur later in the disease, and which may affect the muscles of the pharynx, larynx, and eye, or less frequently the lower limbs (being sometimes of paraplegic type), all these being grouped together under the term " post-diphtheritic paralyses." It may be stated here that all these conditions have been experimentally reproduced by the action of the bacillus of diphtheria, or by its toxines. Other bacteria are, however, concerned in producing various secondary inflammatory complications in the region of the throat, such as ulceration, gangrenous change, and suppura- tion, which may be accompanied by symptoms of general septic poisoning. The detection of the bacillus of Loffler in the false membrane or secretions of the mouth is to be regarded as the only certain means of diagnosis of diphtheria. With the exception of the tubercle bacillus, there is probably no organism which has been the subject of so much routine examination, and the opinion of all who are competent to judge may be said to be unanimous on this subject. Bacillus Diphtheriae — Microscopical Characters. — If a film preparation be made from a piece of diphtheria mem- brane (in the manner described below) and stained with BACILLUS DIPHTHERLsE. 355 methylene-blue, the bacilli are found to have the following characters. They are slender rods, straight or slightly curved, and usually about 3 fx in length, their thickness being a little greater than that of the tubercle bacillus. i ■ 9 m Fig. 89.— Film preparation from diphtheria membrane ; showing numerous diphtheria bacilli. One or two degenerated forms are seen near the centre of the field. (Cultures made from the same piece of membrane showed the organism to be present in practically nure condition.) ; Stained with methylene-blue. x 1000. The size, however, varies somewhat in different cases, and for this reason varieties have been distinguished as small and large, and even of intermediate size. It is sufficient to mention here that in some cases most are about 3 in length, whilst in others they may measure fully 5° a Corresponding differences in size are found in cultures 356 DIPHTHERIA. They stain deeply with the blue, sometimes being uniformly coloured, but often showing, in their substance, little granules more darkly stained, so that a dotted or beaded appearance is presented. Sometimes the ends are swollen and more darkly stained than the rest; often, however, they are rather tapered off (Fig. 89). In some cases the terminal swelling is very marked, so as to amount to clubbing, and with some specimens of methylene-blue these swellings and granules stain of a violet tint. Distinct clubbing, however, is less frequent than in cultures. There is a want of uniformity in the appearance of the bacilli when compared side by side. They usually lie irregularly scattered or in clusters, the individual bacilli being disposed in all direc- tions. Some may be contained within leucocytes. They do not form chains, but occasionally forms longer than those mentioned may be found, and these specially occur in the spaces between the fibrin as seen in sections. Distribution of the Bacilli. — The diphtheria bacilli may be found in the membrane wherever it is formed, and may also occur in the secretions of the pharynx and larynx in the disease. It may be mentioned that distinctions formerly drawn between true diphtheria and non-diphtheritic con- ditions from the appearance and site of the membrane, have no scientific value, the only true criterion being the presence of the diphtheria bacilli. The occurrence of a membranous formation produced by streptococci has already been mentioned (p. 178). In diphtheria the membrane has a somewhat different structure according as it is formed on a surface covered with stratified squamous epithelium as in the pharynx, or on a surface covered by ciliated epithelium as in the trachea. In the former situation necrosis of the epithelium occurs either uniformly or in patches, and along with this there is marked inflammatory reaction in the connective tissue beneath, attended by abundant fibrinous exudation. The necrosed epithelium becomes raised up by the fibrin, and its interstices are also filled by it. The fibrinous exudation also occurs around the vessels in the tissue beneath, and in DISTRIBUTION OF THE B AC I ILL 357 this way the membrane is firmly adherent. In the trachea, on the other hand, the epithelial cells rapidly become shed, and the membrane is found to consist almost exclusively of fibrin with leucocytes, the former arranged in a reticulated or somewhat laminated manner, and varying in density in "V. v Jt 'v. • '. t'"\ . Fig. 90. — Section through a diphtheritic membrane in trachea, show- ing diphtheria bacilli (stained darkly) in clumps, and also scattered amongst the fibrin. Some streptococci are also shown, towards the surface on the left side. Stained by Gram's method and Bismarck-brown. x 1000. different parts. The membrane lies upon the basement membrane, and is less firmly adherent than in the case of the pharynx. The position of the diphtheria bacilli varies somewhat in different cases, but they are most frequently found lying in oval or irregular clumps in the spaces between the fibrin, 358 DIPHTHERIA. towards the superficial, that is, usually, the oldest part of the false membrane (Fig. 90). There they may be in a practically pure condition, though streptococci and occa- sionally some other organisms may be present along with them. They may occur also deeper, but are rarely found in the fibrin around the blood vessels. On the surface of the membrane they may be also seen lying in large numbers, but are there usually accompanied by numerous other organisms of various kinds. Occasionally a few bacilli have been detected in the lymphatic glands. As Loftier first described, they may be found after death in pneumonic patches in the lung, this being a secondary extension by the air passages. They have also been occa- sionally found by various observers in the spleen, liver, and other organs after death. This occurrence is probably to be explained by an entrance into the blood stream shortly before death, similar to what occurs in the case of other organisms, e.g., the bacillus coli communis. With these exceptions, however, it may be stated that the bacillus of diphtheria occurs only locally in the false membrane and in the fluids of the mouth, and does not invade even the subjacent tissues to any extent. Association with other Organisms. — The diphtheria organism is sometimes present alone in the membrane, but more frequently associated with some of the pyogenic organisms, the streptococcus pyogenes being the com- monest. The staphylococci, and occasionally the pneumo- coccus or the bacillus coli, may be present in some cases. Streptococci are often found lying side by side with the diphtheria bacilli in the membrane, and also penetrating more deeply into the tissues. In some cases of tracheal diphtheria, we have found streptococci alone, at a lower level in the trachea than the diphtheria bacilli, where the membrane was thinner and softer, the appearance in these cases being as if the streptococci acted as exciters of in- flammation and prepared the way for the bacilli. It is still a matter of dispute as to whether the association of the diphtheria bacillus with the pyogenic organisms is a CULTIVATION OF THE BACILLUS. 359 favourable sign or the contrary, though on experimental grounds the latter is the more probable. We know, however, that some of the complications of diphtheria may be due to their action. The extensive swelling of the tissues of the neck, sometimes attended by suppuration in the glands, and also various hemorrhagic conditions, have been found to be associated with their presence, in fact, in some cases the diphtheritic lesion enables them to' get a foothold in the tissues, where they exert their usual action and may lead to extensive suppurative change, to septic poisoning or to septicaemia. In cases where a gangrenous process is super- added, a great variety of organisms may be present, some of them being anaerobic. Against such complications anti-diphtheritic serum pro- duces no favourable effect, as its action is specific and only neutralises the toxines of the diphtheria bacillus. In view of this fact, in some cases the anti-streptococcic serum has been used along with it, and it is apparent that in such con- ditions the bacteriological examination of the parts affected may afford valuable indications as to treatment. Cultivation.— The diphtheria bacillus grows best in cultures at the temperature of the body; growth still takes place at 22 C, but ceases at 20° C. The best media are the following: Loffler's original medium (p. 52), solidified blood serum, alkaline blood serum (Lorrain Smith), blood agar, and the ordinary agar media. If inoculations be made on the surface of blood serum with a piece of diphtheria membrane, colonies of the bacillus appear within twenty- four hours, and often before any other growths are visible. Ihe colonies are small circular discs of opaque whitish colour, their centre being thicker and of darker greyish appearance when viewed by transmitted light than the periphery. On the second or third day they may reach 4 mm. in size, but when numerous they remain smaller. On the agar media the colonies have much the same appearance (Fig. 9x) but grow less quickly, and sometimes hoi I f COmParatlvely mi™te, so as rather to resemble those of the streptococcus pyogenes. In stroke cultures 360 DIPHTHERIA. the growth forms a Fig. 91. — Cultures of the diphtheria bacillus on an agar plate; twenty-six hours' growth. (a) Two successive strokes ; (/>) isolated colonies from the same plate. of toxine. Ordinary bouillon becomes acid during the first two or three days, and several days later again acquires an alkaline reaction. If, however, the bouillon is glucose-free (p. 87) the acid reaction does not occur. In these media the bacilli show the same characters as in the membrane, but the irregularity in staining is more marked (Figs. 92, continuous layer of the same dull whitish colour, the margins of which often show single colonies partly or completely separated. On gelatine at 22 J C. a puncture culture shows a line of dots along the needle track, whilst at the surface a small disc forms, rather thicker in the middle. In none of the media does any liquefaction occur. In bouillon the organism produces a turbidity which soon settles to the bottom and forms a powdery layer on the wall of the vessel. By starting the growth on the surface and keep- ing the flasks at rest a distinct scum forms, and this is speci- ally suitable for the development Pig. 92.— Diphtheria bacilli from a twenty- four hours' culture on agar. Stained with methylene-blue. x 1000. CULTIVATION OF THE BACILLUS. 361 93). They are at first fairly uniform in size and shape, but if a culture is examined from day to day it will be found that their appearance gradually becomes irregular. Many become swollen at their ends into club- shaped masses which are stained deeply, and the protoplasm becomes broken up into globules with unstained parts be- tween (Fig. 94). Some become thicker throughout, and seg- Fig 94-- Involution forms of the diph- theria bac.llus ; from an agar culture of seven days growth.' Stained with carbol-thionin-hlue. x 1000. Fig. 93.— Diphtheria bacilli of larger size than in previous figure, showing also irregular staining of protoplasm. From a three days' agar culture. Stained with weak carbol-fuchsin. x 1000. mented so as to ap- pear like large cocci, and others show globules at their ends, the rest of the rod appearing as a faintly- stained line. These are to be regarded as involution forms, and they occur more quickly and abundant- ly on the media less suitable for their growth, e.g., more quickly on glycerine agar than on serum. The bacilli are non- motile, and do not form spores. 362 DIPJITJIENI.]. Staining.— -They take up the basic aniline dyes, e.g., methylene-blue in watery solution, with great readinessj and stain deeply, the granules often giving the meta- chromatic reaction as described. They also retain the colour in Gram's method. Neisser has recently introduced the following stain as an aid to the diagnosis of the diphtheria bacillus. Two solutions are used as follows : {a) i grm. methylene-blue (Griibler) is dissolved in 20 c.c. of 96 per cent alcohol, and to the solution are added 950 c.c. of distilled water and 50 c.c. of glacial acetic acid ; (//) 2 grms. Bismarck-brown (vesuvin) dissolved in a litre of distilled water. Films are stained in (a) for 1-3 seconds or a little longer, washed in water, stained for 3-5 seconds in (£), dried, and mounted. The protoplasm of the diphtheria bacillus is stained a faint brown colour, the granules a blue colour. Neisser considers that this reaction is characteristic of the organism, provided that cultures on Loffler's serum are used and examined 9-24 hours after incubation at 34-350 C. The same satisfactory results are not obtained in the case of films prepared from membrane, etc. Powers of Resistance, etc. — In cultures the bacilli possess long duration of life. Even when kept at 37° C. for one or two months they may be shown by subcultures to be still alive ; at the room temperature they survive still longer. In the moist condition, whether in cultures or in membrane, they have a low power of resistance, being killed at 6o° C. in a few minutes. On the other hand, in the dry condition they have great powers of endurance. In membrane which is perfectly dry, for example, they can resist a temperature of 9 8° C. for an hour. Dried diphtheria membrane, kept in the absence of light and at the room temperature, has been proved to contain diphtheria bacilli still living and virulent at the end of several months. The presence of light, moisture, or a higher temperature, causes them to die out more rapidly. Corresponding results have been obtained with bacilli obtained from cultures and kept on dried threads. These facts, especially with regard to drying, are of great importance, as they show that the con- tagium of diphtheria may be preserved for a long time in the dried membrane. It follows, of course, that cultures can be obtained from membrane even after it has been dried, a fact of some practical importance. INOCULA TION EXPERIMENTS. 363 Effects of Inoculation. — In considering the effects pro- duced in animals by experimental inoculations of pure cultures, we have to keep in view the local changes which occur in diphtheria, and also the symptoms of general poisoning. Loffler in his original paper stated that in the case of rabbits, guinea-pigs, pigeons, and fowls the bacilli taken from pure cultures produced no change on healthy mucous membranes, but when the latter were injured by scarification or otherwise the production of false membrane resulted. A similar result was obtained when the trachea was inoculated after tracheotomy had been performed. In this case the surrounding tissues became the seat of a blood stained oedema, and the lymphatic glands were enlarged, the general picture resembling pretty closely that of laryngeal diphtheria. These results have been amply confirmed by other ob- servers. The membrane produced by such experiments is usually less firm than in human diphtheria, and the bacilli are not generally found in such large numbers in the mem- brane. Rabbits inoculated after tracheotomy often die, and Roux and Yersin were the first to observe that in some cases paralysis may appear before death. Subcutaneous injection in guinea-pigs, of diphtheria bacilli in a suitable dose, produces death within thirty-six hours. On section at the site of inoculation there is seen a small patch of greyish membrane, whilst in the tissues around there is extensive inflammatory cedema, often associated with haemorrhages, and there is also some swelling of the corre- sponding lymphatic glands. The internal organs show general congestion, the suprarenal capsules being especially affected and often showing haemorrhage. The renal epithe- lium may show cloudy swelling, and there is often effusion into the pleural cavities. After injection the bacilli increase in number for a few hours, but multiplication soon ceases, and at the time of death they may be less numerous than when injected. The bacilli remain quite local,1 cultures 1 This may be stated as a general law, though in exceptional cases a few bacilli have been detected in internal organs. 364 DIPHTHERIA. made from the blood and internal organs giving negative results. If a non-fatal dose of a culture be injected, a local necrosis of the skin and subcutaneous tissue may follow at the site of inoculation. In rabbits, after subcutaneous inoculation, results of the same nature follow, but these animals are less susceptible than guinea-pigs, and the dose requires to be proportion- ately larger. The dog and sheep are also susceptible to inoculation with virulent bacilli, but the mouse and rat enjoy a high degree of immunity. Klein found that cats also were susceptible to inoculation. The animals usually die after a few days, and post mortem there is well- marked nephritis. He also found that after subcutaneous injection in cows, a vesicular eruption appeared on the teats of the udder, the fluid in which contained diphtheria bacilli. The animals gradually wasted and died after two or three weeks, the changes in the internal viscera being of the same nature as those in other animals. At the time of death the diphtheria bacilli were still alive and virulent at the site of injection. The striking fact in connection with these experiments is that the diphtheria bacilli passed into the circulation and were present in the eruption on the udder. He considers that this may throw light on certain epidemics of diphtheria in which the contagion was apparently carried by the milk. Further light is required on this subject. Intraperitoneal injection of the bacilli in sufficient quantity in the guinea-pig produces death less rapidly than when the same dose is injected subcutaneously. The bacilli are chiefly confined to the peritoneum and gradually diminish in number. Intravenous injection of virulent cultures in the rabbit often produces death within three days, there being symp- toms of general poisoning with great prostration and muscular feebleness; there may also be well-marked nephritis. In such experiments Roux and Yersin found that the bacilli rapidly disappeared from the blood, and even after the injection of 1 c.c. of a broth culture no trace of the organisms could be detected by culture after twenty- four hours. The Toxines of Diphtheria. — As in the above experiments the symptoms of poisoning and ultimately a fatal result occur THE T0X1NES OF DIPHTHERIA. 365 when the bacilli are diminishing in number, or even after they have practically disappeared, Roux and Yersin inferred that the chief effects were produced by the products of the organisms, and this supposition they proved to be correct. They showed that broth cultures of three or four weeks' growth freed from bacilli by filtration were highly toxic. The filtrate when injected into guinea-pigs and other animals produces practically the same effects* as the living bacilli, with the exception that locally there is no forma- tion of false membrane ; the internal organs show the same changes, and locally there is inflammatory oedema, which may be attended by a certain amount of necrotic change. The toxicity may be so great that .05 c.c. or even less may be fatal to a guinea-pig in twenty-four hours. In rabbits, when the dose is large, the intestines are found to be distended with fluid, and there may be diarrhoea ; if the animals survive for two or three days there is usually albuminuria, and, post mortem, nephritis is found to be present. After injection either of the toxine or of the living bacilli, when the animals such as guinea-pigs, rabbits, dogs, etc., survive long enough, paralytic phenomena may occur. The hind limbs are usually affected first, the paralysis afterwards extending to other parts, though sometimes the fore-limbs and neck first show the condition. Sometimes symptoms of paralysis do not appear till two or three weeks after inoculation. After paralysis has appeared, a fatal result usually follows in the smaller animals, but in dogs recovery may take place. One point of much interest in relation to the relative nature of toxicity is the high degree of resistance to the toxine possessed by mice and rats. Roux and Yersin, for example, found that 2 c.c. of toxine, which was sufficient to kill a rabbit in sixty hours, had no effect on a mouse, whilst of this toxine even T\ c.c. pro- duced extensive necrosis of the skin of the guinea-pig. Preparation of the Toxine.— -The obtaining of a very active toxine in large quantities is an essential in the preparation of anti-diphtheritic serum. Certain 366 DIPHTHERIA. conditions favour the development of a high degree of toxicity, viz., a free supply of oxygen, the presence of a large proportion of peptone or albumin in the medium, and the absence of substances which produce an acid reaction. In the earlier work a current of sterile air was made to pass over the surface of the medium, as it was found that by this means the period of acid reaction was shortened and the toxine formation favoured. This expedient is now considered unnecessary if an alkaline medium free from glucose is used, as in this no acid reaction is developed. It is then sufficient to grow the cultures in shallow flasks and to start the growth on the surface so that a thick pellicle forms. (The latter can be readily effected by having small fragments of cork floating on the surface.) The absence of glucose — an all important point — may be attained by the method described above (p. 87), or by using for the preparation of the meat extract flesh which is just commencing to putrefy (Spronck). L. Martin uses a medium composed of equal parts of freshly prepared peptone (by digesting pigs' stomachs with HC1 at 35° C), and glucose-free veal bouillon. In this medium he has obtained a toxine of which c.c. is the fatal dose to a guinea-pig of 500 grms. He finds that glucose, glycerine, saccharose and galactose lead to the production of an acid reaction, whilst glycogen does not. The latter fact explains how some observers have found that bouillon prepared from quite fresh flesh is suitable for toxine formation. There is in all cases a period at which the toxicity reaches a maximum, beyond this it begins to fall. This varies in different cases, occurring earlier the more rapid the toxine is formed. Martin found that in his medium the maximum was reached on the 8th-ioth day. It may be added that the power of toxine formation varies much in different races of the diphtheria bacillus, and that many may require to be tested ere one suitable is obtained. Properties and Nature of the Toxine.— The toxic sub- stance in filtered cultures is a relatively unstable body. When kept in sealed tubes in the absence of light, it may NATURE OF THE TOXINE. 367 preserve its powers little altered for several months, but on the other hand, it gradually loses them when exposed to the action of light and air. Heating at 58° C. for two hours destroys the toxic properties in great part, but not altogether. When, however, the toxine is evaporated to dryness, it has much greater resistance to heat. One striking fact discovered by Roux and Yersin, is that after an organic acid, such as tartaric acid, is added to the toxine the toxic property disappears, but that it can be in great part restored by again making the fluid alkaline. The toxic body in filtered cultures can be precipitated by alcohol, and is also carried down by calcium phosphate it is, however, soluble in water and dialyses somewhat slowly through animal membranes. By repeated precipita- tion and again dissolving, aided by dialysis, a solution is obtained which, on evaporating to dryness, gives a whitish yellow powder containing the toxic body, though not in a chemically pure condition. From the characters described Roux and Yersin considered that it belonged to the group of diastases or enzymes. H The true chemical nature of the diphtheria toxine is still unknown, and the matter is further complicated by the possibility that if a ferment is formed by the bacilli it may produce other toxic bodies of a non-diastatic nature Guinochet showed that toxine was also formed from the baclh when grown in urine with no proteid bodies present After growth had taken place he could not detect proSd bodies in the fluid, but on account of the very minute eTcluld TT°T thdr abS6nCe "mid T £ excluded Uschinsky also found that toxic bodies were medium. It follows from this that if the true toxinP i« * protend, it may be formed by synthesis wi,h"bo&s of the b^ a. well as by a change in me proteids ofthe magnesium sulphate 2- a • rii nl? \ S"7, ; calc,um chloride, .1; lactate, 6-7 ; so'diu^iaVnarsT"1 ^P^' ^ > ammonil™ 368 DIPHTHERIA. culture fluid. Brieger and Boer have separated from diphtheria cultures a toxic body which gives no proteid reaction (vide p. 157). Toxic bodies have also been obtained from the tissues of those who have died from diphtheria. Roux and Yersin, by using a filtered watery extract from the spleen from very virulent cases of diphtheria, produced in animals death after wasting and paralysis, and also obtained similar results by employing the urine. The subject of toxic bodies in the tissues, however, has been specially worked out by Sidney Martin. He has separated from the tissues and especially from the spleen of patients who have died from diphtheria, by precipitation by alcohol, chemical substances of two kinds, namely, albumoses (proto- and deutero-, but especially the latter), and an organic acid. The albumoses when injected into rabbits especially in repeated doses, produce fever, diarrhoea, paresis, and loss of weight, with ultimately a fatal result. As in the experiments with the toxine from cultures, the posterior limbs are first affected ; afterwards the respiratory muscles, and finally the heart, are impli- cated. He further found that this paresis is due to well- marked changes in the nerves. The medullary sheaths first become affected, breaking up into globules j ultimately the axis cylinders are involved, and may break across, so that degeneration occurs in the peripheral portion of the nerve fibres. Such changes occur irregularly in patches, both sensory and motor fibres being affected. Fatty change takes place in the associated muscle fibres. There may also be a similar condition in the cardiac muscle. The organic acid has a similar but weaker action. Substances obtained from diphtheria membrane have an action like that of the bodies obtained from the spleen, but in higher degree Martin considers that this is due to the presence in the membrane of an enzyme which has a proteolytic action within the body, resulting in the formation of poisonous albumoses. According to this view the actually toxic bodies are not the direct product of the bacillus, but formed by the enzyme which is produced by it locally are VARIATIONS IN VIRULENCE OF THE BACILLUS. 369 in the membrane. Cartwright Wood has also found that when diphtheria cultures in an albumin-containing medium are filtered germ-free and exposed to 650 C. for an hour (the supposed ferments being thus destroyed), there still remain albumoses which produce febrile reaction and are active in developing immunity. The existence of ferments though a possibility, cannot, however, be considered to be yet completely proved. Nor is it certain whether the proteids obtained by precipitation from cultures and from the tissues are in themselves toxic, or whether the toxic bodies are carried down along with them. Immunity.— This is described in the general chapter on immunity. It is sufficient to state here that a hi^h degree of immunity, against both the bacilli and their toxines, can be produced in various animals by gradually increasing doses either of the bacilli or of their filtered toxines {vide Chap. XIX.). Variations in the Virulence of the Diphtheria Bacillus —in cultures on serum the diphtheria bacilli retain their virulence fairly well, but they lose it much more quickly on less suitable media, such as glycerine agar. Roux and Yersin found that, when the bacilli were grown at an luZT 7 t temPerature' namely 39-5° C, and in a current of air, the-virulence so much diminished that they became practically innocuous. When the virulence was much diminished, these observers found that it could be restored if the bacilli were inoculated into animals along with streptococci, inoculation of the bacilli alone not being alien very low, even the presence of the streptococci was n uffic lent to restore it. As a rule, the cultures mos virulent to guinea-pigs are obtained from the gravest cases of diphthena, though to this rule there are LeptLns i° has been abundantly established that after the cure of he disease, the bacilli may persist in the mouth for weeks Zttl°vn q7k,y diSaPPear- R°UX -d ound by making cultures at various stages after the tenmnation of the disease, that these bacilli*,, the mouth 37o DIPHTHERIA. gradually become attenuated. These observations are of importance in relation to the subject of the pseudo- diphtheria bacillus. At present it would scarcely be safe to make a definite statement as regards the relation of virulence to the size of the bacilli. Perhaps the majority of observers have found that the bacilli of the larger form are usually more virulent than those of the shorter form ; but this is not invariably the case, as sometimes short forms are obtained which possess an extremely virulent character. Both the long and the short forms may become attenuated in the same way. The so-called Pseudo-diphtheria Bacillus. — Under this term more than one species of bacillus has been described and considerable confusion has arisen. The name has been applied by some observers to an organism differing from the diphtheria bacillus solely in its want of virulence. Such an organism must be regarded merely as the diphtheria bacillus in an attenuated condition, and should be spoken of as such. On the other hand, there have been cultivated several species of bacilli which resemble the diphtheria bacillus in some respects, but differ from it in certain im- portant points. Some have not the same morphological and staining characters in young cultures, others do not produce an acid reaction in broth containing glucose ; along with these characters the colonies may be whiter and more shining on the surface, or other minor differences in cultures may be present. Such organisms have been cultivated from the throat, both in the healthy condition and in non- diphtheritic affections, rarely in true diphtheria along with the diphtheria bacillus. The term " pseudo-diphtheritic " if used at all should be applied to such organisms, but only as indicating a group with the common character of resemblance to the diphtheria bacillus in certain points, seeing that there are more than one species. Whether some of these may be really diphtheria bacilli modified by certain conditions is a further question which must be con- sidered undecided. The following observations may be mentioned in illustration of these statements. PSEUDO-DIPHTHERIA BACILLUS. 371 Lbffler, in 1887, was the first to describe a bacillus having closely the characters of the diphtheria bacillus, but differing from it in its want of virulence. He looked upon it as a distinct species, and gave it the name of the pseudo-diphtheria bacillus. Hofmann, in 1888, published an account of his investigations on this subject. He obtained the pseudo-diphtheria bacillus from the throat in healthy conditions, and also in non-diphtheritic affections. His conclusions with regard to the distinct character of this bacillus were similar to those of Loffler. Since that time the organism has been the subject of much research and discussion. Roux and Yersin, on the other side, found a " pseudo-diphtheria " bacillus corresponding in all its characters with a greatly attenuated diphtheria bacillus, and concluded that it was really of the same nature. They failed to make it virulent by any method ; but this result was also obtained in the case of artificially attenuated diphtheria bacilli. Biggs has found that there are two varieties of pseudo-diphtheria bacilli, both differing from the true diphtheria bacillus ; one of these produces an acid reaction in broth containing glucose, whilst the other does not. According to his statistics the two varieties appear to occur with about the same frequency, and these observations have been in the main confirmed by Cobbett and Phillips. Hewlett and Knight find evidence that a true diphtheria bacillus may be modified so as to show the microscopic and cultural characters of the pseudo-diphtheria type, this evidence being obtained both by successive examinations of the throat after diphtheria and by modifying cultures artificially. As a rule the appearances of the colonies and the microscopical characters enable a rapid diagnosis to be made in suspected diphtheria cases. In some cases, however, difficulty may be met with ; and in the first place, all the minor cultural characters must be carefully examined, including the reaction produced in broth. By this procedure it may be determined whether the organism in question differs in any points from the diphtheria bacillus. A positive result on inoculating a guinea-pig (say with 1 c.c. of a 24 hours' broth culture) will be conclusive, but we con- sider that for all practical purposes an organism having all the microscopical and cultural characters of the diphtheria bacillus, may be accepted as such. Even if it is non-viru- lent, it is probably only an attenuated diphtheria bacillus. L. Martin, moreover, has recently pointed out that some races of diphtheria bacillus are so attenuated that 1 c.c. of a 24 hours' growth in bouillon does not cause death in a guinea-pig, „, DIPHTHERIA. yet the true nature is shown not only by their microscopical characters, etc., but also by the fact that on more prolonged arowth they form small quantities of toxine, which is neutralised by diphtheria antitoxine. Neisser also, as the result of an extended enquiry, comes to a similar conclusion with regard to the virulence, and considers that the characteristic staining, the morphological characters, and the production of acid in glucose broth, when taken together, afford conclusive evidence as to the identity of the diphtheria bacillus. . The question, however, has a special interest in regard to the origin and spread of the disease. As is well known the disease usually spreads by infection, direct or indirect from patient to patient; but sometimes it appears to start afresh as it were. In the latter case the existence of the non-virulent diphtheria bacilli may afford an explanation of the occurrence, as such bacilli are frequently found even in healthy subjects. For example, Roux and Yersin found the pseudo-diphtheria bacillus in the throats <^**x out of fifty-nine children examined, living in healthy sur ?ound ng If, accordingly, it may become virulent under ^ Circumstances, this may explain the "ce ^f frpsh outbreaks At present, however, we do not know d« S such is the case, still less do we know con- ditions which render it virulent. Diphtheria does not affect the lower animals with the excStfo i of cats, which have sometimes been observed to suffer from a similar disease, in association with human fpfSmics. Klein has found the diphtheria baciUus m the throat of cats in such circumstances. The so-called aipn thena of pigeons, calves, and other animals is produced by entirely different organisms. The term xerosis bacillus has been given to an onanism fiJ observed by Kuschbert and Ne« ^^^'o, SUMMARY OF PATHOGENIC ACTION. 373 according to Eyre, does not produce an acid reaction in neutral bouillon ; in this way it can be distinguished from the diphtheria bacillus. Action of the Diphtheria Bacillus — Summary. — From a study of the morbid changes in diphtheria and of the results produced experimentally by the bacillus and its toxines, the following summary may be given of its action in the body. Locally, the bacillus produces inflammatory change with fibrinous exudation, but at the same time cellular necrosis is also an outstanding feature. Though false membranes have not been produced by the toxines, a necrotic action may result when these are injected sub- cutaneously. The toxines also act upon the blood vessels, and hence oedema and tendency to haemorrhage are pro- duced. This action on the vessels is also exemplified by the general congestion of organs, and sometimes by the occurrence of haemorrhages as in the suprarenal capsules. The hyaline change in the walls of arterioles and capillaries so often met with in diphtheria, is another example of the action of the toxine. The toxines have also a pernicious action on highly-developed cells and on nerve fibres. Thus in the kidney, cloudy swelling occurs, which may be followed by actual, necrosis of the secreting cells, and along with these changes albuminuria is present. The action is also well seen in the case of the muscle fibres of the heart, which may undergo a sort of hyaline change followed by granular disintegration or by an actual fatty degenera- tion. These changes are of great importance in relation to heart failure in the disease. Changes of a somewhat similar nature have been recently observed in the nerve cells of the central nervous system, those lying near the capillaries, it is said, being affected first. There is also the striking change on the peripheral nerves, which is shown first by the disintegration of the medullary sheaths as already described. It is, however, still a matter of dispute whether these nerve lesions are of primary nature or are secondary to changes in the nerve cells. Methods of Diagnosis.— The bacteriological diagnosis 374 DIPHTHERIA. of diphtheria depends on the discovery of the bacillus. As the bacillus occurs in largest numbers in the mem- brane, a portion of this should be obtained whenever it is possible, and transferred to a sterile test-tube. (The tube can be readily sterilised by boiling some water in it.) If, however, membrane cannot be obtained, a scraping of the surface with a platinum loop may be sufficient. Where the membrane is confined to the trachea the bacilli are often present in the secretions of the pharynx, and may be obtained from that situation by swabbing it with cotton wool (non-antiseptic) or by any other means convenient, the swab being put into a sterile tube or bottle for trans- port. The means for identifying the bacillus are (a) By microscopical examination. — For microscopical examination it is sufficient to tease out a piece of the membrane with forceps and rub it on a cover-glass, or if it be somewhat dry a small drop of distilled water should be added. The films are then dried in the usual way and stained with any ordinary basic stain, though methylene-blue is on the whole to be preferred, used either as a saturated watery solution or in the form of Loffier's solution. After staining tor two or three minutes the films are washed in water, dried, and mounted. As a rule no decolorising is necessary, as the blue does not overstain. Any secretion from the pharynx or other part is to be treated in the same way. The value of microscopical examination alone depends much upon the experience of the observer. In some cases the bacilli are present in characteristic form in such numbers as to leave no doubt in the matter. In other cases a few- only may be found, mixed with large quantities of other organisms, and sometimes their characters are not suffi- ciently distinct to render a definite opinion possible. We have frequently obtained the bacillus by means of cultures, when the result of microscopical examination of the same piece of membrane was non-conclusive. As already said, however, microscopical examination alone is more reliable after the observer has had experience in METHODS OF DIAGNOSIS. 375 examining cases of diphtheria and making cultures from them. (b) By making cultures. — For this purpose a piece of the membrane should be separated by forceps from the pharynx or other part when that is possible. It should be then washed well in a tube containing sterile water, most of the surface impurities being removed in this way. A fragment is then fixed in a platinum loop by means of sterile forceps, and a series of stroke cultures are made on the surface of any of the media mentioned, the same por- tion of the membrane being always brought into contact with the surface. The tubes are then placed in the incu- bator at 370 C, and, in the case of the serum media and blood-agar, the circular colonies of the diphtheria bacillus are visible in twenty-four hours. A small portion of a colony is then removed by means of a platinum needle, stained, and examined in the usual way, the characteristic appearance of the organism being readily recognised. In cases where a suspicion arises that the organism found is the pseudo-diphtheria bacillus, a broth containing a trace of glucose should be inoculated and incubated at 3 6° C. The reaction should be tested after one and after two days' growth. If it remains alkaline the diphtheria bacillus may be excluded, but if it becomes acid the organ- ism may still be the so-called pseudo-diphtheria bacillus. All the microscopical and cultural characters must then be carefully observed, and its degree of virulence may be ascertained by inoculating a guinea-pig, say with i c.c. of a broth culture of two days' growth. (See also pp. 370, 3-7*.) CHAPTER XVI. TETANUS.1 SYNONYMS. LOCKJAW. GERMAN, WUNDSTARRKRAMPF. FRENCH, TETANOS. Introductory. — Tetanus is a disease which in natural con- ditions affects chiefly man and the horse. Clinically it is characterised by the gradual onset of general spasms of the voluntary muscles, commencing in those of the jaw and the back of the neck, and extending to all the muscles of the body. These spasms are of a tonic nature, and, as the disease advances, succeed each other with only a slight intermission of time. There are often, towards the end of a case, fever and rise of respiration and pulse rate. The disease is usually associated with a wound received from four to fourteen days previously, and which has been de- filed by earth or dung. Such a wound may be very small. The disease is, in the majority of cases, fatal. Post mortem there is little to be observed on naked eye examination. The most marked feature is the occurrence of patches of congestion in the spinal cord, and especially the medulla. HiBtoricaL-To the pathologist the disease was, till recently a complete mystery ; for, while certain lesions were often met with, they . i This disease is not to be confused with the « « tetany ' ' of infants, which in its essential pathology probably differs from tetanus. I his remark ol course does ^exclude the possibility of the occurrence of true tetanus m very young subjects. BACILLUS TETANL 377 were slight in extent, and no explanation whatever could be given of their occurrence. The general association of the condition with the presence of wounds, suggested that some infection took place through the latter, but nothing was known as to the nature of this infection. Carle and Rattone in 1884 announced that they had produced the disease in a number of animals by inoculation with material from a wound in tetanus. They thus demonstrated the transmissibility of the disease. An important paper by Nicolaier appeared in 1885. This author infected mice and rabbits with garden earth, and found that many of them developed tetanus. Suppuration occurred in the neigh- bourhood of the point of inoculation, and in this pus, besides other organisms, there was always present when tetanus had occurred, a bacillus having certain constant microscopic characters as regards size and staining reaction. Inoculation of fresh animals with such pus reproduced the disease. Nicolaier's attempts at its isolation by the ordinary gelatine plate culture method were, however, unsuccessful. He succeeded in getting it to grow in liquid blood serum, but always in mixture with other organisms. Infection of animals with such a culture produced the disease. These experiments were evidently in- complete, but were confirmed by Rosenbach, who produced the disease in animals by inoculation, and noted the presence of the same bacillus. Though he failed to obtain it in pure culture, he cultivated the other organisms present, and inoculated them with negative results. He further pointed out, as characteristic of the bacillus, its development of terminal spores. In 1889, Kitasato succeeded in isolating from the local suppuration of mice inoculated from a human case, several bacilli, only one of which, when injected in pure culture into animals, caused the disease, and which was now named the B. tetani This organism is the same as that observed by Nicolaier and Rosenbach Kitasato found that the cause of earlier culture failures was the fact that it could only grow in the absence of oxygen. The pathology of the disease was further elucidated by Faber, who, having isolated bacterium-free poisons from cultures, reproduced the symptoms of the disease. Bacillus Tetani.— If in a case of tetanus naturally aris- ing in man, there be a definite wound with pus formation or necrotic change, the bacillus tetani may be recognised in film preparations from the pus, if the characteristic spore formation has occurred (Fig. 95). If, however, the tetanus bacilli have not formed spores, they appear as somewhat slender rods, without presenting any characteristic features. here ,s usually Present in such pus a great variety of other organisms— cocci and bacilli. The characters of the bacillus are, therefore, best studied in cultures. It is then seen to 378 TETANUS. be a slender organism, usually about 4 //. to 5 p in length and .4 [i. in thickness, with somewhat rounded ends. Be sides occurring as short rods it also develops filamentous forms, the latter being more common in fluid media. It 'Mr! t i m Fig q s - Film preparation of discharge from wound in a case of tetanus showing'tveral tetanus bacilli of ■ ■ drumstick " form. The : th.cker bac,, u with oval and not quite terminal spore, m the upper par of the field [towards the right side, is not a tetanus bacillus but a putrefactive anaerobe which was obtained in pure culture from the wound.) Stained with gentian-violet. x 1000. stains readily by any of the usual stains and also by Gram'j method. A feature in it is the uniformity with which the protoplasm stains. It is very slightly motile, and its motility can be best studied in an anaerobic hangmg-dicp preparation (p. 76). When stained by the special method| already described, it is found to possess numerous delicat BACILLUS TETAN1. 379 flagella attached both at the sides and at the ends (Fig. 96). These flagella, though they may be of considerable length, are usually curled up close to the body of the bacillus. The formation of flagella can be best studied in preparations made from surface anaerobic cultures (p. 74)- As is the case with many other anaerobic flagellated Fig. 96. — Tetanus bacilli, showing flagella. Stained by Rd. Muir's method. x 1000.' bacteria the flagella, on becoming detached, often become massed together in the form of spirals of striking ap- pearance (Fig. 97). At incubation temperature B. tetani readily forms spores, and then presents a very characteristic appearance. The spores are round, and in diameter may be three or four times the thickness of the bacilli They are developed at one end of a bacillus, which thus assumes 38° TETANUS. i what is usually described as the drumstick form (Figs. 95, 98). In a speci- f* men stained with a watery solution of gentian-violet or methylene-blue, the spores are un- coloured except at the periphery, so that the appearance of a small ring is produced ; if a powerful stain such as carbol-fuchsin be applied for some time, the spores Fig. 97. —Spiral composed of numerous become deeply twisted flagella of the tetanus bacillus. coloured like the Stained by Rd. Muir's method. x 1000. bacim Further, they may become free in the culture ^ medium. They can - ^ » be stained by the r appropriate methods. _^ \xx *~* \ Isolation. — The ^ ^ 13 — isolation of the te- tanus bacillus is some- what difficult. By r-« "V » \ \ inoculation experi- S ^ V ments in animals, its . / m*~ natural habitat has ( \ been proved to be S . garden soil, and ' especially the con- tents of dung heaps _Tetanus bacilli . some of which where it probably g 9 From a culture in glucose leads a saprophytic agar, incubated for three days at 37 C. existence, though its Stained with carbol-fuchsin. x 1000. ISOLATION OF THE BACILLUS. 38i function as a saprophyte is unknown. From such sources and from the pus of wounds in tetanus, occurring naturally or experimentally produced, it has been isolated by means of the methods appropriate for anaerobic bacteria. The best methods for dealing with such pus are as follows : — (1) The principle is to take advantage of the resistance of the spores of the bacillus to heat. A sloped tube of inspissated serum or a deep tube of glucose agar is inocu- lated with the pus and incubated at 370 C. for forty-eight hours, at the end of which time numerous spore-bearing bacilli can often be observed microscopically. The culture is then kept at 8o° C. for from three-quarters to one hour, with the view of killing all organisms except those which have spored. A loopful is then added to glucose gelatine, and roll-tube cultures are made in the usual way and kept in an atmosphere of hydrogen at 2 2° C. ; after five days the plates are ready for examination. Kitasato compares the colonies in gelatine plates to those of the B. subtilis. They consist of a thick centre with shoots radiating out on all sides. They liquefy the gelatine more slowly than the B. subtilis. This method of isolation is not always successful partly because along with the tetanus bacilli, both in its natural habitats outside the body and in the pus of wounds, other spore-forming obligatory and facultative anaerobes occur, which grow faster than the tetanus bacillus, and thus overgrow it. (2) If in any discharge the spore-bearing tetanus bacilli be seen on microscopic examination, then a method of isolation based on the same principle as the last may be adopted. Inoculations with the suspected material are made in half a dozen deep tubes of glucose agar, previously melted and kept at a temperature of ioo° C. After inocu- lation they are again placed in boiling water and kept for varying times, say for half a minute, for one, three, four five, and six minutes respectively. They are then plunged in cold water till cool, and thereafter placed in the incubator at 370 C, in the hope that in one or other of the tubes all the organisms present will have been killed, except the 382 TETANUS. tetanus spores which can develop in pure culture. Another variation may be adopted by making use of Vignal's method (p. 70) at any stage of the procedure just described. The isolation of the tetanus bacillus is in many cases a difficult matter, and various expedients require to be tried. Characters of Cultures. — Pure cul- tures having been obtained, subcultures can be made in deep upright glucose gelatine or agar tubes. On glucose gela- tine in such a tube there commences, an inch or so below the surface, a growth consisting of fine straight threads, rather longer in the lower than in the upper parts of the tube, radiating out from the needle track (Fig. 99). Slow liquefaction of the gelatine takes place, with slight gas formation. In agar the growth is somewhat similar, consisting of small nodules along the needle track, with irregular short off-shoots passing out into the medium (Fig. 102, a). There is slight formation of gas, but, of course, no liquefaction. Growth also occurs in blood serum and also in glucose bouillon under anaerobic conditions. The latter is the medium usually employed for obtaining the soluble products of the organism. There is in it at first a slight and later a thin layer of a deposit on the walls of the All the cultures give out a burnt odour of rather un- FiG. 99. — Stab culture of the tetanus bacillus in glucose gelatine, showing the lateral shoots (Kita- sato). Natural size. turbidity, powdery vessel, peculiar pleasant character. Conditions of Growth, etc.— The B. tetani grows best at 370 C. The minimum growth temperature is about 140 C, and below 220 C. growth takes place very slowly. Growth takes place only in the absence of oxygen, the organism PA TH0GEN1C EFFECTS. 383 being a strict ancerobe. Sporulation may commence at the end of twenty-four hours in cultures grown at 370 C. — much later at lower temperatures. Like other spores, those of tetanus are extremely resistant. They can usually with- stand boiling for five minutes, and can be kept in a dry condition for many months without being killed, or losing their virulence. They have also high powers of resistance to antiseptics. Pathogenic Effects. — The proof that the B. tetani is the cause of tetanus is complete. It can be isolated in pure culture, and when reinjected in pure culture it reproduces the disease. It may be impossible to isolate it from some cases of the disease, but the cause of this very probably is the small numbers in which it sometimes occurs. (a) The Disease as arising Naturally. — The disease occurs naturally, chiefly in horses and in man. Other animals may, however, be affected. There is usually some wound, often of a ragged character, which has either been made by an object soiled with earth or dung, or which has become contaminated with these substances. There is often purulent or foetid discharge, though this may be absent. Microscopic examination of sections may show at the edges of the wound necrosed tissue in which the tetanus bacilli may be very numerous. If a scraping from the wound be examined microscopically, bacilli resembling the tetanus bacillus may be recognised. If these have spored, there can be practically no doubt as to their identity, as the drumstick appearance which the terminal spore gives to the bacillus is not common among other bacilli. Care must be taken, however, to distinguish it from other thicker bacilli with oval spores placed at a short distance from their extremities, such forms being common in earth, etc., and also met with in contaminated wounds (Fig. 95). 'it is important to note that the wound through which infection has taken place may be very small, in fact, may consist of a mere abrasion. In some cases, especially in the tropics, it may be merely the bite of an insect. The absence of a definite channel of infection has given rise to the term 3»4 7 ETA N US. " idiopathic " tetanus. There is, however, practically no doubt that all such cases are true cases of tetanus, and that in all of them the cause is the B. tetani. The latter has also been found in the bronchial mucous membrane in some cases of the so-called rheumatic tetanus, the cause of which is usually said to be cold. The pathological changes found post mortem are not striking. There may be haemorrhages in the muscles which have been the subject of the spasms. These are probably due to mechanical causes. Naturally it is in the nervous system that we look for the most important lesions. Here there is ordinarily a general redness of the grey matter, and the most striking feature is the occurrence of irregular patches of congestion which are not limited particularly to grey or white matter, or to any tract of the latter. These patches are usually best marked in the grey matter of the medulla and pons. Microscopically there is little of a definite nature to be found. There is congestion, and there may be minute haemorrhages in the areas noted by the naked eye. The ganglion cells may show appearances which have been regarded as degenerative in nature, and similar changes have been described in the white matter. The only marked feature is thus a vascular disturbance in the central nervous system, with a possible tendency to degeneration in its specialised cells. Both of these conditions are probably due to the action of the toxines of the bacillus. In the case of the cellular degenerations the cells have been observed to return to the normal under the curative influence of the antitoxines (v. infra). In the other organs of the body there are no constant changes. We have said that the general distribution of pathogenic bacteria throughout the body is probably a relative pheno- menon, and that bacteria usually found locally may occur generally and vice versa. With regard to the tetanus bacillus it is, however, probably the case that very rarely, ifi ever, are the organisms found anywhere except in the local lesion. (b) The Artificially-produced Disease.— -The disease can EXPERIMENTAL INOCULATION. 385 be communicated to animals by any of the usual methods of inoculation, but does not arise in animals fed with bacilli whether these contain spores or not. Kitasato found that pure cultures, injected subcutaneously or intravenously, caused death in mice, rats, guinea-pigs, and rabbits. In mice, symptoms appear in a day, and death occurs in two or three days, after inoculation with a loopful of a bouillon cul- ture. The other animals mentioned require larger doses, and death does not occur so rapidly. The symptoms generally are those of the natural disease, the spasms beginning in the muscles nearest the site of inoculation. After death there is found slight hyperemia without pus formation, at the seat of inoculation. The bacilli diminish in number, and may be absent at the time of death. The organs generally show little change. Kitasato acknowledges that in these earlier experiments the quantity of culture medium injected along with the bacilli, already contained enough of the poisonous bodies formed by the bacilli to cause death. The symptoms came on sooner than by the improved method mentioned below, and were, therefore, due to the toxines already present. In his subsequent work, therefore, he employed splinters of wood soaked in cultures in which spores were present, and subsequently subjected for one hour to a temperature of 8o° C. The latter treatment not only killed all the bacilli, but, as we shall see, was sufficient to destroy the activity of the toxines. When such splinters are introduced subcutan- eously, death results by the development of the spores which they carry. In this way he completed the proof that the bacilli by themselves can form toxines in the body and produce the disease. Further, if a small quantity of garden earth be placed under the skin of a mouse, death from tetanus takes place in a great many cases. [Sometimes, however, in such circumstances death occurs without tetanic symptoms, and is not due to the tetanus bacillus but to the bacillus of malignant oedema, which also is of common occurrence in the soil (v. infra).] By such experiments, supplemented by the culture experiments mentioned the 25 386 TETANUS. natural habitats of the B. tetani, as given above, have become known. The Toxines of the Tetanus Bacillus. — The tetanus bacillus being thus accepted as the cause of the disease, we have to consider how it produces its pathogenic effects. Almost contemporaneously with the work on diphtheria was the attempt made with regard to tetanus to explain the general symptoms by supposing that the bacillus could excrete soluble poisons. Brieger, for instance, in his earlier work recorded that a base tetanin could be isolated from dead cultures, and this, as well as another base called tetanotoxin, was also obtained by Kitasato and Weyl. When injected into animals, these substances produced spasms and death, but though they may have contained the real toxine they were obtained by the earlier faulty methods. In 1890 Brieger and Fraenkel announced that they had isolated a toxalbumin from tetanus cultures, and this body was independently discovered by Faber in the same year. Brieger and Fraenkel's body consisted practically of an alcoholic precipitate from filtered culture in bouillon, and was undoubtedly toxic. The toxic properties of bacterium- free filtrates of pure cultures of the B. tetani were in- vestigated in 1 89 1 by Kitasato. He found that when the filtrate, in certain doses, was injected subcutaneously or intravenously into mice, tetanic spasms developed, first in muscles contiguous to the site of inoculation and later all over the body. Death resulted. He found that guinea- pigs were more susceptible than mice, and rabbits less so. In order that a strongly toxic bouillon be produced, it must originally have been either neutral or slightly alkaline. Kitasato further found that the toxine was easily injured by heat. Exposure for a few minutes at 650 C. destroyed it. It was also destroyed by twenty minutes' exposure at 60 L. and by one and a half hours' at 55° C. Drying had no effect. It was, however, destroyed by various chemicals such as pyrogallol and also by sunlight. Behring has more recently pointed out that after the filtration of cultures containing toxine, the latter may very rapidly ose i s power, and in a few days may only possess xtnrth or us THE TOXINES OF THE BACILLUS. 387 original toxicity. This he attributes to such factors as temperature and light, and especially to the action of oxygen. Various attempts have been made to find out the nature of this toxine. Sidney Martin derived from the organs of persons dead of tetanus two classes of bodies. One of these consisted of a purified alcoholic precipitate (formed chiefly of albumoses). To these he attributes a fever-producing action. The other bodies were those soluble in alcohol and also in ether. They were non-proteid, and to them he attributed the excitation of the muscular spasms in tetanus. Uschinsky, moreover, has found that the bacillus can pro- duce its toxine when growing in a fluid containing no proteid matter. The toxine may thus be formed independ- ently of the breaking up of the proteids on which the bacillus may be living, though it no doubt has a digestive action on these. Brieger also has now apparently come to the conclusion that the toxicity of the toxalbumins originally described by him is due to the presence of a non-proteid body. In his latest paper he describes the isolation, by a special method, of a toxine which is neither peptone, albumin, nor albuminate, and the nature of which is quite unknown. It is thought by some that a diastase is concerned in the toxic action of the tetanus bacillus. Like a ferment, the toxine is destroyed, as we have seen, by comparatively low temperatures, but it may simply be an unstable chemical compound, for albuminous bodies not diastatic in nature may be changed at similar temperatures. The liquefaction (i.e., probable peptonisation) of gelatine cultures advances pari passu with the development of toxines, and filtered bacterium-free cultures will still liquefy gelatine. It may be, however, that there is developed, in addition, a peptic ferment which will, of course, also pass through the filter. For if equal portions of the filtered culture be left in contact with equal portions of gelatine for various lengths of time, there is no increase of toxicity in those kept longest. There is thus no fresh development of toxine 388 TETANUS. during the advancing liquefaction of the gelatine. Thus peptic digestion and toxine formation are apparently due to different vital processes on the part of the tetanus bacillus.' A strong though not conclusive argument in favour of a ferment being concerned in the toxine production, is derived from the occurrence of a definite incubation period between the introduction of the toxine into an animal's body and the appearance of symptoms. The incubation period varies according to the species of animal employed, and the path of infection. In the guinea-pig it is from thirteen to eighteen hours, in the horse five days, and the incubation is shorter when the poison is introduced into a vein than when injected subcutaneously. The interpreta- tion put on the occurrence of this period of incubation by the upholders of the ferment theory has been that a time is required for the supposed diastase to elaborate from the tissues albumoses, which are the immediately toxic agents. Whatever the nature of the toxine is, it is undoubtedly one of the most powerful poisons known. Even with his probably impure toxalbumin Brieger found that the fatal dose for a mouse was .0005 of a milligramme, if the susceptibility of man be the same as that of a mouse, the fatal dose for an average adult would be .23 of a milli- gramme or about booths of a grain. With regard to the action of the toxine it has been shown to have no effect on the sensory or motor endings of the nerves It acts solely as an exciter of the reflex excitability of the' motor cells in the spinal cord. The motor cells in the pons and medulla are also affected, and to a much greater degree than those in the cerebral cortex. When injected subcutaneously the toxine probably to a certain extent is absorbed into the sheaths of the nerves and thence finds its way to that part of the spinal cord from which these nerves spring. This explains the fact that in an animal the tetanic spasms appear first in the muscles of the part in which the inoculation has taken place It is doubtful whether such absorption takes place in tetanus THE TOXINES OF THE BACILLUS. 389 arising naturally. In artificial injection of toxine part finds its way into the blood stream, and if infected animals be killed during the incubation period there is often evidence of toxine in the blood and solid organs. Rarely, however, during this period, and probably never after symptoms have begun, is there free toxine in the central nervous system. A possible explanation of this will be discussed in the chapter on Immunity. If tetanus toxine be introduced into the stomach or intestine, it is not absorbed. It to a large extent passes through the intestine unchanged. Evidence that any destruction takes place is wanting. There is one question which must arise in connection with tetanus, namely : Granted that the B. tetani is so widely present in the soil, how is it that the disease is not more common than it is, for wounds must constantly be contami- nated with such soil ? Experiments by Vaillard throw light on this point. We have seen that unless suitable precau- tions are adopted, in experimental tetanus in animals death results not from inoculation but from an intoxication with toxine previously existent in the fluid in which the bacilli have been growing. According to Vaillard, if spores rendered toxine-free, by being kept for a sufficient time at 8o° C, are injected into an animal, death does not take place. It was found, however, that such spores can be rendered pathogenic by injecting along with them such chemicals as lactic acid, by injuring the point of inoculation so as to cause effusion of blood, by fracturing an adjacent bone, by introducing a mechanical irritant such as soil or a splinter of wood (as in Kitasato's experiments), or by the simultane- ous injection of other bacteria such as the staphylococcus pyogenes aureus. These facts, especially the last, throw great light on the disease as it occurs naturally, for tetanus results especially from wounds which have been accidentally subjected to conditions such as those enumerated Kita sato now holds that in the natural infection in man, along with tetanus spores, the presence of foreign material or of other bacteria is necessary. Spores alone or tetanus bacilli without spores die in the tissues, and tetanus does not result 39o TETANUS. Summary. — In view of all the facts available we must thus look on tetanus as caused by the B. tetani. The bacillus gains entrance to the body through wounds or abrasions, and, multiplying locally, produces poisons which diffuse into the tissues and have an elective action as stimu- lants especially of the spinal cord. The chemical composi- tion of these poisons is not yet fully known. The enormous potency of such poisons explains how, even in a fatal case, extreme smallness of the wound and difficulty in isolating the bacillus do not detract from the theory that the latter is the cause of the disease. Immunity against Tetanus. — Antitetanic Serum. — The artificial immunisation of animals against tetanus has received much attention. The most complete study of the question is found in the work of Behring and Kitasato m Germany, and of Tizzoni and Cattani in Italy. The former observers found that such an immunity could be conferred by the injection of very small and progressively increasing doses of the tetanus toxine. The degree of immunity attained, however, was not high. More successful was the method of accompanying the early injections of such toxine with the subcutaneous introduction of small doses of iodine terchloride. Tizzoni and Cattani have also used the method of administering progressively increasing doses of living cultures attenuated in various ways, e.g., by heat. By any of these methods susceptible animals can rapidly acquire great immunity, not only against many times the fatal dose of tetanic toxine, but also against injections of the living bacilli. The degree of immunisation acquired by an animal remains in existence for several months. Mot only so, but the injection of the serum of such immune animals can protect susceptible animals against the subse- quent infection with a fatal dose of tetanus bacilli or toxine. Further, if injected subsequently to such infection, the serum can in certain cases prevent a fatal result, even when symptoms have begun to appear. The degree °f success attained depends, however, on the shortness of the time which has elapsed between the infection with the bacilli ANTITETANIC SERUM. 391 or toxine and the injection of the serum. The longer the interval which is allowed to elapse, not only the greater must be the dose of the serum but the less likely is cure to occur. In animals, where symptoms have fully manifested themselves only a small proportion of cases can be saved. As in other cases, there is no evidence that the antitetanic serum has any detrimental effect on the bacilli. It only neutralises the effects of the toxine. The standardisation of the antitetanic serum is of the highest importance. Behring recommends that for protecting animals a serum should be obtained of which one gramme will protect 1,000,000 grammes weight of mice against the minimum fatal dose of the bacillus or toxine. A mouse weighing twenty grammes would thus require .00002 gramme of such a serum to protect it against the minimum lethal dose. In the injection of such a serum subsequent to infection, if symptoms have begun to appear, 1000 times this dose would be necessary j a few hours later 10,000 times, and so on. As the result of his experiments, Behring aimed at obtain- ing a curative effect in the natural disease occurring in man. For this purpose, as for his later laboratory experiments, he obtained serum by the immunisation of such large animals as the horse, the sheep, and the goat. The prin- ciples of the process were the same as in his earlier work, namely, the injection of toxine, accompanied at first with the injection of iodine terchloride. It was found that the greater the degree of the natural susceptibility of an animal to tetanus, the easier was it to obtain a serum of a high antitetanic potency. The horse was, therefore, the most .suitable animal. If now we take for granted that the relative susceptibility of man and the mouse towards tetanus are nearly equal, a man weighing 100 kilogrm. would require .1 grm. of the serum mentioned above, to protect him from inoculation with the minimum lethal dose of bacilli or toxine. If symptoms had begun to appear, 100 cc. at once would be necessary, and as the injection of such a quantity might be inconvenient, Behring recom- mended that for man a more powerful serum should be 392 TETANUS. obtained, viz., a serum of which one gramme would protect 100,000,000 grammes weight of mice.1 The potency is maintained for several months if precautions are taken to avoid putrefaction, exposure to bright light, etc. To this end .5 per cent carbolic acid is usually added. In a case of tetanus in man, 100 c.c. of such a serum should be injected within twenty-four hours in five doses, each at a different part of the body, and this followed up by further injections if no improvement takes place. Many cases of human tetanus have been thus treated, but with only a small measure of success. The improvement in the death-rate has not been nearly so marked as that which has occurred in diphtheria under similar circumstances. As in the case of diphtheria, however, the results would probably be better if more attention were paid to the dosage of the serum. The great difficulty is that, as a matter of fact, we have not the opportunity of recognising the presence of the tetanus bacilli till they have begun to manifest their gravest effects. In diphtheria we have a well-marked clinical feature which draws attention to the probable presence of the bacilli — a presence which can be readily proved, — and the curative agent can thus be early applied. In tetanus, the wound in which the bacilli exist may be, as we have seen, of the most trifling character, and even when a well-marked wound exists, the search for the bacilli is a matter of difficulty. Still, it might be well, when practicable, that every ragged, unhealthy- looking wound, especially when contaminated with soil, should, as a matter of routine, be examined bacteriologically. In such cases, undoubtedly, from time to time cases of tetanus would be detected early, and their treatment could be undertaken with more hope of success than at present. However, in the existing state of matters, whenever the first symptoms of tetanus appear, large doses, such as those above indicated, of a serum whose strength is known, should be at once administered. In giving a prognosis as to the jprob- 1 The antitetanic serum sent out by the Pasteur Institute in Paris has a strength of i : 1,000,000,000. Of this it is recommended that 50 to 100 c.c. should be injected in one or two doses. METHODS OF EXAMINATION. 393 able result, the two clinical observations on which, accord- ing to Behring, chief reliance ought to be placed, are the presence or absence of interference with respiration, and the rapidity with which the groups of muscles usually affected are attacked. If dyspnoea or irregularity in respiration comes on soon, and if group after group of muscles is quickly involved, then the outlook is extremely grave. Of the nature of the antitoxine of tetanus we know little. It is not affected by heat, light, or atmospheric conditions. Brieger and Boer state that they have isolated it from the serum by the methods used to obtain the toxine. The theory as to the nature of antitoxic action will be discussed later in the chapter on Immunity. Here it need only be noted that Wassermann and others have found that there exist, in the nervous system of animals susceptible to tetanus, bodies which seem to neutralise the action of tetanus toxine. Thus i c.c. of an emulsion made by rub- bing up the nervous system of a mouse in water will neutralise an amount of toxine equal to ten times the minimum lethal dose for a mouse if the two substances be mixed together in vitro and injected. Methods of Examination in a case of Tetanus. — The routine bacteriological procedure in a case presenting the clinical features of tetanus ought to be as follows : — (a) Microscopic. — Though tetanus is not a disease in which the discovery of the bacilli is easy, still microscopic examination should be undertaken in every case. From every wound or abrasion from which sufficient discharge can be obtained, film preparations ought to be made and stained with any of the ordinary combinations, e.g., carbol- fuchsin diluted with five parts of water. Drumstick-shaped spore-bearing bacilli are to be looked for. The presence of such, having characters corresponding to those of the tetanus bacilli, though not absolutely conclusive proof of identifica- tion, is yet sufficient for all practical purposes. If only bacilli without spores, resembling the tetanus bacilli, are seen, then the identification can only be provisional. 394 MALIGNANT (EDEMA. The microscopic examination of wounds contaminated by soil, etc., may, as we have said in some cases, lead to the anticipation that tetanus will probably result. (/>) Cultivation. — The methods to be employed in isolating the tetanus bacilli have already been described (p. 381). It may be added, however, that if the character- istic forms are not seen on microscopic examination of the material from the wound, they may often be found by inoculating a deep tube of one of the glucose media with such material, and incubating for forty-eight hours at 37° C. At the end of this period, spore-bearing tetanus bacilli may be detected microscopically, though of course mixed with other organisms. (c) Inoculation. — Mice and guinea-pigs are the most suitable animals. Inoculation with the material from a wound should be made subcutaneously. A loopful of the discharge introduced at the root of the tail in a mouse will soon give rise to the characteristic symptoms, if tetanus bacilli are present. Malignant CEdema (Septicemic de Pasteur). The organism now usually known as the bacillus of malignant oedema is the same as that first discovered by Pasteur and named vibrion septique. He described its characters, distinguishing it from the anthrax bacillus which it somewhat resembles morphologically, and also the lesions produced by it. He found that it grew only in anaerobic conditions, but was able to cultivate it merely in an impure state. It was more fully studied by Koch, who called it the bacillus of malignant oedema, and pointed out that the disease produced by it is not really of the nature of a septicemia, as immediately after death the blood is practically free from the bacilli. " Malignant oedema " in the human subject is usually described as a spreading inflammatory oedema attended with emphysema, and ultimately followed by gangrene ot CHARACTERS OF THE BACILLUS. 395 the skin and subjacent parts. In many cases of this nature the bacillus of malignant oedema is present, associated with other organisms which aid its spread, whilst in others it may be absent. One of us has, however, recently observed a case in which the bacillus was present in pure condition. Here there occurred intense oedema with swelling and in- duration of the tissues, and the formation of vesicles on the skin. Those changes were attended with a reddish dis- coloration, afterwards becoming livid. Emphysema was not recognisable until the limb was incised, when it was detected though in small degree. Further, the tissues had a peculiar heavy but not putrid odour. The bacillus, which was obtained in pure culture, was present in enor- mous numbers in the affected tissues, attended by cellular necrosis, serous exudation, and at places much leucocytic emigration. The picture, in short, corresponded with that seen on inoculating a guinea-pig with a pure culture. The term " malignant oedema " should be limited in its applica- tion to cases in which the bacillus in question is present. In most of these there is a mixed infection ; in some this bacillus may be present alone. This bacillus has a very widespread distribution in nature, being present in garden soil, dung, and various putrefying animal fluids; and it is by contamination of lacerated wounds by such substances that the disease is usually set up in the human subject. Malignant oedema can be readily produced by inoculating susceptible animals, such as guinea-pigs, with garden soil. The bacillus is also often present in the intestine of man and animals, and has been described as being present in some gangrenous conditions originating in connection with the intestine in the human subject. Microscopical Characters. — The bacillus of malignant oedema is a comparatively large organism, being slightly less than t in thickness, that is, thinner than the anthrax bacillus. It occurs in the form of single rods 3 ^ to 10 jx in length, but both in the tissues and in cultures in fluids it frequently grows out into long filaments, which may be 396 MALIGNANT (EDEMA. uniform throughout or segmented at irregular intervals. In cultures on solid media it chiefly occurs in the form of shorter rods with somewhat rounded ends. The rods are motile, possessing several laterally placed flagella, but in a given specimen, as a rule, only a few bacilli show active \ ' \ I < I * i I mam \ Fig. ioo.— Film preparation from the affected tissues in a case of malignant oedema in the human subject, showing the spore-bearing bacilli. Gentian-violet. x 1000. movement. Under suitable conditions they form spores which are usually near the centre of the rods and have an oval shape, their thickness somewhat exceeding that of the bacillus (Figs, i oo, i o i ). The bacillus can be readily stained by any of the basic aniline stains, but loses the colour in Gram's method, in this way differing from the anthrax bacillus. CHARACTERS OF CULTURES. 397 Characters of Cultures. — This organism grows readily at ordinary temperature, but only under ancerobic conditions. In a puncture culture in a deep tube of glucose gelatine, the growth appears as a whitish line giving off minute short processes, the growth, of course, not reaching the surface of the medium. Soon liquefaction occurs, and a long fluid funnel is formed, with turbid contents and flocculent masses of growth at the bottom. At the same time bubbles of gas are given off, which may split up the gelatine. The colonies in gela- tine plates under anae- robic conditions appear first as small whitish points which under the microscope show a radiating appearance at the periphery, re- sembling the colonies of the bacillus subtilis. Soon, however, liquefaction occurs around the colonies, and spheres with turbid contents result • gas is developed around the colonies. In deep tubes of glucose agar at 37° C, growth is ex- tremely rapid. Along the line of puncture, growth appears as a somewhat broad white line with short lateral projec- tions here and there (Fig. 102, b). Here also gas may be formed, but this is most marked in a shake culture, in which the medium becomes cracked in various directions, and may be pushed upwards so high as to displace the cotton- wool plug. The cultures possess a peculiar heavy, though not putrid, odour. Spore formation occurs above 200 C, and is usually well seen within forty-eight hours at 370 C. The spores Fig. ioi. — Bacillus of malignant oedema, showing spores. From a culture in glucose agar, incubated for three days at 370 C. Stained with weak carbol-fuchsin. x 1000. 398 MALIGNANT CEDEMA. have the usual high powers of resistance, and may be kept for months in the dried condition without being killed. Experimental Inoculation. — A considerable number of animals — the guinea-pig, rabbit, sheep, and goat, for ex- A B C Fig. 102.— Stab cultures in agar, five days' growth at 370 C. Natural size. A. Tetanus bacillus. B. Bacillus of malignant cedema. C. Bacillus of quarter-evil (Rauschbrand). ample— are susceptible to inoculation with this organism. The ox is said to be quite immune to experimental inocu- lation, though it can, under certain conditions, contract the disease by natural channels. The guinea-pig is the animal most convenient for experimental inoculation. When the disease is set up in the guinea-pig by sub- cutaneous inoculation with garden soil, death usually occurs EXPERIMENTAL INOCULATION. 399 in about twenty-four to forty-eight hours. There is an intense inflammatory oedema around the site of inoculation, which extends over the wall of the abdomen and thorax. The skin and subcutaneous tissue are infiltrated with a reddish-brown fluid and softened ; they contain bubbles of gas and are at places gangrenous. The superficial muscles are also involved. These parts have a very putrid odour. The internal organs are congested, the spleen soft but not much enlarged. In such conditions the bacillus of malig- nant oedema, both in short and long forms, will be found in the affected tissues along with various other organisms. Spores may be present, especially when the examination is made some time after the death of the animal. If the animal is examined immediately after death, a few of the bacilli may be present in the peritoneum and pleura?, usually in the form of long motile filaments, but they are almost invariably absent from the blood. A short time after death, however, they spread directlyinto the blood and various organs, and may then be found in considerable numbers. Subcutaneous inoculation with pure cultures of the bacillus of malignant oedema produces chiefly a spreading bloody oedema, the muscles being softened and partly necrosed; but there is little formation of gas, and the putrid odour is almost absent. When the bacilli are injected into mice, however they enter and multiply in the blood stream, and they are found in considerable numbers in the various organs, so that a condition not unlike that of anthrax is found. The spleen also is much swollen. The virulence of the bacillus of malignant oedema vanes considerably in different cases, and it always be- comes diminished in cultures grown for some time To produce a fatal disease, a relatively large number of the organisms is necessary, and these must be introduced deeply int0 the tissues> inoculation by scarification b . followed by no result. A smaller dose produces a fatal esul when injected along with various other organisms (bacillus prodigiosus, etc.). 400 MALIGNANT (EDEMA. Immunity. — Malignant oedema was one of the first diseases against which immunity was produced by injec- tion of toxines. The filtered cultures of the bacillus in sufficient doses produce death with the same symptoms as those caused by the living organisms, but a relatively large quantity is necessary. Chamberland and Roux (1887) found that if guinea-pigs were injected with several non-fatal doses of cultures sterilised by heat or freed from the bacilli by filtration, immunity against the living or- ganism could be developed in a comparatively short time. They found that the filtered serum of animals dead of the disease is more highly toxic, and also gives immunity when injected in small doses. These experiments have been confirmed by Sanfelice. Methods of Diagnosis.— In a case of supposed malignant oedema, the fluid from the affected tissues ought first to be examined microscopically, to ascertain the characters of the organisms present. Though it is not possible to identify absolutely the bacillus of malignant oedema without culti- vating it, the presence of spore-bearing bacilli with the characters described above is highly suspicious (Fig. 100). In such a case the fluid containing the bacilli should be first exposed to a temperature of 8o° C. for half an hour, and then a deep glucose agar tube should be inoculated. ^ In this way the spore-free organisms are killed off. Pure cultures may be thus obtained, or this procedure may require to be followed by the roll tube method under anaerobic conditions. An inoculation experiment, if available, may also be made on a guinea-pig. Quarter-evil (German, Rauschbrand ; French, Charbon Symptomatique). The characters of the bacillus need be only briefly describee 1 as so far as is known, it never infects the human subject. I he natmal disease which "spec ally occurs in certain localities, affects chiefly sheep, Srie and eoat Infection takes place by some wound of the surface] 2fS£f5£ in the regie n -nd inflamn^tory^^ attend^ ^tol^r^Xl't SET Hence the QUARTER- EVIL. 401 name 'blackleg" by which the disease is sometimes known. The baallus which produces this condition is present in large numbers in the affected issues, associated with other organisms, and also occurs in small numbers in the blood of internal orgkns The bacillus morphologically closely resembles that of malignant of p-mwfli ! tGr> alS°' U is a Stn'Ct anferobe> and ils conditions ot growth as regards tem- perature are also similar. It is, however, somewhat thicker, and does not usually form such long filaments. Moreover the spores, which are of oval shape and broader than the bacillus, are almost invariably situated close to one extremity, though not actually terminal (Fig. 103). The characters of the cultures, also, resemble those of the bacillus of malignant oedema, but in a stab culture in glucose ' agar there are more numerous and longer lateral off-shoots, the growth being also more luxuriant (Fig. 102, c). This bacillus is actively motile, and possesses numerous lateral flagella. The disease can be readily produced in various animals e sr Snl" ^ ^ rcuIation wiLh the affected tissues of diseased amrn 'orltn y-meann°f PU1'e CUltUreS' th°U^ a considerable amount of the latter is usually necessary. The condition produced in ^ w^yclosel y resembles that in malignant oedema, though there is said to be more formation of gas in the tissues. Rabbits are practically ?S5S££ disease' whiist they are c— * The disease is one against which immunity can be readily produced m various ways, and methods of preventive inoculation ha^e been adopted in the case of animals liable to suffer from it. This subject was Serf y rked r by r°ing' cornevin' and dose of !mmumty ™y be. Produced by injection with a non-fata^ ^l:i aVAUeY\ 7 inJGCti0nTwhh J?** quantities of the viru ducts fJ * I n ' C!rying' £tC- 11 Can be P^duced also by the pro- ducts of the bacilli obtained by filtration of cultures. 1 26 Fig. 103.— Bacillus of quarter-evil, show- ing spores. From a culture in glucose agar, incubated for three days at 37° C. Stained with weak carbol-fuchsin. x 1000. CHAPTER XVII. CHOLERA. Introductory. — It is no exaggeration of the facts to say that previously to 1883 practically nothing of value was known regarding the nature of the virus of cholera. In that year Koch was sent to Egypt, where the disease had broken out, in charge of a commission for the purpose of investigating its nature. In the course of his researches he discovered the organism now generally known as the "comma bacillus" or the "cholera spirillum." He found this organism in the discharges from the intestine, and also post mortem in the intestinal contents and in certain parts of the intestinal mucous membrane. Later he made more extensive observations in India, and also investigated two cases at Toulon, nearly 100 cases in all being examined, and came to the conclusion that the association of this organism with the disease was constant. The organism, moreover, was one which was quite unknown before, and numerous observations made in other diseased conditions failed to show its presence. He also obtained pure cultures of the organism from a large number of cases of cholera, and described their characters. The results of his re- searches were given at the first Cholera Conference at Berlin in 1884. The general conclusions at which Koch arrived received, in the main, confirmation from the in- vestigations of others, though some criticism arose, especially THE CHOLERA SPIRILLUM. 403 as regards the uniformity of the characters of the comma bacillus. Within recent years, and especially during the epidemic in Europe in 1892-93, spirilla have been cultivated from cases of cholera in a great many different localities, and though this extensive investigation has revealed the invari- able presence in true cholera of organisms resembling more or less closely Koch's spirillum, certain difficulties have arisen. For it has been found that the cultures obtained from different places have shown considerable variations in their characters, and, further, spirilla which closely resemble Koch's cholera spirillum have been culti- vated from sources other than cases of true cholera. There has therefore been much controversy, on the one hand, as to the signification of these variations — whether they con- stitute different species, or whether they are to be regarded merely as indicating varieties of the same species — and on the other hand as to the means of distinguishing the cholera spirillum from other species which resemble it. We shall first give an account of the characters of the cholera spirillum, with the evidence for its causal relation- ship to the disease, and afterwards discuss some of the questions just referred to. It may, however, be stated here that no other organism of any kind has been dis- covered which has even the faintest claim to be the cause of the disease. In considering the bacteriology of cholera it is to be borne in mind that in this disease, in addition to the evi- dence of great intestinal irritation, accompanied by profuse watery discharge, and often by vomiting, there are also symptoms of general systemic disturbance which cannot be accounted for merely by the withdrawal of water and certain substances from the system. Such symptoms include the profound general prostration, cramps in the muscles, extreme cardiac depression, the cold and clammy condition of the surface, the subnormal temperature, suppression of urine etc. These taken in their entirety are indications of a general poisoning in which the circulatory and thermo- CHOLERA. regulatory mechanisms are specially involved. In some, though rare, cases known as cholera sicca, general collapse occurs with remarkable suddenness, and is rapidly followed by a fatal result, whilst there is little or no evacuation from the bowel, though post mortem the intestine is distended with fluid contents. As the characteristic organisms in cholera are found only in the intestine, the general dis- turbances are to be regarded as the result of toxic sub- stances absorbed from the bowel. It is also to be noted that cholera is a disease of which the onset and course are much more rapid fCt'Sj *«Jf**^Xi - than is tne case £x ^ T . ' in most infective aV-*V$ -xl *y diseases, such as >P* r&fX-* \ w» typhoid and diph- 7 <7 " theria^ and that t-C ° 1 c^ x "sL~ recovery also, when ^t^VLCL it takes place, does " ? so more quickly. The two factors to be co-related to these facts are (a) a ^j/Pv^iT^ rtjr rapid multiplication J ^ r of organisms, {b) the ' production of rapidly Fig. 104.— Cholera spirilla, from a culture acting toxines. on agar of twenty-four hours' growth. p>,nlpra Smril- Stained with weak carbol-fuchsin. x 1000. The CHolera bpiTll lum. Microscopical Characters.— The cholera spirilla as found in the intestines in cholera are small organisms measuring about 1.5 to 2 fi in length, and rather less than .5 in thickness. They are distinctly curved m one direc- tion, hence the appearance of a comma (Fig. 104); most occur singly, but some are attached in pairs and curved L opposite directions, so that an S-shape results. Longer forms are rarely seen in the intestine, but in cultures in fluids as is especially well seen in hanging-drop prepara- tions'they may grow into longer spiral filaments, showing THE CHOLERA SPIRILLUM. 4°5 a large number of turns. If film preparations be made from the intestinal contents in typical cases, it will be found that these organisms are present in enormous numbers in almost pure culture, and that most of the spirilla lie with their long axis in the same direction, so as to give the appearance which Koch compared to a number of fish in a stream. They possess very active motility, which is most marked in the single forms. When stained by the suitable methods they are seen to be flagellated. Usually a single terminal flagellum is present at one end only I (Fig. 105). It is very delicate, and measures four or five times the length of the organism. In some varieties, however, there may be such a flagellum at both ends, and again, more than one may be present. Cultures obtained at different places have shown considerable varia- tions in this respect. Cholera spirilla do not form spores. In old cultures, how- ever, small rounded and highly refractile bodies may be found in the organisms, which have been regarded by Hueppe as "arthrospores," but which are in reality merely the result of degeneration, as they have no higher powers of resistance than the spirilla themselves, and cultures containing enormous numbers of such bodies may be found to be quite dead. Along with such appearances in old cultures there is found great change in the size and shape of the organisms (Fig. 106). Some are irregularly twisted filaments, sometimes globose, sometimes Fig. 105. —Cholera spirilla stained to show the terminal flagella. x iooo. 406 CHOLERA. clubbed at their extremities, and also showing irregular swellings along their course. Others are short and thick, and may have the appearance of large cocci, often staining faintly. All these changes in appear- ance are to be classed together as involution forms. Staining. — Cholera spirilla stain readily with the usual basic aniline stains, though Loffler's methylene - blue or weak carbol- fuchsin FiG. 106.— Cholera spirilla from an old is specially suitable, agar culture, showing irregularities in size and ^, ]oSQ the stain _i „,;*v. T,.impmii<; fnintlv-stained coccoid . ~ , j in Gram s method. Distribution within the Body. — The chief fact in this connection is that the spirilla are confined to the intestine, and are not present in the blood or internal organs. This was determined by Koch in his earlier work, and his statement has been amply confirmed. In cases in which there is the characteristic "rice-water" fluid in the intestines, they occur in enormous numbers— almost in pure culture. The lower half of the small intestine is the part most affected. Its surface epithelium becomes shed in great part, and the flakes floating in the fluid consist chiefly of masses of epithelial cells and mucus, amongst which are numerous spirilla The spirilla also penetrate the follicles of Lieber- kiihn, and may be seen lying between the basement mem- brane and the epithelial lining, which becomes loosened by their action. They are, however, rarely found m the connective tissue beneath, and never penetrate deeply. shape, with numerous faintly-stained coccoid bodies — involution forms. Stained with fuchsin. x iooo CULTIVATION. 407 Along with these changes there is congestion of the mucosa, especially around the Peyer's patches and solitary glands, which are somewhat swollen and prominent. In some very acute cases the mucosa may show general acute congestion with a rosy pink colour but very little desquamation of epi- thelium, the intestinal contents being a comparatively clear fluid containing the spirilla in large numbers. In other cases of a more chronic type, the intestine may show more extensive necrosis of the mucosa and a considerable amount of haemorrhage into its substance, along with formation of false membrane at places. The intestinal contents in such cases are blood-stained and foul-smelling, there being a great proportion of other organisms present besides the cholera spirilla (Koch). Cultivation. — (For Methods, see p. 425). The cholera spirillum grows readily on all the ordinary media, and with the exception of that on potato, growth takes place at the ordinary room 'temperature. The most suitable temperature, however, is that of the body, and growth usually stops about 160 C, though in some cases it has been obtained at a lower temperature. Peptone gelatine. — On this medium the organism grows well and produces liquefaction. In puncture cultivations at 220 C. a whitish line appears along the needle track, at the upper part of which liquefaction commences, and as evaporation quickly occurs, a small bell-shaped depression forms, which gives the appearance of an air-bubble. On the fourth or fifth day we get the following appearance : there is at the surface the bubble-shaped depression ; below this there is a funnel-shaped area of liquefaction, the fluid being only slightly turbid, but covered on its surface with a more or less complete pellicle, and showing at its lower end thick masses of growth of a more or less spiral shape (Fig. 107). The liquefied portion gradually tapers off down- wards towards the needle track. (This appearance is, however, in some varieties not produced till much later, especially when the gelatine is very stiff, and, in other varieties which liquefy very slowly, may not be met with at CHOLERA. all.) At a later stage, liquefaction spreads and may reach the side of the tube. In gelatine plates the colonies are somewhat characteristic. They appear as minute whitish points, visible in twenty - four to forty - eight hours, which, under a low power of the microscope, do not present a smooth circular outline, but one which is ir- regularly granular or furrowed ; as they become larger their surface has an ap- pearance which has been compared to fragments of broken glass. Later, lique- faction occurs, and the colony sinks into the small cup formed, the plate then showing small sharply - marked rings around the colonies (Fig. 1 08). Under the microscope the outer margin of the cup is circular and sharply marked. Within the cup the liquefied portion forms a ring which has a more or less granular appearance, whilst the mass of growth in the centre is irregular and often broken up at its margins. Later still, liquefaction spreads around and the appearance becomes less characteristic. The growth of the colonies in gelatine plates constitutes one of the most im- Fig. 107. —Puncture tant means of distinguishing the culture of the cholera * , • a spirillum in peptone cholera spirillum from other organisms, gelatine — six days' On the surface of the agar media growth. Natural size. ft semi- transparent greyish white layer forms, which presents no special char- acters. On solidified blood serum the growth has at first the same appearance, but afterwards liquefaction of the medium occurs. On agar plates the superficial colonies under a low power are circular discs of brownish-yellow colour, and more transparent than those of most other organ- isms. On potato at the ordinary temperature, growth does CULTIVATION. 409 not take place, but when it is incubated at a temperature of from 300 to 370 C, a moist layer appears, which assumes a dirty brown colour somewhat like that of the glanders bacillus. It has, however, a greyish-brown rather than a chocolate tint, and moreover the appearance varies some- what in different varieties, and also on different sorts of potatoes. In bouillon with alkaline reaction the organism grows very readily, there occurring in twelve hours at 370 C. a a b Fig. 108. — Colonies of the cholera spirillum in a gelatine plate ; three days' growth. A, shows the granular surface, liquefaction just' com- mencing ; in B liquefaction is well marked. general turbidity, while the surface shows a thin pellicle composed of spirilla in a very actively motile condition. Growth ^ takes place under the same conditions equally rapidly in peptone solution (1 per cent with .5 per cent sodium chloride added). In milk also the organism grows well and produces no coagulation nor any change in its appearance, at least for several days. On all the media the growth of the cholera spirillum is a relatively rapid one, and especially is this the case in the peptone solution named and in bouillon, a circumstance of importance in relation to its separation in cases of cholera {vide p. 425). Another characteristic, though one not peculiar to this CHOLERA. organism, is the so-called cholera-red reaction. If to a culture in peptone bouillon or solution of peptone (i per cent), which has grown for twenty -four hours at 370 C, a few drops of pure sulphuric acid are added, a reddish- pink colour is produced. This is due to the fact that indol and a nitrite are formed by the spirillum in the medium. The addition of sulphuric acid causes a nitroso-indol body to be produced from these, and this gives the red colour. Here, as in the case of the negative indol reaction given by the typhoid organism, it is found that not every specimen of peptone is suitable, and it is advisable to select a peptone which gives the characteristic reaction with a known cholera organism, and to use it for further tests. It is also essential that the sulphuric acid should be pure, for if traces of nitrites are present the reaction might be given by an organ- ism which had not the power of forming nitrites. This is one of the most important tests in the diagnosis of the cholera organism. It is always given by a true cholera spirillum, and though the reaction is not peculiar to it, the number of organisms which give the reaction under the conditions mentioned are comparatively few. The cholera organism is one which grows much more rapidly in the presence of oxygen than in anaerobic con- ditions. Koch, in his earlier work, believed that no growth took place in the absence of oxygen, and it has been recently stated that this is the case in absolutely anaerobic conditions. Growth, however, takes place in the ordinary anaerobic conditions, usually employed in the culture of anaerobic organisms, such as those of tetanus and malignant oedema, though it occurs more slowly than in the presence of oxygen. In the intestines the oxygen supply, though small in amount, is yet sufficient for the growth of the spirilla. Powers of Resistance.— In their resistance against heat cholera spirilla correspond with spore-free organisms, and are killed in an hour by a temperature of 55° C, and much more rapidly at higher temperatures. They have com- paratively high powers of resistance against great cold, POWERS OF RESISTANCE. 411 and have been found alive after being exposed for several hours to the temperature of -io° C. They are, however, killed by being kept in ice for a few days. Against the ordinary antiseptics they have comparatively low powers of resistance, and Pfuhl found that the addition of lime, in the proportion of 1 per cent, to water containing the cholera organisms, was sufficient to kill them in the course of an hour. As regards the powers of resistance in ordinary con- ditions, the following facts may be stated. In cholera stools kept at the ordinary room temperature, the cholera organisms are rapidly outgrown by putrefactive bacteria, but in some cases they have been found alive even after two or three months. In most experiments, however, attempts to cultivate them even after a much shorter time have failed. The general conclusion may be drawn from the work of various observers that the spirilla do not multiply freely in ordinary sewage water, whilst they may remain alive for a considerable period of time. In distilled water they remain alive for several weeks at least, but do not multiply, nor does any considerable growth take place without the presence of a pretty large proportion of organic matter. On moist linen, as Koch showed, they can flourish very rapidly. When the cholera organisms are grown along with other organisms in fluids at a warm temperature, it is found that at first they may multiply more rapidly than the others, but that after a certain time they are outgrown by some of the organisms present, gradually diminish in number, and ultimately disappear. It must not, however, be inferred from such experiments that a similar result will necessarily follow in nature, as any particular saprophytic organism requires a special habitat — that is, certain suitable con- ditions for its growth in competition with other organisms. Though we can state generally that the conditions favour- able for the growth of the cholera spirillum are, a warm temperature, moisture, a good supply of oxygen, and a con- siderable proportion of organic material, we do not know the exact circumstances under which it can flourish for an 412 CHOLERA. indefinite period of time as a saprophyte. The fact that the area in which cholera is an endemic disease is so restricted tends to show that the conditions for a prolonged growth of the spirillum outside the body are not usually supplied. Yet, on the other hand, there is no doubt that in ordinary conditions it can live a sufficient time outside the body and multiply to a sufficient extent, to explain all the facts known with regard to the persistence and spread of cholera epidemics. Numerous experiments show that the cholera organisms are, as a rule, rapidly killed by drying, usually in two or three minutes when the drying has been thorough, and it is inferred from this that they cannot be carried in the living condition for any great distance through the air, a con- clusion which is well supported by observations on the spread of the disease. Cholera is practically always trans- mitted by means of water or food contaminated by the organism, and there is no doubt that contamination of the water supply by choleraic discharges is the chief means by which areas of population are rapidly infected. It has been shown that if flies are fed on material containing cholera organisms, the organisms may be found alive within their bodies twenty-four hours afterwards. And further, Haffkine found that sterilised milk might become con- taminated with cholera organisms, if kept in open jars to which flies had free access, in a locality infected by cholera. It is quite possible that infection may be carried by this method in some cases. Experimental Inoculation.— In considering the effects of inoculation with the cholera organism, we are met with the difficulty that none of the lower animals, so far as is known, suffer from the disease under natural conditions. Even in places where cholera is endemic, no corresponding affection has been observed in any animals. And further, before the discovery of the cholera organism, various efforts had been made to induce the disease in animals by feeding them with cholera dejecta, but without success. It is therefore not surprising that the earlier experiments on animals by EXPERIMENTAL INOCULATION. 413 feeding them with pure cultures were attended with negative results. As the organisms are confined to the alimentary tract in the natural disease, attempts to induce their multi- plication within the intestine of animals by artificially arranging favouring conditions, have occupied a prominent place in the experimental work. We shall give a short account of such experiments. Nikati and Rietsch were the first to inject the organisms directly into the duodenum of clogs and rabbits, and they succeeded in produc- ing, in a considerable proportion of the animals, a choleraic condition of the intestine ; in their earlier experiments the common bile duct was ligatured, but the later were performed without this operation. These experiments were confirmed by other observers, including Koch. Thinking that probably the spirillum, when introduced by the mouth, is destroyed by the action of the hydrochloric acid of the gastric secretion, Koch first neutralised this acidity by administering to guinea-pigs 5 c.c. of a 5 per cent solution of carbonate of soda, and sometime afterwards introduced a pure culture into the stomach by means of a tube. Of nineteen animals treated in this way, only one died with choleraic changes in the small intestine. This animal had aborted a short time previously, and as its abdominal walls were very relaxed, Koch considered that the intestinal peristalsis had been interfered with, and thus opportunity had been afforded to the organ- isms of gaining a foothold and multiplying in the intestine. He accordingly tried the effect of artificially interfering with the intestinal peristalsis by injecting tincture of opium into the peritoneum (1 c.c. per 200 grm. weight), in addition to neutralising as before with the carbonate of sodium solution. The result was remarkable, as thirty, out of thirty-five animals treated, died with the same changes as in the single animal in the previous series of experiments. The animals infected by this method show signs of general prostration, their posterior extremities being especially weakened ; the abdomen becomes tumid, respiration slow, heart's action weak, and the surface cold. Death occurs after a few hours. Post mortem the small intestine is distended, its mucous membrane congested, and it contains a colourless fluid with small flocculi and the cholera organisms in practically pure cultures. There experiments, which have been repeatedly confirmed, therefore demonstrated that the cholera organisms could, under certain con- ditions, set up in animals a condition in some respects resembling cholera. Koch, however, found that when the spirilla of Finkler and Prior, of Deneke, and of Miller {vide infra) were employed by the same method, a certain, though much smaller, proportion of the animals died from an intestinal infection. Though the changes in these cases were not so characteristic, they were sufficient to prevent 414 CHOLERA. the results obtained with the cholera organism from being used as a demonstration of the specific relation of the latter to the disease. Within the last few years some additional facts of high interest have been established with regard to choleraic infection of animals. For example, Sabolotny found that in the marmot an intestinal infection readily takes place by simple feeding with the organism, there result- ing the usual intestinal changes, sometimes with hemorrhagic peritonitis, the organisms, however, being present also in the blood. It was found by Issaeff and Kolle that young rabbits could be infected by merely neutralising the gastric secretion with sodium carbonate, the use of opium being unnecessary ; but of special interest is the fact, discovered by Metchnikoff, that in the case of young rabbits shortly after birth, a large proportion die of choleraic infection when the organisms are simply introduced along with the milk, as may be done by infecting the teats of the mother. Further, from these animals thus infected the disease may be transmitted to others by a natural mode of infection. In this affection of young rabbits many of the symptoms of cholera are present — great prostration, markedly subnormal temperature, some- times anuria, and occasionally slight cramps before death. Most frequently there is diarrhoea, though sometimes this may be absent, the group of phenomena sometimes corresponding, according to Metchni- koff, with cholera sicca. The organisms occur in large numbers in the intestine, and in some cases a few may be found in the blood, and especially in the gall bladder. Many of these experiments were per- formed with the vibrio of Massowah, which is now admitted not to be a true cholera organism, others with a cholera vibrio obtained from the water of the Seine. It will be seen from the above account that the evi- dence obtained from experiments on intestinal infection of animals, though by no means sufficient to establish the specific relationship of the cholera organism, is on the whole favourable to this view, especially when it is borne in mind that animals do not in natural conditions suffer from the disease. Experiments performed by direct inoculation also supply interesting facts. Intraperitoneal injection in guinea-pigs is followed by general symptoms of illness, the most prominent being distension of the abdomen, subnormal temperature, and, ultimately, profound collapse. There is peritoneal effusion, which may be comparatively clear, or may be somewhat turbid and contain flakes of lymph, according to the stage at which death takes place. INTRAPERITONEAL INJECTION. 415 If the dose is large, organisms are found in considerable numbers in the blood and also in the small intestine, but with smaller doses they are practically confined to the peritoneum. Kolle found that when the minimum lethal dose was used in guinea-pigs, the peritoneum might be free from organisms at the time of death, the fatal result having taken place from an intoxication (cf. diphtheria, p. 364). In rabbits, after intravenous injection of comparatively large quantities, death may follow within eighteen hours, with symptoms of general intoxication ; the organisms are present in the blood, though rather diminished in number, and few are to be found in the intestine. If, however, the dose is smaller and the animals live longer, then the organisms may settle and multiply in the intestine, and changes quite analogous to those in cholera are produced — congestion of mucous membrane, and at places desqua- mation of epithelium (Issaeff and Kolle). In the case of animals which die when these changes have occurred, the organisms may have quite disappeared from the blood and internal organs. These experiments show that though the organisms undergo a certain amount of multiplication when introduced by the channels mentioned, still the tendency to invade the tissues is not a marked one. On the other hand the symptoms of general intoxication are always pro- nounced. Hence arise questions as to the nature and mode of action of toxic bodies produced by the cholera organism. Toxines. — Though there is no doubt that there are formed by Koch's spirillum toxic bodies which produce many of the symptoms of cholera, there is at present very little satisfactory knowledge regarding their chemical nature. The following summary may be given. It has been shown, especially by R. Pfeiffer,1 that toxic phenomena can be produced by injection of the dead spirilla into animals. A certain quantity of a young culture 1 Pfeiffer obtained his earlier results with a vibrio from Massowah, which is now known not to be a true cholera organism. The fact shows that the effects described are not specific to the latter. 4i6 CHOLERA. on agar, killed by exposure to the vapour of chloroform, when injected intraperitoneally into a guinea-pig, may cause death in from eight to twelve hours. There is extreme collapse, sometimes clonic spasms occur, and the temperature may fall below 300 C. before death. Pfeiffer considers that the toxic substances are contained in the bodies of the organisms, that is, they are intracellular, and that they are only set free by the disintegration of the latter. This opinion is grounded chiefly on the fact that when bouillon cultures were filtered, he found that the nitrate possessed very feeble toxic properties. The dead cultures administered by the mouth produce no effect unless the intestinal epithelium is injured, in which case poisoning may result. He considers that the desquamation of the epithelium is an essential factor in the production of the phenomena of the disease in the human subject. Pfeiffer found that the toxic bodies were to a great extent destroyed at 6o° C, but even after heating at 1060 C. a small pro- portion of toxine remained, which had the same physio- logical action. On the other hand, other observers (Petri, Ransom, Klein, and others), have obtained toxic bodies from filtered cultures. Recently Metchnikoff, E. Roux, and Taurelli-Salimbeni have demonstrated the formation of diffusible toxic bodies in fluid media in the following manner. Small collodion sacs were prepared, each con- taining 2 to 4 c.c. of bouillon. One sac was inoculated with a living virulent culture of the cholera vibrio ; to the second, two entire cultures on agar of the same organism were added, the cultures being first killed by chloroform. Each sac was then closed and placed with aseptic pre- cautions in the peritoneum of a guinea-pig. The animal which received the sac containing the living vibrios soon showed symptoms of choleraic poisoning, and died in a few days, whilst the animal which received the sac containing large quantities of dead organisms showed only transitory symptoms of illness. These observers therefore concluded that toxic substances are formed by the living organisms, TOXINES OF KOCH'S SPIRILLUM. 417 which quickly diffuse into the medium (and in the experi- ments, through the wall of the sac). By greatly increasing the virulence of the organism, then growing it in bouillon and filtering the cultures on the third and fourth day, they obtained a fluid which was highly toxic to guinea-pigs (the fatal dose usually being \ c.c. per 100 grm. weight). If the dose of the toxine is very large, death follows in an hour or even less. The symptoms closely resemble those obtained by Pfeiffer, the rapid fall of temperature being a striking feature. Post mortem, at the site of inoculation there is a little inflammatory oedema, the internal organs are congested, and the small intestine is distended with fluid contents. The toxicity of the filtrate they found not to be altered by boiling. It is somewhat difficult to reconcile the results of Pfeiffer and Metchnikoff as regards the action of heat, though probably the toxine obtained by Metchnikoff corresponds with the secondary body of Pfeiffer, which he obtained in small quantities. A con- siderable number of observers, however, agree in stating that the toxines obtained from cholera cultures are not destroyed at ioo° C. A great many observers have attempted to obtain toxines in a chemically pure condition, but so far without results which can be re- garded as conclusive. Hueppe and Wood found that the most active toxines were produced by growing the cholera organism in albumin in anoerobic conditions, and considered that this corresponded to the mode of their production in cholera. Scholl confirmed Hueppe's re- sults, and obtained from cultures under such conditions a peptone which possessed highly toxic properties, and which he called cholera toxo- peptone. These results, however, have been adversely criticised by various observers. Wesbrook obtained different substances according o the media on which the cholera organisms were grown, and yet nese produced very much the same effects, chiefly collapse, subnormal temperature, cramps, dyspnoea, etc. Such toxic bodies were even obtained from cultures in asparaginate of soda, which did not contain any proteid substance. He therefore came to the conclusion that so- called toxalbumins etc. are really mixtures of albumins and true toxines the chem.cal nature of the latter not having been yet determined.' Wesbrook also obtained the toxic bodies in small quantity from the T, I" °f P^-P^ kil1^ by the vibrio. Bosc also found that the blood, and to a less extent the urine, of patients who had died 27 4i8 CHOLERA. in the algid stage, produce toxic phenomena and death, when injected intravenously in rabbits. In this case also, nothing is known with regard to the chemical nature of the toxic bodies. Experiments on the Human Subject. — Experiments have also been performed in the case of the human subject, both intentionally and accidentally. In the course of Koch's earlier work, one of the workers in his laboratory shortly after leaving was seized with severe choleraic symptoms. The stools were found to contain cholera spirilla in enor- mous numbers. Recovery, however, took place. In this case there was no other possible source of infection than the cultures with which the man had been working, as no cholera was present in Germany at the time. Within recent years a considerable number of experiments have been per- formed on the human subject, which certainly show that in some cases more or less severe choleraic symptoms may follow ingestion of pure cultures, whilst in others no effects may result. The former was the case, for example, with Emmerich and Pettenkofer, who made experiments on themselves, the former especially becoming seriously ill. In the case of both, diarrhoea was well marked, and numer- ous cholera spirilla were present in the stools, though toxic symptoms were proportionately less pronounced. Metchni- koff also by experiments on himself and others obtained results which convinced him of the specific relation of the cholera spirillum to the disease. Lastly, the case of Dr. Orgel in Hamburg may be noted, who contracted the disease in the course of experiments with the cholera and other spirilla, and died in spite of treatment. It is believed that in sucking up some peritoneal fluid containing cholera spirilla, a little entered his mouth and thus infection was produced. This took place in September 1894 at a time when there was no cholera in Germany. On the other hand, in many cases the experimental ingestion of cholera spirilla by the human subject has given negative results. Still, as the result of observation of what takes place in a cholera epidemic, it is the general opinion of authorities that only a certain proportion of people are susceptible IMMUNITY. 419 to cholera, and the facts mentioned above have, in our opinion, great weight in establishing the relation of the organism to the disease. Immunity. — As this subject is discussed later, only a few facts will be here stated, chiefly for the purpose of making clear what follows with regard to the means of distinguishing the cholera spirillum from other organisms. The guinea-pig or any other animal may be easily immunised against the cholera organism by repeated injections (con- veniently made into the peritoneum) of non-fatal doses of the spirilla. It is better to commence the process with non-fatal doses of cultures killed by the vapour of chloroform or by heat, the doses being gradually increased, and after- wards to proceed with increasing doses of the living organism. In this way a high degree of immunity against the organism is developed, and further, the blood serum of an animal thus immunised (anti-cholera serum) has markedly protective power when injected, even in a small quantity, into a guinea-pig along with five or ten times the fatal dose of the living organism. The blood serum of an animal immunised against the cholera organism has, however, no special pro- tecting power against another species of organism This constitutes the principle of Pfeiffer's method of diagnosis to be described. A curious fact, however, is, that immunity produced by the above method is only exerted against the living organisms, and does not pro- tect against the tonnes, that is, it is. due to certain substances which act as germicides (indirectly), but which are not antitoxic. Further it does not protect the guinea-pig from the intestinal infection by Koch's method (Pfeiffer and Wassermann, Scbernheim, Klein), nor does the anti-cholera serum protect young rabbits against the choleraic affection produced by ingestion of the cholera vibrios (Metchnikoft). The infer ence from these latter results appears to be, that when the vibrios are introduced into the tissues, they are killed by certain substances in he serum, but in the intestine they are in a sense outside of the tissues and can there multiply and produce toxines. Metchnikoff has ore pared a true antitoxic cholera serum by injections of repeated and" gradually increased doses of the toxine, and has found tha tl ?an ox,c serum has a distinct effect against the choleraic affect on of 420 CHOLERA. For Haffkine's method of preventive inoculation vide chapter on Immunity. " Means of Distinguishing the Cholera Organism. — According to Koch the most important points in the dia- gnosis of cholera are : — (a) Microscopical characters of the dejecta, (b) Ap- pearance of the colonies in gelatine plates, (c) Their appearance on agar plates, (d) The growth in peptone solution, (e) The cholera-red reaction. (/) The effect of intraperitoneal inoculation of guinea-pigs with pure cultures. There can be no doubt that in the great majority of cases these points taken collectively are sufficient, but in some cases difficulties have arisen. Pfeiffer has accord- ingly introduced the method of diagnosis described above, which depends on the supposed specific action of an anti- cholera serum. Further, he has found that a striking dis- integrative change is observed microscopically in the spirilla when injected along with the protective serum into the peritoneal cavity of another guinea-pig— Pfeiffer's reaction. The method is as follows: A loopful (2 mgrm.) of recent agar culture of the organism to be tested is added to 1 c.c. of ordinary bouillon containing .001 c.c. of anti-cholera serum. The mixture is then injected into the peritoneal cavity of a young guinea-pig (about 200 erm. in weight), and the peritoneal fluid of this animal (con- veniently obtained by means of capillary glass tubes inserted into the peritoneum) is examined microscopically after a few minutes If the spirilla injected have been cholera spirilla, it will be found that they become motionless, swell up into globules, and ultimately break down and disappear- positive result. If they are found active and motile 'then the possibility of their being true cholera spirilla may be excluded —nemtive result. In the former case (positive result) there is, how- ever still the possibility that the organism is devoid of pathogenic properties and has been destroyed by the normal peritoneal fluid. A con rol experiment should accordingly be made with .001 c.c of normal serum in place of the anti-cholera serum. If no alteration _of the onanism occurs with its use, then it is to be concluded that the ^ S in question has been demonstrated by the specific reaction to be the cholera spirillum. Bordet, and Gruber and Durham, have smce devised methods by which a corresponding reaction can be observed outside the body (see Chap. XIX.). MODES OF DISTINGUISHING THE SPIRILLUM. 421 Further experiments are necessary to show what the exact worth of this reaction is, but extensive observations made up to the present time, especially those of Dunbar conducted on a large series of spirilla, are on the whole distinctly in favour of Pfeiffer's statement being a general law. This method makes the effects of the vital activity of the organism the criterion for distinguishing it from others, and, so far as the production of the disease is concerned, this appears quite rational. It still remains to be seen how far distinction by this means corresponds with differences in cultures. Difficulties may arise when the cholera organ- ism has been grown for a long time outside the body and has lost its virulence. Properties of the Serum of Cholera Patients and Con- valescents.—Lazarus was the first to show that the serum of patients who had suffered from cholera, possessed the power of protecting guinea-pigs, when injected in very minute quantity along with a fatal dose of the cholera organism. These experiments have been confirmed by Klemperer, Issaeff, and Pfeiffer, and the last mentioned found that the serum of such patients gave the character- istic reaction if injected with the vibrios into the peritoneal cavity of a guinea-pig. Further, so far as experiment has gone, this action is not exerted against any other organism, that is, it is specific towards the cholera spirillum. This action of the serum may be present eight or ten days after the attack of the disease, but is most marked four weeks after; it then gradually becomes weaker and disappears in two or three months (Pfeiffer and Issaeff). Specific agglutinative properties have, however been detected in the serum of cholera patients at a much' earlier date, in some cases even on the first day of the disease, though usually a day or two later. The dilutions used were 1:15 to 1:120, and these had no appreciable effect on organisms other than the cholera spirillum (Achard arid Bensande). Needless to say, such facts supply strong additional evidence of the relation of Koch's spirillum to 422 CHOLERA. General Summary. — We may briefly summarise as follows the facts in favour of Koch's spirillum being the cause of cholera. First, there is the constant presence of spirilla in true cases of cholera, which on the whole con- form closely with Koch's description, though variations un- doubtedly occur. Moreover, the facts known with regard to their conditions of growth, etc., are in conformity with the origin and spread of cholera epidemics. Secondly, the experiments on animals with Koch's spirillum or its toxines give as definite results as one can reasonably look for in view of the fact that animals do not suffer naturally from the disease. Thirdly, the experiments on the human subject and the results of accidental infection by means of pure cultures are also strongly in favour of this view. Fourthly, the agglutinative and protective properties of the serum of cholera patients and convalescents constitute another point in its favour. Fifthly, bacteriological methods, which proceed on the assumption that Koch's spirillum is the cause of the disease, have been of the greatest value in the diagnosis of the disease. And lastly, the results of Haffkine's method of preventive inoculation in the human subject, which are on the whole favourable, also supply additional evidence. If all these facts are taken together, we consider the con- clusion must be arrived at that the growth of Koch's spirillum in the intestine is the immediate cause of the disease. This does not exclude the probability of an important part being played by conditions of weather and locality, though such are very imperfectly understood. Pettenkofer, for example, recognises two main factors in the causation of epidemics, which he designates * and y, and considers that these two must be present together in order that cholera may spread. The x is the direct cause of the disease— an organism which he now admits to be Koch's spirillum ; the y in- cludes climatic and local conditions, e.g., state of ground- water, etc. . , Difficulty does not arise, however, so much with regard to the causal relationship of Koch's spirillum to cholera as in connection with various organisms which have been SPIRILLA RESEMBLING CHOLERA SPIRILLUM. 423 cultivated from other sources, and which more or less closely resemble it. Other Spirilla Kesembling the Cholera Organism. — These have been chiefly obtained either from water con- taminated by sewage or from the intestinal discharge in cases with choleraic symptoms. Some of them differ so widely in their cultural and other characters (some, for example, are phosphorescent) that no one would hesitate to classify them as distinct species. Others, however, closely resemble the cholera organism. The vibrio berolinejisis, cultivated by Neisser from Berlin sewage water, differs from the cholera organism only in the appearance of its colonies in gelatine plates, its weak pathogenic action, and its giving a negative result with Pfeiffer's test. It, however, gives the cholera-red reaction. The vibrio Danubicus, cultivated by Heider from canal water, also differs in the appearance of its colonies in plates, and also reacts negatively to Pfeiffer's test ; in most respects it closely resembles the cholera organism. Another spirillum (v. Ivanoff) was cultivated by Ivanoff from the stools of a typhoid patient after these had been diluted with water. This organism differed somewhat in the appear- ance of its colonies and in its great tendency to grow out in the form of long threads, but Pfeiffer found that it reacted to his test in the same way as the cholera organism, and he considered that it was really a variety of the cholera organism. No spirilla could be found micro- scopically in the stools in this case, and Pfeiffer is of the opinion that the organism gained entrance accidentally. These examples will show how differences of opinion, even amongst authorities, might arise as to whether a certain spirillum were really the cholera organism or a distinct species resembling it. A few examples may also be given of organisms cul- tivated from cases in which cholera-like symptoms were present. The vibrio of Massowa/i was cultivated by Pasquale from a case during a small epidemic of cholera. This organism so closely re- sembles Koch's spirillum that it was accepted by several authorities as the true cholera organism, and, as already stated, Metchnikoff pro- duced by it cholera symptoms in the human subject, and also the cholera-like disease in young rabbits. It possesses four flagella, has a high degree of virulence, producing septicemia both in guinea-pigs and pigeons, and its colonies in plates differ somewhat from the cholera organism. Moreover, it reacts negatively to Pfeiffer's test. 424 CHOLERA. Another organism, the v. Gindha, was cultivated by Pasquale from a well, and was at first accepted by Pfeiffer as the cholera organism, but afte rwards rejected, chiefly because it failed to give the specific im- munity reaction. It also differs somewhat from the cholera organism in its pathogenic effects, and it fails to give the cholera-red reaction or gives it very faintly. Pestana and Bettencourt also cultivated a species of spirillum from a number of cases during an epidemic in Lisbon — an epidemic in which there were symptoms of gastro-enteritis, although only in a few instances did the disease resemble cholera. They also cultivated the same organism from the drinking water. It differs from the cholera organism in the appearance of its colonies and of puncture cultures in gelatine. It has very feeble pathogenic effects, and gives a very faint, or no, cholera-red reaction. To Pfeiffer's test it also reacts negatively. Another spirillum [v. Romamis) was obtained by Celli and Santori from twelve out of forty-four cases where there were the symptoms of mild cholera. This organism does not give the cholera-red reaction, nor is it pathogenic for animals. They look upon it as a " transitory variety " of the cholera organism, though sufficient evidence for this view is not adduced. We have mentioned these examples in order to show some of the difficulties which exist in connection with this subject. It is important to note that, on the one hand, spirilla which have been judged to be of different species from the cholera organism, have been cultivated from cases in which cholera-like symptoms were present, and on the other hand, in cases of apparently true cholera considerable variations in the characters of the cholera organisms have been found. Such variations have especially been recorded by Surgeon-Major Cunningham in India. It is therefore quite an open question whether some of the organisms in the former case may not be cholera spirilla which have undergone variations as a result of the conditions of their growth. That such variations may occur we have a con- siderable amount of evidence. The great bulk, however, of evidence goes to show that Asiatic cholera always spreads as an epidemic from places in India where the disease is endemic, and that its direct cause is Koch's spirillum. It is sufficient to bear in mind that choleraic symptoms may be produced by other causes, and that in some of such cases spirilla which have some resemblance to Koch's METHODS OF DIAGNOSIS. 425 organism may be present in the intestinal discharges, though rarely in large numbers. Methods of Diagnosis. — In the first place, the stools ought to be examined microscopically. Dried film prepara- tions should be made and stained by any ordinary stains, though carbol-fuchsin diluted four times with water is specially to be recommended. Hanging-drop preparations, with or without the addition of a weak watery solution of gentian-violet or other stain, should also be made, by which method the motility of the organism can be readily seen. By microscopic examination the presence of spirilla will be ascertained, and an idea as to their number obtained. In some cases the cholera spirilla are so numerous in the stools that a picture is presented which is obtained in no other condition, and a microscopic examination may be sufficient for practical purposes. According to Koch, a diagnosis was made in 50 per cent of the cases during the Hamburg epidemic by microscopic examination alone. If the organisms are very numerous, gelatine or agar plates may be made at once and pure cultures obtained. If the spirilla occur in comparatively small numbers, the best method is to inoculate peptone solution (1 per cent) and incubate for eight to twelve hours. At the end of that time the spirilla will be found on microscopic examination in enormous numbers at the surface, and thereafter plate cultures can readily be made. If the spirilla are very few in number, or if a suspected water is to be examined for cholera organisms, the peptone solution which has been inoculated should be examined at short intervals till the spirilla are found microscopically. A second flask of peptone solution should then be inoculated, and possibly again a third from the second. By this method, properly carried out, a culture may be obtained which, though impure contains a large proportion of the vibrios, and then plate cultures may be made. When a spirillum has been obtained in pure condition by these methods, the appearance of the colonies in plates should be specially noted, the test for the cholera-red reaction 426 CHOLERA. should be applied, and in many cases it is advisable to test the effects of intraperitoneal injection of a portion of a recent agar culture in a guinea-pig, the amount sufficient to cause death being also ascertained. The agglutinating or sedimenting properties of the serum of the patient should be tested against a known cholera organism, and against the spirillum cultivated from the case. In the same way the action of the serum of an immunised guinea-pig may be tested both as regards agglutinating and protective properties. A number of other JjA spirilla have been ryt „ Jfi^^i \ cultivated, which are *' * of interest on account „ 4 JO ff \ * « of their points of re- * j/\ >( semblance to the cholera organism, though probably they produce no patho- logical conditions in the human subject. Metchnikoff's Spi- rillum (vibrio Metch- nikovi). — This organ- ism was obtained by Fig. 109. —Metchnikoff's spirillum, both in Gamaleia from an curved and straight forms ; from an agar epidemic disease of culture of twenty-four hours' growth. , , . 0fi „nfi Stained with weak carbol-fuchsin. x 1000. fowls in Odessa, ana is of special interest on account of its close resemblance to the cholera organism. In the natural disease, which especially affects young fowls, the animals suffer from diarrhoea, pass into a sort of stupor, sitting with their feathers ruffled, and usually die within forty-eight hours. The intestines contain a greyish-yellow fluid, sometimes slightly blood-stained, in which the spirilla are found. A few of these spirilla may also be found m the blood in the younger fowls, though generally absent from the blood in the older. Morphologically the organism is practically identical with VIBRIO ME TCHNIKO VI. 427 Koch's spirillum (Fig. 109). It is actively motile, and has the same staining reactions. Its growth in peptone-gelatine also closely resembles that of the cholera organism, though it produces liquefaction more rapidly (Fig. 1 1 o, A). In gelatine plates the young colonies are, however, smoother and more circular. After liquefac- tion occurs, some of the colonies are almost identical in appearance with those of the cholera organism, whilst others show more uniformly turbid contents. In puncture cultures the growth takes place more rapidly, but in appear- ance closely resembles that of the cholera organism a few days older. Its growth in peptone solution too is closely similar, and it also gives the cholera-red reaction. This organism can, however, be readily distinguished from the cholera organism by the effects of inoculation on animals, especially on pigeons and guinea-pigs. Subcutaneous inoculation of small quantities of pure culture in pigeons is followed by acute inflammatory swelling with degeneration of the subjacent muscles ; and septicaemia occurs, which produces a fatal result usually within twenty-four hours. Inoculation with the same quantity of cholera organism produces practically no a b Fig. 110. — Puncture cultures in pep- tone-gelatine. A. Metchnikoflf's spirillum. Five days' growth. B. Finkler and Prior's spirillum. Four days' growth. Natural size. 428 CHOLERA. •V result ; even with large quantities death is rarely produced. The vibrio Mctchnikovi produces somewhat similar effects in the guinea-pig to those in the pigeon, subcutaneous inocula- tion being followed by extensive hemorrhagic cedema, and a rapidly fatal septicaemia. Young fowls can be infected by feeding with virulent cultures. We have evidence from the work of Gamaleia that the toxines of this organism have some- what the same action as those of the cholera organism. The organism is therefore one which very closely resembles the cholera organism, the results on inoculating the pigeon offering the most ready means of distinction. It gives a negative re- action to Pfeiffer's test; that is, the properties of an anti- cholera serum are not exerted against it. It may also be men- tioned that an or- ganism which is ap- parently the same as the vibrio Metchni- kovi was cultivated by Pfuhl from water, and named v. Nordhafen. Finkler and Prior's Spirillum. — These observers, shortly after* Koch's discovery of the cholera organism, separated a spirillum, in a case of cholera nostras, from the stools after they had been allowed to decompose for several days. There is, however, no evidence that the spirillum has any causal relationship to this or any other disease in the human subject. Morphologically it closely resembles Koch's spirillum, and cannot be distinguished from it by its microscopical characters, although, on the whole, it tends % \ w - Fig. hi. — Finkler and Prior's spirillum, from an agar culture of twenty-four hours' growth. Stained with carbol-fuchsin. x iooo. FINKLER AND PRIOR'S SPIRILLUM. 429 to be rather thicker in the centre and more pointed at the ends (Fig. 111). In cultures, however, it presents marked differences. In puncture cultures on gelatine it grows much more quickly, and liquefaction is generally visible within twenty-four hours. The liquefaction spreads rapidly, and usually in forty-eight hours it has produced a funnel- shaped tube with turbid contents, denser below (Fig. 110, B). In plate cultures the growth of the colonies is proportion- ately rapid. Before they have produced liquefaction around them, they appear, unlike those of the cholera organism, as minute spheres with smooth margins. When liquefaction occurs, they appear as little spheres with turbid contents, which rapidly increase in size. The individual colonies may reach the third of an inch in diameter, and ultimately general liquefaction occurs. On potatoes this organism grows well at the ordinary temperature, and in two or three days has formed a slimy layer of greyish-yellow colour, which rapidly spreads over the potato. On all the media the growth has a distinctly foetid odour. A growth in peptone solution fails to give the cholera-red reaction at the end of twenty-four hours, though later a faint reaction may appear. As stated above, Koch succeeded in producing, by this organism; an intestinal affection in guinea-pigs after neutralising the stomach contents and paralysing the intestine with opium. This occurs in a small proportion of the animals experimented on, and the contents of the intestine, unlike what was found in the case of the cholera organism, were turbid in appearance, and had a markedly foetid odour. When tested by intraperitoneal injection, its effects are somewhat of the same nature as those of the cholera organism, but its virulence is of a much lower order. An organism cultivated by Miller from the cavity of a decayed tooth in a human subject is almost certainly the same organism as Finkler and Prior's spirillum. Deneke's Spirillum.— This organism was obtained from old cheese, and is also known as the spirillum tyrogenum. It closely resembles Koch's spirillum in microscopic appear- 43° CHOLERA. ances, though it is rather thinner and smaller. Its growth in gelatine is also somewhat similar, but liquefaction pro- ceeds more rapidly, and the bell-shaped depression on the surface is larger and shallower, whilst the growth has a more distinctly yellowish tint. The colonies in plates also show points of resemblance, though the youngest colonies are rather smoother and more regular on the surface, and liquefaction occurs more rapidly than in the case of the cholera organism. The colonies have, on naked-eye ex- amination, a distinctly yellowish colour. The organism does not give the cholera-red reaction, and on potato it forms a thin yellowish layer when incubated above 300 C. When tested by intraperitoneal injection and by other methods it is found to possess very feeble, or almost no, pathogenic properties. Koch found that this organism, when administered through the stomach in the same way as the cholera organism, produced a fatal result in three cases out of fifteen. Deneke's spirillum is usually regarded as a comparatively harmless saprophyte. CHAPTER XVIII. INFLUENZA, PLAGUE, RELAPSING FEVER, MALTA FEVER, YELLOW FEVER. Influenza. The first account of the organism now known as the influenza bacillus was published simultaneously by Pfeiffer, Kitasato, and Canon, in January 1892. The - \^Vj * 4**, two first - mentioned * * * £ 1 observers found it in / the bronchial sputum, ", \ -<~ ^ me Droncniai sputum, ' ' , ..'"yfrf* *'a and obtained pure cul- J* 'r ' ' tP^" ' > ' 't , tures, and Canon ob- ,-1 iy " ' ,> served it in the blood ' m „*_ v//X s • /'.^ in a few cases of the . , ^ * '" -U t \ 4" ^\ " disease. It is, however, ' -„ •4'- - : 7*-^ to Pfeiffer's work that we owe most of our '» •. V knowledge regarding " * '/ V , » . its characters and ac- v * , 4 ' ' . ' £ *" tion. From the facts ; ' which have been FlG' II2-— InAuenza bacilli from a culture , 1 1 • 1 1 . on blood agar. established concerning stained with carbol-fuchsin. x 1000. it, this organism has strong claims to be considered the specific agent in the disease, though absolute proof is still wanting. Microscopical Characters.— The influenza bacilli as seen 432 INFLUENZA. in the sputum are very minute rods not exceeding 1.5 u in length and .3 /x in thickness. They are straight, with rounded ends, and sometimes stain more deeply at the ex- tremities (Fig. 112). The bacilli occur singly or form clumps by their aggregation, but do not grow into chains. They show no capsule. They take up the basic aniline stains somewhat feebly, and are best stained by a weak solution (1 : 10) of carbol-fuchsin applied for 5 to 10 minutes. They lose the stain in Gram's method. They are non-motile, and do not form spores. Cultivation. — The best medium for the growth of the influenza bacillus is blood agar (see page 49), which was introduced by Pfeiffer. He obtained growths of the bacilli on agar which had been smeared with influenza sputum, but he failed to get any ^-cultures on the agar media or on serum. The growth in the first cultures he considered to be probably due to the presence of certain organic substances in the sputum, and accordingly he tried the expedient of smearing the agar with drops of blood before making the inoculations. The blood of the lower animals is suitable, as well as human blood. In this way he com- pletely succeeded in attaining his object. The colonies of the influenza bacilli on blood agar, incubated at 370 C, ap- pear within twenty-four hours, in the form of minute circular dots almost completely transparent. When numerous, the colonies are scarcely visible to the naked eye, but when sparsely arranged they may reach the size of a small pin's head. This size is generally reached on the second day. The bacilli die out somewhat quickly in cultures, and in order to keep them alive sub-cultures should be made every four to five days. By this method the cultures may be maintained for an indefinite period. They also grow well on agar smeared with a solution of haemoglobin ; growth on the ordinary agar media is slight and somewhat uncertain. A very small amount of growth takes place in bouillon, but it is more marked when a little fresh blood is added. The growth forms a thin whitish deposit at the bottom of the flask. The limits of growth are from 250 to 42 0 C, the DISTRIBUTION OF BACILLI. 433 optimum temperature being that of the body. The influenza bacillus is a strictly aerobic organism. The powers of resistance of this organism are of a low- order. Pfeiffer found that dried cultures kept at the ordinary temperature were usually dead in twenty hours, and that if sputum were kept in a dry condition for two days, all the influenza bacilli were dead, or rather cultures could be no longer obtained. Their duration of life in ordinary water is also short, the bacilli usually being dead within two days. From these experiments Pfeiffer concludes that outside the body in ordinary conditions they cannot multiply, and can remain alive only for a short time. The mode of infection in the disease he accordingly considers to be chiefly by direct contact by means of mucus, etc. Distribution in the Body. — The bacilli are found chiefly in the respiratory passages in influenza. They may be present in large numbers in the nasal secretion, generally mixed with a considerable number of other organisms, but it is in the small masses of greenish-yellow sputum from the bronchi that they occur in largest numbers, and in many cases almost in a state of purity. They occur in clumps which may contain as many as 100 bacilli, and in the early stages of the disease are chiefly lying free. As the disease advances, they may be found in considerable numbers within the leucocytes, and towards the end of the disease a large proportion have this position. It is a matter of considerable importance, however, that they may persist for weeks after symptoms of the disease have dis- appeared, and may still be detected in the sputum. Especially is this the case when there is any chronic pul- monary disease. They also occur in large numbers in the capillary bronchitis and catarrhal pneumonia of influenza, as Pfeiffer showed by means of sections of the affected parts. In these sections he found the bacilli lying amongst the leucocytes which filled the minute bronchi, and also penetrating between the epithelial cells and into the super- ficial parts of the mucous membrane. Their presence sets 28 434 INFLUENZA. up a marked leucocytic emigration in the peribronchial tissue, the leucocytes passing in large numbers into the lumen of the tubes and sometimes taking up the bacilli. Other organisms also, especially Fraenkel's pneumococcus, may be concerned in the pneumonic conditions following influenza. In some cases influenza occurs in tubercular subjects, or is followed by tubercular affection, in which cases both influenza and tubercle bacilli may be found in the sputum. In such a condition the prognosis is very grave. Regard- ing the presence of influenza bacilli in the other pulmonary complications following influenza, much information is still required. Occasionally in the foci of suppurative softening in the lung the influenza bacilli have been found in a practically pure condition. In cases of empyema the organ- isms present would appear to be chiefly streptococci and pneumococci ; whilst in the gangrenous conditions, which sometimes occur, a great variety of organisms has been found. As above stated, Canon described the bacilli as occur- ring in the blood during life, and Pfeiffer, on examining Canon's preparations, admits that the bacilli shown re- sembled the influenza bacilli. His own observations on a large series of cases convinced him that the organism was very rarely present in the blood, that in fact its occurrence there must be looked upon as exceptional. The conclu- sions of other observers have, on the whole, confirmed this statement. It has been regularly found in enormous numbers in the sputum in influenza, but only occasionally and in small numbers in the blood. It is probable that the chief symptoms in the disease are due to toxines absorbed from the respiratory tract (vide infra). We cannot yet speak definitely with regard to the relation of the bacillus to other complications in influenza. Pfeiffer found it in inflammation of the middle ear, but in a case of meningitis following influenza Fraenkel's diplo- coccus was present. In a few cases of meningitis, however, the influenza bacillus has been found, sometimes alone, EXPERIMENTAL INOCULATION. 435 sometimes along with pyogenic cocci (Pfuhl and Walter, Cornil and Durante) ; Pfuhl considers that in these the path of infection is usually a direct one through the roof of the nasal cavity. This observer also found post mortem in a rapidly fatal case with profound general symptoms, influenza bacilli in various organs, both within and outside of the vessels. Experimental Inoculation. — There is no satisfactory evidence that any of the lower animals suffer from influenza in natural conditions, and accordingly we cannot look for very definite results from experimental inoculation. Pfeiffer, by injecting living cultures of the organism into the lungs of monkeys, in three cases produced a condition of fever of a remittent type. Somewhat similar results were obtained in one animal by smearing the uninjured mucous mem- brane of the nose with a pure culture. The fever appeared about twenty-four hours after the injection, and lasted for from three to five days. In another case in which large quantities of the bacilli were injected into the trachea, marked prostration and high temperature occurred, death following in twenty-four hours. There was, however, little evidence that the bacilli had undergone multiplication, the symptoms being apparently produced by their toxines. ' In the case of rabbits, intravenous injection of living cultures produces dyspnoea, muscular weakness, and slight rise of temperature, but the bacilli rapidly disappear in the body, and exactly similar symptoms are produced by injection of cultures killed by the vapour of chloroform. Pfeiffer, therefore, came to the conclusion that the influenza bacilli contain toxic substances which can produce in animals some of the substances of the disease, but that animals are not liable to infection, the bacilli not having power of multiplying to any extent in their tissues. Cantani in a recent work succeeded in producing infec- tion to some extent in rabbits, by injecting the bacilli directly into the anterior portion of the brain. In these experiments the organisms spread to the ventricles, and then through the spinal cord by means of the central canal 436 INFLUENZA. afterwards infecting the substance of the cord. An acute encephalitis was thus produced, and sometimes a purulent condition in the lateral ventricles. The bacilli were, how- ever, never found in the blood or in other organs. The symptoms produced were great dyspnoea, cardiac weakness, and also a paralytic condition which appeared first in the posterior limbs, and then spread to the rest of the body. The temperature was at first elevated, but before death fell below normal. Similar symptoms were also produced by injection of dead cultures, though in this case the dose required to be five or six times larger. Cantani therefore concludes that the brain substance is the most suitable nidus for their growth, but agrees with Pfeiffer in believing that the chief symptoms are produced by toxines resident in the bodies of the bacilli. He made control experiments by injecting other organisms, and also by injecting inert substances into the cerebral tissue. The evidence, accordingly, that the influenza bacillus is the cause of the disease rests chiefly on the well-established fact that it is always present in the secretions of the respiratory tract in true cases of influenza, and that it is an organism which has not been found in any other condition. Moreover, it is an organism which is practically restricted by its conditions of growth to the animal body. A certain amount of confirmatory evidence has been supplied by the results of experiment. Methods of Examination— (a) Microscope— A portion of the greenish-yellow purulent material which often occurs in little round masses in the sputum should be selected, and film preparations should be made in the usual way. Films are best stained by Ziehl - Neelsen carbol - fuchsin diluted with ten parts of water, the films being stained for ten minutes at least. In sections of the tissues, such as the lungs, the bacilli are bejst brought out, according to I feiffer, by staining with the same solution as above for half an hour The sections are then placed in alcohol containing a few drops of acetic acid, in which they are dehydrated and slightly decolorised at the same time. They should PLAGUE. 437 be allowed to remain till they have a moderately light colour, the time varying according to their appearance. They are then placed in xylol and afterwards mounted in balsam. (l>) Cultures. — A suitable portion of the greenish-yellow material having been selected from the sputum, it should be washed well in several changes of sterilised water. A portion should then be taken on a platinum needle, and successive strokes made on the surface of blood-agar tubes. The tubes should then be incubated at 370 C, when the transparent colonies of the influenza bacillus will appear, usually within twenty-four hours. These should give a negative result on inoculation on ordinary agar media. Plague. The bacillus of oriental plague or bubonic pest was discovered independently by Kitasato and Yersin during the epidemic at Hong Kong in 1894. The ^ *,~"~\ \ < *-v results of their investi- 1 . *'t»v /m gations, which were \\i w > ' * - / published almost at the same time, agree in most of the im- portant points. They cultivated the same organism from a large number of cases of • *»j • *\ ' x ''-J * ^ ' plague, and reproduced T-'l s N~* %m " ~ 2 t the disease in suscept- ^ " f ' 7 s ' ' " x ible animals by inocula- » — • i tion of pure cultures. * *" It is to be noted that FlGl "3-— Bacillus of plague from a , . . , . young culture on agar. during an epidemic of Stained with weak carbol-fuchsin. x 1000. plague, sometimes even preceding it, a high mortality has been observed amongst certain animals, especially rats and mice, and that 438 PLAGUE. from the bodies of these animals found dead in the plague- stricken district, the same bacillus was obtained by Kitasato and also by Yersin. Bacillus of Plague — Microscopical Characters. — As seen in the affected glands or buboes in this disease the bacilli are small oval rods, somewhat shorter than the typhoid bacillus, and about the same thickness (Fig. 113). They have rounded ends, and in stained preparations a portion is sometimes left unstained in the middle of the bacilli, giving the so-called " pole -staining." In the tissues they are found scattered amongst the cells, . for the most part lying singly, though pairs are also seen ; but in cultures, especially in fluids, they have a tendency to grow into chains, the form known as a strepto- bacillus resulting Fig. 114.— Bacillus of plague in chains (pjg_ 114). Some • • — 1-- From a young . showing polar staining, culture in bouillon. Stained with thionin-blue. x 1000. times in the tissues they are seen to be surrounded by an unstained capsule, though this appearance is by no means invariable. They do not form spores. Gordon, who has shewn that they possess flagella which, however, stain with difficulty, states that they are motile. They stain readily with the basic aniline stains, but are decolorised by Gram s method. , Anatomical Changes and Distribution of Bacilli.— 1 he disease occurs in several forms, the bubonic and the pulmonary being the best recognised ; to these may be added the septicemic. The most striking feature in the DISTRIBUTION OF BACILLI. 439 bubonic form is the affection of the lymphatic glands, which undergo intense inflammatory swelling, attended with hemorrhage, and generally ending in a greater or less degree of necrotic softening if the patient lives long enough. The connective tissue around the glands is similarly affected. True suppuration is rare. Usually one group of glands is affected first, constituting the primary bubo— in the great majority the inguinal or the axillary glands — and afterwards other groups may become involved, though to a much less extent. Along with these changes there is great swelling of the spleen, and often intense cloudy swelling of the cells of the kidneys, liver, and other organs. There may also occur secondary areas of haemorrhage and necrosis, chiefly in the lungs, liver, and spleen. The bacilli occur in enormous numbers in the swollen glands, being often so numerous that a film preparation made from a scraping almost resembles a pure culture; they lie irregularly arranged between the cellular elements. In the spleen they are fairly numerous, and in the secondary lesions mentioned they are often abundant. In the pulmonary form the lesion is the well-recognised "plague pneumonia." This is of broncho-pneumonic type, though large areas may be formed by confluence of the consolidated patches, and the inflammatory process is attended usually by much haemorrhage; the bronchial glands show inflammatory swelling. Clinically there may be little or no cough or expectoration ; in the sputum the bacilli may be found in large numbers. The disease in this form is said to be invariably fatal. In the septicemic form there is no primary bubo discoverable, though there may be a general enlargement of lymphatic glands ; here also the disease is of specially grave character. An intestinal form with extensive affection of the mesenteric glands has been described, but it is exceedingly rare — so much so that many observers with extensive experience have doubted its occurrence. In the various forms of the disease the bacilli occur also in the blood, in which they may be found during life by microscopic examination, chiefly, how- 44Q PLAGUE. ever, just before death in very severe and rapidly fatal cases. In most cases, however, they cannot be detected in the blood by this means, though in some of these they may be obtained by means of cultures. Cultivation. — From the affected glands the bacillus can readily be cultivated on the ordinary media. It grows best at the temperature of the body, though growth occurs as low as 1 8° C. On agar and on blood serum the colonies are circular discs of somewhat transparent appearance and smooth shining surface. When examined with a lens, their borders appear slightly wavy. In stroke cultures on agar there forms a continuous line of growth with the same appearance, showing partly separated colonies at its margins. When cultures on agar with a dry surface and slightly alkaline reaction are incubated at the body temperature involution forms rapidly appear. These are well seen on the third day and present the most varied forms — rounded, oval, pyriform, etc. ; some reach a size many times that of the healthy bacillus (Haffkine). In stab cultures in peptone gelatine, growth takes place along the needle track as a white line, composed of small spherical colonies, whilst on the surface of the gelatine a thin semi-transparent layer is formed, which is usually restricted to the region of puncture, though sometimes it may spread to the wall of the tube. In bouillon the growth usually forms a slightly granular or powdery deposit at the foot and sides of the flask, somewhat resembling that of a streptococcus. If, however, the flasks containing the bouillon be kept absolutely at rest flakes form at the surface, and from these growth extends downwards forming the so-called "stalactites." The columns of growth are easily broken when the flasks are moved, and then fall to the bottom in the form of a powdery deposit. Haffkine con- siders this feature along with the involution forms on dry agar to be of great importance in the diagnosis of the organism. The organism in its powers of resistance corresponds with other spore-free bacilli, and is readily killed by heat. EXPERIMENTAL INOCULATION. 441 It resists drying for four days at latest, and exposure to direct sunlight for three or four hours kills it. When cultivated outside the body the organism rapidly loses its virulence. Experimental Inoculation. — Rats, mice, guinea-pigs, and rabbits are susceptible to inoculation. After subcutaneous injection there occurs a local inflammatory oedema, which is followed by inflammatory swelling of the lymphatic glands, and thereafter by a general infection. The animals die usually in from one to five days, the chief changes, in addition to the glandular enlargement, being congestion of internal organs, sometimes with haemorrhages, and enlargement of the spleen ; the bacilli are present in the lymphatic glands and also, though in smaller numbers, throughout the blood. Rats and mice can also be infected by feeding either with pure cultures or with pieces of organs from cases of the disease, and animals infected by inoculation may transmit the disease to healthy animals kept along with them. Monkeys also are highly susceptible to infection, and it has been shown in the case of these animals that, when inoculation is made on the skin surface, for example, by means of a spine charged with -the bacillus, the glands in relation to the part may show the characteristic lesion and a fatal result may follow without there being any noticeable lesion at the primary seat. This fact throws important light on infection by the skin in the human subject. The disease may also extensively affect monkeys by natural means during an epidemic. There can be no doubt that this bacillus is the immediate cause of the disease, and the bacteriological observations throw much light on its method of spread. The affection of the lower animals by the same bacilli has been abundantly proved, and large numbers of dead animals in the infected localities were found to contain the organism. The disease was produced also by inoculation with dust from infected houses, and Yersin found the organism in' large numbers in the bodies of dead flies in the infected locality. Ogata also has furnished evidence that flies and mosquitoes may play a part in the spread of the disease. 442 PLAGUE. There can be little doubt, however, that rats play the most important part in distributing it over wide areas of a town or district when once it has broken out. This has been abundantly proved in the case of Bombay, where observations have shown that the migration of plague- infected rats to districts comparatively free from the disease has been attended by extensive outbreak in these places. The disease can also be transmitted by contagion from persons affected, but this method of transmission can be much more easily controlled than the previous. The bacillus enters the human body by lesions of the skin surface, by the respiratory passages, and possibly also by the alimentary canal. The first mentioned is the commonest mode, cracks and abrasions of the skin apparently supplying the means of easy entrance ; in this connection the fact that there may be no local lesion at the site of inoculation is of much im- portance. The occurrence of infection by the respiratory passages is less common, but is proved by the anatomical changes as detailed above. In addition to the sputum, bacilli have been demonstrated also in the urine and the fasces of those suffering from the -disease. From the facts stated with regard to the powers of comparatively rapid multiplication of the bacillus, its wide dissemination by affected rats, human excreta, etc., it may be understood* how extensively the soil and dwellings may become infected, and how difficult it may be to arrest the ravages of the disease. How important a part such infection of a locality plays is strikingly shown by the rapid fall in the number of cases where the people go into tents. Immunity. — Yersin, Calmette, and Borrel succeeded in producing a certain amount of immunity in rabbits against the organism by injection of cultures killed by heat at 5 8° C. They further found that the serum of such animals had certain protective powers when tested in mice. Later, they immunised a horse by intravenous injection of the living bacilli, and obtained a serum which had more powerful properties. This anti-plague serum has been employed by Yersin in cases of the disease at Canton, IMMUNITY. 443 Amoy, and, more recently, Bombay. The earlier reports of the results obtained were of a distinctly favourable character, the later less so, although no doubt there has been much difficulty in obtaining in India a serum of the requisite strength. The system of preventive inoculation against plague devised by Haffkine has been carried out on a pretty extensive scale in India. In this method the vaccine used is a bouillon culture of the plague bacillus killed by exposure to 700 C. for an hour. To obtain a very abundant growth clarified butter (" ghee ") is added to the bouillon and, after inoculation, from the drops of fat floating on the surface growth takes place in the stalactite form. When this has occurred the " stalactites " are broken by shaking and fall to the bottom, and then fresh growth occurs from above downwards. In this manner six crops may be obtained in about four weeks, the final result being a very abundant deposit with clear supernatant fluid. The former when injected into an animal causes a local inflammatory swelling, the latter a rise in temperature. A mixture of the clear fluid and deposit is used for inoculation against plague, about 3 c.c. in the sterile condition being injected into the flank. The method has been systematically tested by inocu- lating a certain proportion of the inhabitants of districts exposed to infection, leaving others uninoculated, and then observing the proportion of cases of the disease and of fatal results amongst the two classes. The results have been dis- tinctly satisfactory, there being a distinct diminution in the number of cases among the inoculated, and a still greater di- ■ munition in the mortality. In the most favourable series reported by Haffkine and Bannerman the mortality amongst the inoculated was diminished by over 80 per cent. Further observations, which will no doubt be shortly forthcoming, will supply definite results as to the exact value of the method. 444 RELAPSING FEVER. Relapsing Fever. At a comparatively early date, namely in 1873, when practically nothing was known with regard to the production of disease by bacteria, a highly characteristic organism was discovered in the blood of patients suffering from relapsing fever. This discovery was made by Obermeier, and the organism is usually known as the spirillum or spirachcete Obermeieri, or the spirillum of relapsing fever. He described its microscopical characters, and found that its presence in the blood had a definite relation to the time of the fever, as the organism rapidly disappeared about the time of the crisis, and reappeared when a relapse occurred. He failed to find such an organism in any other disease. His observations were fully confirmed, and his views as to its causal relationship to the disease were generally accepted. Later, the disease was produced in the human subject by inoculations with blood containing the organisms, and a similar condition has been produced in y apes. y ^ Characters of the Spirillum. — The or- ganisms as seen in the blood during the fever are delicate spiral ... ^rsJ v filaments which have f^jlL a length of 2 to 6 % . times the diameter f \ of a red blood cor- v 1 1 puscle. They are, \ however, exceedingly thin, their thickness Fig. 115.— Spirilla of relapsing fever in hejng much less than human blood Film preparation. (After Qf ^ Koch.) x about 1000. . spirillum. They show several regular sharp curves or windings, of number varying according to the length of the spirilla, and their extremities RELATIONS OF SPIRILLUM TO DISEASE. 445 are finely pointed (Fig. 115). They are actively motile, and may be seen moving quickly across the microscopic field with a peculiar movement which is partly twisting and partly undulatory, and disturbing the blood corpuscles in their course. They stain with watery solutions of the basic aniline dyes, though somewhat faintly, and are best coloured in film preparations by Loffler's or Klihne's methylene-blue solutions. When thus stained they usually have a uniform appearance throughout, or may be slightly granular at places, but they show no division into short segments. They lose the stain in Gram's method. In blood outside the body the organisms have a con- siderable degree of vitality, and when kept in sealed tubes have been found alive and active after many days. They are readily killed at a temperature of 6o° C., but may be exposed to o° C. without being killed. There is no evidence that they form spores. Relations to the Disease. — In relapsing fever, after a period of incubation there occurs a rapid rise of tempera- ture which lasts for about five to seven days. At the end of this time a crisis occurs, the temperature falling quickly to normal. In the course of about another seven days a sharp rise of temperature again takes place, but on this occa- sion the fevef lasts a shorter time, again suddenly disappear- ing. A second or even third relapse may occur after a similar interval. The spirilla begin to appear in the blood shortly before the onset of the pyrexia, and during the rise of temperature rapidly increase in number. They are very numerous during the fever, a large number being often present in every field of the microscope when the blood is examined at this stage. They begin to disappear shortly before the crisis : after the crisis they are entirely absent from the circulating blood. A similar relation between the presence of the spirilla in the blood and the fever is found in the case of the relapses, whilst between these they are entirely absent. Munch in 1876 produced the disease in the human subject by injecting blood containing the spirilla, 446 RIU.M'SING /■ A / 'EN. and this experiment has been several times repeated with the same result ; after a period of incubation the spirilla begin to appear in the circulating blood, and their appear- ance is soon followed by the occurrence of pyrexia. Numerous attempts to cultivate this organism outside the body have all been attended with failure, and it has been abundantly shown that it does not grow on any of the media ordinarily in use. Koch found that on blood serum the filaments of the spirilla increased somewhat in length, and formed a sort of felted mass, but that no multi- plication took place. Additional proof, however, that the organism is the cause of the disease has been afforded by experiments on monkeys, and facts of considerable interest have been thus established. Carter in 1879 was the first to show that the disease could be readily produced in these animals, and his experiments were confirmed by Koch. In such experiments the blood taken from patients and containing the spirilla was injected subcutaneously. In the disease thus produced there is an incubation period which usually lasts about three days. At the end of that time the spirilla rapidly appea^ in the blood, and shortly afterwards the temperature quickly rises. The period of pyrexia usually lasts for two or three days, and is followed by a marked crisis. As a rule there is no relapse, but occasionally one of short duration occurs. The presence of spirilla in the blood has the same relation to the pyrexial period as in the human subject. For a long time the place and mode of destruction of the spirilla were quite unknown, but valuable light was thrown on these points by Metchnikoff, who produced the disease in monkeys and killed them at various stages of the fever. He found that during the fever the spirilla were practically never taken up by the leucocytes in the circulating blood, but at the time of the crisis the spirilla, on disappearing from the blood, accumulated in the spleen and were ingested in large numbers by the microphages or polymorphonuclear leucocytes. Within these they rapidly underwent degeneration and disappeared. Metchnikoff also RELATIONS OF SPIRILLUM TO DISEASE. 447 found that after the spirilla had disappeared from the blood, the disease could be produced in another animal by inocu- lations with spleen pulp, in which the spirilla were contained within the leucocytes, thus showing that they were living and active in the spleen. It is to be noted in this con- nection that swelling of the spleen is a very marked feature in relapsing fever. These observations have been entirely confirmed by Soudakewitch, who also showed that the destruction of the spirilla in the spleen was an extremely rapid one, as they were all destroyed ten hours after their disappearance from the blood. He also produced the disease in two monkeys from which the spleen had been previously removed, the animals having been allowed to recover completely from the operation. In these cases the spirilla did not disappear from the blood at the usual time, but rather increased in number, and a fatal result followed on the eighth and ninth days respectively. Post mortem he found the spirilla in enormous numbers throughout the blood vessels, and in the portal vein they almost equalled the red blood corpuscles in number. By these experiments it would appear to be established that the spleen has an important function in the destruction of the organisms. It has not been shown, however, why the organisms disappear from the blood at a particular time and accumulate in the spleen. In the case of the human subject it has been found that a second attack of the disease can follow the first after a comparatively short period of time, and it is often said that one attack does not confer immunity. It is probably rather the case that the immunity conferred is of very short duration. The course of events in the disease might be explained by supposing that immunity of short duration is produced during the first period of pyrexia, but that it does not last until all the spirilla have been destroyed, some still surviving in internal organs. With the disappearance of the immunity the organisms reappear in the blood, the relapse being, however, of shorter duration and less severe than the first attack. This is repeated till the immunity lasts long enough 44s MALTA FEVER. to allow all the organisms to be killed. On these points, however, further information is still necessary. Any anti- microbic power which the serum may possess after the crisis has not yet been demonstrated. It is further to be noted that relapsing fever is unique amongst bacterial diseases affecting the human subject, in respect of the enormous numbers of organisms which can be observed in the circu- lating blood during life. Malta Fever. Synonyms — Mediterranean Fever: Rock Fever of Gibraltar : Neapolitan Fever, etc. This disease is of common occurrence along the shores of the Mediterranean and in its islands. Although from its symptomatology and pathological anatomy it had been recognised as a distinct affection, and was known under various names, its precise etiology was unknown till the publication of the researches of Surgeon-Major Bruce in 1887. From the spleen of patients dead of the disease he cultivated a characteristic organism, now known as the micrococcus melitensis, and by means of inoculation experi- ments established its causal relationship to the disease. His results have been confirmed by other observers, and additional confirmatory evidence has been supplied by means of serum diagnosis, as will be described below. Bacteriological methods have therefore been the means of differentiating the disease, and also of affording a more exact basis for diagnosis. By means of the agglutinating test Wright and Smith have shown that it occurs also in some parts of India, and there can be little doubt that its distribution will be found to be much wider than was formerly supposed. The duration of the disease is usually long— often two or three months, though shorter and much longer periods are met with. Its course is very variable, the fever being of the continued type with irregular remissions. In addi- MICROCOCCUS ME LI TENS IS. 449 tion to the usual symptoms of pyrexia there occur profuse perspirations, pains and sometimes swellings in the joints, occasionally orchitis, whilst constipation is usually a marked feature. The mortality is low — about 2 per cent (Bruce). In fatal cases the most striking post-mortem change is in the spleen. This organ is enlarged, often weighing slightly over a pound, and in a condition of acute congestion ; the pulp is soft and may be diffluent, and the Malpighian bodies are swollen and indistinct. In the other organs the chief change is cloudy swelling ; in the kidneys there may- be in addition glomerular nephritis. The lymphoid tissue of the intestines shows none of the changes characteristic of typhoid fever. Micrococcus Melitensis. — This is a small, rounded or slightly oval organism about .5 p in diameter which is specially abundant in the spleen. It usually occurs singly or in pairs, but in cultures short chains are also met with (Fig. 116). (Durham has shown that in old cultures kept at the room tem- perature b'acillary forms appear, and we have noticed indica- tions of such in com- paratively young cul- tures ; the usual form ic V.^oro,^^ fk^f „ FlG- II6- — Micrococcus melitensis, from is, however, that of a a two days, culture on agar at 3f c COCCUS.) It Stains Stained with fuchsin. x 1000. fairly readily with the ordinary basic aniline stains, but loses the stain in Gram's method. It is generally said to be a non-motile organism. Gordon, however, is of a contrary opinion, and has recently demonstrated that it possesses from one to four flagella which, however, are difficult to stain. In the spleen 29 45° MALTA FEVER. of a patient dead of the disease it occurs irregularly scattered through the congested pulp. It may also be found in small numbers post mortem in the capillaries of various organs, but examination of the blood during life gives negative results. It can, however, be obtained by puncture of the spleen during life. Cultivation. — This can usually readily be effected by making stroke cultures on agar tubes from the spleen pulp and incubating at 370 C. The colonies, which are usually not visible before the third day, appear as small round discs, slightly raised and of somewhat transparent appear- ance. The maximum size — 2-3 mm. in diameter— is reached about the ninth day j at this period by reflected light they appear pearly white, while by transmitted light they have a yellowish tint in the centre, bluish white at the periphery. A stroke culture shows a layer of growth of similar appearance with somewhat serrated margins. Old cultures assume a buff tint. The optimum temperature is 37c C, but growth still occurs down to about 200 C. On gelatine at summer temperature growth is extremely slow after two or three weeks, in a puncture culture, there is a delicate line of growth along the needle track and a small fiat expansion of growth on the surface. There is no liquefaction of the medium. In bouillon there occurs a general turbidity with floccu- lent deposit at the bottom; on the surface there is no formation of a pellicle. On potatoes no visible growth takes place even at the body temperature, though the organism multiplies to a certain extent. Relations to the Disease.— There is in the first place ample evidence, from examination of the spleen, both post mortem and during life, that this organism is always present in the disease. The experiments of Bruce and Hughes show that by inoculation with even comparatively small doses of pure cultures the disease can be produced in monkeys In these experiments seven animals in all were used in every case with a positive result. Four died at varying periods of time, after showing well-marked fever, RELATIONS TO THE DISEASE. 45i closely resembling in character that occurring in the human subject, and the same organism was obtained from the organs post mortem. The other three animals recovered after suffering from illness with corresponding pyrexia — in two cases extending over two months. The disease has also been produced in the human subject, in one instance by accidental inoculation with a pure culture of the micrococcus. Rabbits, guinea-pigs and mice are insusceptible to inoculation by the ordinary method. Durham, by using the intracerebral method of inoculation, has, however, succeeded in raising the virulence so that the organism is capable of producing in guinea-pigs on intra-peritoneal injection illness with sometimes a fatal result many weeks afterwards. An interesting point brought out by these experiments is that in the case of animals which survive the micrococcus may be cultivated from the urine several months after inoculation. Agglutinative Action of Serum. — The blood serum of patients suffering from Malta fever possesses the power of agglutinating the micrococcus melitensis in a manner analog- ous to what has been described in the case of typhoid fever. This action is manifested throughout the disease, and also for a considerable time after recovery. Wright and Smith found it well marked a year afterwards. They found that the greatest dilution which gives distinct agglutinative effects varies in different cases from 1:10 to 1 : 1000. It is needless to point out that the method affords a satisfactory means of diagnosis from other fevers, such as typhoid — a disease for which it is often mistaken. Methods of Diagnosis. — During life the best means of diagnosis is supplied by the agglutinative test just described (for technique vide p. 118). Cultures are most easily obtained from the spleen, either during life or post mortem. Inoculate a number of agar tubes by successive strokes and incubate at 370 C. Film preparations should also be made from the spleen pulp 452 YELLOW FEVER. and stained with carbol-thionin-blue or diluted carbol- fuchsin (i : 10). Yellow Fever. Yellow fever is an infectious disease which is endemic in the West Indies, in Brazil, in Sierra Leone and the adjacent parts of West Africa, though it is probable that it was from the first- named region that the others were originally in- fected. From time to time serious outbreaks occur, during which neighbouring countries also suffer, and the disease may be carried to other parts of the world. In this way epidemics have occurred in the United States, in Spain, a^id even in England, infection usually being carried by cases occurring among the crews of ships. In the parts where it is endemic, though usually a few cases may occur from time to time, there is some evidence that occasionally the disease may remain in abeyance for many years and then originate de novo. There is, therefore, reason to suspect that the infective agent can exist for considerable periods outside the human body. It is possible, however, that continuity may be maintained by the occurrence of a mild type of the disease which maybe grouped with the "bilious fevers " prevalent in yellow fever regions. This would ex- plain the degree of immunity which is shewn during a serious epidemic by the older immigrants. Great variations are observed in the clinical types under which the disease presents itself. Usually after two to four days' incubation a sudden onset in thejorm of a rigor occurs The temperature rises to 1040 F. to 105 *. The person is livid, with outstanding bloodshot eyes. There is great prostration, pain in the back, and vomiting, at first of mucus, later of bile. The urine is diminished and contains albumin. About the fifth day an apparent im- provement takes place, and this may lead on to recovery. Frequently, however, the remission, which may last from a few hours to two days, is followed by an aggravation of all the symptoms. The temperature rises, jaundice is observed, haemorrhages occur from all the mucous surfaces, causing, in BACTERIA IN YELLOW FEVER. 453 the case of the stomach, the " black vomit " — one of the clinical signs of the disease in its worst form. Anuria, coma, and cardiac collapse usher in a fatal issue. The mortality varies in different epidemics from about 35 to 99 per cent of those attacked. Both white and black races are susceptible, but those who have resided long in a country are less susceptible than new immigrants. An attack of the disease usually confers complete immunity against subsequent infection. On making a post-mortem examination the stomach is found in a state of acute gastritis, and contains much altered blood derived from haemorrhages which have occurred in the mucous and submucous coats. The intestine may be normal, but is often congested and may be ulcerated ; the mesenteric glands are enlarged. The liver is in a state of fatty degeneration of greater or less degree, but often resembling the condition found in phosphorus poisoning. The kidneys are in a state of intense glomerulo - nephritis, with fatty degeneration of the epithelium. There is congestion of the meninges, especially in the lumbar region, and haemorrhages may occur. The other organs do not show much change, though small haemorrhages under the skin and into all the tissues of the body are not infrequent. In the blood a feature is the excess of urea present, amounting, it may be, to nearly 4 per cent. Bacteria in Yellow Fever. — A very full research into the bacteriology of yellow fever was that of Sternberg (1890), who made cultures from various organs which had been kept for some days at room temperature. Of the organisms isolated one which he named " bacillus x " appeared possibly to have some causal relationship to the disease. Sanarelli (in 1897) obtained cultures of the staphy- lococcus aureus and albus, B. coli, and of an organism which he named bacillus icteroides, and which he considers to be the cause of yellow fever. It is probably identical with the " bacillus x." Bacillus Icteroides. — This is a bacillus belonging to the 454 YELLOW FEVER. B. coli group. It has rounded ends, is 2 to 4 /x long, about .5 [x, broad, often occurring in pairs (Fig. 117), staining by V WSJ. ' s *. Fig. 117. — Bacillus Icteroides, from a young culture on agar (Sanarelli). x 1000. the ordinary stains but decolorised in Gram's method. It is motile and possesses 4 to 8 flagella. It grows on all the usual media ; on gelatine plates after twenty-four hours the colonies are minute points some- what transparent to the naked eye, and under a low power have a finely granular appearance. After six or seven days there appears somewhere in the colony a focus of more active growth, forming an opaque centre, from which granular stria? radiate to the periphery. The gelatine is not liquefied. The most characteristic growth is that in sloped agar. After twenty-four hours at 37° C. there is a grey iridescent and somewhat transparent growth. On being transferred to a temperature of 200 C. to 2 8° C. this Fig. 118. — Culture of Bacillus Icteroides on agar, showing the characteristic appear- ance when incubated at the two temperatures mentioned (Sanarelli). Natural size. BA C1LL US IC TER 01 D ES. 455 becomes in twelve hours surrounded by a halo of white, opaque, pearly growth of higher level than the central part. In a few days the growth at the lower temperature becomes liquid in character and runs slowly down the medium as a drop of melted paraffin would do (Fig. 118). Growth also takes place in bouillon and blood serum. On potatoes there is a fine transparent pellicle which does not alter its colour with age. Milk is curdled, but only after some weeks. The bacillus ferments glucose but not lactose. It gives a feeble indol reaction. Sanarelli investigated twelve cases of yellow fever and found the B. icteroides present in relatively small numbers in six. It appeared chiefly in the capillaries of the liver and kidneys, rarely in other parts of the body. The methods applied in the case of the gastro- intestinal tract are not given, but it was never found there. Mice, guinea-pigs and rabbits succumbed to subcu- taneous injection of the bacilli, but the results were not characteristic. In the dog sometimes death occurred after intravenous injection in a few hours, sometimes in from eight to twenty-one days. The symptoms were sickness, continuous loss of weight, anuria, ■ diarrhoea, it might be sanguineous, and jaundice. Post mortem there were fatty degeneration of the liver and a grave glomerulo- nephritis, the intestinal walls were hypersemic, and the lumen filled with a coffee-coloured matter. The blood contained an exxess of urea and the B. icteroides was more or less widely present in the body. Bouillon cultures twenty days old when filtered germ- free and injected subcutaneously do not give rise in the dog to a characteristic illness. Cultures killed by ether and injected intravenously cause practically the same effects as living cultures. Sanarelli states that sterile bouillon cultures wfien injected- in man subcutaneously or intravenously give rise to all the symptoms of yellow fever. If the latter statement be correct there can be little 456 YELLOW FEVER. doubt that the bacillus icteroides is the cause of yellow fever. It must be looked on as settling chiefly in the liver and kidneys and there producing very powerful toxines whose chief effects are on the cells of these organs and on the small blood vessels of the body, thus causing the rupture of vessel walls, which frequently results. These grave actions of the toxines open up a path for infection by the other organisms we have noted as frequently occurring in conjunction with the B. icteroides in the bodies of those dead of the disease. To such secondary infection is due the variability in the clinical types met with, though probably another cause is interference with the functions of the kidneys when the affection of these organs predominates. There is evidence that when the secondary infections commence their results are inimical to the vitality of the B. icteroides, a fact which would to some extent explain the many failures to find the latter in cases of yellow fever. These results have not thrown any light on the channels of natural infection. Sanarelli has shown that between moulds and the B. icteroides there exists a symbiosis favour- able to the latter. This is a possible explanation of the well-known belief that the infection has a special tendency to hang about the holds of ships. The non-occurrence of the B. icteroides in the intestinal canal is interesting, as several observers had tried unsuccessfully to inoculate themselves by means of the black vomit. Immunity against the B. Icteroides. — The serum of yellow fever patients is said by Sanarelli to clump the B. icteroides. Other observers have confirmed the observation, the most appropriate dilution being i : 40. The reaction is said to appear on the second day. The smaller animals are unsuitable for immunisation on account of their great susceptibility to toxic action ; but horses have been immunised by careful injections of filtered cultures, cultures killed by ether and living cultures suc- cessively. The process is, however, a very tedious one. The serum of such a horse was found capable of saving BACILLUS ICTER01DES. 457 a guinea-pig even when symptoms had begun to appear. In a record of eight cases where the serum was used in man six recoveries took place. This is a very hopeful result, and there is also considerable ground for believing that such serum may be even more useful as a prophylactic agent. CHAPTER XIX. IMMUNITY. Introductory. — By immunity is meant non-susceptibility to a given disease or to a given organism, either under natural conditions or under conditions experimentally produced. The term is also used in relation to the toxines of an organ- ism. Immunity may be possessed by an animal naturally, and is then usually called natural immunity, or it may be acquired by an animal either by passing through an attack of the disease, or by artificial means of inoculation. It has been shown that certain diseases affect the lower animals but never occur in the human subject, e.g., swine plague ; and, on the other hand, diseases such as typhoid fever and cholera do not under natural conditions affect any of the lower animals, so far as is known. That is to say, man and the lower animals respectively enjoy immunity against certain diseases, when exposed to infection under ordinary conditions. From this fact, however, it does not follow that when the organisms of the respective diseases are introduced into the body by artificial methods of inoculation, pathological effects will not follow. We have seen above, for example, that the organisms of cholera and typhoid may artificially be made to infect guinea-pigs, though they do not do so naturally. Immunity may thus be of very varying degrees, and accordingly the use of the term has a correspondingly relative significance. Such a thing as absolute immunity is scarcely known, just as we ACQUIRED IMMUNITY IN THE HUMAN SUBJECT. 459 have seen is the case with absolute susceptibility. This is not only true of infection by bacteria, but of toxines also ; — when the resistance of an animal to these is of high degree, the resistance may be overcome by a very large dose of the toxic agent. For example, the common fowl may be able to resist as much as 20 c.c. of powerful tetanus toxine, but on this amount being exceeded may be affected by tetanic spasms (Klemperer). On the other hand, in cases where the natural powers of resistance are very high, these can be still further exalted by artificial means, that is, the natural immunity may be artificially intensified. Acquired Immunity in the Human Subject. — The following facts are supplied by a study of the natural diseases which affect the human subject. First, in the case of certain diseases, one attack protects against another for many years, sometimes practically for a lifetime, e.g., smallpox, typhoid, scarlet fever, etc. Secondly, in the case of other diseases, e.g., erysipelas, diphtheria, influenza, and pneumonia, a patient may suffer from several attacks. In the case of the diseases of the second group, however, experimental research has shown that in many of them a certain degree of immunity does follow ; and, though we cannot definitely state it as a universal law, it must be considered highly probable that the attack of an acute disease produced by an organism, confers immunity for a longer or shorter period. The facts known regarding vaccination and smallpox- exemplify another principle. We may take it as practically proved that vaccinia is variola or smallpox in the cow, and that when vaccination is performed, the patient is inocu- lated with a modified variola (vide Smallpox, in Appendix). Vaccination produces certain pathogenic effects which are of trifling degree as compared with those of smallpox, and we find that the degree of protection is less complete and lasts a shorter time than that produced by the natural disease. Again, inoculation with lymph from a smallpox pustule produces a form of smallpox less severe than the natural disease but a much more severe condition than 460 IMMUNITY. that produced by vaccination, and it is found that the degree of protection or immunity resulting occupies an intermediate position. The corresponding general con- clusion from experiments is that the more virulent the organism injected, provided that the animal recovers satis- factorily, the higher is the degree of immunity acquired by it against that organism. Thus in developing immunity of the highest degree the most virulent organisms are ultimately employed. A corresponding principle, with certain restrictions {vide p. 472), obtains in the case of toxines. Immunity and Recovery from Disease. — Recovery from an acute infective disease shows that in natural conditions the virus may be exhausted after a time, the period of time varying in different diseases. How this is accomplished we do not yet fully know, but it has been found in the case of diphtheria, typhoid, cholera, pneumonia, etc., that in the course of the disease certain substances (called by German writers Antikorper) appear in the blood, which are antagon- istic either to the toxine or to the vital activity of the organism. In such cases a process of immunisation would appear to be going on during the progress of the disease, and when this immunisation has reached a certain height, the disease naturally comes to an end. It cannot, how- ever, be said at present that such antagonistic substances are developed in all cases. Artificial Immunity. Varieties. — A number of facts regarding immunity have been given in the description of the pathogenic organisms in previous chapters. We shall here give a general systematic description of the methods, and discuss the principles involved. According to the means by which it is produced, immunity may be said to be of two kinds, to which the terms active and passive are generally applied, or we may speak of immunity directly, or indirectly produced. ARTIFICIAL IMMUNITY. 461 Active immunity is obtained by (a) injections of the organisms either in an attenuated condition or in sub-lethal doses, or (b) by sub-lethal doses of their products, i.e., of their "toxines," the word being used in the widest sense. By repeated injections at suitable intervals the dose of organisms or of the products can be gradually increased, and a proportionate degree of resistance or immunity can be developed, which degree in course of time may reach a very high level. Such a method can be preventive, but it can never be curative, as the immunity must be developed before the onset of the disease. Immunity of this kind is comparatively slowly produced and lasts a considerable time, the duration varying in different cases. Passive immunity depends upon the fact that if an animal be immunised to a very high degree by the previous method, its serum may have distinctly antagonistic or neutralising effects when injected into another animal along with the organisms, or with their products, as the case may be. Here the serum of the highly-immunised animal may confer immunity on another animal, if introduced at the same time as infection occurs or even a short time after- wards ; the method can, therefore, be employed as a curative agent. The serum is also preventive, i.e., protects an animal from subsequent infection, but the immunity thus conferred lasts a comparatively short time. These facts form the basis of serum therapeutics. When such a serum has the power of neutralising a toxine it is called antitoxic ; when, with little or no antitoxic power it pro- tects against the living bacterium in a virulent condition it is called antimicrobic or antibacterial (vide infra). In the accompanying table a sketch of the chief methods by which an immunity may be artificially produced is given. It has been arranged for purposes of convenience and to aid subsequent description, and it is not to be inferred that all the different methods imply essentially different principles. There is still some doubt as regards the relation of A 2, for example, to A 1 and A 3. 462 IMMUNITY. Artificial Immunity. A. Active Immunity — i.e., produced in an animal by an injection, or by a series of injections, of non-lethal doses of an organism or its toxines. 1. By injection of the living organisms. (a) Attenuated in various ways. Examples : — (1) By growing in the presence of oxygen, or in a current of air. (2) By passing through the tissues of one species of animal (becomes attenuated for another species). (3) By growing at abnormal temperatures, etc. (4) By growing in the presence of weak anti- septics, or by injecting the latter along with the organism, etc. (b) In a virulent condition, in non-lethal doses. 2. By injection of the dead organisms. 3. By injection of filtered bacterial cultures, i.e., toxines ; or of chemical substances derived from these. These methods may also be combined in various ways. B. Passive Immunity, i.e., produced in one animal by injec- tion of the serum of another animal highly immunised by the methods of A. 1. By antitoxic serum, i.e., the serum of an animal highly immunised against a particular toxine. 2. By antimicrobic serum, i.e., the serum of an animal highly immunised against a particular organism in the living and virulent condition. A. Active Immunity. 1 By Living- Cultures.— (a) Attenuated.— In the earlier work on immunity in the case of anthrax, chicken cholera, swine plague, etc., the methods consisted in the employ- ACTIVE IMMUNITY. 463 ment of cultures of the living organisms, the virulence of which was so diminished that on inoculation they did not produce a fatal disease, but yet had effects sufficient for protection. The principle is therefore the same as that of vaccination, and the attenuated cultures are often spoken of as vaccines. The virulence of an organism may be diminished in various ways, of which the following examples may be given. (1) In the first place, practically every organism when cultivated for some time outside the body, loses its virulence, and in the case of some this is very marked indeed, e.g., the pneumococcus. Pasteur found in the case of chicken cholera, that when cultures were kept for a long time in ordinary conditions, they gradually lost their virulence, and that when sub-cultures were made, the diminished virulence persisted. Such attenuated cultures could be used for pro- tective inoculation. He considered the loss of virulence to be due to the action of the oxygen of the air, as he found that in tubes sealed in the absence of oxygen the virulence was not lost. Haffkine attenuated cultures of the cholera spirillum by growing them in a current of air. (2) The virulence of an organism for a particular animal may be lessened by passing the organism through the body of another animal. Duguid and Burdon Sanderson found that the virulence of the anthrax bacillus for bovine animals was lessened by being passed through guinea-pigs, the dis- ease produced in the ox by inoculation from the guinea-pig being a non-fatal one. This discovery was confirmed by Greenfield, who found that the bacilli cultivated from guinea- pigs preserved their property in cultures, and could therefore be used for protective inoculation of cattle. A similar principle was applied in the case of swine plague by Pasteur, who found that if the organism producing this disease was inoculated from rabbit to rabbit, its virulence was increased for rabbits but was diminished for pigs. Organisms which had been passed through a series of rabbits produced in the pig illness, but not death, and protection for at least a year resulted. The method 464 IMMUNITY. of vaccination against smallpox depends upon the same principle. (3) Many organisms become diminished in virulence when grown at an abnormally high temperature. The method of Pasteur, already described (p. 313), for producing immunity in sheep against anthrax bacilli, depends upon this fact. A virulent organism may also be attenuated by being exposed to an elevated temperature which is insufficient to kill it. Toussaint at an early date obtained protective inoculation against anthrax by means of cultures which had been exposed for a certain time to a temperature of 550 C, though it is possible that in some cases the bacilli were really killed, and immunity resulted from their chemical products. (4) Still another method may be mentioned, namely, the attenuation of the virulence by growing the organism in the presence of weak antiseptics. Chamberland and Roux, for example, succeeded in attenuating the anthrax bacillus by growing it in a medium containing carbolic acid in the proportion of 1 : 600. The virulence may also sometimes be attenuated by injecting certain chemical substances along with the bacteria into the body. Iodine terchloride was found by Behring to modify in this way the virulence of the diphtheria bacillus. These examples will serve to show the principles under- lying attenuation of the virulence of an organism. There are, however, still other methods, most of which consist in growing the organism in conditions somewhat unfavourable to its growth, e.g., under compressed air, etc. (p) By living Virulent Cultures in non-lethal Doses. — Immunity may also be produced by employing virulent cultures in small, that is, non-lethal doses. In subsequent inoculations the doses may be increased in amount. For example, immunity may thus be obtained in rabbits against the bacillus pyocyaneus. Such a method, however, has had a limited application in the case of virulent organisms, as it has been found more convenient to commence the process by attenuated cultures. BY LIVING CULTURES. 465 Exaltation of the Virulence. — The converse process to attenuation, i.e., the exaltation of the virulence, is obtained chiefly by the method of cultivating the organism from animal to animal — the method of passage discovered by Pasteur (first, we believe, in the case of an organism obtained from the saliva in hydrophobia, though having no causal relationship to that disease). This is most con- veniently done by intraperitoneal injections, as there is less risk of contamination. The organisms in the peritoneal fluid may be used for the subsequent injection, or a culture may be made between each inoculation. The virulence of a great number of organisms can be increased in this way, the animals most frequently used being rabbits and guinea-pigs. This method can be applied to the organisms of typhoid, cholera, pneumonia, to streptococci, and staphylo- cocci, and in fact to those organisms generally which invade the tissues. The virulence of an organism, especially when in a relatively attenuated condition, can also be raised by inject- ing along with it a quantity of a culture of another organism either in the living or dead condition. A few examples may be mentioned. An attenuated diphtheria culture may have its virulence raised by being injected into an animal along with the streptococcus pyogenes ; an attenuated culture of the bacillus of malignant oedema by being injected with the bacillus prodigiosus ; an attenuated streptococcus by being injected with the bacillus coli, etc. A culture of the typhoid bacillus may be increased in virulence, as already stated, by being injected along with a dead culture of the bacillus coli. In such cases the ac- companying injection enables the attenuated organism to gain a foothold in' the tissues, and it may be stated as a general rule that the virulence of an organism for a particular animal is raised by its growing in the tissues of that animal. Combination of Methods. — The above methods may be combined in various ways. By repeated injections of cultures at first attenuated and afterwards more virulent, 30 466 IMMUNITY. and by increasing the doses, a high degree of immunity may be arrived at. Anti-Cholera Inoculation. — Haffkine's method for in- oculation against cholera exemplifies the above principles. It depends upon (a) attenuation of the virus, that is, the cholera organism, and (b) exaltation of the virus. The virulence of the organism is diminished by passing a current of sterile air over the surface of the cultures, or by various other methods. The virulence is exalted by the method of passage, that is, by growing the organism in the peritoneum in a series of guinea-pigs. By the latter method the virulence after a time is increased twenty-fold, that is, the fatal dose has been reduced to a twentieth of the original. Cultures treated in this way constitute the virus exalte. Subcutaneous injection of the virus exalte produces a local necrosis, and may be followed by the death of the animal, but if the animal be treated first with the attenuated virus, the subsequent injection of the virus exalte produces only a local oedema. After inoculation first by attenuated and afterwards by exalted virus, the guinea-pig has acquired a high degree of immunity, and Haffkine believed that this immunity was effective in the case of every method of inoculation, that is, by the mouth as well as by injection into the tissues. After trying his method on the human subject and finding it free from risk, he extended it in practice on a large scale in India in 1894, and these experiments are still going on. So far the results are, on the whole, encouraging. In the human subject two or sometimes three inoculations are made with attenuated virus before the virus exalte is used. Wasser- mann and Pfeiffer, and also Klein, have found, however, that guinea-pigs immunised by Haffkine's method are not immunised against intestinal infection when the animal is treated by Koch's method (that is, by paralysing the intestines with opium, vide p. 413). Notwithstanding this fact Haffkine's method may still have a beneficial effect, though it may not be preventive in all cases. 2. Immunity by Dead Cultures of Bacteria. — In some BY DEAD CULTURES. 467 cases a high degree of immunity against infection by a given microbe may be developed by repeated and gradually increasing doses of the dead cultures, the cultures being killed sometimes by heat, sometimes by exposure to the vapour of chloroform. Some consider that in this method only the intracellular toxic substances of the organism are introduced when the cultures have been taken from the surface of a solid medium, such as agar, but as the surface is moist, some of the extracellular products must be present also. The cultures when dead produce, of course, less effect than when living, and this method may be con- veniently used in the initial stages of active immunisation, to be afterwards followed by injections of the living cultures. The method has been extensively used by Pfeiffer and others in the production of a high degree of immunity in guinea-pigs against the typhoid, cholera, and other organisms. 3. Immunity by the Separated Bacterial Products or Toxines. — The organisms in a virulent condition are grown in a fluid medium for a certain time, and the fluid is then filtered through a Chamberland or other porcelain filter. The filtrate contains the toxines, and it may be used unaltered, or may be reduced in bulk by evaporation, or may be evaporated to dryness. The process of immunisation by the toxine is started by small non-lethal doses of the strong toxine, or by larger doses of toxine the power of which has been weakened by various methods (vide infra). After- wards the doses are gradually increased. Immunity pro- duced in this way is effective not only against the toxine, but also against large doses of the virulent organism in a living condition. This method was carried out with a great degree of success in the case of diphtheria, tetanus, malignant oedema, etc. It appears capable of very general application, though, in the case of many organisms, it is difficult to get a very active toxine from the filtered cultures. It has also been applied in the case of snake poisons by Calmette and Fraser, and a high degree of immunity has been produced. Immunity may also be obtained by means of certain 468 IMMUNITY. chemical substances separated from filtered bacterial cul- tures, though these substances are generally in a more or less impure condition. Hankin was the first to obtain this result by means of an albumose separated from anthrax cultures. Though, as already stated, none of these methods can be used directly as curative agents, seeing that they imply previous treatment before exposure to infection, yet they supply the means of developing a very high degree of immunity, which is the first stage in the production of an active curative serum. Active Immunity by Feeding.— Ehrlich found that mice could be gradually immunised against ricin and abrin by feeding them with increasing quantities of these substances {vide p. 1 60). In the course of some weeks' treatment in this way the resulting immunity was of so high a degree that the animals could tolerate 400 times the originally fatal dose by subcutaneous inoculation. Fraser also found in the case of snake poison that rabbits could be immunised, by feeding with the poisons, against several times the lethal dose of venom injected into the tissues. By feeding animals with dead cultures of bacteria or with their separated toxines, a certain degree of immunity may in certain cases be gradually developed. But this method is so much less certain in results, and so much more tedious than the others, that it has obtained no practical applications. Active immunity of high degree developed by the methods described maybe regarded as specific, that is, is exerted only towards the organism or toxine by means of which it has been produced. A certain degree of immunity, or rather ot increased general resistance of parts of the body (for example the peritoneum), can, however, be produced by the injection of various substances— bouillon, blood serum, solution of nuclein, etc. (Issaeff). Also increased resistance to one organism can be thus produced by injections of another organism. Immunity of this kind, however, never reaches a high degree. PASSIVE IMMUNITY. 469 B. Passive Immunity. Action of the Serum of Highly-Immunised Animals. — 1. The serum of an animal A, treated by repeated and gradually increased doses of the toxine of a particular microbe, may protect an animal B against a certain amount of the same toxine when injected along with the latter, or a short time before it. As would be expected, it has less effect when injected some time afterwards, but even then within certain limits it has a degree of protective or palliative power. Seeing that the serum of animal A appears to neutralise the toxine, the term antitoxic has been applied to it. It also protects under like conditions from infection with the corresponding microbe. Thus an anti- diphtheritic serum prepared by injections of the toxine protects also from injections of the living virulent bacillus. 2. The serum of an animal A, highly immunised against a microbe by repeated and gradually increasing doses of the living organism, protects an animal B against an infec- tion by the living organism when injected under conditions similar to the above. This serum is therefore antimicrobic, or preventive against invasion by a particular organism. When spoken of in relation to the bacterium by means of which it has been prepared, a serum is usually called homologous ; in relation to any other bacterium, heterologous. In a considerable number of instances, an antimicrobic serum has been found to possess little effect against the toxine — that is, to possess little or no antitoxic power. This fact, if taken alone, would leave it still doubtful whether the difference between the two kinds of sera were one of quality or one merely of quantity. It has, however, been shown in many cases that an antimicrobic serum has a distinct action on the vital activity of the corresponding bacterium, and may even produce alteration in its structure. It is manifest that such a serum differs fundamentally in its point of attack, so to speak, from an antitoxic serum. It must not be supposed, however, that a serum must be 47o IMMUNITY. purely antitoxic or purely antimicrobic according to the method by which it is prepared. For example, an anti- toxic serum can be obtained by injecting living diphtheria bacilli into the tissues of an animal, the antitoxic property being in all probability developed by means of toxines formed by the bacilli within the body. Having given this explanation, we shall consider the two kinds of serum separately. i. Antitoxic Serum. — The best examples are the anti- toxic sera of diphtheria and tetanus, though similar principles and methods are involved in the preparation of the sera protective against ricin and abrin, and against snake poison. We shall here speak of diphtheria and tetanus. The steps in the process of preparation may be said to be the follow- ing : First, the preparation of a powerful toxine. Second, the estimation of the power of the toxine. Third, the development of antitoxine in the blood of a suitable animal by gradually increasing doses of the toxine. Fourth, the estimation from time to time of the antitoxic power of the serum of the animal thus treated. 1. Preparation of the Toxine.— -The mode of preparation and the conditions affecting the development of diphtheria toxine have already been described (p. 365)- In the case of tetanus, the growth takes place in glucose bouillon under an atmosphere of hydrogen {vide p. 72). In either case the culture is filtered through a Chamberland filter when the maximum degree of toxicity has been reached. The term "toxine" is usually applied for convenience to the filtered (sterile) culture. 2. Estimation of the Toxine.— The power of the toxine is estimated by the subcutaneous injection of varying amounts in a number of guinea-pigs, and the minimum dose which will produce death is thus obtained. This, ot course, varies in proportion to the weight of the animal, and is expressed accordingly. In the case of diphtheria, in Ehrlich's standard, the minimum lethal dose is the smallest amount which will certainly cause death in a guinea-pig of 250 grms. within four days. Behring uses the term ANTITOXIC SERUM. 47 1 " normal diphtheria toxine of simple strength " (DTN1), as indicating a toxine of which .01 c.c. is the minimum lethal dose under these conditions. A toxine of which the minimum lethal dose is .02 will be of half normal strength (DTN-5); and so on. The testing of a toxine directly is a tedious process, and in actual practice, where many toxines have to be dealt with, it is found more convenient to test them by finding how much will be neutralised by a certain amount of a standard antitoxic serum, viz., an " immunity unit." 3. Development of Antitoxine. — The earlier experiments on tetanus and diphtheria were performed on the small animals, such as guinea-pigs, but afterwards the sheep and the goat were used, and finally horses. In the case of the small animals it was found advisable to use in the first stages of the process either a weak toxine or a powerful toxine modified by certain methods. Such methods are the addition to the toxine of terchloride of iodine (Behring and Kitasato), the addition of Gram's iodine solution in the proportion of one to three (Roux and Vaillard), and the plan, adopted by Vaillard in the case of tetanus, of using a series of toxines weakened to varying degrees by being exposed to different temperatures, viz. 6o°, 550, and 500 C. But in the case of large animals, such as the horse, the first injections are simply made with small doses of the ordinary toxine. The toxine is at first injected into the subcutaneous tissues, the dose being gradually increased according to the results of the toxine injected. As pointed out by Behring, immunisation proceeds best when each injection produces a reaction in the form of localised inflammatory swelling ; in other words, the dose should be as large as- possible, so long as general injurious effects are not produced. Later, when large doses of toxine injected subcutaneously are well borne, the toxine is injected directly into the jugular vein of the animal. Ultimately 300 c.c, or more, of active diphtheria toxine thus injected may be borne by a horse, such a degree of resistance being developed after the treatment has been carried out for two or three months. In all cases of 472 IMMUNITY. immunising the general health of the animal ought not to suffer. If the process is pushed too rapidly the antitoxic power of the serum may diminish instead of increasing, and a condition of marasmus may set in and may even lead to the death of the animal. (In immunisation of small animals an indication of their general condition may be obtained by weighing them from time to time.) Up till recently, the preparation from a horse of an antitoxic serum of high value involved very prolonged treatment, usually lasting for eight or ten months. Cartwright Wood has, however, devised a method by which the period of immunisation is much shortened, and which promises to give serum of very high antitoxic powers. In this method he used two "toxines." The one is the ordinary toxine obtained from bouillon cultures as above described, which is believed to contain the "ferments"; the other is obtained by growing the diphtheria bacillus in a mixture of bouillon and 20 per cent of blood serum (the latter is prevented from coagulating by having its lime salts precipitated by oxalic or citric acid). Such a culture when filtered contains the supposed ferments along with a large proportion of albu- moses, produced by the action of the bacillus on the albumin. The former are destroyed by exposure to 650 C. for an hour, and the fluid is then known as "serum toxine" in contradistinction to the ordinary " broth toxine." (It would be of importance to know whether or not in this method the toxines are merely modified by the heating, i.e., changed into toxoids, vide p. 162.) The serum toxine gives rise to little local irritation but to marked febrile reaction. By its use the early period of immunisation is much shortened, so that a horse can tolerate a large dose of ordinary broth toxine in a shorter time than was formerly possible ; and by combining its use with that of broth toxine a serum of remarkably high antitoxic powers may be obtained in a month or two. 4. Estimating the Antitoxic Power of, or ' 'standardising," the Serum.— This is done by testing the effect of various quantities of the serum of the immunised animal against a certain amount of toxine. Various standards have been used, of which the two chief are that of Behring and Ehrlich and that of Roux. Behring adopted as the im- munity unit the amount of antitoxic serum which will neutralise completely1 100 times the minimum lethal dose 1 Bv this is meant not only that a fatal result does not follow but also that there is an absence of local swelling on the fourth day, and the animal does not lose weight. STANDARDISING OF SERA. 473 of toxine, serum and toxine being mixed, diluted up to 4 c.c. and injected together subcutaneously. Ten times the lethal dose was used in testing, and a " normal " antitoxic serum is one of which .1 c.c. neutralises this amount, i.e., of which 1 c.c. contains an immunity unit. Owing to the difficulty of estimating the occurrence of local infiltra- tion at the site of injection, the prevention of the death of the animal is now used as the indication of neutralisation. Ehrlich now uses 100 times the lethal dose as the test amount of toxine. As a standard in testing, Ehrlich employs quantities of serum of known antitoxic power in a dry condition, preserved in a vacuum in a cool place, and in the absence of light. A thoroughly dry condition is ensured by having the glass bulb containing the dried serum connected with another bulb containing anhydrous phosphoric acid. Thus 1 c.c. of a serum of which .002 c.c. will protect from ten times the lethal dose, will possess fifty immunity units, and 20 c.c. of this serum 1000 immunity units. Serum has been prepared of which 1 c.c. has the value of 800 units or even more. Roux adopts a standard which represents the animal weight in grammes protected by 1 c.c. of serum against the dose of virulent bacilli lethal to a control guinea-pig in thirty hours, the serum being injected twelve hours previously. Thus, if .01 c.c. of a serum will protect a guinea-pig of 500 grms. against the lethal dose, 1 c.c. (1 grm.) will protect 50,000 grms. of guinea-pig, and the value of the serum will be 50,000. During the process of development of antitoxine a small quantity of the blood of the animal is withdrawn from time to time, and the antitoxic power tested in the manner described above. After a sufficiently high degree of antitoxic power has been reached, the animal is bled under aseptic precautions, and the serum is allowed to separate in the usual manner. It is then ready for use, but some weak antiseptic, such as .5 per cent carbolic acid, is usually added to prevent its decomposing. Other anti- toxic sera are prepared in a corresponding manner. 474 IMMUNITY. Some further facts about antitctanic serum are given on page 390. Use of Antitoxic Sera. — In all cases the antitoxic serum ought to be injected as early in the disease as possible, and in large doses. In the case of diphtheria 1500 immunity units of antitoxic serum was the amount first recommended for the treatment of a bad case, but the advisability of using larger doses has gradually become more and more evident. Sidney Martin recommends that as much as 4000 units should be administered at once, and that if necessary this quantity should be repeated. The strongest serum prepared at present by Behring contains 3000 units in 5-6 ex., but, as already stated, there is now a good prospect of obtaining a more powerful serum easily. Even very large doses of antitoxic serum are without any harmful effects beyond the occasional production of urticarial and erythematous rashes. Where large quantities of serum require to be administered, as is always the case with antitetanic serum, injections must be made at different parts of the body ; preferably not more than 20 c.c. should be injected at one place. The immunity conferred by injection of antitoxic serum lasts a comparatively short time, usually a few weeks at longest. Sera of Animals immunised against Vegetable and Animal Poisons. — It was found by Ehrlich in the case of the vege- table toxines, ricin and abrin, and also by Calmette and Fraser in the case of the snake poisons, that the serum of animals immunised against these respective substances had a protective effect when injected along with them into other animals. Ehrlich found, for example, that the serum of a mouse which had been highly immunised against ricin by feeding as described above, could protect another mouse against forty times the fatal dose of that substance. He considered that in the case of the two poisons, antagonistic substances — "anti-ricin" and "anti-abrin" — were developed in the blood of the highly-immunised animals. A cor- responding antagonistic body, to which Fraser has given the name " antivenene," appears in the blood of animals in the process of immunisation against snake poison. NATURE OF ANTITOXIC ACTION. 475 These investigations are specially instructive, as the poisons, both as regards their local action and the general toxic phenomena produced by them, present, as we have seen, an analogy to various toxines of bacteria. Nature of 'Antitoxic Action. — On this subject there has been much diversity of opinion. Some observers consider that the antagonism between toxine and antitoxine depends upon a chemical union between the two substances, whilst others consider that it is of a physiological nature, the anti- toxine acting through the medium of the cells of the organism. Again, with regard to the source of the anti- toxine, some hold that it is produced by the living cells under the stimulus of the toxine, whilst others look upon it as a modified toxine. The bulk of evidence recently brought forward is, however, strongly in favour of the view that the two bodies unite in vitro to form a compound inert towards the living tissues, there probably being in the toxine molecule an atom group which has a specific affinity for the antitoxine molecule or part of it, and that in no sense is the antitoxine molecule a modified toxine mole- cule. We shall consider the facts in favour of this view, and in doing so we must also take into account the anti- sera of the vegetable toxines, of snake poisons, etc. When toxine and antitoxine are brought together in vitro it can be proved that their behaviour towards each other resembles what is observed in a simple chemical union. The test which is the indication of the neutralisa- tion of the toxine by the antitoxine, is that when the resultant body is injected into a susceptible animal no symptoms occur. As in chemical union a definite period of time elapses before combination is complete. C. J. Martin and Cherry and also Brodie have shown, that in the case of diphtheria toxine and in that of an Australian snake poison the molecules will pass through a colloid membrane (p. 157), whilst those of the corresponding antitoxines will not. Now if a mixture of equivalent parts of toxine and antitoxine is freshly prepared and at once filtered, a certain amount of toxine will pass through, but the longer such 476 IMMUNITY. mixtures are allowed to stand before filtration the less toxine passes, till a time is reached when no toxine is found in the filtrate. Further, if the portion of fluid which at this stage has not passed through the filter be injected into an animal, no symptoms take place. This shows that after a time neutralisation is complete. Other points of re- semblance to simple chemical union are found in the facts that neutralisation takes place more rapidly in strong solu- tions than in weak, and that it is hastened by warmth and delayed by cold. It has been found that if these factors be taken into account and a standard toxine of definite strength be employed, a toxine can be titrated against an antitoxine with corresponding accuracy to what obtains in the case of an acid and an alkali. These facts are strongly in favour of toxine neutralisation consisting in a chemical union, and such a view would also throw light on the other- wise somewhat puzzling fact that while, e.g., by lapse of time the toxicity of a toxine may become diminished, it may still require the same proportion of antitoxine to neutralise it as it did before. On the chemical theory this, according to Ehrlich, is due to the disintegration of the toxophorous atom group of the toxine molecule {vide p. 161), while the combining (haptophorous) group still remains unaltered. Quite analogous cases could be cited from pure chemistry. The next question to be considered is the mode of pro- duction of antitoxines. In the first place, we have evidence of naturally existing antitoxines. Wassermann and Takaki and others have shown, that in the grey matter of the central nervous system of animals susceptible to tetanus there nor- mally exist bodies which have the power of neutralising a certain amount of tetanus toxine, presumably by combining with it. This neutralising power is of course demonstrated by injections in another animal. Similarly in some horses the serum has been found to have antitoxic properties against diphtheria toxine, and Fraser has shown that ox- bile has a degree of antitoxic action against serpents' venom. It may further be noted here that in the nervous system of the common fowl, on which tetanus toxine has MODE OF PRODUCTION OF ANTI TO XI NES. 477 little effect, Ransom has found neutralising capacity almost entirely absent. A definite theory, in accordance with these and other facts, has been formulated by Ehrlich. He supposes that there normally exist in the cells of an animal capable of supplying antitoxine certain atom-groups which, while ordinarily performing a physiological function, are also capable of combining with the toxine molecule. Thus, for example, when diphtheria toxine is injected into a horse in relatively small doses the toxine combines with these atom groups and their function in the cell economy is lost. There then occurs a regeneration of new molecules to take up this function, and when these are again used up by fresh toxine molecules introduced a further regeneration takes place. Ultimately there occurs an over-regeneration, and the appearance of these molecules (antitoxine) in the blood. It is to be noted that when the symptoms of poisoning occur, in tetanus for example, these combining molecules do not act as antitoxines, but constitute the means by which the toxine is bound to the nerve cell, and thus produces its effects. It is only when they are freed from their attachment to the cell protoplasm that they act as antitoxines. Whilst this theory cannot be regarded as completely established, we may state that it seems to us to be in harmony with the conditions affecting the rapid and suc- cessful production of antitoxines. It also explains why modified toxines, e.g., tetanus toxine treated with carbon bisulphide, may be efficient immunising agents. It need scarcely be pointed out that the direct proof of combination of tetanus poison with nerve cells, as mentioned above, is another argument in its favour. Two other facts of im- portance may be mentioned. The first is that an animal, after having been successfully treated with toxine, may supply an amount of antitoxine much greater than the total quantity of toxine employed in the process — the quantity of each being reckoned by thek neutralisation value. The second is that after a horse with antitoxic serum has been bled, the antitoxic value of its serum, which of course falls at first, 478 IMMUNITY. may afterwards be partially restored, thereby indicating that there has been an actual regeneration of antitoxine though no fresh toxine has been injected. These facts disprove the theory held by some that the antitoxine is a modified toxine — a theory which, besides, is scarcely intelligible on biological grounds. According to Ehrlich's theory, the cells of the organism have acquired under the stimulus of the toxine a secretory or regenerating habit which lasts for some time after the stimulus has been removed. It may further be mentioned that after the antitoxic property has dis- appeared from the blood the animal still possesses a degree of immunity to the toxine. Whether or not this is due to the fact that there are still surplus antitoxine molecules in the cells, but not in such excess that they pass into the blood, we cannot definitely say. Of the ■ chemical nature of atttitoxines we know little. From their experiments C. J. Martin and Cherry deduce that while toxines are probably of the nature of albumoses, the antitoxines probably have a molecule of greater size, and may be allied to the globulines. Such a supposed difference in the sizes of the molecules might explain the following facts, observed by Fraser and also by C. J. Martin. If a certain amount of toxine be injected subcutaneously.. and at the same time an amount of antitoxine sufficient to neutralise it in vitro be injected intravenously, the latter will nearly neutralise the former. If, however, the antitoxine also be introduced subcutaneously (into a different part of the body), from ten to twenty times as much is required to effect neutralisation. The explanation of this would be that the large molecule of the antitoxine was absorbed relatively more slowly than that of the toxine ; thus in a given time there was more of the latter free in the circula- tion and ready to produce pathogenic effects. In the case of the intravenous injection of antitoxine, the toxine on reaching the blood stream was neutralised by the antitoxine already circulating there. Antitoxine, when present in the serum, leaves the body by the various secretions, and in these it has been found, ANTIMICROBIC SERUM. 479 though in much less concentration than in the blood. It is present in the milk, and a certain degree of immunity can be conferred on animals by feeding them with such milk, as has been shown by Ehrlich, Klemperer, and others. Klemperer also found traces of antitoxine in the yolk of eggs of hens whose serum contained antitoxine. Antimicrobic Serum. — The stages in preparation of antimicrobic sera correspond to those in the case of antitoxic sera, but living, or, in the early stages, dead cultures are used instead of the toxine separated by filtration, and in order to obtain a serum of high antimicrobic power, a very virulent culture in large doses must be ultimately tolerated by the animal. For this purpose a fairly virulent culture is obtained fresh from a case of the particular disease, and its virulence may be further increased by the method of passage. This method of obtaining a high degree of immunity against the microbe is specially applicable in the case of those organisms which invade the tissues and multiply to a great extent within the body, and of which the toxic effects, though always existent, are proportionately small in relation to the number of organisms present. The method has been applied in the case of the typhoid and cholera organisms, the bacillus of bubonic plague, the bacillus coli communis, the pneumococcus, streptococcus (Marmorek), and many others. In fact, it seems capable of very general application. The important result obtained by such experiments is, that if an animal be highly immunised by the method mentioned, the development of the immunity is accom- panied by the appearance in the blood of protective sub- stances, which can be transferred to another animal. The law enunciated by Behring regarding immunity against toxines thus holds good in the case of the living organisms, as was first shown by Pfeiffer. The latter found, for example, that in the case of the cholera organism, so high a degree of immunity could be produced in the guinea-pig, that .002 c.c. of its serum would protect another guinea- pig against ten times the lethal dose of the organisms, when 480 IMMUNITY. injected along with them. Here again is presented the remarkable potency of the antagonising substances in the serum, which in this case lead to the destruction of the corresponding microbe. The antistreptococcic serum of Marmorek may be briefly described, as it has come into extensive practical use. This observer found that he could intensify the virulence of a streptococcus by growing it alternately in the peritoneal cavity of a guinea-pig, and in a mixture of human blood serum and bouillon (vide p. 53). The virulence became so enormously increased by this method, that when only one or two organisms were introduced into the tissues of a rabbit a rapidly fatal septicaemia was produced. Strepto- cocci of this high degree of virulence were used first by subcutaneous, afterwards by intravenous injection, to develop a high degree of resistance in the horse. Injections were continued over a considerable period of time, and the protective power of the serum was tested by mixing it with a certain dose of the virulent organisms, and then injecting into a rabbit. The serum of a horse highly immunised in this way constitutes the anti-streptococcic serum which has been extensively used with success in many cases of streptococcic invasion in the human subject. Marmorek, however, found that this serum had little antitoxic power, that is, could only protect from a comparatively small dose of toxine obtained by filtration of cultures. Anti-typhoid, anti- cholera,1 anti-pneumococcic, anti- plague, and other sera are all prepared in an analogous manner. Properties of Antimicrobic Serum. — Within the last two or three years important objective reactions presented by antimicrobic serum against the corresponding organism have been discovered, and these are of high importance, as they afford valuable aid in the study of the nature of the preventive power. Of such actions the two chief are the lysogenic and the agglutinative. 1 A true antitoxic cholera serum has been prepared by Metchnikoff, E. Roux, and Taurelli-Salimbini. PFEIFFER'S PHENOMENON. 481 Lysogenic Action. — Pfeiffer found that if certain organisms, e.g., the cholera spirillum, were injected into the peritoneal cavity of a guinea-pig highly immunised against these organisms they lost their motility almost immediately, gradually became granular and swollen up in places into droplets, and then disappeared in the fluid, all these changes sometimes occurring within half an hour — lysogenic action. Further, he found that the same phenomenon was witnessed if a minute quantity of the anti-serum (that is, the serum of an animal highly immunised against the corresponding organism) was added to a certain quantity of the organisms, and the mixture injected into the peritoneal cavity of another animal. In both cases the organisms die an extracellular death, and their destruction is brought about by the medium of a specific substance in the anti-serum. Pfeiffer found that the serum of convalescent cholera patients gave the same reaction as that of immunised animals, that is, it possesses specific antagonising substances. He obtained the same reaction also in the case of the typhoid bacillus and other organisms. From his observations he concluded that the reaction was specific and could be used as a means of distinguishing organisms which resemble one another. He considered that the specific substance in the serum existed chiefly in an inert form, and that it became actively bactericidal 1 by the aid of living cells, probably those of the peritoneal endothelium. Thus he found that if the anti-serum was injected into the peritoneal cavity of a fresh animal, and if, after a time, some of the peritoneal fluid was withdrawn and the corresponding organism added to it, the reaction could be observed outside the body. Metchnikoff, however, showed that the same result was obtained when the organism was simply placed in some fresh peritoneal fluid to which the anti-serum had been added outside the body. In this case, accordingly, the action of the endothelial cells was excluded. Bordet confirmed this observation and showed further that a small 1 In some cases an antimicrobic serum possesses specific directly bac- tericidal powers, but this is not a general law. 3T 482 IMMUNITY. quantity of normal serum played the same part as fresh peritoneal fluid. His method was the following : — (a) An emulsion of the living organisms (for example of the cholera vibrio) was made by adding a young culture to about 5 c.c. of bouillon ; (b) two drops of this emulsion were taken, and mixed with a small drop of anti-cholera serum ; (f) a drop of this mixture was taken, and there was added to it a drop of equal size of fresh serum from a normal guinea-pig. A hanging-drop preparation was made, and a change similar to that described by Pfeifl'er was observed within one to two hours if the preparation was kept at the temperature of the body. Bordet found that in every case in which Pfeiffer's reaction took place within the body of an animal, a similar lysogenic reaction could be observed by his method outside the body. He considers that the reaction depends upon the presence of a specific immunising substance in the anti- serum which greatly increases the bactericidal power of the normal serum, but that in most cases this specific substance cannot lead to the destruction of the organisms without the aid of healthy serum. Bordet and Metchnikoff hold the source of the specific protective substance as well as that of bactericidal substances to be in the leucocytes. Agglutination.— Charrin and Roger had previously (1 889) observed that when the bacillus pyocyaneus was grown in the serum of an animal immunised against this organism, the growth formed a deposit at the foot of the vessel; whereas a growth in normal serum produced a uniform turbidity. Gruber and Durham, in investigating Pfeiffer's reaction, discovered an analogous phenomenon. They found that when a small quantity of the serum of an animal highly immunised against a particular motile organism (cholera vibrio, typhoid bacillus, etc.) is added to an emulsion of the organisms, the latter lose their motility, and become agglutinated into clumps. In a small test-tube a reaction in this way occurs which is visible to the naked eye, a sort of precipitate forming which consists of masses of non-motile organisms. The higher the degree of immunity, the smaller is the amount of serum necessary to bring about this phenomenon. The specific substance in the serum, therefore, has a direct action on the organisms, AGGLUTINATION. 483 probably attended with a physical change in their envelopes, and this they consider forms the essential part of Pfeiffer's reaction. When the organisms are thus weakened by the specific body, the bactericidal power of the normal peritoneal fluid or blood serum, as the case may be, comes into action and completes the process. The observations just described have led to the dis- covery of the method of serum diagnosis of disease, which has been applied especially to typhoid fever, as already detailed (vide p. 343). It had been already found that the serum of convalescents from typhoid fever could protect animals to a certain extent against typhoid fever, and, in view of the facts experimentally established, it appeared a natural proceeding to enquire whether such serum possessed an agglutinative action and at what stage of the disease it appeared. The result, first published by Widal, was to show that the serum possessed this specific action long before the cure of the disease, in fact shortly after infection had taken place. It is probable that it depends upon a process of immunisation developing from an early stage of the disease. Agglutination is also observed in the case of cholera, Malta fever, glanders, infection by Gartner's bacillus, B. coli, etc. Besides those stated above, other phenomena have been observed in the inter-action of anti-sera and the correspond- ing bacteria. For example, it has been shown that when certain bacteria, e.g., the typhoid bacillus, B. coli and B. proteus, are grown in bouillon containing a small propor- tion of the homologous serum, their morphological characters may be altered, growth faking place in the form of threads or chains, which are not observed in ordinary conditions. In other instances a serum may inhibit some of the vital functions of the corresponding bacterium. When we come to enquire into the relation of the various properties of an antimicrobic serum to one another, we find that there still prevails considerable doubt. As will be seen from the above, practically all observers are agreed that in the destruction of a bacterium — either within 484 IMMUNITY. or outside the animal body — the action of the homologous serum has a dual character, in other words, two substances are concerned. One of these is the specific substance — " immune-body " — developed in the serum, the other a sub- stance present in normal serum ; by a temperature of 6o° C. the latter is readily destroyed, while the former is practically unaffected, at least for some time. A serum thus heated also retains its agglutinative action, but there is still a dispute as to whether the agglutinine and the immune- body are the same. Some observers, e.g., Pfeiffer, Kolle, and Ehrlich consider that the agglutinative and preventive action are independent of one another, whilst Gruber and Durham consider them intimately related and probably due to the same substance. It appears that agglutination is not essential to the possession of preventive power, but on the other hand it is very doubtful whether high agglutina- tive power ever occurs quite apart from the latter. In view of all the facts the appearance of the agglutinative property is most probably to be regarded as a phenomenon pre- judicial to the corresponding organism, and thus in its nature is to be associated with the process of immunisation. If Ehrlich's theory with regard to lysogenic action (vide infra) is correct, it will in all probability apply to the de- velopment of agglutinines. It will remain to be determined whether the immune-body sometimes ^ acts as an agglutinine or whether the two substances are distinct. Lysogenic Action towards Blood Corpuscles. These striking pro- perties "are, however, not peculiar to bacterial immunity. Bordet has recently shown that similar reactions can be acquired by the serum of one animal towards the red corpuscles of another of different species instances had previously been known of the serum of one animal c^Sn' agglutination of the red corpuscles of another or producing "solution, but these powers are not generally possessed. The experiments of Bordet, however, show that such properties may be acqu S aid may reach a high degree. This was effected by mject- K defibdnated blood of the rabbit into the peritoneum of a guinea- II After a number of injections the serum of the latter when added to defib rinated rabbit's blood dissolves the red corpuscles with great °aptit 1 point of special interest is that ^ f^^^Z are concerned. When the serum is heated to 55 C. it loses its d.s THE OR Y OF LYSO GEN1C A C TION. 48 5 solving power, though it still agglutinates ; but if a small quantity of normal serum is added to it the active hsemolytic power is restored. The action is not absolutely restricted to the red corpuscles of the species of animal employed, though it is most marked in this case. Theory of Lysogenic Action. — Ehrlich has recently applied his theory of antitoxines to the lysogenic action of sera towards bacteria and red corpuscles. He has confirmed Bordet's experiments with regard to haemolysis in the case of the goat treated with injections of sheep's corpuscles, though here he observed no agglutina- tive property. His observations show that the body specially developed in the blood of the animal treated — the "immune-body," enters into firm combination with the red corpuscles, but is unable of itself to cause their solution. The other substance, present in normal blood and probably a ferment, has no chemical affinity for red corpuscles but forms a loose compound with the "immune-body." The injection of red corpuscles stimulates the production of the " immune-body," which is to be regarded as a " side-chain " of the normal cells with an affinity for the protoplasm of the red corpuscles. The process is thus of the same nature as in the case of antitoxines, the difference being that here the body developed does not act alone, but is attached by a second atom complex to a ferment naturally existing in the serum. ' Probably it is the same ferment which effects the solution both of red corpuscles and of bacteria, the peculiar or specific element being the immune-body which binds, by means of the affinities of its atom groups, this ferment to a red corpuscle or to a bacterium, as the case may be. Substances contained in the bacterial protoplasm thus stimulate, as toxines do, the over -regeneration of molecules for which they have a specific chemical affinity. It may be added that evidence has recently been brought forward that in cholera (Pfeiffer and Marx) and in typhoid (Wassermann) the specific immune-bodies are chiefly formed in the spleen, bone-marrow and lymphatic glands. This result was obtained by examining the various organs during the earlier stages of immunisation. 486 IMMUNITY. Summary with regard to Anti-Sera. — In a former chapter it has been shown that in the production of disease by bacteria there are two main factors concerned, viz., the multiplication of the living organisms in the tissues and the production by them of toxines. The facts which have been stated above show that in the blood serum of highly immunised animals there are present substances of remark- able potency, which may act against either of these two factors. In the first place, a serum may protect against the separated toxine, or, in other words, may be antitoxic. In this case there is immunity also against the living organisms, as might naturally be expected ; for when their toxines are neutralised their harmful action on the tissues is removed, and they are then destroyed probably by the same means as ordinary non -pathogenic organisms. In the second place, a serum may lead to the destruction of the organisms. In this case, it is usually indirectly bacteri- cidal, i.e., becomes bactericidal in certain conditions, though in many instances a directly paralysing action on the organ- isms has also been demonstrated. The term antimicrobic is, therefore, conveniently applied to such a serum. In many instances an antimicrobic serum has little or no effect against the toxines ; this is the case with the anti- streptococcic serum (Marmorek), the anti-cholera and anti- typhoid sera (Pfeiffer), and many others. The action of both varieties of anti-sera is, within certain limits, specific, being exerted only against the particular organism or toxine which has been used in its preparation. In the case of both, immunity can be transferred to another animal by means of a certain quantity of the serum, the latter having a definite value which can be ascertained by experiment. It does not follow from what has been said that a serum may not act in both of the ways described. A given serum might, for example, be powerfully anti- microbic and feebly antitoxic at the same time. Therapeutic effects of Anti-Sera.— As will have been gathered, the human diseases treated by anti-sera are diph- theria, tetanus, streptococcus infection, pneumonia, plague THERAPEUTIC EFFECTS OF ANTI-SEN J. 487 and snake bite. Of the results of such treatment most is known in the case of diphtheria. Here a very great diminution in the mortality has resulted. The diphtheria antitoxine came into general use about October 1894, and the statistics published by Behring towards the end of 1895 indicate results which have since been confirmed. In the Berlin Hospitals the average mortality for the years 1891-93 was 36.1 per cent, in 1894 it was 21.1 per cent, and in January-July 1895, T4-9 Per cent The objection that in some epidemics a very mild type of disease prevails is met by the fact that similar diminutions of mortality have occurred all over the world. Loddo collected the results of 7000 cases in Europe, America, Australia, and Japan, in which the mortality was 20 per cent as compared with a former mortality in the same hospitals of 44 per cent. It has also been observed that if during an epidemic the supply of serum fails, the mortality at once rises; and in two cases recorded it was doubled. It must here be remembered that from the spread of bacteriological knowledge the diagnosis of diphtheria is now much more accurate than formerly. Again, the antitoxic treatment has made trache- otomy less frequently necessary. The American Pediatric Society has collected statistics showing" that only 39.2 per cent of laryngeal cases were operated on in 1897 instead of 90 per cent, the calculated previous figure. When tracheotomy is necessary the percentage of recoveries is now much higher, being 73 per cent instead of 27 per cent in the American group of cases. In the London fever hospitals since 1894 the recoveries after tracheotomy have been 56.4 as compared with 32.1 per cent previous to the introduction of antitoxine. One of the most strik- ing results obtained in the same hospitals is a reduction of the death rate in post-scarlatinal diphtheria from 50 per cent to between 4 per cent and 5 per cent. As the disease occurs while the patient is under observation the treatment is nearly always begun on the first day. It is a matter of prime importance that the treatment should be commenced whenever the disease is recognised. Behring 488 IMMUNITY. showed that in cases treated on the first and second days of the disease the mortality was only 7.3 per cent, and this has been generally confirmed. After the fifth day it is of little service to apply the treatment. In order to obtain such results it cannot be too strongly insisted on that attention should be given to the dosage. When bad results are obtained it may be strongly suspected that this precaution has not been observed. In the treatment of acute tetanus by the antitoxine the improvement in results has not been marked, but some chronic cases have been benefited. Recently there has been practised with slightly more hopeful result a drop by drop injection (repeated daily if necessary) of from 2^-10 c.c. of antitoxine into the substance of the frontal lobe by a syringe inserted through a dental drill hole over the frontal eminence. In the case of Yersin's anti-plague serum, though benefit has appeared to follow its use, experience with its effects has been too limited to enable a judgment to be formed. The same may be said to be true of the antistreptococcic and anti- pneumonic sera, and also of antivenene, though in the case of the first mentioned numerous cases of apparently successful result have been recorded. Theories as to Acquired Immunity. The advances made within recent years in our knowledge regarding artificial immunity and the methods by which it may be produced have demonstrated the insufficiency of various theories which had been propounded. They also show the futility of attempting, even now, to make a general statement which would be applicable to all cases. One or two of these theories may, however, be mentioned, as they are of interest in connection with the development of this subject. 1. The Theory of Exhaustion, with which Pasteur's name is associated, supposes that in the body of the living animal there are substances necessary for the existence of a par- ticular organism, which become used up during the sojourn THEORIES AS TO ACQUIRED I AIM UNITY. 489 of that organism in the tissues ; this pabulum being ex- hausted, the organisms die out. Such a supposition is, of course, quite disproved by the fact of passive immunity, as a small quantity of serum in which the pabulum has been exhausted cannot lead to its exhaustion in the serum of another animal. 2. The Theory of Retention supposes that the organisms within the body produce substances which are inimical to their growth, so that they die out, just as they do in a test- tube culture before the medium is really exhausted. In its simple form the theory is scarcely tenable, as it would be difficult to conceive how such substances could be retained in the body for so many years as acquired immunity some- times lasts. In a modified form, however, it might include theories still held and which are founded on the facts of passive immunity. It might, for example, include the theory of Buchner, according to which the antitoxic sub- stance in the serum is merely a modified toxine, which has the power of producing in another animal a rapid reaction resulting in immunity. The facts stated above with regard to the production of antitoxine are, however, quite opposed to such a supposition. . 3. The Theory of Phagocytosis. — This is the theory which was brought forward by Metchnikoff to explain the facts of natural and acquired immunity, and which has been of enormous influence in stimulating research on this subject. Looking at the subject from the standpoint of the compara- tive anatomist, he saw that it was a very general property possessed by certain cells throughout the animal kingdom that they should take up foreign bodies into their interior and in many cases destroy them. On extending his obser- vations to what occurred in disease, he came to the conclu- sion that the successful resistance of an animal against bacteria depended on the activity of certain cells called phagocytes. In the human subject he distinguished two chief varieties, namely (a) the microphages, which are the " polymorpho-nuclear " leucocytes of the blood, and (b) the macrophages, which include the larger hyaline leucocytes, 490 IMMUNITY. endothelial cells, connective tissue corpuscles, and, in short, any of the larger cells which have the power of ingesting bacteria. Insusceptibility to a given disease is indicated by a great activity on the part of the phagocytes, different varieties being concerned in different cases, — an activity which may rapidly destroy the bacteria and prevent even local damage. If the animal is moderately susceptible and the organisms are introduced into the subcutaneous tissue, there occurs an inflammatory reaction with local leucocytosis, which results in the intracellular destruction of the invading organisms. Phagocytosis is regarded by Metchnikoff as the essence of inflammation. He also showed that the bacteria may be in a living and active state when they are ingested by leucocytes. On the other hand, he found that in a susceptible animal phagocytosis did not occur or was only imperfect. He also found that when a naturally susceptible animal was immunised, the process was accompanied by the appearance of an active phagocytosis. The leucocytes and other cells are guided in their attack on the organisms by chemiotaxis, a process which has already been explained. According to this theory, in the process of immunisation by attenuated cultures the phagocytes are so educated by dealing with the bacteria in an attenuated condition that they can ultimately destroy them even in a highly virulent state. The work of Metchnikoff has been of great importance in demonstrating one of the chief means possessed by the body of dealing with invading organisms, and for a time his theory obtained considerable support as an explanation of the facts of immunity. The insufficiency of the theory, however, was at once apparent when the method of im- munising against a toxine was discovered; and the facts discovered later, with regard to the action of antimicrobic sera, showed that the cellular ingestion of bacteria was not the most important factor in immunity against the living organisms. The theory as originally propounded is, ac- cordingly, no longer tenable, and even if it were consistent with facts it only removes the property of immunity a step THE HUMORAL THEORY. 491 farther back, namely to the phagocytes. The phenomena of phagocytosis so admirably demonstrated by Metchnikoff may be regarded as the results of immunity, but cannot be accepted as its cause. 5. The Humoral Theory. — This theory, which ascribed immunity to changes in the serum and other fluids of the body, was chiefly developed by Behring and by others of the German school. It may be said to have originated with the discovery of bactericidal power possessed by normal blood serum ; and the earlier work consisted in an attempt to explain natural and acquired immunity by supposing changes to take place in this bactericidal power. It is, however, unnecessary to state the various phases through which the theory has passed, as these are now chiefly of historic interest. So far as active immunity is concerned, it may be held as proved that certainly the appearance of immunity is accompanied by changes in the serum, as described above, that is, by the development of antimicrobic or antitoxic substances. No doubt, how- ever, such substances are not produced simply by chemical changes in the body fluids, but are products of cellular action brought about by the presence of the bacteria or their toxines. The question remains as to which cells chiefly generate such substances. A certain amount of evidence, which it is unnecessary to detail, has been brought forward by Metchnikoff, Bordet and others to show that both bactericidal substances of normal serum and specific substances of antimicrobic sera are derived from leuco- cytes. In this way the theory of phagocytosis has under- gone modification. Similar evidence with regard to the origin of antitoxic substances is wanting. The whole question, however, is still an open one, and there is evidence that the formation of these bodies is not restricted to one class of cells but takes place in various tissues. 492 IMMUNITY. Natural Immunity. We have placed the consideration of this subject after that of acquired immunity, as the latter supplies facts which indicate in what direction an explanation of the former may be looked for. There may be said to be two main facts with regard to natural immunity. The first is, that there is a large number of bacteria — the so-called non-pathogenic organisms — which are practically incapable, unless perhaps in very large doses, of producing patho- genic effects in any animal; when these are introduced into the body, they rapidly die out. This fact accordingly shows that the animal tissues generally have a remarkable power of destroying living bacteria. The second fact is, that there are other bacteria which are very virulent to some species of animals, whilst they are almost harmless to other species ; the anthrax bacillus may be taken as an example. Now it is manifest that natural immunity against such an organism might be due to a special power pos- sessed by an animal of destroying the organisms when introduced into its tissues. It might also, however, be due to an insusceptibility to, or power of neutralising, the toxines of the organism. For the study of the various diseases shows that the toxines (in the widest sense) are the weapons by which morbid changes are produced, and that toxine-formation is a property common to all patho- genic bacteria. No doubt, as we have seen, the power of toxine production does not go hand in hand with the power of multiplying throughout the body. In the case of organ- isms which multiply in the blood and produce septicaemia, the amount of toxine formed relatively to the number of the organisms is small, and it would appear as if these or- ganisms had especially a power of destroying the normal preventive power resident in the blood and tissues. There is however, no such thing known as an organism multiply- ing in the living tissues without producing local or general changes, though, theoretically, there might be. We may NATURAL IMMUNITY. 493 infer from this that if the toxines are completely neutralised or rendered powerless in the case of any animal, that animal will be immune against the particular organism. This is also borne out by the fact that immunity against a particular organism can be artificially obtained by injections of the toxines of that organism. (a) Variations in Natural Susceptibility to Toxines. — We may consider, then, the question in the first instance from the point of view of toxines. Now we must start with the fundamental fact, incapable of explanation, that toxicity is a relative thing, or in other words that different animals have different degrees of resistance or non-susceptibility to toxic bodies. In every case a certain dose must be reached before effects can be observed, and up to that point the animal has. resistance. This natural resistance is found to present very remarkable degrees of variation in different animals. The great resistance of the common fowl to the toxine of the tetanus bacillus may be here mentioned ; the high resistance of the pigeon to morphia is a striking example in the case of vegetable poisons. This variation in resistance to toxines applies also to those which produce local effects, as well as to those which cause symptoms of general poisoning. Instances of this are furnished, for example, by the vegetable poisons ricin and abrin, by the snake poisons, and by bacterial toxines such as that of diphtheria. We must take this natural resistance for granted, though it is possible that ere long it will be ex- plained. It is possible then that an animal might be immune against the anthrax bacillus, for example, if the toxines of the latter were simply inert towards the animal tissues, or, in other words, if its tissues enjoyed a natural insusceptibility to the toxines. In such a case the anthrax bacillus would be in the position of the bacillus subtilis, and would be destroyed in the tissues by the same means. (b) Natural Bactericidal Powers. — The second factor may now be considered, namely, the power of killing the organism, though it appears to us that natural immunity has been too exclusively looked at from this side. Special 494 IMMUNITY. powers of destroying organisms in natural immunity have been ascribed to (a) phagocytosis, and (b) the action of the serum. (a) The chief factors with regard to phagocytosis have been given above. The bacteria in a naturally immune animal, for example, the anthrax bacillus in the tissues of the white rat, are undoubtedly taken up in large numbers by the phagocytes, whereas in a susceptible animal this only occurs to a small extent ; and Metchnikoff has shown that they are taken up in a living condition, and are still virulent when tested in a susceptible animal. The presence or absence of chemiotaxis is also no doubt of importance. But is the phagocytosis the cause or the effect of immunity? The facts of artificial immunity would rather point to its being the latter. The following experiment performed by Metchnikoff, though belonging to the subject of artificial immunity, may be given here. He injected into a guinea- pig a virulent culture of the bacillus of hog-cholera, and at the same time injected the anti-serum of the same organism into a vein. At the end of a few hours a local swelling formed at the site of injection, in which there were enormous numbers of bacilli but no leucocytes. After another injec- tion of the serum, however, the leucocytes gathered around and attacked the bacilli. From this experiment he infers that the serum induced a hyperactivity of phagocytes. The matter, however, may be interpreted from another point of view, namely, that it was not until the toxines of the bacilli were neutralised, or at least till the bacilli were weakened by the action of the serum, that the phagocytes could attack them. All the striking phenomena of phago- cytic action in the case of natural immunity can be looked at from the latter point of view, and it appears to us that the evidence of the essence of natural immunity depending upon special properties of the phagocytes, is quite in- sufficient. Variations in phagocytic activity no doubt play a part, but may themselves be found capable of explanation. (b) When it had been shown that normal serum possessed certain bactericidal powers against different organisms, the NATURAL BACTERICIDAL POWERS. 495 question naturally arose as to whether this bactericidal power varied in different animals in proportion to the natural immunity enjoyed by them. The earlier experi- ments of Behring appeared to give grounds for the belief that this was the case. He found, for example, that the serum of the white rat, which has a remarkable immunity to anthrax, had greater bactericidal powers than that of other animals investigated. He found also that the serum of guinea-pigs immunised against the vibrio Metchnikovi had a bactericidal action, whereas in that of susceptible animals no such action was found. Further investigation, however, has shown that these are not examples of a general law, and that this bactericidal action of the serum does not vary pari passu with immunity either in the natural condi- tion or when artificially produced. The bactericidal action of the serum was specially studied by Buchner and Hankin, who believe that the serum owes its power to certain sub- stances in it derived from the spleen, lymphatic glands, thymus, and other tissues rich in leucocytes. To these substances Buchner gave the name of alexines. These substances are somewhat unstable compounds, and are destroyed by the action of light, and also by a temperature of 6o° C. They can be precipitated by alcohol and by am- monium sulphate, and correspond in their general behaviour with enzymes or unorganised ferments. Regarding the existence in the serum of bactericidal substances which are very easily destroyed by heat there can be no doubt, but their properties can only be studied outside the body, and it must not be assumed that the serum in such conditions has always the same property as in the living body. In some cases, for example, the bactericidal power of the serum in vitro has been found to be greater than in a living animal. The bactericidal action, moreover, is mani- fested towards some organisms and not towards others, and this variation does not always correspond with the immunity of the animal against these organisms. At present, therefore, the facts of natural immunity can- not be fully explained. In some cases the insusceptibility 496 IMMUNITY. to toxic substances may explain the degrees of immunity possessed by different animals, whilst in others immunity may be due to special bactericidal powers possessed by them. What these bactericidal powers really are cannot be explained on any single theory. A vital activity of the tissues and fluids is, no doubt, brought about by the pres- ence of the bacteria, and this cannot be fully imitated in experiments outside the body. The facts given above with regard to the action of antimicrobic serum, show how com- plicated a matter the bactericidal process may be. Further, in natural immunity a direct killing of the organisms by the fluids of the serum is not necessary. It may be sufficient that their growth is prevented, so that they ultimately die out or are taken up by the phagocytes. APPENDIX A. SMALLPOX AND VACCINATION. Smallpox is a disease to which much study has been devoted, owing, on the one hand, to the havoc which it formerly wrought among the nations of Europe — a havoc which at the present day it is difficult to realise, — and on the other hand, to the controversies which have arisen in connection with the active immunisation against it intro- duced by Jenner. Though there is little doubt that a contagium vivum is concerned in its occurrence, the etio- logical relationship of any particular organism to smallpox has still to be proved, and with regard to Jennerian vac- cination, it is only the advance of bacteriological knowledge which is now enabling us to understand the principles which underlie the treatment, and which is furnishing methods whereby, in the near future, the vexed questions concerned will probably be satisfactorily settled. We can- not here do more than touch on some of the results of investigation with regard to the disease. Jennerian Vaccination. — Up to Jenner's time the only means adopted to mitigate the disease had been by in- oculation (by scarification) of virus taken from a smallpox pustule, especially from a mild case. By this means it was shown that in the great majority of cases a mild form of the disease was originated. It had previously been known that one attack of the disease protected against future infection, and that the mild attack produced by inoculation 32 498 SMALLPOX AND VACCINATION. also had this effect. This inoculation method had long been practised in various parts of the world, and had con- siderable popularity all over Europe during the eighteenth century. Its disadvantage was that the resulting disease, though mild, was still infectious, and thus might be the starting-point of a virulent form among unprotected persons. Jenner's discovery was published when inoculation was still considerably practised. It was founded on the popular belief that those who had contracted cowpox from an affected animal were insusceptible to subsequent infection from smallpox. In the horse there occurs a disease known as horsepox, especially tending to arise in wet cold springs, which consists in an inflammatory condition about the hocks, giving rise to ulceration. Jenner believed that the matter from these ulcers, when transferred by the hands of men who dressed the sores to the teats of cows subsequently milked by them, gave rise to cowpox in the latter. This disease was thus identical with horsepox in epidemics of which it had its origin. Jenner was, however, probably in error in confounding horsepox with another disease of horses, namely grease. Cowpox manifests itself as a papular eruption on the teats; the papules become pustules ; their contents dry up to form scabs, ca more or less deep ulcers are formed at their sites. From such a lesion the hands of the milkers may become affected through abrasions, and a similar local eruption occurs, with general symptoms in the form of slight fever, malaise, and Toss of appetite. It is this illness, which, according to Jenner, gives rise to immunity from smallpox infection. He showed experimentally that persons who had suffered from such attacks did not react to inoculation with small- pox, and further, that persons to whom he communicated cowpox artificially, were similarly immune. The results of Jenner's observations and experiments were published in 1798 under the title An Inquiry into the Causes and Effects of the Variola Vaccines. Though from the first Jennerian vaccination had many opponents, it gradually gained the confidence of the unprejudiced, and became extensively jENNERIAN VACCINATION. 499 practised all over the world, as it is at the present day. The evidence in favour of vaccination is very strong. There is no doubt that inoculation with lymph properly taken from a case of cowpox, can be maintained with very little variation in strength for a long time by passage from calf to calf, and such calves are now the favourite source of the lymph used for human vaccination. When lymph derived from them is used for the latter purpose, immunity against smallpox is conferred on the vaccinated individual. It has been objected that some of the lymph which has been used has been derived from calves inoculated, not with cowpox, but with human smallpox. It is possible that this may have occurred in some of the strains of lymph in use shortly after the publication of Jenner's discovery, but there is no doubt that most of the strains at present in use have been derived originally from cowpox. The most striking evidence in favour of vaccination is derived from its effects among the staffs of smallpox hospitals, for here, in numerous instances, it is only the unvaccinated in- dividuals who have contracted the disease. While vaccina- tion is undoubtedly efficacious in protecting against small- pox, Jenner was wrong in supposing that a vaccination in infancy afforded protection for more than a certain number of years thereafter. It has been noted in smallpox epi- demics which have occurred since the introduction of vaccination, that whereas young unprotected subjects readily contract the disease, those vaccinated as infants escape more or less till after the 13th to the 15th years. It has become, therefore, more and more evident that re- vaccination is necessary if immunity is to continue, and where this is done in any population, smallpox becomes a rare disease, as has happened in the German army, where the mortality is practically nil. The whole question of the efficacy of vaccination has recently been investigated in this country by a Royal Commission, whose general con- clusions are as follows. Vaccination diminishes the liability to attack by smallpox, and when the latter does 500 SMALLPOX AND VACCINATION. occur, the disease is milder and less fatal. Protection against attack is greatest during nine or ten years after vaccination. It is still efficacious for a further period of five years, and possibly never wholly ceases. The power of vaccination to modify attack outlasts its power wholly to ward it off. Revaccination restores protection, but this operation must be from time to time repeated. Vaccina- tion is beneficial according to the thoroughness with which it is performed. The Relationship of Smallpox (Variola) to Cowpox (Vaccinia). — This is the question regarding which, since the introduction of vaccination, the greatest controversy has taken place ; a subsidiary point has been the inter- relationships within the group of animal diseases which includes cowpox, horsepox, sheep-pox, and cattle plague. With reference to smallpox and cowpox the problem has been, Are they identical or not? There is no doubt that cowpox can be communicated to man, in whom it produces the eruption limited to the point of inoculation, and the slight general symptoms which vaccination with calf lymph has made familiar. Apparently against the view that cowpox is a modified smallpox are the facts that it never reproduces in man a general eruption, and that the local eruption is only infectious when matter from it is intro- duced into an abrasion. The loss of infectiveness by transmission through the body of a relatively insusceptible animal is a condition of which we have already seen many instances in other diseases, and the uniformity of the type of the affection resulting from vaccination with calf lymph finds a parallel in such a disease as hydrophobia, where, after passage through a series of monkeys, a virus of attenuated but constant virulence can be obtained. We have seen that there are good grounds for believing that the virus of calf lymph confers immunity against human smallpox. In considering the relationships of cowpox and smallpox, this is an important though subsidiary point ; for at present it is questionable whether there are any well- authenticated instances of one disease having the capacity RELATIONS OF SMALLrOX AND COWPOX. 501 of conferring immunity against another. The most diffi- cult question in this connection is what happens when inoculations of smallpox matter are made on cattle. Chauveau denies that in such circumstances cowpox is obtained. He, however, only experimented on adult cows. The transformation has been accomplished by many observers, including, in this country, Simpson, Klein, Hime, and Copeman. The general result of these ex- periments has been that if a series of calves is inoculated with variolous matter, in the first there may not be much local reaction, though redness and swelling appear at the point of inoculation, and some general symptoms manifest themselves. On squeezing some of the lymph from such reaction as occurs, and using it to continue the passages through other calves, after a very few transfers a local reaction indistinguishable from that caused by cowpox lymph generally takes place, and the animals are now found to be immune against the latter. Not only so, but on using for human vaccination the lymph from such variolated calves, results indistinguishable from those pro- duced by vaccine lymph are obtained, and the transitory illness which follows, unlike that produced in man by inoculation with smallpox lymph, is no longer infectious. In fact many of the strains of lymph in use in Germany at present have been derived thus from the variolation of calves. The criticism of these experiments which has been offered, namely, that since many of them wTere performed in vaccine establishments, the calves were prob- ably at the same time infected with vaccinia, is not of great weight, as in all the recent cases at least, very elaborate precautions have been adopted against such a contingency. And at any rate it would be rather extra- ordinary that this accident should happen to occur in every case. We can, therefore, say that at present there is the very strongest ground for holding not only that vaccinia confers immunity against variola, but that variola confers immunity against vaccinia. The experimentum cruris for establishing the identity of the two diseases would of 502 SMA L I. POX AND VA CC1NA TION. course be the isolation of the same micro-organism from both, and the obtaining of all the results just detailed by means of pure cultures or the products of such. In the absence of this evidence we are at present justified in considering that there is strong reason for believing that vaccinia and variola are the same disease, and that the differences between them result from the relative sus- ceptibilities of the two species of animals in which they naturally occur. With regard to the relation of cowpox to horsepox, it is extremely probable that they are the same disease. Some epidemics of the former have originated from the horse, but in other cases such a source has not been traced. Cattle plague from the clinical standpoint, and also from that of pathological anatomy, resembles very closely human smallpox. Though each of the two diseases is extremely infectious to its appropriate animal, there is no record of cattle plague giving rise to smallpox in man or vice versa. When matter from a cattle plague pustule is inoculated in man, a pustule resembling a vaccine pustule occurs, and further, the individual is asserted to be now immune to vaccination ; but vaccination of cattle with cowpox lymph offers no protection against cattle plague, though some have looked on the latter as merely a malignant cowpox. Sheep-pox also has many clinical and pathological analogies with human smallpox, and facts as to its relation to cowpox vaccination similar to those observed in cattle plague, have been reported. Smallpox, cowpox, cattle plague, horse- pox, and sheep-pox, in short, constitute an interesting group of analogous diseases, of the true relationships of which to one another we are, however, still ignorant. Micro-organisms associated with Smallpox. — Burdon Sanderson was among the first to show that in vaccine lymph there were certain bodies which he recognised as bacteria. Since then numerous observations have been made as to the occurrence of such in matter derived from variolous and vaccine pustules. In especially the later stages of the latter, many of the pyogenic organisms are BACTERIA IN SA/ALLPOX. 503 always present, e.g., staphylococcus aureus and staphylococcus cereus flavus, and many of the ordinary skin saprophytes also are often present, but no organism has ever been isolated which on transference to animals has been shown to have any specific relationship to the disease. A bacillus, however, discovered independently by Klein and Copeman, and at present sub judice, may afford better results. Klein observed this organism in lymph taken from a vaccine pustule in a calf on the fifth and sixth days, in human vaccine lymph on the eighth day, and in lymph from a smallpox pustule on the fourth day. To demonstrate the bacilli, cover-glass films are dried and placed for five minutes in acetic acid (1 in 2), washed in distilled water, dried, and placed in alcoholic gentian-violet for from twenty- four to forty-eight hours, after which they are washed in water and mounted. Copeman and Kent also found the bacilli in sections of vaccine pustules stained by Loffler's methylene-blue, or by Gram's method. The organisms are .4 to .8 jj, in length, and one-third to a half of this in thickness. They are generally thinner and stain better at the ends than at the middle. They occur in groups of from three to ten in both the lymph and the tissues. In the centre of their protoplasm there is often a clear globule, which is looked on as a spore. They have hitherto resisted the ordinary isolation methods, a fact which is rather in favour of their non-saprophytic nature. By in- oculating fresh eggs with the crusts of smallpox pustules Copeman has, however, obtained a growth of a bacillus resembling that found by him in the tissues. Though subcultures on ordinary media have been obtained, the pathogenic effects of these have not yet been fully investi- gated, and thus the identity of this bacillus with that seen in the tissues is not yet proved. The facts that the latter is one hitherto not recognised microscopically, that it exists in the pustules, the contents of which are probably the means by which the disease naturally spreads, that it resists artificial cultivation, that the possession by it of spores explains some of the characteristics of vaccine lymph 5°4 SMA L L POX A ND VA CC/NA TIQN. (resistance to drying, etc.), make its further investigation a matter of considerable interest. Va rious observers have described appearances in the epithelial cells in the neighbourhood of the smallpox or vaccine pustules, which they have interpreted as being protozoa. Thus Ruffer and Plimmer describe as occurring in clear vacuoles in the cells of the rete Malpighii at the edge of the pustule, in paraffin sections of vaccine and smallpox pustules carefully hardened in alcohol, and stained by the Ehrlich-Biondi mixture, small round bodies about four times the size of a staphylococcus pyogenes, coloured red by the acid fuchsin, sometimes with a central part stained by the methyl-green. These appear to multiply by simple division, and in the living condition exhibit amoeboid movement. Similar bodies have been described by Reed in the blood of smallpox patients and of vaccinated children and calves. The significance of such appearances is unknown. The Nature of Vaccination. — As we are ignorant of the cause of smallpox, we can only conjecture what the nature of vaccination is. From what we know of other like processes, however, we have some ground for believing that it consists in an active immunisation by means of an attenuated form of the causal organism. As to how im- munity is maintained after vaccination, we do not know much. Some, including Beclere, Chambon, and Menard (who jointly investigated the subject), maintain that in the blood of vaccinated animals substances exist which, when transferred to other animals, can confer a certain degree of passive immunity against vaccination, and which have also a degree of curative action in animals already vaccinated. Beumer and Peiper, on the other hand, could not find evidence of the existence of such bodies. If they do exist, we cannot as yet say whether they are antitoxic or antimicrobic. APPENDIX B. HYDROPHOBIA. SYNONYMS. RABIES : FRENCH, LA RAGE : GERMAN, LYSSA, DIE HUNDSWUTH, DIE TOLLWUTH. Introductory. — Hydrophobia is an infectious disease which in nature occurs epidemically chiefly among the carnivora, especially in the dog and the wolf. Infection is carried by the bite of a rabid animal Or by a wound or abrasion being licked by such. The disease can be transferred to other species, and when once started can be spread from in- dividual to individual by the same paths of infection. Thus it occurs epidemically from time to time in cattle, sheep, horse's, and deer, and can be communicated to man ; but in modern times at least, infection practically never takes place from man to man, though such an occurrence is quite possible. In Western Europe the disease is most frequently observed in the dog ; but in Eastern Europe, especially in Russia, epidemics among wolves constitute a serious danger both to other animals and to man. All the mani- festations of the disease point to a serious affection of the nervous system ; but inasmuch as symptoms of excitement or of depression may predominate, it is customary to describe clinically two varieties of rabies, (i) rabies proper, or furious rabies (la rage vraie, la rage furieuse : die rasende Wuth) ; and (2) dumb madness or paralytic rabies (la rage mue : die HYDROPHOBIA. stilk Wnth). The disease, however, is essentially the same in both cases. In the dog the furious form is the more common. After a period of incubation of from three to six weeks, the first symptom noticed is a change in the animal's aspect ; it becomes restless, it snaps at anything which it touches, and tears up and swallows unwonted objects ; it has a peculiar high-toned bark. Spasms of the throat muscles come on, especially in swallowing, and there is abundant secretion of saliva ; its supposed fear of water is, however, a myth. Gradually convulsions, paralysis, and coma come on ; and death supervenes. In the paralytic form, the early symptoms are the same, but paralysis appears sooner. The lower jaw of the animal drops, from implication of the elevator muscles, all the muscles of the body become more or less weakened, and death ensues without any very marked irritative symptoms. In man the incubation period after infection varies from fifteen days to seven or eight months, or even longer, but is usually about forty days. When symptoms of rabies are about to appear, certain prodromata, such as pains in the wound and along the nerves of the limb in which the wound has been received, may be observed. To this succeeds a stage of nervous irritability, during which all the leflexes are augmented— the victim starting at the slightest sound, for example. There are spasms, especially of the muscles of deglutition and respiration, and cortical excitement evidenced by delirium may occur. On this follows a period in which all the reflexes are diminished, weakness and paralysis are observed, convulsions occur, and finally coma and death supervene. The duration of the acute illness is usually from four to eight days, and death invariably results. The existence of paralytic rabies in man has been denied by some, but it undoubtedly occurs. This is usually manifested by paralysis of the limb in which the infection has been received, and of the neighbouring parts; but while in such cases this is often the first symptom observed, during the whole of the illness the occurrence of wide- spread and progressive paralysis is the outstanding feature. PATHOLOGY OF HYDROPHOBIA. 507 The Pathology of Hydrophobia. — In hydrophobia as in tetanus, to which it bears more than a superficial re- semblance, the appearances presented in. the nervous system, to which all the symptoms are naturally referred, are comparatively unimportant. On naked-eye examination, congestions, and, it may be, minute haemorrhages in the central nervous system, are the only features noticeable. Microscopically, leucocytic exudation into the perivascular lymphatic spaces in the nerve centres has been observed, and in the cells of the anterior cornua of the grey matter in the spinal cord, and also in the nuclei of the cranial nerves, various degenerations have been described. The latter include pigmentation, atrophy, and vacuolation of the protoplasm, and the occurrence of a deposit of granules in the nucleus. In the white matter, especially in the posterior columns, swelling of the axis cylinders and breaking up of the myeline sheaths have been noted, and similar changes occur also in the spinal nerves, especially of the part of the body through which infection has come. In the nervous system also some have seen minute bodies which they have considered to be cocci, but that they are really such there is no evidence. The changes in the other parts of the body are unimportant. Experimental pathology confirms the view that the nervous system is the centre of the disease by finding in it a special concentration of what, from want of a more exact term, we must call the hydrophobic virus. Earlier inoculation experiments made by subcutaneous injection of material from various parts of animals dead of rabies had not given uniform results, as, whatever was the source of the material, the disease was not invariably produced. Pasteur's first contribution to the subject was to show that the most certain method of infection was by inserting the infective matter beneath the dura mater. He found that in the case of any animal or man dead of the disease, injection by this method of emulsions of any part of the central nervous system, of the cerebro- spinal fluid, or of the saliva, invariably gave rise to rabies, and also that HYDROPHOBIA. the natural period of incubation was shortened. Further, the identity of the furious and paralytic forms was proved, as sometimes the one, sometimes the other, was produced, whatever form had been present in the original case. Inoculation into the anterior chamber of the eye is nearly as efficacious as subdural infection. Infection with the blood of rabic animals does not reproduce the disease. There is evidence, however, that the poison also exists in such glands as the pancreas and mamma. Subcutaneous infection with part of the nervous system of an animal dead of rabies usually gives rise to the disease. In consequence of the introduction of such reliable inoculation methods, further information has been acquired regarding the spread and distribution of the virus in the body. Gaining entrance by the infected wound, it early manifests its affinity for the nervous tissues. It reaches the central nervous system chiefly by spreading up the peripheral nerves. This can be shown by inoculating an animal subcutaneously in one of its limbs, with virulent material. If now the animal be killed before symptoms have manifested themselves, rabies can be produced by subdural inoculation from the nerves of the limb which was infected. Further, rabies can often be produced from such a case by subdural infection with the part of the spinal cord into which these nerves pass, while the other parts of the animal's nervous system do not give rise to the disease. This explains how the initial symptoms of the disease (pains along nerves, paralyses, etc.) so often appear in the infected part of the body, and it probably also explains the fact that bites in such richly nervous parts as the face and head are much more likely to be followed by hydrophobia than bites in other parts of the body. Again, injection into a peripheral nerve, such as the sciatic, is almost as certain a method of infection as injection into the subdural space, and gives rise to the same type of symptoms as injection into the corresponding limb. Intravenous injec- tion of the virus, on the other hand, differs from the other modes of infection in that it more frequently gives rise to THE VIRUS OF HYDROPHOBIA. 509 paralytic rabies. This fact Pasteur explained by supposing that the whole of the nervous system in such a case becomes simultaneously affected. The virus seems to have an elective affinity for the salivary glands, as well as for the nervous system. Roux and Nocard found that the saliva of the dog became virulent three days before the first appearance of symptoms of the disease. The Virus of Hydrophobia. — While a source of infection undoubtedly occurs in all cases of hydrophobia, and can usually be traced, all attempts to determine the actual morbific cause have been unsatisfactory. In this connec- tion various organisms have been described as being associated with the disease. Undoubtedly several occur not infrequently in the brains of animals and men dead of rabies, and for two of these a causal connection has been claimed. Thus Memmo has isolated an organism which resembles a yeast, but which he places amongst the blastomycetes, and with which he states he has produced both types of rabies in rabbits and dogs. Bruschettini also, by using media containing brain substance, has grown a bacillus having some resemblances to the members of the diphtheria group, and with which he claims to have produced paralytic rabies in rabbits. In the case of the work of neither of these observers has there been confirmation from independent- sources, and in neither case is there evidence of the crucial test having been applied, namely, that of immunising animals against the ordinary hydrophobic virus by means of pure cultures of the alleged causal organism. With regard to other possible causal agents, Grigorjew thinks such may be found in a protozoon which he has constantly observed after inoculation in the cornea. There is no doubt that between rabies and the bacterial diseases we have studied there are at every point analogies, the most striking being the protective inoculation methods which constitute the great work of Pasteur, and everything points to a micro-organism being the cause. Judging from our knowledge of similar diseases we would strongly suspect that it is actually present in a living condition in the central HYDROPHOBIA. nervous system, the saliva, etc., which yield what we have called the hydrophobic virus, for by no mere toxine could the disease be transmitted through a series of animals, as we shall presently see can be done. The resistance of the virus to external agents varies. Thus a nervous system containing it is virulent till destroyed by putrefaction ; it can resist the prolonged application of a temperature of from - to0 to - 2o° C. but, on the other hand it is rendered non-virulent by one hour's exposure at 50° C. Again, its potency probably varies in nature according to the source. Thus, while the death rate among persons bitten by mad dogs is about 16 per cent, the corresponding death rate after the bites of wolves is 80 per cent. Here, however, it must be kept in view that, as the wolf is naturally the more savage animal, the number and extent of the bites, i.e., the number of channels of entrance of the virus into the body, and the total dose, are greater than in the case of persons bitten by dogs. As we shall see, alterations in the potency of the virus can certainly be effected by artificial means. The Prophylactic Treatment of Hydrophobia. — Until the publication of Pasteur's researches in 1885, the only means adopted to prevent the development of hydrophobia in a person bitten by a rabid animal, had consisted in the cauterisation of the wound. Such a procedure was un- doubtedly not without effect. It has been shown that cauterisation within five minutes of the infliction of a rabic wound prevents the disease from developing, and that if done within half an hour, it saves a proportion of the cases. After this time, cauterisation only lengthens the period of incubation; but, as we shall see presently, this is an extremely important effect. The work of Pasteur has, however, revolutionised the whole treatment of wounds inflicted by hydrophobic animals. Pasteur started with the idea that, since the period of incubation in the case of animals infected subdurally from the nervous systems of mad clogs, is constant in the dog, the virus has been from time immemorial of constant strength. Such a virus, of what might be called natural PROPHYLAXIS OF HYDROPHOBIA. 511 strength, is usually referred to in his works as the virus of la rage des rues, in the writings of German authors as the virus of die Strasswuth. Pasteur found on inoculating a monkey subdurally with such a virus, and then inoculating a second monkey from the first, and so on with a series of monkeys, that it gradually lost its virulence, as evidenced by lengthened periods of incubation on subdural inocula- tion of dogs, until it wholly lost the power of producing rabies in dogs, when introduced subcutaneously. When this point had been attained, its virulence was not diminished by further passage through the monkey. On the other hand, if the virus of la rage des rues were similarly passed through a series of rabbits or guinea-pigs, its virulence was increased till a constant strength (the virus fixe) was attained. Pasteur had thus at command three varieties of virus — that of natural strength, that which had been attenuated, and that which had been exalted. He further found that, commencing with the subcutaneous injection of a weak virus and following this up with the injection of the stronger varieties, he could ultimately, in a very short time, immunise dogs against subdural infection with a virus which, under ordinary conditions, would certainly have caused a fatal result. He also elucidated the fact that the exalted virus contained in the spinal cords of rabbits such as those referred to, could be attenuated so as no longer to produce rabies in dogs by subcutaneous injection. This was done by drying the cords in air over caustic potash (to absorb the moisture), the diminution of virulence being proportional to the length of time during which the cords were kept. Accordingly, by taking a series of such spinal cords kept for various periods of time, he was supplied with a series of vaccines of different strengths. Pasteur at once applied himself to find whether the com- paratively long period of incubation in man could • not be taken advantage of to " vaccinate " him against the disease before its gravest manifestation took place. The following is the record of the first case thus treated. The technique was to rub up in a little sterile bouillon a small piece of 512 HYDROPHOBIA. the cord used, and inject it under die skin by means of a hypodermic syringe. The first injection was made with a very attenuated virus, i.e., a cord fourteen days old. In subsequent injections the strength of the virus was gradu- ally increased, as shown in the table : — July 7. 1885, 9 A.M., cord of June 23. i.e. 14 days old. > » 7 J l 6 P.M. 25 j j 12 8 1 » 9 A.M. ) j 27 i » II ) ) 8 )» 6 P.M. 1 ) 29 n 9 > > 9 ) 1 1 1 A.M. cord of July 1 » ) 8 » J IO 5 ) 3 >? 7 1 » 1 1 > J 5 6 ) J 12 J ) 7 > > 5 3 ) 13 ) ) ) t 9 >) 4 J > 14 i ) > 1 1 1 j 7 3 >> J ) 15 )) j » 13 » 5 2 1 ) 16 > » 1 > 15 5S 1 day old. The patient never manifested the slightest symptom of hydrophobia. Other similarly favourable results followed ; and this prophylactic treatment of the disease quickly gained the confidence of the scientific world, which it still maintains. (The principle is, of course, the same as in artificially developing a high degree of active immunity against a bacterial infection.) The only modification which the method has undergone, has been in the treatment of serious cases, such as multiple bites from wolves, extensive bites about the head, especially in children, cases which come under treatment at a late period of the incubation stage, and cases where the wounds have not cicatrised. In such cases the stages of the treatment are condensed. Thus on the first day, say at 1 1 a.m. and 4 P.M. and 9 P.M., cords of 12, 10, and 8 days respectively are used ; on the second day, cords of 6, 4, and 2 days ; on the third day, a cord of I day ; on the fourth day, cords of 8, 6, and 4 days ; on the fifth, cords of 3 and 2 days ; on the sixth, cords of 1 day ; and so on for 10 days. In each case the average dose is about 2 c.c. of the emulsion. . The success of the treatment has been very marked. 1 he statistics of the cases treated in Paris are -published quarterly in the Annales de i Inslilut Pasteur, and general summaries of the results of each year are also prepared. As we have said, the ordinary mortality formerly was 16 per cent of all persons bitten. During the ten years 1886-95, ANTI RABIC SERUM. 513 17,337 cases were treated, with a mortality of .48 per cent. It has been alleged that many people are treated who have been bitten by dogs that were not mad. This, however, is not more true of the cases treated by Pasteur's method than it was of those on which the ordinary mortality of 16 per cent was based, and care is taken in making up the statistics to distinguish the cases into three classes. Class A includes only persons bitten by dogs proved to have had rabies, by inoculation in healthy animals of parts of their central nervous system. Class B includes those bitten by dogs that a com- petent veterinary surgeon has pronounced to be mad. Class C includes all other cases. During 1895, 122 cases belonging to Class A were treated, with no deaths ; 949 belonging to Class B, with two deaths ; and 449 belonging to Class C, with no deaths. Besides the Institute in Paris, similar institutions exist in other parts of France, in Italy, and especially in Russia, as well as in other parts of the world ; and in these similar success has been experienced. It may be now taken as established, that a very grave responsibility rests on those concerned, if a person bitten by a mad animal is not subjected to the Pasteur treatment. Antirabic Serum. — In the early part of the present century an Italian physician, Valli, showed that immunity against rabies could be conferred by administering through the stomach progressively increasing doses of hydrophobic virus. Following up this observation, Tizzoni and Cen- tanni have attenuated rabic virus by submitting it to peptic digestion, and have immunised animals by injecting gradu- ally increasing strengths of such virus. This method is usually referred to as the Italian method of immunisation. The latter workers showed from this that the serum of animals thus immunised could give rise to passive im- munity in other animals ; and further, that if injected into animals from 7 to 14 days after infection with the virus, it prevented the latter from producing its fatal effects, even when symptoms had begun to manifest themselves. They further succeeded in producing in the sheep and the dog an immunity equal to from 1-25,000 to 1-50,000 {vide P- 473)j an<3 tney recommended the use, in severe cases, of the serum of such animals in addition to the treatment of the patient by the Pasteur method. We do not, of course, know whether the serum contains antitoxic or anti- microbic bodies. 33 514 HYDROPHOBIA. Methods (a) Diagnosis. — When a person is bitten by an animal suspected to be rabid, the latter must under no circumstances be killed. Much more can be learned by watching it while alive than by post-mortem examination. In the latter case only such things as the occurrence of broken teeth, marked congestion of the fauces, or the presence of unwonted material in the stomach throw any light on the condition ; nothing of a positive nature can be learned from examining the nervous system. On the other hand, in the living animal the development of the character- istic symptoms can be watched, and death will occur in not more than 5 days. If the suspected animal has been killed, then a small piece of its medulla or cord must be taken, with all aseptic precautions, rubbed up in a little sterile .75 per cent sodium chloride solution, and injected by means of a syringe beneath the dura mater of a rabbit, the latter having been trephined over the cerebrum by means of the small trephine which is made for the purpose. Symptoms usually occur in 8 to 1 2 days, though some- times not for three weeks, and death a day or two later. When such inoculation has to be practised it is evident that the diagnosis is delayed. When the material for inoculation has to be sent any distance this is best effected by packing the head of the animal in ice. The virulence of organs is not lost, however, if they are simply placed in sterile water or glycerine in well-stoppered bottles^ (b) Treatment. — Every wound inflicted by a rabid animal ought to be cauterised with the actual cautery as soon as possible. By such treatment the incubation period will at any rate be lengthened, and therefore there will be better opportunity for the Pasteur inoculation method being efficacious. The person ought then to be sent to the nearest Pasteur Institute for treatment. It is of great importance that in such a case the nervous system of the animal should also be sent, in order that the diagnosis may be certainly verified. APPENDIX C. MALARIAL FEVER. The organism which is now almost universally believed to be the causal agent in malarial fever was discovered by Laveran in 1880. This organism does not belong to the vegetable but to the animal kingdom ; it is not a bacterium but a protozoon. It is usually known as the hcematozoon or Plasmodium malarice. The use of the term plaslnodium is, however, incorrect. Laveran's discovery received con- firmation from the independent researches of Marchiafava and Celli, and later from the researches of many others in various parts of the world. Valuable additional information on the subject was supplied by the work of Golgi, who specially has the credit of first distinguishing certain varieties of the organism in the different types of malarial fever. In this country valuable work on the subject has been done by Manson. Regarding the invariable presence of this organism in the blood, and the cycle of changes which it undergoes in relation to the paroxysms of fever, practically all are agreed. On the other hand, some doubt still prevails regarding certain stages in its development, and especially regarding the number of varieties of the organism and their relations to one another. We shall first give an account of the different forms in which the organisms are met with, and afterwards state some facts with regard to the varieties which have been described. The description will be simplified by stating that the parasites in all the types of malarial fever pass through a definite cycle of 5i6 MALARIAL FEVER. development, which is completed in a period of time corresponding to the type of the fever; that is, in the quotidian 1 in twenty-four hours, in the tertian in forty-eight hours, in the quartan in seventy-two hours. In this cycle the youngest forms of the parasite appear as minute rounded protoplasmic bodies, which are at first free in the blood plasma but afterwards become attached to and invade the red corpuscles. Within the latter they gradually increase in size, till at a certain period multiplication by division or sporulation takes place, which results in the setting free of a number of young forms ; thus the cycle is completed. We may state then that there is a stage of gradual growth of the parasite, which is followed by a stage of sporulation or the formation of a new generation of young forms. The latter stage corresponds more or less closely with the rigor at the onset of the attack of fever. The parasites are always most abundant in the blood during the attack of fever, and in the intervals become greatly diminished in number or may actually disappear. They are also as a rule more abundant in internal organs than in the peri- pheral blood, and in some types of fever the process of sporulation is practically confined to the former. In addition to the forms which appear to constitute stages in this regular cycle of development within the human body there are two others, namely, the crescentic bodies and the flagellated organisms. These different forms may now be described in more detail, i. The spores are the youngest and smallest forms, which result from the segmentation of the adult parasite. They are rounded or oval protoplasmic bodies of minute size, usually not measuring much more than i /x in diameter their exact size, however, varying in the different types of fever They possess little or no amoeboid movement. They remain free in the serum for a short time, but soon attack the red corpuscles, when they become the intra- corpuscular amoeboid bodies. ^ i A quotidian type may, however, be produced by two generations of tertian parasites with a twenty-four hours' mterval, etc. FORMS OF THE MALARIAL PARASITE. 5'7 2. Epi- or Intra- corpuscular Bodies. — These include the parasites which have attacked the red corpuscles j they are at first situated on the surface of the latter but after- wards penetrate their substance. They usually occur singly in the red corpuscles, but sometimes two or more may be present together. The youngest or smallest forms appear as minute colourless specks, scarcely exceeding i /* in diameter. As seen in fresh blood, they exhibit more or less active amoeboid movement, showing marked varia- tions in shape. The amount and character of the amoe- boid movement varies somewhat in different types of fever. As they increase in size, pigment appears in their interior as minute dark brown or black specks, and gradually becomes more abundant (Figs. 119, 120). The pigment may be scattered through their substance, or concentrated at one or more points, and often shows vibratory or oscillating movements. This pigment is no doubt derived from the haemoglobin of the red corpuscles, the parasites growing at the expense of the latter. The red corpuscles thus invaded may remain unaltered in appearance, may become swollen and pale, or somewhat shrivelled and of darker tint. In stained specimens a nucleus may be seen in the parasite as a pale spot containing a minute and deeply-stained nucleolus, the nucleus being more distinct at some stages than at others. Sometimes, namely in the quotidian and malignant fevers, the parasite passes into a quiescent "ring form." The organisms in this condition show a well-defined outer circular margin and a central spot which is less sharply marked off, the pigment being usually collected in a small clump at one side (Fig. 122). These ring forms may again assume amoeboid movement. Within the red corpuscles the parasites gradually increase in size till the full adult form is reached (Fig. 120). In the latter stage the parasite loses its amoeboid movement more or less completely, has a somewhat rounded form, and contains a considerable amount of pigment. Some- times, for example in the quotidian form, it only occupies a fraction of the red corpuscle. The adult parasites may 5x8 MALARIAL FEVER. then undergo segmentation, i.e., sporulation, but not all of them do so ; many become degenerated and ultimately break down. 3. Segmentation or Sporulation Forms. — In the process of segmentation or sporulation, the pigment becomes col- lected into a small central mass, and from it as a centre, lines radiate outwards and divide the protoplasm into regular segments (Fig. 121). In this way a characteristic appearance is produced which has given the name of " rosette form " to this stage. The segments or spores thus formed vary in number and also in size, in different types of fever. They become more or less rounded in shape and are set free in the blood plasma. The pigment granules remain apart from the spores, sometimes sur- rounded by a portion of the substance of the parasite, and are chiefly taken up by leucocytes. The process of segmentation, however, does not occur ' in all forms of malarial fever in this radiate fashion, but in some takes place more or less irregularly. 4. Peculiar forms are those known as crescents or crescentic bodies. These are non-amoeboid, and of cres- centic or sausage shape, usually measuring 8 to 9 fi in length. Occasionally a fine curved line is seen joining the extremities on their concave aspect, which probably re- presents the remains of the envelope of a red corpuscle (Fig. 123). They are colourless and transparent, have a distinct enclosing membrane, and usually show a small collection of granular pigment about their middle. Man- naberg's view regarding the origin of the crescentic bodies is that they are conjugation-forms, resulting from the fusion of two intra-corpuscular bodies. He gives to them the name of "syzygies." Of this view Surgeon-Major Ross supplies important confirmation by actually tracing the process of conjugation in the infection of birds by a closely similar protozoon, the proteosoma. Bodies of the crescent series are not found in all the types of malarial fever, but especially in the quotidian and malignant types, and apparently do not represent a stage in the ordinary cycle Fig. 123. Fig. 124. Figs, i 19-124.— From dried blood-films showing parasites of malarial fever. Magnified about 1000 diameters. Fie no. Early intra-corpuscular form of the "mild tertian" parasite. Fig. 120. Large intra-corpuscular form of the same parasite showing scattered pigment granules; the invaded red corpuscle is much enlarged Fig. 121. Two members of the "rosette" series of the same parasite; that to the right shows the radiate segmentation, that to the left is an earlier stage. In both the pigment is collected in the centre. Fig. 122. Two "ring-forms" of the quotidian parasite, within red corpuscles. Fig. 123 shows a "crescentic body" ; from a case of malignant tertian fever Fig. 124. A flagellated organism, derived outside the body from a crescentic form. (All these figures are from negatives or preparations kindly lent by Dr. Patrick Manson.) 520 MA LA HI A 1. EE VER. of development. They appear in the blood after the fever has lasted for some time, apparently remain unchanged through the attacks of pyrexia, and may persist for a con- siderable period after the fever has gone, being often present in the cachexia or anaemia following these feVers. 5. Flagellated Organisms. — If a drop of blood be ex- amined under the microscope for some time, flagellated organisms may be found. So far as is known, they do not occur as such in the circulating blood, but only appear in the blood outside the body. They are derived either from the crescents or from the larger pigmented intra-corpuscular bodies. In the former case, when watched under the microscope the crescents alter their shape, becoming straight, then oval, and ultimately spherical. The pigment granules first become arranged as a ring, and afterwards show a peculiar vibratory movement, which is apparently produced by flagella which have formed within the sphere. When this stage is reached, the flagella, usually three or four, though sometimes more, are shot through the enve- lope, sometimes simultaneously, sometimes one after the other, and present a very rapid lashing action. The flagella are very delicate filaments, with sometimes a slight bulbous swelling at their free extremity (Fig. 124). They may afterwards become detached, and move away with an active independent movement. In the case of their development from the large intra-corpuscular bodies, the pigment shows an agitated movement in the same way, and ultimately the flagella are suddenly shot out. There has been, and still is, great diversity of opinion concerning the nature of the crescentic and the flagellated bodies. The view which appears to be best supported is, that the former represent a sort of resting form for the life of the organisms outside the body, the first stage of which is the flagellated condition. This view has been advanced notably by Manson, who considers that the flagella are really flagellated spores which undergo further change, and that this probably occurs in the body of mosquitoes which have taken up blood containing the parasite. Ross supports FLA GELLA TED ORGANISMS. 521 this view, and has found that in the stomach of the mosquito many of the crescents become spherical, and develop into the flagellated forms. Furthermore, in the case of certain, mosquitoes when allowed to suck malarial blood there appear in the walls of the stomach certain rounded cells containing pigment like that seen in the malarial parasite in the blood. Beyond this stage he has not yet traced the development of the malarial parasite in the mosquito, but he has done so in the case of the proteosoma infection of birds above referred to. The stages of development, which are of a highly interesting character, are as follows. On the second day after mosquitoes have been allowed to suck the blood of infected birds there appear in the walls of the insects' stomachs small oval pigmented bodies, chiefly situated between the muscle fibres. These bodies, which measure at this stage about 6 p, in diameter, rapidly increase in size, so that on the fourth or fifth day they may reach a diameter of 60 fx and may cause little bulgings on the outer wall of the stomach. They have a well-defined outer capsule, and their protoplasm shows finely striated mark- ings ; they may be called coccidium cysts. The cysts in course of time burst and set free in the body cavity an enormous number of minute spindle-shaped and somewhat curved rods- — " germinal rods." These rods are carried to various parts of the body and are especially taken up by the epithelial cells in a branching gland at the base of the insect's proboscis*. The striking result has been obtained that when a mosquito with the rods in this position is allowed to bite a healthy bird the latter becomes infected, and the proteosoma appears in large numbers in its blood after an incubation period of six or seven days. These experiments which have thus resulted in tracing the life- history of this parasite, have been carefully checked by controls and have also been recently confirmed in all essential details by the observations of Koch on the same parasite. There can be little doubt that a similar cycle, the earlier- part of which has already been observed, will be 522 MALARIAL FEVER. completely worked out in the case of the malarial parasite. If such a result is established it of course does not follow that the disease is always transmitted by the bite of a mosquito, as there are other means by whjch the extra- corporeal forms of the parasite may gain entrance to the human body, e.g. by drinking water, possibly by inhalation, etc. The origin of the pigmented cells in the stomach of the mosquito has not yet been traced, but light is thrown on the subject by the observations of MacCallum on halteridium, another protozoon infecting birds. He found that outside the body some of the flagellated forms entered certain of the parasites which had escaped from the corpuscles and assumed a rounded form. As a result these latter, impreg- nated as it were, became vermicules with powers of inde- pendent movement and containing pigment in their interior. A similar process probably occurs in the stomach of the mosquito, the vermicules afterwards penetrating the gastric wall. This confirms Manson's view, mentioned above, that the flagella are really flagellated spores. Koch regards them in a similar light, calling them spermatozoa. Varieties of trie Malarial Parasite. — The view pro- pounded by Laveran is that there is only one species of malarial parasite, which is polymorphous, and presents slight differences in structural character in the different types of fever. This view is now held by only a small minority of authorities, and it is generally believed that there are several distinct varieties, though there is still diversity of opinion as to their exact number. There is, however, a fairly general agreement as to the division of the varieties of malarial fever, according to the characters of the parasite, into two main classes ; the first including the milder forms, tertian and quartan, and the second including the quotidian, malignant, and certain irregular forms. The following arrangement follows closely that of Marchiafava and Bignami, and of Mannaberg. (a) Milder forms, in which crescentic bodies do not occur. —The parasites of the quartan and tertian fevers (" winter- VARIETIES OF THE MALARIAL PARASITE. 523 spring " fevers of Italian writers) were first distinguished by Golgi. Their characters are the following : — 1. Quartan— The parasite passes through its cycle of development in three days, and all the various stages are found in the blood. Only the smaller forms within the red corpuscles show amoeboid movements, and these are not of very active character. The red corpuscles invaded by the parasite do not become decolorised or altered in size ; the pigment granules are somewhat coarse. Typical rosette-forms are seen in the process of sporulation, which results in the formation of six to twelve segments or spores. The spores in the fresh blood show a central clear spot which is not seen in the spores of the tertian parasite. 2. Tertian — The cycle of development of the parasite is completed in forty-eight hours. The young forms within the red corpuscles show much more active movement than in the quartan type, and give off longer and more slender processes, whilst the pigment granules are finer. The infected corpuscles become swollen and pale. Sporulation, resulting in the formation of from fifteen to twenty round spores, takes place by means of a rosette or rather a sunflower formation, the lines of segmentation being at the periphery, and a portion remaining around the central collection of pigment {vide Figs. 11 9-1 21). (b) The more severe forms (sestivo-autumnal fevers). — In these the crescent forms are found (Fig. 123). 1. Quotidian. — This is the form most commonly assumed by malarial fever in the tropics. The parasite passes through its cycle of development in twenty -four hours. Within the red corpuscles the parasite is of small size, and even in its adult condition, immediately before sporulation, does not usually occupy more than a third of the corpuscle. The amoeboid forms often pass into the " ring-form " described above. In their course of develop- ment they acquire very fine dust-like pigment, and in the adult quiescent form the pigment becomes collected into a small dark body. The spores (usually six to eight) are formed by irregular segmentation, and are very minute ; the 524 MA L A R I A L EE VER. process takes place almost exclusively in internal organs — spleen, etc. — so that as a rule no sporulating forms can be found in the blood taken in the usual way. 2. A non-pigmented quotidian parasite has been differ- entiated and described by'Marchiafava, which differs from the previous only in the absence of pigment. 3. Malignant Tertian or Summer-autumn Tertian (Mar- chiafava and Bignami). — The pafasite closely resembles that of the quotidian. Its cycle of development, however, occupies forty-eight hours, and the young parasites may be without pigment for twenty-four hours. The amoeboid activity is maintained even in the adult pigmented forms. There are also some minor points of difference. In this variety also ring-forms occur. In these three varieties the red corpuscles invaded by the parasite have a certain tendency to shrivel and become of deeper or coppery tint. The disease sometimes assumes a malignant character, and when a fatal result occurs, large numbers of parasites, many in process of segmentation, may be found in the brain and internal organs. In some fatal cases with coma, the cerebral capillaries may appear to be almost filled with them. Irregular types of fever, sometimes of a continued character, may be produced by infection with different generations of the same variety of parasite, or by different varieties (mixed infections). In the various tropical malarial fevers it is quite possible that there are still other varieties of parasites whose characters have not yet been worked out. Kelations to the Disease. — Though the malarial para- sites have in no form been cultivated outside the body, the evidence that they are the cause of the disease amounts to a practical certainty. They are always present in the disease, and have been found in no condition apart from malaria. Their cycle of development also corresponds in a remarkable manner with the course of the fever, each febrile attack being accompanied by the appearance of a new generation of parasites in the blood. In all probability METHODS OF EXAMINATION. 525 the fever is produced by toxic bodies set free by the young parasites, but that cannot yet be directly proved. 1 he presence of the parasites in the red corpuscles and the destruction of their substance which takes place explain the occurrence of the anaemia which so often results ; the subsequent distribution and storage of the altered haemo- globin producing the pigmentary changes in the various organs. The disease can also be communicated from one person to another by injecting the blood containing the parasites. Several experiments of this kind have been performed (usually about \ to i c.c. of blood has been used), and the result is more certain in intravenous than in subcutaneous injection. In such cases there is an incubation period, usually of seven to fourteen days, after which the fever occurs. The bulk of evidence goes to show that the same type of fever is reproduced as was present in the patient from whom the blood was taken. It may also be mentioned that in certain affections both of birds (as already referred to) and of reptiles, parasites of somewhat similar character to those in malaria, though of distinct species, occur in the blood of these animals. Methods of Examination. — The parasites may be studied by examining the blood in the fresh condition, or by permanent preparations. In the former case, a slide and cover-glass having been thoroughly cleaned, a small drop of blood from the finger or lobe of the ear is caught by the cover-glass, and allowed to spread out between it and the slide. It ought to be of such a size that only a thin layer is formed. A ring of vaseline is placed round the edge of the cover-glass to prevent evaporation. For satisfactory examination an immersion lens is to be pre- ferred. The amoeboid movements are visible at the ordinary room temperature, though they are more active on a warm stage. With an Abbe condenser a small aperture of the diaphragm should be used. Permanent preparations are best made by means of dried films. A small drop of blood is allowed to spread 526 MALARIAL FEVER. itself out between two cover-glasses, which are separated by sliding the one on the other. The films are then allowed to dry. A very good method is that of Manson, who catches the drop of blood on a piece of gutta-percha tissue, and then makes a film on a clean slide by drawing the blood over the surface. The dried films are then fixed by one of the methods already given (p. 96), or by placing in absolute alcohol for five minutes (Manson). They may be stained by a saturated watery solution of methylene-blue for five minutes, or by Ehrlich-Biondi fluid for half an hour. A double stain may be obtained by staining first with a 1 per cent watery solution of eosin for five minutes, then washing well in water, and thereafter staining for about a minute with a saturated watery solution of methylene-blue ; the red corpuscles are red, the parasites and nuclei of leucocytes are coloured with the blue. After being stained the films are washed in water, dried, and mounted in balsam ; those stained with Ehrlich-Biondi fluid are for some purposes best examined in the dry condition, the cover-glass being fixed at its margins to the slide by balsam, the film downwards, but not in contact with the slide. APPENDIX D. DYSENTERY. Amongst the early researches on the relation of organisms to this disease probably the most important are those of Loschj who noted the presence and described the characters of amoeba; in the stools of a person suffering from dysen- tery, and considered that they were probably the causal agents. Further observations on a more extended scale were made by Kartulis with confirmatory results, this observer finding the same organisms also in liver abscesses associated with dysentery. The subject was, however, complicated by the fact that the same or closely similar organisms had been previously found in the intestine in normal conditions and in other diseases than dysentery (by Cunningham and Lewis and others), and additional re- search confirmed these results. Two questions thus arose. In the first place, Is there an amoeba peculiar to dysentery (amoeba dysenteriae) and distinguishable from the amoeba? present in other conditions ? In the second place, Is this organism the cause of the disease ? Both of these questions may now be said to be practically answered in the affirma- tive. It has, moreover, been found that so far as etiology is concerned there are several forms of dysentery, and that it is the endemic dysentery of the tropics and of some sub- tropical countries which is in all probability produced by amoeba?. Hence this form is often now called amoebic dysentery, and Councilman and Lafleur, working in Balti- more, have found that it can be distinguished from other 528 D YS ENTER V. forms not only by the presence of amoebae but also by its pathological anatomy. The results of these observers have been confirmed by those obtained in Egypt by Kruse and Pasquale, who have also supplied important facts regarding the pathogenic effects of the amoebae when inoculated into animals. The following description is chiefly taken from the monographs of the four writers last mentioned. Amoebic Dysentery — Characters of the Amoeba. — The amoebae, as seen in the stools of a case of dysentery, are rounded or somewhat irregular protoplasmic masses, usually c CL b Fig. 125. — Amoebae of dysentery. a and b, amoebae as seen in the fresh stools, showing blunt amoeboid processes of ectoplasm. The endoplasm of a shows a nucleus, three red corpuscles and numerous vacuoles ; that of b, numerous red corpuscles and a few vacuoles. c, an amoeba as seen in a fixed film preparation, showing a small rounded nucleus (Kruse and Pasquale). X 600. measuring about 25 to 35 /z in diameter, though both larger and smaller forms are met with. When the parasite is at rest it has a more or less rounded shape ; the protoplasm is finely granular and of refractile appearance, and is without differentiation into layers. The organism may show sluggish amoebic move- ments at the ordinary temperature, but these become much more active when a warm stage is used. When they occur, the amoeba shows differentiation into a central granular endoplasm and an outer hyaline layer or ectoplasm DISTRIBUTION OF THE AMCEBsE. 529 which is very thin and well marked off from the former. The blunt processes which are protruded in amoebic move- ment are composed of the ectoplasm (Fig. 125). By the amoebic movements slow locomotion may be produced. The amoeba? often show vacuoles in their substance, and may contain numerous red corpuscles (which appear to undergo digestive liquefaction), also bacteria, etc. There is a single nucleus which lies in the central part of the organism and usually measures about 6 to 8 /a in diameter. It is round or oval and contains a nucleolus. In the living condition the nucleus is invisible or is faintly seen, but becomes very evident on the addition of acetic acid, etc. The amoebae break down pretty rapidly outside the body, and examination of the dysenteric stools twenty-four hours after being passed usually fails to detect any of them. It is only on one or two rare occasions that the process of division of the amoebae has been observed and described. By some there have also been described encysted forms. These are of smaller size, about 10 to 15 /x, with a well- marked capsule, sometimes showing a double contour and a central protoplasm in which a nucleus may or may not be visible. It is still doubtful, however, whether these structures really constitute a stage in the development of the organism, as direct transformation from the one form into the other has not been observed. Distribution of the Amoebae. — As already stated, they are usually found in large numbers in the contents of the large intestine in tropical dysentery. They also, however, penetrate into the tissues, where they appear to exert a well-marked action. They are found in the mucous membrane when ulcers are being formed, but their most characteristic site is beyond the ulcerated area, where they may be seen penetrating deeply into the submucous, and even into the muscular coats. In these positions they may be unattended by any other organisms, and the tissues around them show more or less necrotic change without much accompanying inflammatory reaction. In this way the ulcers are lined by sloughing tissue, and have often an 34 53° D YSENTER V. undermined character. These lesions are considered by Councilman and Lafleur to be characteristic of amoebic dysentery. In the or two later. The action here on the tissues is of an analogous nature, namely, a necrosis with softening and partial liquefaction, attended by little or no suppurative change. The amoebae have also been found in the sputum when a liver abscess has ruptured into the lung, as not very infrequently happens. Relations to the Disease. — It may be stated in the first place that cultures of these amoebae outside the body have not been obtained. Kartulis announced that he had cultivated the organism on straw infusion, but it is now recognised that his results are erroneous, the amoebae observed by him being probably derived from the infusion itself. In fact, everything seems to show that the amoebae in their usual form rapidly disintegrate outside the body, and it is still unknown in what form they survive and lead to the propagation of the disease. The points of distinction between the amoeba of dysentery and the ordinary amoeba coli, so far as the morphology is concerned, are that the liver abscesses associ- ated with dysentery the amoebae are usually to be found, and not infrequently are the only organisms present (Fig. 126). They are most numerous at the spreading margin, and this probably explains a fact pointed out by Manson, that examina- tion of the contents first removed may give a negative result, while they may be detected in the discharge a day Fig. 126. — Section of wall of liver abscess, showing an amoeba of spherical form with vacuolated protoplasm. x 1000. RELATIONS TO THE DISEASE. 53i latter is on the whole of smaller size, its protoplasm is more finely granular, and it does not appear to take up red corpuscles, etc., as is the case with the former. The distinction, however, can only be definitely drawn by the result of experiment. Injection of certain quantities of dysenteric stools containing the amcebse into various animals per rectum has been carried out by different observers, especially by Kruse and Pasquale. In cats, in the majority of cases, a hasmorrhagic enteritis is pro- duced, amoeba? being present in the stools and also in- vading the mucous membrane of the • intestine in the ulcerated areas which are sometimes formed. The deep infiltration of the submucous coat by the amcebse, which is so characteristic a feature in the human disease, does not occur in these animals. Not infrequently death follows. Kruse and Pasquale obtained corresponding results when the material from a liver abscess containing amoebae without any other organisms was injected. In the absence of cultures of amoebae outside the body, this evidence must be taken as conclusive that the disease produced in cats is really caused by the amoebae. Similar injections with material containing amoebae derived from other sources are unattended by any pathogenic effects of similar nature. Feeding the animals with material containing the amoebae is much more uncertain in its effect. Quincke and Roos obtained no effects when the amoebae were administered, but they obtained a fatal result in two out of four cases when the cyst-like forms were given. From this fact they infer that the latter are probably a cystic stage of the former and that the former are destroyed in the gastric contents. This practically constitutes the only important evidence that a cystic stage of the organism has really been observed. These observers found that the cyst-like bodies were still present even after the material had been kept for two or three weeks. From the above facts, all of which have received ample confirmation, with the exception of the statements regarding the cyst-like forms, there can be. little or no doubt that the 532 DYSENTERY. amoeba; described are the causes of the form of dysentery with which they are associated. We are still ignorant, however, as to their life-history outside the body, and the modes by which infection is produced. Further, in any case where they act as the primary agent, secondary in- flammatory changes in the intestine may be produced by the action of various bacteria. Varieties of Dysentery. — We have already pointed out that all dysenteric conditions are not of the same nature, and that in certain forms amoebae are not present. Ogata, for example, investigated an extensive epidemic in Japan without detecting amoebae. He found, however, in sections of the affected tissues enormous numbers of small bacilli about the same thickness as the tubercle bacillus, but very much shorter. These bacilli were sometimes found in a practically pure condition. They were actively motile and could be stained by Gram's method. He also obtained pure cultures from various cases and tested their patho- genic effects. They grew well on gelatine at the ordinary temperature producing liquefaction, the growth somewhat resembling that of the cholera spirillum. By injection into cats and guinea-pigs, as well as by feeding them, this organism was found to have distinct pathogenic effects; these were chiefly confined to the large intestine, hemor- rhagic inflammation and ulceration being produced. Kruse and Pasquale conclude that so far as our present knowledge of the etiology of dysentery goes, there are several varieties which may be arranged as follows : — First, the amoebic or tropical dysentery, having the characters as above described; second, the various diphtheritic and catarrhal forms without amoeba;, possibly produced by bacteria of different kinds, but the nature of which has not been fully investigated; third, the Japanese form of dysentery as investigated by Ogata. Methods of Examination.— The fasces in a case of sus- pected dysentery ought to be examined microscopically as soon as possible after being passed, as the amoebae dis- appear rapidly, especially when the reaction becomes acid. METHODS OF EXAMINATION. 533 A drop is placed on a slide without the addition of any reagent, a cover-glass is placed over it and the preparation is examined in the ordinary way or on a hot stage, prefer- ably by the latter method, as the movements of the amoebre are more active and it is difficult to recognise them when they are at rest. Dried films are not suitable, as in the preparation of these the amoebae become broken down; but films may be fixed with corrosive sublimate or other fixative {vide p. 97). In sections of tissue the amoebae may be stained by methylene-blue, by safranin, by haematoxylin, and eosin, etc. p BIBLIOGRAPHY. General Text-books.— In English the student may consult the following : " Micro-organisms and Disease," E. Klein, 3rd ed. London, 1896 " Bacteriology and Infective Diseases," Edgar M. Crookshank, London, 1896. " A Manual of Bacteriology," George M. Sternberg, New York, isted. 1893, 2nd ed. 1896 (this book contains a full biblio- graphy) " Textbook upon the Pathogenic Bacteria," Joseph M 'Far- land, London, 1896. " Practical Bacteriology," A. A. Kanthack and J H. Drysdale, London, 1895. " Bacteria and their Products," G. S. Woodhead, London, 1891. The articles on bacteriological subjects in Clifford Allbutt's " System of Medicine," London, are of the highest excellence, and have full bibliographies appended. For the hygienic aspects of bacteriology see *" System of Hygiene," Stevenson and Murphy, London, 1892-94. In German: "Die Mikroorganismen," by Dr. C. Fliigge, 3rd ed. Leipzig, 1896. (The first edition of this book, published in 1886, was a monograph by Fliigge. The third is practically a new work edited by Fliigge and written by Frosch, Gotschlich, Kolle, Kruse, and R. Pfeiffer. It contains a very full treatment of the subject and has a complete list of references.) " Lehrbuch der patholog- ischen Mykologie," by Baumgarten, Braunschweig, 1890. " Grun- driss der Bakterienkunde," C. Fraenkel, Berlin, 1890. "Die Methoden der Bakterien-Forschung," F. Hueppe, Wiesbaden, 1 891. " Naturwissenschaftliche Einfuhrung in die Bakteriologie," F. Hueppe, Wiesbaden, 1896. "Einfuhrung in das Studium der Bakterio- logie," C. Giinther, Leipzig, 1893 (4th ed. 1895). "Lehrbuch der bakteriologischen Untersuchung unci Diagnostik," L. Heim, Stutt- gart, 1894. , . In French : For students, two extremely useful books are " Precis de microbie," by Thoinot and Masselin, 3rd ed. Paris, 1896, and "Precis de bacteriologie clinique," Wurtz, Paris, 1895. Periodicals. — For references to current work see Centralbl. f. Bakteriol. u. Parasitenk., Jena. This publication commenced in 1887. Two volumes, each containing 26 weekly numbers, are issued yearly. 536 BIBLIOGRAniY. In 1S95 it was divided into two parts. Abtheilung I. deals with Medizinisch-hygienische Bakteriologie und thierische Parasitenkunde. The volumes of this part are numbered consecutively with those of the former series, the first issued thus being vol. xvii. Abtheilung II. deals with Ailgemeine landwirlschaftlich-tcchnoloqische Bakteriologie Gii rungsphysiologie und Pflanzcnpalhologie. Twenty-six numbers forming one volume are issued yearly. The first volume is entitled Zweite Abtheilung, Bd. I. Both parts contain original articles, Referale, an account of new methods, progress of questions relating to immunity, and a catalogue of new literature. The most complete account of the work of the year is found in the Jahresb. ii. d. Fortschr. . . . d. path. Milwoorganismen, conducted by Baumgarten, and published in Braunschweig. This publication commenced in 1887. Its disadvantage is that the volume for any year does not usually appear till two years later. Bacteriology is also dealt with in the Index Medicus. For valuable lists of papers by particular authors see Royal Society Catalogue of Scientific Papers. The chief bacteriological periodicals are the Jotirn. Path, and Bacteria/., edited by Sims Woodhead, Edinburgh and London ; the Ztschr. f. Hyg. v. Lnfectionskrankh., edited by Koch and Fliigge, Leipzig ; and the Ann. de PInst. Pasteur, edited by Duclaux, Paris. Valuable papers also from time to time appear in the Lancet, Brit. Med. Journ., Deutsche med. Wclmschr., Berl. klin. Wchnschr., Seinaine mid., Arch. f Hyg., Arch. f. exper. Path. u. Phar?nakol. Besides these periodicals the student may have to consult the Supple- mental volumes of the Reports of the Local Government Board which contain the reports of the medical officers, also the Proc. Poy. Soc. London, the Compt. rend. Acad. d. sc. , Paris, the Compt. rend. Soc. de biol., Paris, and the Arb. a. d. k. Gsndhlsamle (the first two volumes of the last were denominated Mittheihingen). CHAPTER I. — General Morphology and Biology. Consult here especially Fliigge, "Die Mikroorganismen." De Bary, "Bacteria," translated by Garnsey and Bayley Balfour, Oxford, 1887. Zopf, "Zur Morphologie der Spaltpflanzen," Leipzig, 1882; " Beitr. z. Physiologie und Morphologie niederer Organismen,"' 5th ed., Leipzig, 1895. Cohn, Beitr. z. Biol. d. Pflanz., Bresl., (1876) ii. v. Niigeli, " Die niederen Pilze," Munich, 1877: " Unter- suchungen liber niedere Pilze," Munich, 1882. Migula, "System der Bakterien," Jena, 1897. Duclaux, " Traite de microbiologie,'' Paris, 1898-99. For general morphological relations see Ray Lankester, art. "Bacteria," Encyc. Brit., 9th ed. Engler and Prantl, "Die nalitrlichen Pflanzenfamilien," Lieferung 129. — " Schizophyta" (by BIBLIOGRAPHY. 537 W Miaula). Structure of Bacterial Cell.— Butschli, " Uber den Ban der Bakterien," Leipzig, 1890; « Weitere Ausfuhrungen Uber den Bau der Cyanophyceen und Bakterien," Le.pz g, 1896. ith ■ V- dt. in text' Buchner, Longard and Riedlin, Lcntralbl ffSlertol u. Parasitenk., ii. 1. Ernst, Ztschr /Hyg, - 4 * , Babes ibid v. 173. Neisser, ibid., iv. 165. Motili iy.— Klein, BulSl. FiUer/cohn, loc. cit. L5ffler, Centralbl f Ba^L^ ParasitmL, vi. 209 ; vii. 625. Pigments.- Zopf, loc at Galeott , Ref. in CentraM.f. Bakteriol. u. Parasztenk., ™??6-J™%?' Ztschr f. Hyg, xx. 3. SPORULATION.-Prazmowski Bwl Central., ^ 30 . \ Koch, Botan. Ztg, (1888) Nos 18-22 Buchner SitJigsb. d. math.-phys. CI. d. k. bayer. Akad. d Wrssensch. zu Munchen, 7th Feb. 1880. R. Koch, Mitth. a. d. k Gsndhtsamte i 65. Chemical Structure of Bacteria. — Nencki, Ber. d. deulsch. chem. Gesellsch., (1884) xvii. 2605. Cjamer Arc h.J Hyg, xvi 154. Buchner, Bert. klin. Wchnsckr., (1890) 673. 1084 ; FlUece, op. cit. Classification of Bacteria. -For general review see Marshall Ward, Ann. of Botany, vi. 103 ; Migula ,loc. at. supra. Food of Bacteria. Nageli, Cohn, oP at. P^teur, " Etudes sur la biere," 1876. Hueppe, Mitth. a. d. k. Gsndhtsamte ii log Relations to Oxygen.— Pasteur, Compt. rend. Acaa. a. s'c HL 344, 1142 ; Kitasato and Weyl, Ztschr. f. Hyg., vui. 41, 404; ix 97. Temperature.— Vide Fliigge, op. cit.; for thermophilic bacteria, Rabinowitsch, Ztschr. f. Hyg., xx. 154; Macfadyen and Blaxall, /WA. and Bacterial, iii. 87. Action of Bacterial Ferments. -Salkowski, Ztschr. f. Biol., N.F., vn. 92 > Pafur and Toubert, GwyV. rwirf. ^«** ^ lxxxllL 5 5 Sheridan L,ea, Journ. Physiol, vi. 136; Beijerinck, Centralbl f. Baktenol u. Parasitenk., Abth. II. i. 221 ; E. Fischer, Ber. d. deutsch. chem. Gesellsch., xxviii. 143° 5 Liborius, Ztschr. f. Hyg, 1. 1 15 J see also Pasteur, " Royal Society Catalogue of Scientific Papers. varia- bility.—Cohn, Nageli, Fliigge, op. cit. Winogradski, " Beitr. z. Morph. u. Physiol, d. Bakt.," Leipzig, 1888; Ray Lankester, Quart Journ. Micr. Sc., N.S., (1873) xiii. 408; (1876) xvi. 27, 278. Nitrifying Organisms.— Winogradski, Ann. de Vlnst. Pasteur,™. 213, 257, 760; v. 92, 577- Maze> xi. 44; xu. 1, 263. Death of Bacteria.— R. Koch, Mitth. a. d. k. Gsndhtsamte., 1. 234; Behring, Ztschr. f. Hyg., ix. 395. CHAPTER II. — Methods of Cultivation of Bacteria. For General Principles.— Pasteur, Compt. rend. Acad. d. sc., 1. 303; li. 348, 675; Ann. de chem., Ixiii. 5; Tyndall, "Floating Matter of the Air in relation to putrefaction and infection," London, 1881 ; H. C. Bastian, "The Beginnings of Life," London, 1872. 538 BIBLIOG RAPHY. METHODS of Sterilisation.— R. Koch, Gaffky and Loftier, Mitth. a. d. k. Gsndhtsamte'., i. 322; Koch and Wolffhiigel, ibid., i. 301. Culture Media. — See text-books, especially Kanthack and Drys- dale ; Pasteur, " Etudes sur la biere," Paris, 1876; R. Koch, Mitth. a. d. k. Gsndhtsamte., i. 1 ; Roux et Nocard, Ann. de Plnst. Pasteur, i. 1; Roux, ibid., ii. 28; Marmorek, ibid., ix. 593; Kitasato and Weyl, op. cit. supra; P. and Mrs. Percy Frankland, "Micro- organisms in water," London, 1894. Fuller, Rep. Amer. rub. Health Ass., xx. 381. Theobald Smith, Centralbl. f. Bakteriol. u. Parasi- tenk.,m\. 502 ; xiv. 864. Durham, Brit. Med. Journ. 1898, i. 1387. "Report of American Committee on Bacteriological Methods," Concord, 1898. CHAPTER III. — Microscopic Methods, etc. Consult text-books, especially Klein, Kanthack and Drysdale, Hueppe, Gunther, Heim, Thoinot et Masselin ; also Bolles Lee, " The Microtomist's Vademecum," 4th ed., London, 1896 (this is the most complete treatise on the subject). Rawitz, op. cit. in text ; Koch, Mitth. a. d. k. Gsndhtsamte., i. 1 ; Ehrlich, Ztschr.f. klin. Med., i. 553 ; ii. 710. Gram, Fortschr. d. Med., (1884) ii. No. 6; Nicholle, Ann. de Plnst. Pasteur, ix. 666 ; Kiihne, " Praktische Anleitung zum mikroscopischen Nachweis der Bakterien im tierischen Gewebe," Leipzig, 1888 ; van Ermengem, ref. Centralbl. f. Bakteriol. v. Parasitenk., xv. 969; Richard Muir, Journ. Path, and Bacleriol, v. 374- Agglutination. — Delepine, Brit. Med. Joum., (1897) ii 529, 967. Widal and Sicard, Ann. de Plnst. Pasteur, xi. 353. Wright, Brit. Med. Journ., (1897) i. 139; (1898) i. 355. CHAPTER IV. — Non-Pathogenic Organisms, etc. For non- pathogenic bacteria usually occurring in man consult Heim, op. cit. For fungi see De Bary, " Comparative Morphology and Biology of the Fungi, Mycetozoa and Bacteria," transl. by Garnsey and Balfour, Oxford, 1887; Sachs, "Text-book of Botany," transl. by Garnsey and Balfour, Oxford, 1887, ii. CHAPTER V. — Relations of Bacteria to Disease, etc. As the observations on which this chapter is based are scattered through the rest of the book, the references to them will be found under the different diseases. BIBLIOGRAPHY. 539 CHATTER VI.— Suppuration and Allied Diseases. Ogston, Brit. Med. Journ., (1881) L 369- or-anismen bei den Wundinfectionskrankheiten des Menschen, Wies- Kn, 1884. Passet, Fortschr. d. fed., {iSS5) ^ » W. Watson Cheyne, << Suppuration and Septic Diseases ^dinbuigh, 1889. Grawitz, VirMs Archiv, cxvi. 116 ; Deutsche med Wchnschr., (1889) No. 23. Steinhaus, Ztschr. f. Hyg., v. 518 (micro- coccus tetragenus; "Die Aetiologie der acuten Eiterung," Leipzig, 1889. Christmas-Dirckinck-Holmfeld>«'Recherchesexpenmentalessm la suppuration," Paris, 1888. Gairf, Fortschr. d. Med., (1885) No. 6 Marmorek, Ann. de Vinst. Pasteur, ix. 593- Petnischky, ,ZtseAi . f. Hy*, xvii. 59; xviii. 413; xxiii- T42! (with Koch, xxm. 477)- Liibbert, "Biologische Spaltpilzuntersuchung," Wurzburg 1886. Krause, Fortschr. d. Med., (1884) Nos. 7 and 8. Ribbert, fortschr d Med., (1886) No. 1. Widal and Besancon, Ann. de I Inst. Pasteur, ix. 1 04. v. Lingelsheim, Ztschr. f. Hyg, x. 331 ; xn. 30b. Behring, Centmlbl. f. Bakteriol. u. Parasitenk., xn. 192. Thoinot et Masselin, Rev. de mid., (1894) 449- Orth and Wyssokowitsch, CentraM. f. d. med. Wissensch., (1885) 577- Netter, Arch, de physiol. norm, et path., (1886) 106. Weichselbaum, Wien. med Wchnschr., (1885) No. 41 5 (1888) Nos. 28-32; Centralbl. f. Bakteriol. u. Parasitenk., ii. 209 ; Beitr. z. path. Anat. u. z. allg.Pat/i., iv 127. Becker, Deutsche med. Wchnschr., (1883) No. 46. Lanne- longue et Achard, Ann. de Vinst. Pasteur, v. 209. Fehleisen, " Die Aetiologie des Erysipels," Berlin, 1883. Lemoine, Ann. de I Inst. Pasteur, ix. 877. Kurth, Arb. a. d. k. Gsndhtsamte., vii. 3»9- Knorr, Ztschr. f. Hyg, xiii. 427- Bullock, Lancet, (1896) 1. 982, 1216. Bordet, Ann. de Vinst. Pasteur, xi. 177. Booker (strepto- coccus enteritis), Johns Hopkins Hasp. Rep., vi. 159- Hirscj1' Centralbl. f. Bakteriol. u. Parasitenk., xxii. 369. Libman, ibid. , xxii. 376. Weichselbaum (meningitis), Fortschr. d. Med., (1887) v. 573, 620. Jaeger, Ztschr. f. Hyg, xix. 351. CHAPTER VII. -Gonorrhoea, Soft Sore, Syphilis. Gonorrhoea.— Neisser, Centmlbl. f. d. med. Wissensch., (1879) 497; Deutsche med. Wchnschr., (1882) 279; (1894) 335- Bumm, " Der Mikroorganismus der gonorrhoischen Schleimhauterkrankungen, Wiesbaden, 1885, 2nd ed. 1887; Munchen. med. Wchnschr., (1886) No. 27 ; (1 89 1) Nos. 50 and 51 ; Centralbl. f. Gyndk., (1891) No. 22; Wien. med. Presse, (1891) No. 24. Bockhart, Monatsh. f. prakt. Dermat., (1886) v. No. 4 ; (1887) vi. No. 19. Steinschneider, Berl. klin. Wchnschr., (1890) No. 24; (1893). No. 29; Verhandl. d. deutsch. dermat. Gesellsch. L. Congress, Wien, 1889, 159. Wertheim, Wien. klin. Wchnschr., (1890) 25; Deutsche med. 54° BIBLIOGRAPHY. Wchnschr., (i 891) No. 50; Arch. f. Gynaek., xli. Heft 1; Cenlralbl. f. Gyndk., (1891) No. 24; (1892) No. 20; Wicn. klin. Wchnschr., (1894)441. Gerhardt, Charili'-Ann., (1889) 241. Leyden, Ztschr. f. klin. Med., xxi. 607; Deutsche me,/. Wchnschr., (1893) 9°9- Bordoni-Uflreduzzi, ibid., (1894) 484. Councilman, Am. Journ. Med. Sc., cvi. 277. Finger, Ghon, and Schlagenhaufer, Arch. f. Dermal, u. Syph., xxviii. 3, 276. Lang, ibid., (1892) 1007; Wien. vied. Wchnschr., ( 1 891) No. 7; "Der Venerisclie Katarrh, dessen Pathologie und Therapie," Wiesbaden, 1893. Klein, Monatschr. f. Geburlsh. u. Cynaek., (1895) 33- Michaelis, Ztschr. f. klin. Med., xxix. 556. Heiman, New York Med. Rec, (1895) 769, (1896) Dec. 19. Foulerton, Trans. Brit. Inst. Prcvcn. Med., i. 40. De Christmas, Ann. del Inst. Pasteur, xi. 609. Nicolaysen, Cenlralbl. f. Bakteriol. u. Parasilenk., xxii. 305. Rendu, Perl. klin. Wchnschr., (1898) 431. Wasser- mann, Ztschr. f. Hyg., xxvii. 298. Soft Sore. — Ducrey, Monatsh. f. prakt. Dermal., ix. 221. Krefting, Arch. f. Dermat. u. Syph., (1892) 263. Jullien, Journ. d. mal. cutan. et syph., (1892) 330. Unna, Monatsh. f. prakt. Dermat., (1892) 475 ; (1895) 6l- Quinquand, Semaine mid., (1892) 278. Petersen, Cenlralbl. f. Bakteriol. u. Parasilenk., xiii. 743 ; Arch. f. Dermat. u. Syph., (1894) 419. Audrey, Monatsh. f. prakt. Dermat., (1895) 267. Syphilis. — Lustgarten, Wien. med. Wchnschr., (1884) No. 47. Doutrelepont and Schiitz, Deutsche med. Wchnschr., (1885) No. 19. Gottstein, Fortschr. d. Med., (1885) No. 16. De Michele and Radice, Gior. internaz. disc, med., (1892) 535. Sabouraud, Ann. de I' Inst. Pasteur, vi. 184. Golasz, Journ. d. mal. cutan. et syph., (1894) 170. Markuse, Vrtljschr. f. Dermat. u. Syph., (1883) No. 3. v. Niessen, Cenlralbl. f. Bakteriol. u. Parasilenk., xxiii. 49. CHAPTER VIII.— Acute Pneumonia. Friedlander, Fortschr. d. Med., i. No. 22 ; ii. 287 ; Virchoiii's Archiv, Ixxxvii. 319. A. Fraenkel, Ztschr. f. klin. Med., (1886) 401. Salvioli and Zaslein, Cenlralbl. f. d. med. Wissensch., (1883) 721. Ziehl, ibid., (1883) 433; (1884) 97. Klein, ibid., (1S84) 529. Jiirgensen, Berl. klin. Wchnschr., (1884) 270. Seibert, ibid., (1884) 272, 292. Senger, Arch. f. exper. Path. 11. Pharmakol., (1886) 389. Weichselbaum, Wien. med. Wchnschr., xxxvi. 1301, J339> 1367 ; Monatschr. f. Ohrenh., (1888) Nos. 8 and 9 ; Cenlralbl. f. Bakteriol. u. Parasilenk., v. 33. Gamaleia, Ann. de VInst. Pasteur, ii. 440. Guarnieri, Atti d. r. Accad. med. di Roma, (1888), ser. ii. iv. Kruse and Pansini, Ztschr. f. Hyg., xi. 279. E. Fraenkel and Reiche, Ztschr. f. klin. Med., xxv. 230. Sanarelli, Cenlralbl. f. Bakteriol. u. Parasitenk., x. 817. Lannelongue, Gaz. d. hSp., (1 89 1) 379. Netter, Bull, et nihil. Soc. mid. d. hop. de BIBLIOGRAPHY. 54 » n ? ° / 9""f,S86i No % Emmerich, ilfltoAm. *a* ^A*^-. Sno ( f ^aeff,3^^ " P Prist. Pasteur, vii. 260. Grim- ffi L«?*3^ SSU xi. 840. Washbourn Brit. M'd.Journ, (1897) L 510 ; (1897) ii- 1849. Eyre and Washbourn, /«m. /WA. and Bacteriol., iv. 394 ; v. 13- CHAPTER IX.— Tuberculosis. Klencke, " Untersuchungen und Erfuhrungen im Gebiet der Anatomie, etc." Leipzig, i843- Villemin, «De la virulence et de la fpecificite de la tuberculosa" Paris, 1868. Cohnheim and Fraenke , « Experimentelle Untersuchungen iiber der Ubertragbarkeit der Tuber- culose auf Thiere." Cohnheim, "Die Tuberculose vom Standpunkt derlnfectionslehre," 1879. Various Authors, » Discussion sur la tuber- culose," Bull. Acad, de mid., (1867) xxxii., xxxm. Armanni, Novi- mento med.-chir." Naples, 1872. Baumgarten « Lehrb. d. path. Mvk " 1890. Straus, " La tuberculose et son bacille, Pans, 1895. Koch! Berl. klin. Wchnschr., (1882) 221 ; Mitth. a. d. k. Gsndhts- amtc 1884; Deutsche med. Wchnschr., (1890) No. 46a; 1891) Nos.'3 and 43 ; (1897) No. 14. Nocard, « The Animal Tuberculoses (trans ), London, 1895. Cornet, Ztschr. /. Hyg, y. 191. Nocard and Roux, Ann. de Vbist. Pasteur, i. 19. Pawlowsky, ibid., u. 3°3- Sander, Arch. /. Hyg, xvi. 238. Coppen Jones, Centrum./. Bafc- teriol. u. Parasitenk., xvii. 1. Prudden and Hodenpyl, New York Med .far ,(1891)636. Vissman, V ire fund's Archiv, exxix. 163. Straus and Gamaleia, Arch, de mid. e'xpir. et d'anat. path. , iii. No. 4- Cour- mont, Semaine mid., (1893) 53 5 ^evue de mid., (1891) No 10. Hericourt and Richet, Bull, mid., (1892) 741, 9&6. Williams, Lancet, (1883) i. 312. Pawlowksy, Ann. deVLnst. Pasteur, vi. 116. Maffuc'ci, "Sull azione tossica dei prodotti del bacillo della tuber- culosi " ; Centralblf. allg. Path. u. path. Anat., i. 404- Kruse, Beitr z.palh. Anat. it. z. allg. Path., xii. 221. Bollinger, Miinchcn. med. Wchnschr., (1889) No. 37 ; Verhandl. d. Gesellsch. deutsch. Naturf. u. Aertze, (1890) ii. 187. Hofmann, Wien. med. Wchnschr., (1894) No. 38 Straus and Wiirtz, Cong. p. Vitude de la tuberculose, Paris, July 1888. Gilbert and Roger, Mini. Soc. de bioL, (1891). Diem, Monatsh. f. prakt. Thierh., iii. 481. Weyl, Deutsche med. Wchnschr., (1891) 256. Buchner, CentralU. f. Bakteriol. u. Parasitenk., xi. 488. Cour- mont and Dor, Province mid., (1890) No. 50. Tizzoni and Centanni, Cenlralbl. f. Bakteriol. u. Parasitenk., xi. 82. Ribbert, Deutsche med. Wchnschr., (1892) 353- Virchow, ibid., (1891) 131. Hunter, Brit. Med.Journ., (1891), July 25. Kiihne, Ztschr. f. Biol., xxix. 1 ; xxx. 221. Krehl, Arch. /, exper. Path. u. Pharmakol, xxxv. 222. Krehl 542 BIBLIOGRAPHY, and Matthes, ibid xxxvi. 437. Bang, « La lutte contre la tuberculose en Danemark, Geneva, 1895. Maragliano, << Le serum antituber- culeux et son ant.tox.ne," Paris, 1896; Berl. klin. Wchnschr., (1896) 409, 437, 773- Nocard, Ann. de I'Inst. Pasteur, xii. 561. Stock- man, Brit Med. Journ., (,898) ii. 601. Maragliano, re/. Brit. Med fount., Epitome, (1896) i. 63. Baumgarten and Walz, Centra/6/, f. Bakteriol. u. Parasitenk., xxiii. 587. CHAPTER X.— Leprosy. Hansen, Norsk Mag. f. Lagevidensk., 1874; Virchow's Ar- chiv, lxxix. 32; xc. 542; ciii. 388; Virchow's Feslschr., (1892) iii See papers by Neisser and Cornil and Suchard in « Microparasites in Disease ' ' (New Sydenham Soc. ,1886). Hansen and Looft, ' ' Leprosy, " Bristol, 1895. Doutrelepont and Wolters, Arch. f. Dermal, u. Syph., (1892) 55. Thoma, Sitzungsb. d. Dorpater Natur/orsch. , 1889.' Unna, Dermat. Stud., Hamburg, (1887) iv. Bordoni - Uffreduzzi, Ztschr. f. Hyg., iii. 178; Bert. klin. Wchnschr., (1885) No. 11. Aming and Nonne, Virchozu's Archiv, exxxiv. 319. Gairdnei, Brit Med. Journ., (1887) U 1269. Hutchinson, Arch. Surg, (1889) i. V. Torok, " Summary of Discussion on Leprosy at the 1st Internat. Congr. for Dermatol, and Syph.," v. Monatsh. f. prakt. Dermat., ix. 238. Profeta, Gior. internaz. d. sc. med., 1889. See Journal of the Leprosy Investigation Committee, 1890-91. Philippson, Vij-chow's Archiv, exxxii. 529. Danielssen, Monatsh. f. prakt. Dermat., (1891) 85, 142. Wesener, Centralb/. f. Bakteriol. u. Parasitenk., ii. 450; Miinchen. vied. Wchnschr., (1887) No. 18. CHAPTER XL — Glanders— Rhinoscleroma. Loffler and Schultz, Deutsche med. Wchnschr., (1882) No. 52. Lbffler, Mitlh. a. d. k. Gsndhtsamte., i. 134. Weichselbaum, Wien. med. Wchnschr., (1885) Nos. 21-24. Preusse, Berl thierarztt. Wchnschr., (1889) Nos. 3, 5, n ; ibid., (i894)Nos. 39, 51. Gama- leia, Ann. de I'Inst. Pasteur, iv. 103. A. Babes, Arch, de med. cxpir. et if anal, path., (1892) 450. Straus, Compt. rend. Acad. d. sc., cviii., 530. M'Fadyean and Woodhead, Rep. National Vet. Assoc., 1888. Baumgarten, Centralb/. f. Bakteriol. u. Parasitenk., iii. 397. Silviera, Semaine med., (1891) No. 31. Bonome, Deutsche med. Wchnschr., (1894) 703, 725, 744. Raining, Arch. f. Veteriniiiwisscnsch., (St. Petersburg) i. Apr. May. Foth, Centralb/. f. Bakteriol. u. Para- sitenk., xvi. 508, 550. M'Fadyean, Journ. Comp. Path, and Therap., 1892, 1893, 1894. Leclaiche and Montane, Ann. de I 'Inst. Pasteur, vii. 481. Leo, Ztschr. f. Hyg., vii. 505. Rhinoscleroma.— Frisch, Wien. med. Wchnschr., (1882) No. 32. Cornil and Alvarez, Arch, de physiol. norm, et path., 1895, 3rd series, BIBLIOGRAPHY. 543 vL u. Paltaufand Eiselsberg, Fortschr d Med., Wolkowitsch, Centralbl. f. d. med. Wissensch., 1886. Dit- 20. trich, . Babes, ?- AnUri., ii. 617. Pawbwsk!, ix 742, etiologie et la pathologie du rhinosclerome," Berlin, 1891. ^ltaut, Wien. Ld. Wchnschr., (189O No, 5*, 53; (1892) No, I, 2. Wilde, Semaine mid., (1896) 336. CHAPTER XII.— Actinomycosis. Bollinger, Centralbl. f. d. med. Wissensch., i&7 7- J. Israel vgLhAnto, ixxiv. 15; l-viii 421. ^g|^ ?sTae ' ZtoAr., 1879; "Die Aktinomykose desMenschen 1882. O. Isiael, Virchow's Archiv, xcvi. i75- Chiari, Prag^ted. Wchnschr., 1884. Langhans, Cbr.-2W. / «M Aerzte, xvm. (1888). Luning and Hanau, ibid., xix. (1889). Shattock, Trans. Path. Sac London, I88S. Adand/iW., 1886. Delepine, ^.,1889. Harley ,Med.- Chir. Trans. London, 1886. Crookshank, ibzd 1889 1; "Manual of Bacteriology," London, 1896. Ransome Med-Chtr. Tran? London, 1891. M'Fadyean, Journ. Comfi. Path and Theraf ..1889. Bostrom, AO* «. Anat. u. z. allg. Path., 1890. Wolff and Israel, Virchow's Archiv, cxxvi. II. Mich, " Beitrage zur Klinik der Aktinomykose," Wien, 1 892. Grainger Stewart and Muir, Edin. Hasp. Ret., 1893. Leith, ibid., 1894- Gasperini, Centralbl.f. Bakterwl. u. Parasitenk., xv. 684. Hummel, Beitr. z. klin. Chir., xm. No. 3. Pawlowsky and Maksutoff, Ann. de VInst. Pasteur, vu. 544- Madura Disease.— Carter, " On Mycetoma or the Fungus Disease of India," London. Bassini, Ref. in Centralbl.f. Bakterwl. u. Para- sitenk iv 652. Lewis and Cunningham, nth Ann. Rep. San. Com. India. Kobner, Fortschr. d. Med., (1886) No. 17. Kanthack, Toum. Path.- and Bacterial, i. 140. Boyce and Surveyor, Proc. Roy. Soc. London, 1893. Vandyke Carter, Trans. Path. Soc. London, 1886. Vincent, Ann. de VInst. Pasteur, viii. 129. CHAPTER XIII.— Anthrax. Bollinger in Ziemssen's " Cyclopaedia of Medicine." Greenfield, "Malignant Pustule" in Quain's "Dictionary of Medicine," London, 1 894 Pollender, Vrtljschr. f. gerichtl. Med. , viii. ; Davaine, Compt. rend Acad. d. sc., lvii. 220, 351, 386; lix. 393- Koch, Cohn's Beitr. z. Biol. d. PJlanz., ii. Heft 2 (1876). Mitth. a. d. k. Gsndhtsamte., 1. 49. Pasteur, Compt. rend. Acad. d. sc., xci. 86, 455, 531, 697 ; xcii. 209. Buchner, Virchow's Archiv, xci. Chamberland, Ann. de I' Inst. Pasteur, viii. 161. Chauveau, Compt. rend. Acad. d. sc., xci. 33, 648, 880; xcvi. 553. Czaplewski, Beitr. z. path. Anal. u. s. allg. Path., vii. 47. Gamalcia, Ann. del' Inst. Pasteur, ii. 5 17. Marshall Ward, Proc. Roy. Soc. 544 BIBLIOGRAPHY. London, Feb. 1893. Petruschky, Beitr. 2. path. Anal. u. z. allg. Path., iii. 357. Weyl, ZtscHr. f. Hyg, xi. 381. Behring, ibid., vi. 117 ; vii.' 171. Osborne, Arch. f. Hyg.,\\. 51. Roux, Ann. de I' Inst. Pasteur, iv. 25. Ilankin, Brit. Med. Joum., (1889) ii. 810; (1890) ii. 65. Hankin and Wesbrook, Ann. de V Lnst. Pasteur, vi. 633. Sidney Martin, Supplem. Loc. Govt. Board Rep., (1890-91) 255. Marmier, Ann. de VInst. Pasteur, ix. 533. Rd. Muir, Joum. Path, and Bacteriol., v. 374. CHAPTER XIV.— Typhoid Fever, etc. Eberth, Virchow's Archiv, lxxxi. 58 ; lxxxiii. 486. Koch, Mitlh. a. d. k. Gsndhtsamte., i. 46. Gaffky, ibid., ii. 80. Klebs, Arch. f. exper. Path. u. Pharmakol., xii. 231 ; xiii. 381. Escherich, Fortschr. d. Med., (1885) Nos. 16, 17. 'Emmerich, Arch. f. Hyg., iii. 291. Rodet and Roux, Arch, de me'd. expe"r. et d'auat. path., iv. 317. Weisser, Ztschr. f. Hyg., i. 315. Klein, "Micro-organisms and Dis- ease," London, 1896; Supplem. Loc. Govt. Board Rep., (1892-93) 345 ; (1893-94) 457 ; (1894-95) 399, 407, 4"- Babes, Ztschr. f. Hyg., ix. 323. Vincent, Coiupt. rend. Soc. de biol., ser. ix. ii. 62. Birch- Hirschfeld, Arch. f. Hyg., vii. 341. Buchner, Centralbl. f. Bak- teriol. u. Parasitenk., iv. 353. Pfuhl, ibid., iv. 769. Petruschky, ibid. , vi. 660. Kitasato, Ztschr. f. Hyg., vii. 515. Chantemesse and Widal, Bull, me'd., (1891) No. 82; Ann. de VInst. Pasteur, vi- 755 ; v'ii- 14 1- Pere, Ann. de VInst. Pasteur, vi. 512. Neisser, Ztschr. f. klin. Aled., xxiii. 93. Nicholle, Ann. de VInst. Pasteur, viii. 853. Quincke and Stiihlen, Berl. klin. Wchnschr., (1894) 351. A. Fraenkel, Centralbl. f. klin. Med., (1886) No. 10. E. Fraenkel and Simmonds, ibid., (1886) No. 39. Achalme, Semaine med., (1890) No. 27. Grawitz, Chariti - Ann. , xvii. 228. Beumer and Peiper, Centralbl. f. klin. Med., (1887) No. 4 ; Ztschr. f. Hyg., i. 489 ; ii. no, 382. Sirotinin, ibid., i. 465. R. Pfeiffer and Kolle, Ztschr. f. Hyg., xxi. 203. R. Pfeiffer, Deutsche med. Wchnschr., (1894) 898. Sanarelli, Ann. de VInst. Pasteur, vi. 721 ; viii. 193, 353. Brieger and Fraenkel, Berl. klin. Wchnschr., (1890) 241, 268. Brieger, Kitasato, and Wassermann, Ztschr. f. Hyg., xii. 137. Widal, Semaine me'd., (1896) 295, 303. Achard, ibid., 295, 303. Griinbaum, Lancet, Sept. 1896. Delepine, Brit. Med. Joum., (1897) i- 529> 967; Lancet, Dec. 1896. Remlinger and Schneider, Ann. de VInst. Pasteur, xi. 55, 829. Widal and Sicard, ibid., xi. 353. reckham, Joum. Exper. Med., ii. 549. Richardson, ibid., iii. 329. Wright and Semple, Brit. Med. Joum., (1897) i. 256. Sidney Martin, ibid., (1898), i. 1569, 1644 ; ii. 1 1, 73. Bokenham, Trans. Path. Soc. London, (1898) xlix. 373. Christophers, Brit. Med. Joum., (1898) i. 71. Wyatt Johnson, ibid., (1897) i. 231 ; Lancet, (1S97) ii. 1746. Durham, Lancet, (1898) i. 154; ibid., ii. 446. Lorrain Smith and BIBLIOGRAPHY. 545 Tennant, Brit. Med. Journ., (1899) i. 193- Gordon, Jotirn. Path, and Bacteriol., iv. 438. (Bacillus Enteritidis Gaertner) refs. vide Baum- garten's Jahresbericht, iv. 249 ; vii. 297 ; xii. 508. (Psittacosis), ibid., xii. 496. CHAPTER XV.— Diphtheria. Klebs, Verhandl. d. Cong. f. innere Med., (1883) ii. Lbffler, Mitth. a. d. k. Gsndhlsamtc., (1884) 421. Roux and Yersin, Ann. de Plnst. Pasteur, ii. 629 ; iii. 273 ; iv. 385. Brieger and Fraenkelt Be/-/, klin. Wchnschr., (1890) 241, 268. Spronck, Centralbl. f. allg. Path. u. path. Anat., i. No. 25 ; iii. No. 1. Welch and Abbott, Johns Hopkins Hosp. Bull., 1891. Behring and Wernicke, Ztschr. f. Hyg., xii. 10. Loffler, Centralbl./. Bakteriol. u. Parasitenk., ii. 105. v. Hofmann, Wien. vied. Wchnschr.., (1888) Nos. 3 and 4. Cobbet, and Phillips, Journ. Path, and Bacteriol, iv. 193. Peters, ibid., iv. 181. Wright, Boston Med. and S. Journ., (1894) 329, 357. Kanth- ack and Stephens, Journ. Path, and Bacteriol., iv. 45. Klein, Brit. Med. Journ., (1894) ii. 1393; (1895) IO°- Supplem. Loc. Govt. Board Rep., (1890-1) 219; (1891-2) 125. Guinochet, Compt. rend. Soc. de biol., (1892) 480. Roux and Martin, Ann. de Plnst. Pasteur. viii. 609. Cartwright Wood, Lancet, (1896) i. 980, 1076; ii. 1145, Sidney Martin, " Goulstonian Lectures," Brit. Med. Journ., (1.892) i. 641, 696, 755 ; Supplem. Loc. Govt. Board Rep., (1891-2) 147 ; (1892-3) 427. Escherich, Wien. med. Wchnschr., (1893) Nos. 47- 50; Wien. klin. Wchnschr., (1893) Nos. 7-10; (1894) No. 22; Berl. klin. Wchnschr., (1893) Nos. 21, 22, 23. Behring, "Die Geschichte der Diphtherie," Leipzig, 1893 ; " Abhandlungen z. atiol. Therap. v. anst. Krankh.," Leipzig, 1893; " Bekampfung der Infections-krankheiien," Leipzig, 1894. Ehrlich and Wassermann, Ztschr. f. Hyg., xviii. 239. Ehrlich and Kossel, ibid., xvii. 486. Ehrlich, Kossel, and Wassermann, Deutsche med. Wchnschr., (1894) 353. Funck, Ztschr. f. Hyg., xvii. 401. Prochaska, Ztschr. f. Hyg., xxiv. 373. Madsen, ibid., xxiv. 425. Neisser, ibid., xxiv. 443. L. Martin, Ann. de Plnst. Pasteur, xii. 26. Salomonsen and Madsen, ibid., xii. 763. Woodhead, Brit. Med. Journ., (1898) ii. 893. CHAPTER XVI.— Tetanus. Nicolaier, " Eeitr'age zur Aetiologie des Wundstarrkrampfes," Inaug. Diss. Gbttingen, 1885. Rosenbach, Arch. f. klin. Chir., xxxiv. 306. Carle and Rattone, Gior. d. r. Accad. di med. di Torino, 1884. Kitasato, Ztschr. f. Hyg., vii. 225; x. 267; xii. 256. Kitasato and Weyl, ibid., viii. 41, 404. Vaillard, Ann. de Plnst. Pasteur, vi. 224, 676. Vaillard and Rouget, ibid., vi. 385. Behring, "Ab- handlungen z. atiol. Therap. v. anst. Krankh.," Leipzig, 1893 ; Ztschr. f. Hyg., xii. 1, 45; " Blutserumtherapie," Leipzig, 1892; "Das 35 546 BIBLIOGRAPHY. Tetanusheilserum," Leipzig, 1892. Brieger and Fraenkel, Berl. Idin. Wchnschr., (1890) 241, 268. Sidney Martin, Supplem. Loc. Govt. Board Rep., (1893-94) 497 ; (1894-95) 505. Uschinsky, Cenlralbl.f. Baktcriol. u. Parasitenk., xiv. 316. Tizzoni and Cattani, Arch. f. exper. Path. u. Pharmakol., xxvii. 432 ; Cenlralbl. f. Balderiol. u. Parasitenk., ix. 189, 685; x. 33, 576 (Ref.); xi. 325; Berl. klin. Wchnschr., (1894) 732. Malignant (Edema. — Pasteur, Bull. Acad, de vied., 1881, 1887. Koch, Mitth. a. d. k. Gsndhlsamle., i. 54. Kitt, Jahresb. d. It. Centr.- Thierarznei-Schule in Miinchen, 1883-84. W. R. Hesse, Deutsche med. Wchnschr., (1885) No. 14. Chauveau and Arloing, Arch, vet., (1884) 366, 817. Liborius, Ztschr. f. Hyg., i. 115. Roux and Chamberland, Ann. de I'Bist. Pasteur, i. 562. Charrin and Roger, Compt. rend. Soc. de biol., (1877) ser. VIII. iv. 408. Kerry and S. Fraenkel, Ztschr. f. Hyg., xii. 204. Sanfelice, ibid., xiv. 339. Quarter Evil. — See Nocard and Leclainche, " Les maladies microbiennes des animaux," Paris (1896). Arloing, Cornevin, et Thomas, " Le charbon symptomatique du bceuf," Paris (1887). Nocard and Roux, Ann. de Tlnst. Pasteur, i. 256. Roux, ibid., ii. 49. See also Journ. Co/up. Path, and Therap., iii. 253, 346; viii. 166, 233- CHAPTER XVII.— Cholera. Koch, Rep. of 1st Cholera Conference, 1884 (v. " Microparasites in Disease," New Sydenham Soc, 1886). Nikati and Rietsch, Compt. rend. Acad. d. sc., xcix. 928, 1 145. Bosk, Ann. de Tlnst. Pasteur, ix. 507. Pettenkofer, Miinchen. med. Wchnschr., (1892) No. 46; ( 1 894) No. 1 o. Sawtschenko, Cenlralbl. f. Baktcriol. u. Parasitenk. , xii. 893. Pfeiffer, Ztschr. f. Hyg., xi. 393. Kolle, ibid., xvi. 329. Isaeff and Kolle, ibid., xviii. 17. Wassermann, ibid., xiv. 35. Sobernheim, ibid., xiv. 485. Metchnikoff, Ann. de Tlnst. Pasteur, vii. 403, 562 ; viii. 257, 529. Fraenkel and Sobernheim, Hyg. Rundschau, iv. 97. Dunbar, Arb. a. d. k. Gsndhtsamtc., ix. 379. Pfeiffer and Wasser- mann, Ztschr. f. Hyg, xiv. 46. Wesbrook, Ann. de PInst. Pasteur, viii. 318. Scholl, Berl. klin. Wchnschr., (1890) No. 41. Gruber and Wiener, Arch. f. Hyg., xv. 241. Cunningham, Scient. mem. med. off. India, 1890 and 1S94. Plueppe, Deutsche med. Wchnschr., (1889) No. 33. Klemperer, ibid., (1894), 435 5 Berl. klin. Wchnschr., (1892) 969. Lazarus, ibid., (1892) 1071. Reincke, Deutsche mid. Wchnschr., (1894) 795- Koch, Ztschr. f. Hyg., xiv. 319. Voges, Cenlralbl. f. Bakteriol. u. Parasitenk., xv. 453. Pastana and Betten- court, Centralbl. f. Bakteriol. u. Parasitenk., xvi. 401. Dieudonne, ibid., xiv. 323. Celli and Santori, ibid., xv. 289. Neisser, ibid., xiv. 666. Sanarelli, Ann. de PInst. Pasteur, vii. 693. Ivanoff, Ztschr. f. Hyg., xv. 485. Issaeff, ibid., xvi. 286. Pfuhl, ibid., x. 510. Rumpel, Deutsche med. Wchnschr., (1S93) 160. Klein, Supplem. Loc. BIBLIOGRAPHY. 547 Govt. Board Rep., 1893; "Micro-organisms and Disease," London, 1896. Haffkine, Brit. Med. fount., (1895) ii. 1541- , pfeiffer in Fliig^e, "Die Micro-organismen," 3rd ed., 1896 ; Gamaleia, Ann. de Ilnst. Pasteur, ii. 482, 552. Achard and Bensande, Semaine mdd., (1897) 151- CHAPTER XVIIL— Influenza, etc. Influenza. — Pfeiffer, Kitasato, and Canon, Deutsche wed. Wchnschr., xviii. 28, and Brit. Med, Journ., (1892) i. 128. Babes, Deutsche med. Wchnschr., xviii. 113. Pfeiffer and Beck, ibid., (1892) 465. Pfuhl, Centralbl. f. Bakteriol. u. Parasitenk., xi. 397. Klein, Supplem. Loc. Govt. Board Rep., (1893) 85. Pfeiffer, Ztschr. f. Hyg., xiii. 357. Huber, Ztschr. f. Hyg., xv. 454. Kruse, Deutsche med. Wchnschr., (1894) 513. Pelicke, Berl. /din. Wchnschr., (1894) 524. Pfuhl and Walter, Deutsche med. Wchnschr., (1896) 82, 105. Cantani, Ztschr. f. Hyg., xxiii. 265. Pfuhl, Ztschr. f. Hyg., xxvi. 112. Plague.— Kitasato, Lancet, (1894) ii. 428. Yersin, Ann. de VInst. Pasteur, viii. 662. Lowson, Lancet, (1895) ii. 199. Yersin, Calmette, and Borrel, Ann. de VInst. Pasteur, ix. 589. Aoyama, Centralbl. f. Bakteriol. u. Parasitenk., xix. 481. Zettnow, Ztschr. f. Hyg., xxi. 164. Yersin, Ann. de VInst. Pasteur, xi. 81. Gordon, Lancet, (1899) i. 688. Haffkine, Brit. Med. Journ., (1897) i. 424. Wyssokowitz and Zabolotay, Ann. de VInst. Pasteur, xi. 663. Ogata, Centralbl. f. Bakteriol. u.. Parasitenk., xxi. 769. Childe, Brit. Med. Journ., '(1898) ii. 858. See also Brit. Med. Journ. and Lancet, 1897-99. Relapsing Fever. — Obermeier, Centralbl./. d. med. Wissensch., (1873) 145; and Berl. klin. Wchnschr., (1873) No. 35. Munch, Centralbl. f. d. med. Wissensch., 1876. Koch, Deutsche med. Wchnschr., (1879) 327. Moczutkowsky, Deutsches Arch. f. klin. Med., xxiv. 192. Vandyke Carter, Med.-Chir. Trans., London, (1880) 78. Lubinoff, Virchoiu's Archiv, xcviii. 160. Metchnikoff, ibid., cix. 176. Soudake- witch, Ann. de VInst. Pasteur, v. 545. Malta Fever. — Bruce, Practitioner, xxxix. 160; xl. 241 ; Ann. de VInst. Pasteur, vii. 291. Bruce, Hughes and Westcott, Brit. Med. Journ., (1887) ii. 58. Hughes, Ann. de VInst. Pasteur, vii. 628; Lancet (1892) ii. 1265. Wright and Semple, Brit. Med. Journ., (1897) i. 1214. Wright and Smith, ibid., (1897) i. 911 ; Lancet, (1897), i. 656. Welch, ibid., (1897) i. 15 12. Gordon, ibid., (1899) i. 688. Durham, Journ. Path, and Bacteriol., v. 377. Bruce in Davidson's "Hygiene and Diseases of Warm Climates," Edinburgh and London, 1893. Yellow Fever. — Sternberg, Rep. Am. Pub. Health Ass., xv. 170. Sanarelli, Ann. de VInst. Pasteur, xi. 433, 673, 753 ; xii. 348. Davidson, art. in Clifford Allbutt's "System of Medicine," vol. ii. London, 1897. Sternberg, Centralbl. f. Bakteriol. u. Parasitenk., 548 BIBLIOGRAPHY. xxii. 145; xxiii. 769. Sanarelli, ibid., xxii. 668. Archmard, Wood- son, and Archinard (ref.), ibid., xxv. 393. Mendoza (ref.), ibid., xxv. 390. CHAPTER XIX.— Immunity. For early inoculation methods {e.g. against anthrax, chicken cholera, etc.), see " Microparasites in Disease," New Syd. Soc. 1886. Duguid and Sanderson, Journ. Roy. Agric. Soc, (1880) 267. Greenfield, ibid., (1880) 273 ; Proc. Roy. Soc. London, June 1880. Toussaint, Compt. rend. Acad. d. sc., xci. 135. Haffkine, Brit. Med. journ., (1891) ii. 1278. Klein, ibid., (1893) 632> 639. Klemperer, Arch. f. exper. Path. u. Pharmakol., xxxi. 356. Buchner, Munchen. med. Wchnschr.) (1893) 449. Ehrlich, Deutsche med. Wchnschr., (1891) 976, 1218. R. Pfeiffer, Ztschr. f. Hyg., xviii. 1; xx. 198. Pfeiffer and Kolle, ibid., xxi. 203. Bordet, Ann. de VLnst. Pasteur, ix. 462 ; xi. 106. Metchnikoff, Virchow's Archiv, xcvi. 177 ; xcvii. 502 ; cvii. 209 ; cix. 176 ; Ann. de I' Inst. Pasteur, iii. 289 ; iv. 65 ; iv. 193; iv. 493; v. 465; vi. 289; vii. 402; vii. 562 ; viii. 257; viii. 529 ; ix. 433. Calmette, Ann. de I'Lnst. Pasteur, viii. 275 > xi. 95. Fraser, Proc. Roy. Soc. Edin., xx. 448. Marmorek, Ann. de YInst. Pasteur, ix. 593. Metchnikoff, Roux, and Taurelli-Salimbeni, ibid., x. 257. Charrin and Roger, Compt. rend. Soc. de bio/., (1887) 667. G ruber and Durham, Munchen. vied. Wchnschr., (1896), March. Durham, Journ. Path, and BacterioL, iv. 13. Cartjvright Wood, Lancet, (1896) i. 980; ii. 1 145. Sidney Martin, "Serum Treatment of Diphtheria," Lancet, (1896) ii. 1059. Ransome, "On Immunity to Disease," London, 1896. Burdon Sanderson, " Croonian Lectures," Brit. Med. Journ., (1891) ii. 983, 1033, 1083, 1 1 35. Discussion on Immunity, Path. Soc. London, Brit. Med. Journ., (1892) i. 373. Fodor, Deutsche vied. Wchnschr., (1887) No. 34. Hueppe, Bert, klin. Wchnschr., (1892) No. 17. Bordet, Ann. de V Inst. Pasteur, xii. 837. Nicholle, ibid., xii. 161. Salomonsen and Madsen, ibid., xi. 315 ; xii. 763. Roux and Borrell, ibid., xii. 225. Salimbeni, ibid., xi. 277. Wassermann and Takaki, Berl. klin. Wchnschr,, (1898) xxxv. 4. Blumenthal, Deutsche med. Wchnschr., xxiv. 185. Ransom, ibid., xxiv. 117. Meade Bolton, Journ. Exper. Med., i. 543. Fraser, T. R., Brit. Med. Journ., (1895) i. 1309; ii. 415, 416; (1896) i. 957; (1896) ii. 910; (1897) ii. 125, 595. Calmette, Ann. de Vlnst. Pasteur, vi. 160, 604; viii. 275 ; ix. 225 ; x. 675 ; xi. 214 ; xii. 343. C. J- Martin, Journ. Physiol., xx. 364 ; Proc. Roy. Soc. London, lxiv. 88." C. J. Martin and Cherry, ibid., lxiii. 420. Ehrlich, Deutsche med. Wchnschr., (1898) xxiv. 597. "Die Wert- bemessung des Diphtherieheilserums," Jena, 1897. Gautier, " Les Toxines microbiennes et animates, " Paris, 1896. Wassermann, Berl. klin. Wchnschr., (1898) 1209. Pfeiffer and Marx, Ztschr. f. Hyg., xxvii. 272. Bordet, Ann. de V Inst. Pasteur, xii. 688. BIBLIOGRAPHY. 549 Appendix A. — Smat.t.pox. Tenner, "An Inquiry into the Causes and Effects of the Variolse Vaccina;," London, 1798. Creighton, art. "Vaccination" in Encyc.^ Brit., 9th ed. Crookshank, "Bacteriology and Infective Diseases. McVail, "Vaccination Vindicated." Chauveau, Viennois et Mairet, " Vaccine et variole, nouvelle etude experimentale sur la question de l'identite de ces deux affections," Paris, 1865. Klein, Supplem. Loc. Govt. Board Rep., (1892-93) 391 5 (i893"94) 493- Copeman, Brit. Med. Journ., (1894) ii. 631 ; Journ. Path, and Bacteriol., 11. 407 ; art. in Clifford Allbutt's " System of Medicine," vol. ii. L. Pfeiffer, "Die Protozoen als Krankheitserreger," Jena, 1891. Ruffer, Brit. Med. Journ., (1894) June 30. Beclere, Chambon, and Menard, Ann. de PInst. Pasteur, x. 1 ; xii. 837. Copeman, "Vaccination," London, 1899. Appendix B. — Hydrophobia. Pasteur, Compt. rend. Acad. d. sc., xcii. 1259 ; xcv. 1187 ; xcviii. 457, 1229; ci. 765; cii. 459, 835; ciii. 777. Schaffer, Ann. de VInst. Pasteur, iii. 644. Fleming, Trans. *jth Internal. Cong. Hyg. and Demog., iii. 16. Iielman, Ann. de VInst. Pasteur, ii. 274; iii. 15. Babes and Lepp, ibid.., iii. 384. Nocard and Roux, ibid., ii. 341. Roux, ibid., i. 87 ; ii. 479. ' Bruschettini, Centralbl. f. Bakteriol. u. Parasitenk., xx. 214; xxi. 203. Memmo, ibid., xx. 209; xxi. 657. Frantzius, ibid., xxiii. 782 ; xxiv. 971. Appendix C. — Malarial Fever. Laveran, Bull. Acad, de mid., (1880) ser. II. ix. 1346; " Trait e des fievres palustres," Paris, 1884; " Du paludisme et de son hema- tozoaire," Paris, 1891. Marchiafava and Celli, Fortschr. d. Med., 1883 and 1885 ; also in Virchozv's Festschrift. Golgi, Arch, per le sc. vied., 1886 and 1889 ; Fortschr. d. Med., (1889) No. 3 ; Ztschr.f. Hyg., x. 136 ; Deutsche vied. Wchnschr., (1892) 663, 685, 707, 729 ; Sternberg, New York Med. Rec, xxix. No. 18. James, ibid., xxxiii. No. 10. Councilman, Fortschr. d. Med., (1888) Nos. 12, 13. Osier, Trans. Path. Soc. Philadelphia, xii. xiii. Grassi and Feletti, Riforvia vied., (1890) ii. No. 50. Canalis, Fortschr. d. Med., (1890) Nos. 8, 9. Danilewsky, Ann. de Plnst. Pasteur, xi. 758. " Parasites of Malarial Fevers," New. Syden. Soc. 1894 (Monographs by Marchiafava and Bignami, and by Mannaberg, with Bibliography). Manson, Brit. Med. Journ., (1894) i. 1252, 1307; Lancet, (1895) ii. 302; Brit. Med. Journ., (1898) ii. 849. Koch, Berl. klin. IVchnschr., (1899) 69. Ross, Indian Med. Gaz., xxxiii. 14, 133, 401, 448. 55o IUIUJ0GRAP11Y. Appendix D. — Dysentery. Losch, Virchvuts Archiv, lxv. 196. Cunningham, Quart. Journ. Micr. Sc., N.S. xxi. 234. Kartulis, Virchow's Archiv, cv. 118; Centralbl. f. Bakteriol. u. Parasiienh., ii. 745 ; ix. 365. Koch, Arb. a. d. k. Gsndhtsamte., iii. 65. Councilman and Laflcur, Johns Hopkins Hosp. Hep., (1891) ii. 395. Maggiora, Centralbl. f. Bakteriol.u. Parasitenk., xi. 173. Ogata, ibid., xi. 264. Schuberg, ibid., xiii. 598, 701. Quincke and Roos, Berl. Mill. Wchnschr., (1893) 1089. Kruse and Pasquale, Ztschr. f. Hyg., xvi. i. Cic- chanowski and Nowak, Centralbl. f. Bakteriol. u. Parasitenk., xxiii. 445- INDEX INDEX. Abrin, 160 ,, immunity against, 468, 474 Abscesses (v. also Suppuration) bacteria in, . .165 ,, in dysentery, Absolute alcohol, fixing by Acquired immunity in man, Actinomyces, theories of, 488 20 ! 5 characters of, . 283 ,, cultivation of, . 290 ,, inoculation with, 291 ,, varieties of, • 291 Actinomycosis, . . • 282 ' diagnosis of, . 292 lesions in, . 287 origin of, . 289 Active immunity, . . 461, 462 Aerobes, .... 23 ,, culture of, . . 58 ,, separation of, . . 61 /Estivo-autumnal fevers, . 523 Agar media (v. also Culture media), 48 ,, ,, separation by, . 66 Agglutination by sera, . . 482 ,, methods, . 118 ,, of B. mallei, . 278 ,, of B. typhosus, etc., 343 of cholera vibrio, 421 of M. melitensis, 451 530 98 459 >» Agglutination of yellow fever bacillus, . Albumose of anthrax, immun ity by, Albumoses, . ,, in diphtheria, ,, in tetanus, . ,, in tubercle, . Alexines, Amoebic dysentery, Anserobes, ,, cultures of, . ,, separation of, ,, toxines of, Ansesthetic leprosy, Aniline oil, dehydrating by, ,, ,, water, ,, stains, list of, . Animals, autopsies on, . inoculation of, bacillus, PAGE 456 468 154 368 387 251 495 526 23 7i 64 72 260 107 109 103 m 127 Anthrax, . . . 295 296 biology of, 300 cultivation of, 297 ,, inoculation with, . 305 ,, toxines of, 309 diagnosis of, . . 315 in animals, . . 302 in man, . . . 306 protective inoculation, 313 spread of, . 311 554 JNDEX. l'AGE 474 314 466 470 460 479 Anti-abrin, .... Anti-anthrax serum, Anti-cholera vaccination, Antidiphtheritic serum, Antikorper, .... Antimicrobic serum, >> properties of, . Anti-plague serum, Antipneumococcic serum, Antirabic serum, . Anti-ricin, .... Antiseptics, .... Antisera (summary), ,, therapeutic action of, Antistreptococcic serum, Antitetanic serum, > i , , preparation of, 471 et seq. Antitoxic action, nature of, . 475 ,, bodies in normal tissues, sera, use of, . ,, serum, . cholera, standard- isation of, Antitoxines, production of, . ,, chemical nature of, . . . Antitubercular serum, . Antityphoid serum, Appendicitis, Arthrospores, question of occurrence of, . . 9, 405 Artificial immunity, varieties of, . . . 462 et seq. Ascomycetse, . . . 136 Aspergillus, . . . .136 Attenuation of virulence, . 463 Autoclave, 39 Autopsies on animals, . . 131 Avian tuberculosis, . . 243 Bacilli, characters of, . 18 Bacillus acidi lactici, , . 27 ,, anthracis, . . 296 ,, coli communis, lesions caused by, 176 et seq. ,, „ characters of, . 324 13 480 442 220 513 474 32 386 486 480 390 476 474 470 419 472 477 478 253 350 179 Bacillus coli, comparison with B. typhosus, . 326 ,1 ,, pathogenicity of, 341 ,, diphtheriae, . . 354 ,, enteritidis (Gaertner), 330 ,, icteroides, . . 453 lactis acrogenes, . 170 ,, of glanders, . . 271 ,, of influenza, . . 430 ,, of leprosy, . .261 ,, of malignant oedema, 394 ,, Neapolitanus, . . 318 ,, ozcense, . . . 281 ,, of plague, . . 438 ,, of psittacosis, . . 331 ,, pneumoniae, . .211 ,, pseudo-diphthericus, 370 „ pyocyaneus, . .170 >, ,, agglomer- ation of, 446 ,, immunity against, 482 >, ,, occurrence of, ,, pyogenes foetidus, ,, of quarter-evil, ,, of rhinoscleroma, ,, of smegma, ,, of soft-sore, , , smegmatis, ,, subtilis, . ,, of syphilis, ,, tetani, ,, of tubercle, ,, of typhoid, ,, of xerosis, Bacteria, action of dead ,, aerobic [v. Ae , , anaerobic (v. aerobes), . . 23 ,, biology of, .21 ,, capsulated, . . 4 ,, chemical action of, 27 , , , , composi- tion of, 13 ,, classification of, . 15 ,, cultivation of, . 34 ,, death of, . . 32 ,, effects of light on, . 24 ,, food supply of, . 21 . 180 . 166 . 400 . 278 202, 255 • 199 • 255 . 68 . 202 • 377 . 227 • 318 • 372 146 23 obes), An- INDEX. 555 PAGE Bacteria, higher, . . 19 ,, lower, . . . 16 ,, microscopic examin- ation of, . . 93 ,, morphological rela- tions of, . . 3 motility of, . 9 movements of, . 25 multiplication of, . 5 nitrifying, . . 3° parasitic, . • 28 pathogenic, action of, • 139 ,, effects of, 147 purpurin-containing, 31 saprophytic, . . 28 separation of, . 61 et seq. species of, . . 31 spore formation in {v. also Spores), 6, 68 structure of, . . 3 sulphur-containing, 13 ,, temperature of growth of, . . 23 ,, toxines of, . . 153 ,, variability among, . 31 ,, virulence of, 140, 465 et seq. Bacterial ferments, . 29,158 ,, pigments, . . 13 ,, protoplasm, structure of, 10 Bactericidal powers of serum, 494 Bacteriological diagnosis, 121, 125 ,, examination of discharges, . 121 Beggiatoa, .... 20 Behring on immunity, 390, 464, 471 Bismarck-brown, . . 104, 112 Blackleg, . . . . 401 Blood agar, v. Culture media, 49 ,, examination of, . ' 78, 96 ,, in malarial fever, 515,520 ,, in relapsing fever, . 404 Bordet's phenomenon, . .481 Bouillon (v. also Culture media), 43 Bread paste, .... 56 Brieger and Boer, . . 1 57 ,, Fraenkel, . 154 Buboes, . . -195, 201 Bubonic pest, . . . 437 PAGE Buchner on alexines, . . 495 ,, antitoxine, . . 489 Butschli on bacterial structure, 12 Calmette, . . 442, 467, 474 Canon on influenza, . . 434 Cantani on influenza, . . 435 Capsules, staining of . . 115 Carbol-fuchsin, . . .110 „ -methylene-blue, . 109 ,, -thionin-blue, . . 109 Carmine stains, . . .112 Carter on relapsing fever, . 446 Cattle plague, . . . 502 Chamberland and Roux, attenua- tion of B. anthracis, . . 464 Chamberland's filter, . . 80 Chemiotaxis, . 26, 164, 490 Cholera, . . . .402 ,, immunity against, . 419 ,, inoculation of man with, 418 ,, methods of diagnosis of, 425 , , preventive inocula- tion against, . 466 ,, -red reaction, . .410 Cholera spirillum, . . . 404 ,, ,, distribution of, . . 406 ,, ,, inoculation with, . 412 ,, ,, methods of distinguish- ing, . 420 ,, ,, powers of resistance of, . . 410 ,, ,, relations to disease, . 422 ,, ,, toxines of, 415 Cladothrix, .... 20 Clubs in actinomyces, . . 285 Cocci, characters of, . . 16 Cohn, . . . -31 Colonies, counting of, . . 77 Comma bacillus, . . . 402 Commission on tuberculosis, . 248 ,, vaccination, . 499 Contrast stains, . . .112 Copeman on smallpox, . . 503 Cornet's forceps, 95 556 JNDEX. PAGE Corrosive sublimate, fixing by, 98 ,, films of blood, etc., 97 Councilman and Lafleur on dysentery, .... 530 Counting of colonics, . . 77 Cover-glasses, cleaning of, . 95 Cowpox, relation to smallpox, 500 Crescentic bodies in malaria, . 518 Cultivation of anaerobes, . 69 Culture media, preparation of : agar, . . 48 ,, ,, alkaline blood serum, . 53 ,, ,, blood agar, . 49 serum,. 56 ,, ,, bouillon, . 43 ,, ,, bread paste, . 56 ,, ,, glucose agar, . 49 ,, ,, ,, broth, 46 ,, ,, ,, gelatine, 48 ,, ,, glycerine agar, 48 ,, ,, broth, 46 ,, ,, litmus whey, 327 ,, ,, Loffler's serum medium, . 52 ,, ,, Marmorek's serum media, 53 ,, ,, meat extract, . 41 ,, ,, peptone gela- tine, . . 46 ,, ,, peptone solu- tion, . . 50 ,, ,, potatoes, . S3 ,, ,, serum agar, . 192 Cullures, destruction of, . 91 ,, filtration of, . . 80 ,, from organs, . 121, 132 ,, hanging-drop, aerobic, 74 ,, hanging-drop, an- aerobic, . . 76 ,, incubation of, . . 88 ,, microscopic examina- tion of, . . 94 ,, plate, ... 61 ,, pure, ... 58 "shake," . . 85 Cutting of sections, . . 99 Cystitis, . . .179, 195 De Bary, definition of species, 32 I'AGE Decolorising agents, . . 107 Deep cultures, • • • 57 Dehydration of sections, . 107 Delepine 120 Deneke's spirillum, . . 429 Diagnosis, bacteriological, 121, 125 Diphtheria, . . . • 3^3 ,, diagnosis of, . 373 ,, immunity against, 471 ,, origin and spread of, . . . 372 ,, paralysis in, . 368 ,, results of treatment, 487 Diphtheria bacillus, action of, 373 ,, ,, characters . °f> • 354 ,, ,, distribu- tion of, 356 ,, inocula- tion with, 363 ,, isolation of, 375 ,, ,, powers of resist- ance of, 362 ,, . ,, toxines of, 154, 159, 364 , , , , variations in virul- ence of, 369 Diplococcus, ... .16 ,, endocarditidis en- capsulate, . 184 ,, intracellularis meningitidis, . 172 ,, pneumoniae, . 206 Ducrey's bacillus, . . 199 Durham's fermentation tubes, 86 Dysentery, amoebic, . . 527 ,, characters of amceba of, . 528 ,, ' methods of ex- amination in, . 532 ,, varieties of, . . 532 Eberth's bacillus, . . . 317 Ehrlich on ricin and abrin, 468, 474 ,, on toxines, . . 161 ,, stain for tubercle, . 230 ,, theory of antitoxine formation, . . 477 INDEX. 557 21 Embedding in paraffin, . Emmerich's bacillus, Empyema, . Endocarditis, bacteria in, Enteric fever, Enteritis, dysenteric, Ermengem's stain for flagella Erysipelas, . Escherich's bacillus, Esmarch's roll-tubes, Exaltation of virulence, . Examination of water, . Exhaust-pump, PAGE IOO 318 4, 434 , 184 ■ 317 ■ 53i , ii7 . 187 • 3i8 • 65 • 465 • 79 . 80 False membrane, . .178, 356 Farcy, . . • • .269 Feeding, immunity by, . . 468 Fermentation by pneumo- bacillus, . 212 ,, by bacillus coli, 326 ,, methods of ob- serving, . 84 ,, tubes, . _ • 86 Ferments formed by bacteria, 29, 158, 472 ,, in diphtheria, . 367 ,, in tetanus, . . 387 Film preparations, dry, . . 95 wet, . 97 ,, staining of, 105 Filtration of cultures, . . 80 Finkler and Prior's spirillum, 428 Fixation of tissues, . • 98 Flagella, nature of, . . 9 ,, staining of, . . 115 Flagellated organisms in mal- aria, 520 Fltigge, . . . -19 Forceps for cover-gksses, . 95 Foth's dry mallein, . . 278 Fraenkel's pneumococcus, 207, 209 ,, stain for tubercle, . 114 Frankland, .... 3° Fraser, T. R., . 467, 468, 474 Friedlander's pneumobacillus, 208, 211 Frisch on rhinoscleroma, . 280 Fuchsin, carbol-, . . 1 13 Fungi, .... 133^^. PAGE Gamaleia on pneumonia, . 215 Gangrenous emphysema, • 394 pneumonia, • 434 Gas-regulator, 88 Geissler's exhaust-pump, . 80 Gelatine media, 46 „ ,, separation by, 01 ,, phenolated, . ■ 329 Gentian-violet, . • .109 Germicides, 33 Glanders bacillus, . .271 „ agglutina- ( tion of, . 27b ,, inoculation with, . 274 Glanders, . ... .268 ,, diagnosis of, . . 279 ,, in horses, . • 269 ,, in man, . ■ 270 lesions in, . . 276 Glucose media, . . 46 et seq. Glycerine „ . . 4.6 et seq. Golgi on malaria, . . 5 1 5» 523 Gonidia, .... 20 Gonococcus, characters of, . 189 „ inoculation with, 193 ,, toxine of, . 194 Gonorrhcea, .... 189 Gonorrhoea! conjunctivitis, . 196 Gram's method, . . .110 Grease, .... 498 Greenfield on anthrax, . 308, 463 Griiber and Durham's pheno- menon, .... 482 Gulland (methods), . 97, 102 HEematozoon malarias, . . 514 Haffkine on anti-cholera in- oculation, .... 466 Hanging-drop cultures, _ . 74 }, ,, examination of, . 94 Hankin, . • • 3°9. 468 Hansen, leprosy bacilli, . 261 Hog-cholera, immunity in, . 494 Horsepox, .... 498 Hueppe, . . • • 9> x8 Hydrogen, supply of, . . 68 Hydrophobia, . . . 504 ,, diagnosis of, . 514 558 INDEX. PAGE 1 [yclrophobia, prophylactic treatment of, 510 >> the virus of, . 509 Hypodermic syringes, . 127,12s Immunity {v. also Special Dis- eases), . . 458 acquired, theories of, 488 461, 462 460 468 467 462 492 469 472 124 89 87 140 144 43i ,, active, ,, artificial, ,, by feeding, . ,, by toxines, . , , methods, . , natural, , , passive, ,, unit of, Impression preparations, Incubators, . Indol, formation of, Infection, conditions modifyin ,, nature of, Influenza, ,, bacillus, cultivation of, 432 ,, inoculation, 435 ,, lesions in, ,, sputum in, Inoculation, methods of, ,, of animals, ,, ,, separa tion by, ,, protective, 462 et seq. Intestinal changes in cholera, 403 » dysentery, 530 „ ,, typhoid fever, ,, infection in cholera (experimental), Involution forms in bacteria, Iodine solution, Gram's, ,, terchloride, Issaeff, .... Ivanofl's vibrio, 433 436 128 127 67 332 413 5 1 1 1 464, 471 . 468 423 532 Japanese dysentery, Jenner on vaccination, . Joints, gonococci in, Kanthack, on madura disease, Kipp's apparatus, . Kitasato on bacillus of influenza, 431 „ ,j of plague, 437 497 197 294 68 Kitasato on bacillus of tetanus, 377 ct seq. Klehs-LofBer bacillus, . . 353 Klein, . . . 352, 466, 503 klempcrer on pneumonia, Koch on avian tuberculosis. ,, bacillus of malignant oedema, . ,, cholera spirillum, ,, cultivation of B. an thracis, . ,, leveller for plates, ,, tubercle bacillus, . ,, tuberculin, . „ "tuberculin O," and "R, Kruse and Pasquale on dysen- tery, Kiihne on tuberculin, Kuhne's methylene-blue, ,, modification of Gram's method. Laveran's malarial parasite, . Lenses, .... Lepra cells, .... Leprosy, .... ,, bacillus, . ,, ,, distribution of, ,, ,, staining, 113, ,, diagnosis of, ,, etiology of, Leptothrix, . Lesions produced by bacteria Leucomaines, Litmus whey, Liver abscess in dysentery, Lockjaw, Loffler's bacillus, . ,, methylene-blue, ,, serum medium, ,, stain for flagella, ,, and Schutz', glanders bacillus, Losch, arnceba of, . Lustgarten's bacillus, Lymphangeitis, Lymph, vaccine, . Lysogenic action of serum, ,, ,, towards blood corpuscles, 220 244 394 402 296 63 225 249 '254 528 251 109 112 514 93 260 258 261 263 262 267 264 20 174 153 327 53o 376 353 108 52 116 269 527 202 1 78 499 481 484 INDEX. 559 TAGE PAGE Lysogenic action, theory of, . 485 M'Fadyean, . . • • 278 Macrophages, . • • 4°9 Madura disease, . ■ ■ • 292 Malarial fever, examination of blood in, • 525 M ,, mosquitoes in, 520 parasite, . • 5 1 5 ,, inoculation of, 525 ,, varieties of, . 522 Malignant oedema, bacillus of, 394 ,, diagnosis of, 400 n ,, immunity against, . 4°° pustule, . f 3°7 Mallein, . . . • • 27° Malta fever, . . . • 44» ,, ,, methods of diagnosis, 452 Mann's method of fixing sections, . . • • 102 Mannaberg (malaria), . • 522 Manson, ,, • • 52° Maragliano's anti - tubercular serum, t 253 Marchiafava and Celli on mal- aria, . . • . • 215 Marmorek on streptococci, ; 174 ,, anti - streptococcic serum, . . 480 ,, serum media, . 53 Martin, Sidney, on albumoses, etc., . 156 (anthrax), 310 (diphtheria), 368 (tetanus), 387 Martin, C. T. (toxines), . • T57 ,, (antitoxines), 475> 478 Massowah vibrio, . . • 423 Measuring bacteria, . .126 Meat extract, 41 Mediterranean fever, . ■ 44$ Meningitis cerebrospinal, . 172 in influenza,^ ; • 434 ,, . pneumococci in, . 214 Metachromatic granules, . 1 1 Metchnikoff on cholera in rabbits, . 4T4 ,, phagocytosis, 489, 494 Metchnikoff on Pfeiffer's phe- nomenon, . 482 relapsing fever, 446 108, l65, Metchnikoff's spirillum, Methylene-blue, . 104 Methyl-violet, Micrococci of suppuration Micrococcus, ,, of gonorrhoea, . n melitensis, _ • pyogenes tenuis, ,, tetragenus, lesions 426 109 103 178 16 188 449 165 171 106 ,, urea;, Microphages, Microscope, use of, Microtomes, . Migula, Mikulicz, cells of, . Milk as culture medium, Moller's stain for spores, Mordants, . . , • Mosquitoes in malaria, . Mould fungi, Mucorinse, . Muencke's filter, . Mycelium, . Mycetoma, . Mycoderma, . Mycoprotein, N'ageli, Natural immunity, Neelsen's stain for tubercle, Neisser's gonococcus, . Nencki's mycoprotein, . Nicolaier, tetanus bacillus, Nicolle's modification c Gram's method, Nikati and Rietsch (cholera) Nitrifying bacteria, Nitroso-indol body, _ . Non-pathogenic organisms, Nordhafen vibrio, . Obermeier's spirillum, . CEdema, malignant, Ogston, O'ldium lactis, caused by, 178 27, 29 489 f INDEX. I'AGF. Oil, aniline, for dehydrating, etc., 107 ,, immersion lens, . . 93 ( >rganisms, non-pathogenic, . 133 < )riental plague, . . 4™ Osteomyelitis, ^5 Otitis, . Ozcena bacillus, . 2I5> 434 . 281 Paraffin embedding, . . IOO Passage, . . . '. 46$ Passive immunity, . 461, 469 Pasteur on exaltation of viru- lence of bacteria, . ,, on hydrophobia, ,, septicemic de, . ,, on vaccination against anthrax, Pathogenic bacteria, Penicillium, .... Peptone gelatine (v. Culture media), ,, solution, . 50 Periostitis, acute suppurative, Perisporiaceas, Perithecium, .... Peritonitis, . Perlsucht, Petri's capsules, Petruschky's litmus-whey, Pettenkofer on cholera, Pfeffer, Pfeiffer on anti-serum, ,, cholera, ,, influenza, typhoid, Pfeiffer's phenomenon, Phagocytosis, Phenol-phthalein as indicator, Phenomenon of Bordet, 465 507 394 313 139 137 46 410 186 136 136 196 226 . 62 • 327 418, 422 . 26 179. 4i5> 479 420 43i • 34i 420, 481 489, 494 45 481 , , Grtiber and Durham, . 482 >> jj Pfeiffer, 420, 481 Pigments, bacterial, . . 13 Pipettes, .... 122 Pitlield's flagella stain, . .116 Plague, bacillus of, . 437 et set/. ,, immunity against, . 442 ,, preventive inocula- tion against, . . 443 Plasmolysis, . . . .12 PAGE I late cultures, agar, . . c>6 » » gelatine, . 61 " >> gonococcus, . 192 Platinum needles, . . -59 Pneumobacillus (Friedliinder's), 211 et sea. Pneumococcus (Fraenkel's), 209 et set/. ,, immunity against, 220 in endocarditis, ,, lesions caused by ,, toxines of, Pneumonia, bacteria in, ,, gangrenous, , , in influenza, ,, methods of ex- amination of, . varieties of, Polar granules, Potatoes as culture material, . Preparations, impression, Protective inoculation, 462 et seq Protozoa described in hydro- phobia, 509 smallpox, 504 184 213 220 206 434 433 222 204 1 1 53 124 Protozoon malarias, Pseudo-diphtheria bacillus, Psittacosis bacillus, Ptomaines, . Puerperal septicaemia, . Pus, examination of, Pustule, malignant Pyaemia, ,, nature of, 121. Quartan fever, Quarter-evil, bacillus of, Quotidian fever, . 514 370 331 153 179 . 188 • 307 178 et set/. . 164 • 523 . 401 • 523 Rabies, .... 505 Rauschbrand bacillus, . . 401 Ray-fungus (actinomyces), . 282 Reaction of media, standard- ising of, .... 43 Recovery from disease, . . 468 Red stains, .... 104 Reichert's gas regulator, . 88 Relapsing fever, spirillum of, etc., 444 INDEX. 561 TAGE Rhinoscleroma, bacillus of, . 279 Ricin, 160 immunity against, 466,474 Robin, l6° Rock fever, . • • -44* Roll-tubes, Esmarch's, . • °5 Rosenbach (bacteria in sup- puration), . • • ' o Ross on malaria, . • • 5 18 Roux on antitoxic sera, . 473 ,, and Yersin (diphtheria), 364 ct seq. Saccharomyces, . . -137 Safranin, . • • 112 Sanarelli (typhoid fever), . 33° Sanderson, Burdon, . 463» 5°2 Saprophytes, 2° Sarcina, . • • . l° Schizomycetes, ... 3 Schizophycere, ... 3 Schizophyta, • . 3 Scorpion poison, . . .100 Section-cutting, 99 Sections, dehydration of, . 107 Sedimentation methods, . 118 ,, test for typhoid, 344 Separation of aerobes, . • 61 ,, anaerobes, . 64 Septicaemia, nature of, . .164 puerperal. . .179 sputum, . . 206 Septicemic de Pasteur, . . 394 Serum, agglutinative action of, 482 ,, anticharbonneux, . 314 ,, anti-cholera, . • 4X9 ,, antidiphtheritic, . 47 1 ,, antimicrobic, . • 480 ,, anti-plague, . . 442 ,, antipneumococcic, . 220 ,, antirabic, . . • 5*3 ,, antistreptococcic, . 480 ,, antitetanic, . _ . 39° ,, antitoxic, preparation of, . . . 47 1 et seq. ,, antitubercular, . ■ 253 ,, antityphoid, . . 35° ,, bactericidal action of, 493 ,, blood {v. Culture media), 50 ,, diagnosis, . . • 483 PAGB Serum diagnosis, methods . n8 of typhoid, . 343 ,, inspissator, . • 51 ,, lysogenic action of, . 481 towards blood corpuscles, 484 ,, media, 5° Shake cultures, . .85 Sheep-pox 5°2 Slides for hanging-drops, 74, 7° Sloped cultures, aerobic, . 57 anaerobic, . 74 Smallpox, . • • -497 bacilli in, . • 5°3 Smegma bacillus, . . • 255 Smith's, Lorrain, serum medium, 53 Snake poisons, . . .160 J} ,, immunity against, 447 Soft sore, . . • • l99 Soudakewitch on relapsing fever, . ■ ' • -447 Spinal cord, lesions by pyo- genic organisms, . .176 Spirilla, characters of {v. also Vibrio), . . 18 , , like cholera spirillum , 423 Spirillum Metchnikovi, . . 426 ,, of cholera, . . 4°4 Deneke, . • 429 „ Finkler and Prior, 428 Miller . • 429 ,, relapsing fever, in- oculation with, etc. , 445 Spirochreta, . . ■ l9 Splenic fever, . • • 294 Spore formation, arthrosporous, 9 ,, endogenous, 6 JS ,, B. anthracis, 300 Spores, staining of, . • "4 Sporulation of malarial parasite, 518 Sputum, amcebse in, . . 53° „ influenza, . 433. 43° phthisical, . 24°. 256 ,, in plague, . . 439 ,, in pneumonia, . . 207 ,, septicaemia, . . 206 Staining methods, . . 105 et seq. offlagella, . .116 ,, of leprosy bacilli, . 113 562 INDEX. PAGE Staining of spores, . . ,, of tubercle bacilli, . 113 ,, principles, . .103 Stains, basic aniline, . -103 contrast, . . .112 Standard of immunity, . ' . 473 Standardising reaction of media, .... 43 Staphylococci, lesions caused by> 178 Staphylococcus, . . .16 ,, cereus albus, . 168 j> ,, flavus, 168 " pyogenes albus, 168 ,, pyogenes aureus, characters of, . 166 )> inoculation with, . 173 pyogenes citreus, 168 Steam steriliser, Koch's . 37 Sterilisation by heat, . 36 et seq. ,, at low temperatures, 40 ,, by steam at high pressure, . 39 Streptococci in false membrane, 178 ,, in diphtheria, . 358 ,, lesions caused by, 178 ,, varieties of, . 175 Streptococcus, ... 16 ,, brevis, . . 175 ,, conglomerate, 170, 176 ,, erysipelatis, . 175 ,, longus, . .175 ,, pneumonias, . 206 j.j pyogenes, charac- ters of, 168 ,, inocu- lation with, 174 Streptothrix, ... 20 ,, actinomyces, . 283 ,, madurre, . . 292 Subcultures, ... 59 Substances, bactericidal, . 495 Suppuration, bacteria of, . 165 ,, gonococci in, . 197 ,, methods of exa- mination of, . 188 ,, nature of, . .163 PAGE Suppuration, origin of, . . 180 ,, pneumococci in, 214 j j without bacteria, 177 ., typhoid bacillus c in' • • 334 Symptoms caused by bacteria, 152 Syphilis, bacillus of, . . 202 Syringes for inoculation, 127,128 Syzygies, . . . .518 Tabes mesenterica, . . 248 Tertian fever, . . 523, 524 Test-tubes for cultures, . . 56 Tetanus, .... 376 ,, anti-serum of, 470 et seq. >> intracerebral injection of, 488 ,, immunity against, . 390 methods of examina- tion in, . . 393 treatment of, . 392, 488 Tetanus bacillus, . . . 377 >j inoculation with, . 384 >) isolation of, 380 >> spores of, . 379 >) toxines of, 159, 286 Tetrads, .... 16 Theory of exhaustion, . . 488 ,, of phagocytosis, . 489 ,, of retention, . . 489 ,, humoral, . . . 491 Thionin-blue, . . 104, 109 Thiothri.x, .... 20 Tissues, action of bacteria on, 147 ,, fixation of, . . 98 Tizzoni and Cattani on tetanus, 390 Torulse, . . . 137 Toxalbumins, . . .154 Toxic action, theory of, . 161 Toxicity, estimation of, . . 470 Toxines, early work on, . 153 ,, effects of, . . 151 ,, immunisation by, . 467 ,, intra- and extra- cellular, . .155 ,, nature of, . -156 ,, non-proteid, . . 157 ,, of anthrax, cholera, INDEX. 563 PAGE etc. {vide Special Diseases) Toxines, production of, susceptibility to, ,, vegetable, Tubercle bacillus, . _ ■ action PAGE 145 493 160 227 of dead, • 246 )} avian, • 243 cultivation of, " " 231 distribution of, 237 immunity against, . 253 inoculation with, • 241 powers of re- sistance of, 233 insputum,etc, 240 toxinesof, 251,254 „ stains for, . "3 giant cells, methods of examma tion of, Tubercles, structure of, Tubercular leprosy, Tuberculin, '"O" and "R," • Tuberculosis, avian, diagnosis _ by tuberculin, . 'in animals, " modes of infec- tion, Tubes, cultures in, Typhoid bacillus, . comparison with B. coli, < • examination for, . )5 immunity against, . inoculation with, toxines of, . serum diag- nosis, 235 256 228 ■259 249 254 224 243 253 226 247 56 3i8 326 35° 339 334 337 343 Typhoid bacillus, vaccination n against, . 349 fever, . • . J pathological changes in, 332 Ulcerative endocarditis, _ • l84 experi- " mental, 186 gono- cocciin, 198 Unit of immunity, • • 472 Urine, examination of, . • / „ staining of,. • • 9 tubercle bacilli in, 240, 236 typhoid bacilli in, . 351 Vaccination {vide also anthrax, cholera, etc.) • 497 against hydro- phobia, . • 511 against typhoid, . 349 nature of, . • 5°4 Variola, . • • 5°o * seg Venenes, ■:.,,'» ' To Vibrio {see also Spirillum), • *9 berolinensis, . • 423 of cholera, . . • 4 423 „ Metchnikovi, . • 42& „ Nordhafen, . • 42» of Pestana and Betten- court, . . -424 Romanus, . • • 424 Vibrion septique, . • • 394 Virulence, attenuation of, . 4°3 (J exaltation of, . 4°5 of bacteria, . .14° suppurations in, 334 Warington, . • • . • , 3° Water, bacteriological examina- tion of, . • ; 79 ,, supplies, typhoid bacilli in,-. . ..• 351 Weichselbaum on pneumonia, 200 564 INDEX. Weigert's method of dehydra- tion, . , , m o d i fi c a t i o n of Gram's method, . Wertheim's medium, . 191 Widal on serum diagnosis, ,, test for typhoid, . Winogradski, Wood, Cartwright, preparation of antitoxine, Woodhead on tuberculosis, . Woody tongue, Woolsorter's disease, PACE 407 I I I 192 483 347 30 472 248 289 308 Xerosis bacillus, Xylol, . Yeasts, . Yellow fever, bact ena in, " >> immunity against Yersin (v. also Roux), on plague, PACE 372 I02 '37 452 453 456 Ziehl-Neelsen stain, Zooglcea, Zygospore, . 437 el se