i • a • i 1 jffi. ; • • M m m - * jp^l ;•' Pentland's Students Manuals. MANUAL OF BACTERIOLOGY. NUNQU*M ALIUD NATURA, ALIUD SAPIENT.A HI MANUAL OF BACTERIOLOGY BY EGBERT 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. SUPERINTENDENT OF THE ROYAL COLLEGE OF PHYSICIANS' LABORATORY, EDINBURGH FORMERLY PROFESSOR OF PATHOLOGY IN THE UNIVERSITY OF OXFORD FOURTH EDITION WITH ONE HUNDRED & SEVENTY-ONE ILLUSTRATIONS EDINBURGH AND LONDON YOUNG J. PENTLAND 1907 vi PREFACE TO THE FOURTH EDITION the diseases in question in the original arrangement. In the appendix will be found an additional chapter dealing with trypanosomiasis and allied affections. A number of new illustrations have been added throughout the book, and the bibliography has been brought up to date. 0,-fnber 1907. PREFACE TO THE FIEST EDITION. 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 consider the effects in particular instances of various modifying circumstances. Much research on certain subjects is so recent that conclusions on many points must necessarily 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 vii viii PREFACE TO THE FIRST EDITION included on account of their own importance 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 pre- parations from which Figs. 160-165 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 published on each of the subjects considered June 1897. CONTENTS. CHAPTER I. GENERAL MORPHOLOGY AND BIOLOGY. PAGE INTRODUCTORY — Terminology — Structure of the bacterial cell — Reproduction of bacteria — Spore formation — Motility — - Minuter structure of the bacterial protoplasm — Chemical composition of bacteria — Classification — Food supply — Re- lation of bacteria to moisture, gaseous environment, tempera- ture, and light — Conditions affecting bacterial motility — Effects of bacteria in nature — Methods of bacterial action — Variability among bacteria . . . . .1 CHAPTER II. METHODS OF CULTIVATION OP BACTERIA. Introductory— Methods of sterilisation— Preparation of culture media — Use of the culture media — Methods of the separation of aerobic organisms — Principles of the culture of anaerobic organisms — Miscellaneous methods — General laboratory rules ........ 25 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 I ix CONTENTS f'ACK of sections — Staining principles — Mordants and decolorisers — Formulae of stains — Gram's method and its modifications — Stain for tubercle and other acid-fast bacilli — Staining of spores and flagella — The Romanowsky stains — Observation of agglutination and sedimentation — Method of measuring the phagocytic capacity of the leucocytes — Routine bacteriological examination — Methods of inoculation — Autopsies on animals 85 CHAPTER IV. BACTERIA IN AIR, SOIL, AND WATER. ANTISEPTICS. Air : Methods of examination — Results. Soil : Methods of Examination — Varieties of bacteria in soil. Water : Methods of examination — Bacteria in water — Bacterial treatment of sewage. Antiseptics : Methods of investigation — The action of antiseptics — Certain particular antiseptics . . . 126 CHAPTER V. RELATIONS OF BACTERIA TO DISEASE — THE PRODUCTION OF TOXINS BY BACTERIA. Introductory — Conditions modifying pathogenicity — Modes of bacterial action — Tissue changes produced by bacteria— Local lesions — General lesions — Disturbance of metabolism by bacterial action — The production of toxins by bacteria, and the nature of these — Allied vegetable and animal poisons — The theory of toxic action ..... 149 CHAPTER VI. INFLAMMATORY AND SUPPURATIVE CONDITIONS. The relations of inflammation and suppuration — The bacteria of inflammation and suppuration — Experimental inoculation — Lesions in the human subject — Mode of entrance and spread of pyogenic bacteria — Ulcerative endocarditis — Acute suppur- ative periostitis — Erysipelas — Conjunctivitis — Acute rheu- matism— Vaccination treatment of infections by the pyogenic cocci — Methods of examination in inflammatory and suppur- ative conditions ...... 172 CONTENTS xi CHAPTER VII. INFLAMMATORY AND SUPPURATIVE CONDITIONS, CONTINUED : THE ACUTE PNEUMONIAS, EPIDEMIC CEREBRO-SPINAL MENINGITIS. PAGE Introductory — Historical — Bacteria in pneumonia — Fraenkel's pneumococcus — Friedlaender 's pneumococcus — Distribution of pneumobacteria — Experimental inoculation — Pathology of pneumococcus — Methods of examination. Epidemic cerebro- spinal meningitis ...... 196 CHAPTER VIII. GONORRHCEA, SOFT SORE, SYPHILIS. The gonococcus — Microscopical characters — Cultivation — Rela- tions to the disease — Its toxin — Distribution — Gonococcus in joint affections — Methods of diagnosis — Soft sore — Syphilis — Spirochsete pallida — Transmission of the disease to animals . 219 CHAPTER IX. TUBERCULOSIS. Historical — Tuberculosis in animals — Tubercle bacillus — Staining reactions — Cultivation of tubercle bacillus — Powers of resist- ance— Action on the tissues — Histology of tuberculous nodules — Distribution of bacilli — Bacilli in tuberculous discharges — Experimental inoculation — Varieties of tuberculosis — Other acid-fast bacilli — Action of dead tubercle bacilli — Sources of human tuberculosis — Toxins of the tubercle bacillus — Koch's tuberculin — Active immunisation against the tubercle bacillus — Koch's Tuberculin-R — Agglutinative phenomena — Methods of examination ....... 235 CHAPTER X. LEPROSY. Pathological changes — Bacillus of leprosy — Position of the bacilli — Relations to the disease — Methods of diagnosis . . 267 xii CONTENTS CHAPTER XL GLANDERS AND RHINOSCLEROMA. PAGE Glanders : The natural disease — The glanders bacillus — Cultiva- tion of glanders bacillus — Powers of resistance — Experimental inoculation — Action on the tissues — Mode of spread — Mallein and its preparation — Methods of examination. Rhinoscleroma 275 CHAPTER XII. ACTINOMYCOSIS AND ALLIED DISEASES. Characters of the actinomyces — Tissue lesions — Distribution of lesions — Cultivation of actinomyces — Varieties of actinomyces and allied forms — Experimental inoculation — Methods of examination and diagnosis — Madura disease . . .286 CHAPTER XIII. ANTHRAX. Historical summary — Bacillus anthracis — Appearances of cultures — Biology — Sporulation — Natural anthrax in animals — Ex- perimental anthrax — Anthrax in man — Pathology — Toxins of the bacillus anthracis — Mode of spread in nature — Immunisa- tion of animals against anthrax — Methods of examination . 300 CHAPTER XIV. TYPHOID FEVER — BACILLI ALLIED TO THE TYPHOID BACILLUS. Bacillus typhosus — Morphological characters — Characters of cul- tures— Bacillus coli communis — Reactions of b. typhosus and b. coli — Pathological changes in typhoid fever — Suppuration in typhoid fever — Pathogenic effects produced in animals — The toxic products of typhoid bacillus — Immunisation of animals — Relations of bacilli to the disease — Paratyphoid bacillus — Bacillus enteritidis (Gaertner) — Psittacosis bacillus — Serum diagnosis — Vaccination against typhoid— 'Methods of examination — Bacteria in dysentery — Bacillus enteritidis sporogenes — Summer diarrhoea . . .319 CONTENTS xiii CHAPTER XV. DIPHTHERIA. PAGE Historical — General facts — Bacillus diphtherias — Microscopical characters — Distribution — Cultivation — Inoculation experi- ments— The toxins of diphtheria — Variations in virulence of bacilli — Bacilli allied to the diphtheria bacillus — Summary of pathogenic action — Methods of diagnosis . . . 352 CHAPTER XVI. TETANUS. Introductory — Historical — Bacillus tetani — Isolation of bacillus tetani — Characters of cultures — Conditions of growth — Patho- genic effects — Experimental inoculation — Tetanus toxins — Antitetanic serum — Methods of examination — Malignant oedema — Characters of bacjllus — Experimental inoculation — Methods of diagnosis — Bacillus botulinus — Quarter - evil — Bacillus serogenes capsulatus ... . . . 371 CHAPTER XVII. CHOLERA. ^ Introductory — The cholera spirillum — Distribution of the spirilla — Cultivation — Powers of resistance — Experimental inoculation Toxins of cholera spirillum — Inoculation of human subject — Immunity — Methods of diagnosis — General summary — Other spirilla resembling the cholera organism — MetchnikofFs spirillum — Finkler and Prior's spirillum — Deneke's spirillum 399 CHAPTER XVIII. INFLUENZA, PLAGUE, RELAPSING FEVER, MALTA FEVER, YELLOW FEVER. Influenza bacillus — Microscopical characters — Cultivation — Dis- tribution— Experimental inoculation — Methods of examina- xiv CONTENTS PAGE tion — Bacillus of plague — Microscopical characters — Cultiva- tion— Anatomical changes produced and distribution of bacilli — Experimental inoculation — Paths and . mode of in- fection — Toxins, immunity, etc. — Methods of diagnosis — Relapsing fever and African tick fever — Characters of the spirillum — Relations to the disease — Immunity — African tick fever — Malta fever — Micrococcus melitensis — Relations to the disease — Mode of spread of the disease — Methods of diagnosis — Yellow fever — Etiology of yellow fever . . 420 CHAPTER XIX. IMMUNITY. Introductory — Acquired immunity — Artificial immunity — Varieties — Active immunity — Methods of production — At- tenuation and exaltation of virulence — Passive immunity — Action of the serum — Antitoxic serum — Standardising of toxins and of antisera — Nature of antitoxic action — Ehrlich's theory of the constitution of toxins — Antibacterial serum — Bactericidal and lysogenic action — Haemolytic and other sera — Methods of hsemolytic tests — Opsonic action — Ag- glutination— Precipitins — Therapeutic effects of anti-sera — Theories as to acquired immunity — Ehrlich's side-chain theory L- — Serum anaphylaxis — Theory of phagocytosis — Natural immunity — Natural bactericidal powers — Natural suscepti- bility to toxins ....... 456 APPENDIX A. SMALLPOX AND VACCINATION. Jennerian vaccination — Relationship of smallpox to cowpox — Micro-organisms associated with smallpox — The nature of vaccination ....... 503 APPENDIX B. HYDROPHOBIA. Introductory — Pathology — The virus of hydrophobia — Prophylaxis — Antirabic serum — Methods . . 510 CONTENTS xv APPENDIX C. MALARIAL FEVER. PAGE The malarial parasite — The cycle of the malarial parasite in man — The cycle in the mosquito — Varieties of the malarial para- site— General considerations — The pathology of malaria — Methods of examination ..... 521 APPENDIX D. AMCEBIC DYSENTERY. Amoebic dysentery — Characters of the amoeba — Distribution of the amoebae — Experimental inoculation — Methods of examination 537 APPENDIX E. TRYPANOSOMIASIS — KALA-AZAR — PIROPLASMOSIS. The pathogenic trypanosomes — General morphology of the trypano- somata — Trypanosoma Lewisi — Nagana or tse-tse fly disease — Trypanosoma of sleeping sickness — Trypanosoma gambiense — Kdla-dzar — Dehli sore — Piroplasmosis . . . 544 BIBLIOGRAPHY . . .571 INDEX 593 LIST OF ILLUSTRATIONS. FIG. PAGE 1. Forms of bacteria . . . . . .13 2. Hot-air steriliser ... 27 3. Koch's steam steriliser ...... 27 4. Autoclave . . ... . . .29 5. Steriliser for blood serum . . . . .30 6. Meat press . . . . . . .31 7. Hot-water funnel ...... 35 8. Blood serum inspissator . . . . .40 9. Potato jar ....... 45 10. Cylinder of potato cut obliquely . . . .45 11. Ehrlich's tube containing piece of potato . . .45 12. Apparatus for filling tubes . . . . .48 13. Tubes of media ...... 48 14. Platinum wires in glass handles . . . .49 15. Method of inoculating solid tubes . . . .50 16. Rack for platinum needles . . . . .50 17. Petri's capsule . . . . . . .51 18. Koch's levelling apparatus for use in preparing plates . 54 19. Koch's levelling apparatus ..... 54 20. Esmarch's tube for roll culture . . . .55 21. Apparatus for supplying hydrogen for anaerobic cultures . 58 22. Esmarch's roll-tube adapted for culture containingi'anaerobes 59 23. Bulloch's apparatus for anaerobic plate cultures . . 59 24. Flask for anaerobes in liquid media . . . .61 25. Flask arranged for culture of anaerobes which develop gas . 62 26. Tubes for anaerobic cultures on the surface of solid media . 62 27. Slides for hanging-drop cultures . . . . 63 28. Graham Brown's chamber for anaerobic hanging-drops . 64 29. Apparatus for counting colonies . . . .65 30. Wright's 250 c.mm. pipette fitted with nipple . . 66 31. Geissler's vacuum pump for filtering cultures . . .70 32. Chamberland's candle and flask arranged for filtration . 70 xvii .\viii LIST OK [ILLUSTRATIONS no, 33. Chamberland's bou^i'1 with Ump funnel . 71 34. Bougie inserted througli rubber .st(>i>]»ci . . 71 35. Muenck^'s inodilication of Ch;Liiil>ci!ainr ; liltrr . . 72 36. Flask fitted \\ it b poroelain bougie for fllterii [uantitie of fluid ....... 7-'* 37. Flask for filtering smaU quantities of fluid . . . 73 38. Tubes for demonstrating gas-formation by bacteri.i . 70 39. Goryk air-pump for drying in vacua . . .79 40. Reichert's gas regulator . . . . .80 41. Hearson's incubator for use at 37° C. . . . .81 42. Cornet's forceps for holding rover-glasses . . .87 43. Needle with square of paper on end for manipulating puniflin sections ....... 92 44. Syphon wash-bottle for distilled water . . .96 45. Wright's 5 c.mm. pipette . 108 46. Tubes used in testing agglutinating and sedimenting properties of serum ....... 110 47. Wright's blood -capsi i !•• . . . . .114 48. Test-tube and pipette arranged for obtaining fluids containing bactnit . . . . . 116 49. Hollow needle for intraperitoneal inoculation . .121 50. Hesse's tube . ..... 127 61. Petri's sand filter . . . . . .128 52. Staphylococcus pyogenes aureus, young culture 011 agar. xlOOO . . . . . . .176 53. Two stab cultures of staphylococcus pyogenes aureus in gelatin 175 54. Streptococcus pyogenes, young culture on agar. x 1000 . 176 55. Culture of the streptococcus pyogenes on an agar plate . 177 56. Bacillus pyocyaneus ; young culture on agar. xlOOO . 177 57. Micrococcus tetragenus. x 1000 . . .181 58. Streptococci in acute suppuration, x 1000 . . 184 59. Minute focus of commencing suppuration in brain, x 50 . 1 86 60. Secondary infection of a glomerulus of kidney by the staphylo- coccus aureus. x 300 . . . .187 61. Section of a vegetation in ulcerative endocarditis, x 600 . 189 62. Film preparation from a case of acute conjunctivitis, showing the Koch-Weeks bacilli, x 1000 . . 192 63. Film preparation of conjunctival secretion showing the diplo- bacillus of conjunctivitis, x 1000 . .192 64. Film preparation of pneumonic sputum, showing imineroii* pneumococci (Fraenkel's). x 1000 . . . 199 65. Friedlander's pneumobacillus, from exudate in a <;as<- of pneumonia, x 1000 ... . 200 66. Fraenkel's pneumococcus in serous exudation, x 1000 . 200 67. Stroke culture of Fraenkel's pneumococcus on blood agar . 201 LIST OK ILLCSTKATIONS i i«. I-AI;K 68. Kraenkel's pneumococcus from a pure culture on blood agar. x 1000 . 202 69. Stab culture at' 1'Yiefllaii'ler • |.in:iiiii<>Li.<:i I Ills . . 203 70. Kric'llaii'lei' (.neiiiiiobaeillu.,, from a young culture on agar. xlOOO . .203 71. CapHulated pneumococci in blood taken from the heart of a r;ibl,it. XlOOO . . 206 72. FilmjWparatjonofY^MlationfVomacasconiieningitis. .< 1000 21tf 7-'>. I'ure culture of diploeoceus infraccllularis . . . 214 74. Portion of lilrn of ^onorrho-al pus. xlOOO . . . 220 75. Gonococoi, from a pva cnltiire oo blood agar. xlOOO . 221 76. Film preparations of pus from .soft chancre, showing Ducrey's bacillus. x!500 ... .227 77. Ducrcy's bacillus, x 1500 . 228 78 and 7S>. Film preparations from juice of hard chancre showing piroehBtc paiiid;i. xlOOO . . 230 80. Section of spleen from a case of congenital syphilis, showing spiroeh:etc pallida. x 1000 .... iWl 81. SpiiochfEterofringeiiH. x 1000 .... 231 82. Tubercle bacilli, from a pure culture onglycerinagar. x 1000 2:57 «:;. Tubercle bacilli in phthisical sputum, x 1000 . . 238 84. Cultures of tubercle bacilli on glycerin agar . . . 240 86. Tubercle bacilli in section of human lun 90. Smegma bacilli, x 1000 ..... 2f>4 91. Section l.linnitfh leprous skin, sho \vin- I In- m:i •• ..f i-.-llul;ir granulation tissue in the cutis. x 80 . . 268 92. Superficial part of leprous skin. x f)0<) . . . 289 98. Colonies of actinomyces. x 60 . . . . 290 99. Cultures of the actinomyces on glycerin agar . . 293 100. Actinomyees, from a ml! m-e on ^lyeri in agar. x 1000 . 294 101. Shake en If ures of actinomyces in glucose agar . . 295 102. Seel inn ,,j' ,, ,-olony of ael; from a culture, in blood x!500 295 xx LIST OF ILLUSTRATIONS FIG. PAGE 103. Streptothrix Madura, x 1000 . . . .298 104. Surface colony of the anthrax bacillus on an agar plate. x30 . . . . . . . 302 105. Anthrax bacilli, arranged in chains, from a twenty-four hours' culture on agar at 37° C. x 1000 . . . 303 106. Stab culture of the anthrax bacillus in peptone-gelatin . 303 107. Anthrax bacilli containing spores, x 1000 . . . 305 108. Scraping from spleen of guinea-pig dead of anthrax, x 1000 307 109. Portion of kidney of a guinea-pig dead of anthrax, x 300 . 309 110. A large clump of typhoid bacilli in a spleen. x 500 . 320 111. Typhoid bacilli, from a young culture on agar, showing some filamentous forms, x 1000 . . . .321 112. Typhoid bacilli, from a young culture on agar, showing flagella. x 1000 ...... 322 113. Culture of the typhoid bacillus and of the bacillus coli . 323 114. Colonies of the typhoid bacillus in a gelatin plate, x 15 . 324 115. Bacillus coli communis. x 1000 .... 325 116. Film preparation from diphtheria membrane ; showing numerous diphtheria bacilli, x 1000 . . . 354 117. Section through a diphtheritic membrane in trachea, show- ing diphtheria bacilli, x 1000 .... 355 118. Cultures of the diphtheria bacillus on an agar plate . 357 119. Diphtheria bacilli from a twenty-four hours' culture on agar. x 1000 ...... 357 120. Diphtheria bacilli, from a three days' agar culture. x 1000 358 121. Involution forms of the diphtheria bacillus, x 1000 . 358 122. Pseudo-diphtheria bacillus (Hofmann's). x 1000 . . 366 123. Xerosis bacillus from a young agar culture, x 1000 . 367 124. Film preparation of discharge from wound in a case of tetanus, showing several tetanus bacilli of "drumstick " form. xlOOO . . . . . .373 125. Tetanus bacilli, showing flagella. x 1000 . • . . 374 126. Spiral composed of numerous twisted flagella of the tetanus bacillus, x 1000 . . . . . 375 127. Tetanus bacilli, some of which possess spores, x 1000 . 375 128. Stab culture of the tetanus bacillus in glucose gelatin . 376 129. Film preparation from the affected tissues in a case of malignant cedema. x 1000 .... 389 130. Bacillus of malignant cedema, showing spores. x 1000 . 390 131. Stab cultures in agar — tetanus bacillus, bacillus of malignant cedema, and bacillus of quarter-evil . . . 391 132. Bacillus of quarter-evil, showing spores. x 1000 . . 397 133. Bacillus serogenes capsulatus .... 398 134. Cholera spirilla, from a culture on agar of twenty- four hours' growth. xlOOO . ' . . . . . 400 LIST OF ILLUSTRATIONS xxi FIG. PAGE 135. Cholera spirilla stained to show the terminal flagella. xlOOO ....... 401 136. Cholera spirilla from an old agar culture. x 1000 . . 401 137. Puncture culture of the cholera spirillum . . . 403 138. Colonies of the cholera spirillum on a gelatin plate . . 404 139. MetchnikofFs spirillum. x 1000 . . . . ' 417 140. Puncture cultures in peptone-gelatin . . .418 141. Finkler and Prior's spirillum, x 1000 . . . 419 142. Influenza bacilli from a culture on blood agar. x 1000 . 420 143. Film preparation from a plague bubo. x 1000 . . 426 144. Bacillus of plague from a young culture on agar. x 1000 . 427 145. Bacillus of plague in chains, x 1000 . . . 427 146. Culture of the bacillus of plague on 4 per cent salt agar. xlOOO ....... 428 147. Section of a human lymphatic gland in plague, x 50 . 430 148. Film preparation of spleen of rat after inoculation with the bacillus of plague, x 1000 , . . . 432 149. Spirilla of relapsing fever in human blood, x about 1000 . 439 150. Spirillum Obermeieri in blood of infected mouse, x 1000 . 441 151. Film of human blood containing spirillum of tick fever. xlOOO ....... 444 152. Spirillum of human tick fever (Spirillum Duttonij in blood of infected mouse. x 1000 .... 445 153. Micrococcus melitensis. x 1000 .... 448 154-159. Various phases of the benign tertian parasite . . 525 160-165. Exemplifying phases of the malignant parasite . . 526 166. Amoebae of dysentery ...... 538 167. Section of wall of liver abscess, showing an amoeba of spherical form with vacuolated protoplasm. x 1000 . . 540 168. Trypanosoma Brucei from blood of infected rat. Note in two of the organisms commencing division of micronucleus and undulating membrane, x 1000 .... 554 169. Trypanosoma gambiense from blood of guinea-pig. x 1000 . 557 170. Leishman-Donovan bodies from spleen smear, x 1000 . 564 171. Leishman-Donovan bodies within endothelial cell in spleen. x 1000 565 MANUAL OF BACTERIOLOGY MANUAL OF BACTEBIOLOGY. 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 chlorophyll, 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 1 /z (35Soo incn)- These forms can be classified according to their shapes into three main groups — (1) 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 pro- portion 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 full description of the characters of these groups will be more conveniently taken later (p. 11). 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. (1) On the one hand they consist of filaments made up of simple elements such as occur in the lower forms. These 1 GENERAL MORPHOLOGY AXD BIOLOGY filaments may be more or less septate, may be provided 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 inter- dependence 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 strictness 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 (though the occurrence of such is now suspected), 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. Morphological Relations. — Whe relations of the bacteria to the animal kingdom on the one hand and to the vegetable on the other constitute a somewhat difficult question. It is best to think of there being a group of small, unicellular organisms, which may represent the most primitive forms of life before differentiation into animal and vegetable types had occurred. This would include the flagellata and infusoria, the myxomy- cetes, the lower algae, and the bacteria. To the lower algae the bacteria possess many similarities. These algae are unicellular masses of proto- plasm, having generally the same shapes as the bacteria, and largely multiply by fission. Endogenous 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 phyfigpyan. 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 STRUCTURE OF THE BACTERIAL CELL 3 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 schizophycese (German, Spaltalgen), while the bacteria or splitting fungi are called the schizomycetes (German, Spaltpilzen). The bacteria are, therefore, often spoken of as the schizomycetes. Certain bacteria which have been described as containing chlorophyll ought probably to be grouped among the schizophycese. 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 appeal- 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 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 — though from this fact we cannot deduce that the two are identical in composition. A healthy bacterium when thus stained presents the appearance of a finely granular or almost homogeneous structure. The protoplasm is surrounded by an envelope which can in some cases be demonstrated by over- staining 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|0i 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 iap:nown as a capsulated bacterium (vide Fig. 1, No. 3 ; and Fig. 64). The cohesion of bacteria into masses depends largely on the character of the envelope. If the latter is glutinous, tlien 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 knownas a zoogloea mass. On the other hand, if the envelope lias not this cohesive property the separation of * 4 GENERAL MORPHOLOGY AND BIOLOGY 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 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. — When a bacterial cell is placed in favourable surroundings it multiplies ; 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 multiplication go on with great rapidity. A bacterium may reach maturity and divide in from twenty minutes to half an hour. If division takes place only every hour, from one individual after twenty-four hours 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 unfavour- able 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 maintaining 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, homo- geneous appearance, while in an old culture only a small proportion of the bacteria may stain at all. Sometimes, 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 being again transferred to favourable conditions, only slight growth at first takes place. Many individuals have undoubtedly died, and the remainder which SPORE FORMATION 5 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 thread-like 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 rod-like in shape, sometimes 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 protoplasm 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. 1, No. 11). In structure the spore consists of a mass of protoplasm surrounded by a dense membrane. This can be demonstrated by methods which will be described, the underlying principle of which is the prolonged application 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 experi- ments, found that while the bacillus anthracis in the unspored , form was killed by a two minutes' exposure to 1 per cent carbolic! acid, spores of the same organism resisted an exposure of from* one to fifteen days. When a spore is placed in suitable surroundings for growth 6 GENERAL MORPHOLOGY AND BIOLOGY it again assumes the original bacillary or spiral form. Thei capsule dehisces either longitudinally, or terminally, or trans- versely. 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 dehiscence 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 formation 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 wThen active multiplication takes place. The latter is usually referred to as the vegetative 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, spore formation only occurs at temperatures specially favourable for growth and multiplication. There is often a temperature below which, while vegetative growth still takes place, sporulation will not occur ; and in the case of b. anthracis, if the organism 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 preferring the absence of oxygen for vegetative growth, the presence of this gas favours sporulation. Most bacteriologists 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 SPORE FORMATION 7 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 continuance of growth, since not only will the food supply around the growing bacteria be rapidly exhausted, but the excretion 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 (1) its staining reaction, namely, resistance to ordinary staining fluids, but capacity of being stained by the special methods devised for the purpose (vide p. 102) ; (2) the fact that the bacterium containing the spore has higher powers of resistance 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 Arthrosporo.us Bacteria.— It is stated by Hueppe that among certain organisms, e.g. some streptococci, certain individuals may, without endogenous sporulation, 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 arthrospores. 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 preparations (vide p. 63). The movements are of a darting, rolling, or vibratile character. The degree of motility depends on the species, the temperature, the age of the growth, and on the medium in which the bacteria are growing. Sometimes the movements are most active just after the cell has multiplied, 8 GENERAL MORPHOLOGY AND BIOLOGY 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. 112). 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 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 dependent 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 information 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 appear- ances 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 appearance of a short chain of cocci. They are often called metachromatic granules (vide Fig. 1, 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. For the demonstration of the metachromatic granules two methods have been advanced. Ernst recommends that a few drops of Loffler's methylene blue (vide p. 98) be placed on a cover-glass preparation and the 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 in watery Bismarck- brown. The granules are here stained blue, the protoplasm brown. STRUCTURE OF BACTERIAL PROTOPLASM 9 Neisser stains a similar preparation in warm carbol-fuchsin, washes with 1 per cent sulphuric acid, and counter-stains with Loffler's blue. Here the granules are magenta, the protoplasm blue. The 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 franules (vide Fig. 1, No. 16, the bacillus most to the right), (German, olkornchen or Polkorner). With regard to the significance that is to be attached to such appearances, much depends on whether they are constantly present under all circumstances, or only occasionally, when the organism is grown in special media or under special growth conditions. Some bacteria, however stained, show evidence of having the protoplasm somewhat granular, e.g. the diphtheria bacillus. In other cases this granular condition is only seen when the organism has been grown under bad conditions, or where the food supply is becoming exhausted. Some have thought that the appearances might be due to a process allied to mitosis and might signify approaching division, but of this there is no evidence. In perfectly healthy and young bacteria, appearances of granule formation and of vacuolation may be accidentally produced by physical means in the occurrence of wha*t is known as plasmolysis. To speak generally, when a mass of protoplasm surrounded by a fairly firm envelope of a colloidal nature is placed in a solution containing 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 making 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, in certain cases, of Polkorner may thus be brought about. Plasmolysis in bacteria has been extensively investigated,1 and has been found to occur in some species more readily than in others. Furthermore it is often most readily observed in old or otherwise enfeebled cultures. Btitschli, from a study of some large sulphur-containing forms, con- cludes 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 escapes notice, unless when, as in the bacilli, it can be made out at the ends of the cells. Fischer, it may be said, looks on the appearances seen in Biitschli's preparations as due to plasmolysis. 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 schizophyceaB. 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, 1 Consult Fischer, " Untersuchuugen iiber Bakterien," Berlin, 1894; "Ueber den Bau der Cyanophyceeu und Bakterien," Jena, 1897. 10 GENERAL MORPHOLOGY AND BIOLOGY are brilliantly coloured, though few bacteria associated with the production 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 ; but it is usually 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; but 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° 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, however, has found that many of them belong to a group of colouring matters which occur widely in the vegetable 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. 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 isolated from the bodies of certain putrefactive bacteria proteid bodies which, according to Ruppel, appear to have been allied to peptone, and which differed from nucleo- proteids in not containing phosphorus, but many of the proteids isolated by other chemists have been allied in their nature to the protoplasm of the nuclei of cells. Buchner in certain researches obtained bodies of this nature allied to the vegetable caseins, and he adduces evide to show that it is to these that the characteristic staini properties are due. Various observers have isolated similar phosphorus-containing proteids from different bacteria. Besides proteids, however, substances of a different nature have been isolated. Thus cellulose, fatty material, chitin, wax-like bodies, and other substances have been observed. There are also found THE CLASSIFICATION OF BACTERIA 11 various mineral salts, especially those of sodium, potassium, and magnesium. The amount of different constituents varies ac- cording to the age of the culture and the medium used for growth, and certainly great variation takes place in the com- position of different species. The Classification of Bacteria. — There have been numerous schemes set forth for the classification of bacteria, the funda- mental 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 suggested that in every species there should be studied the habitat, best food supply, condition as to gaseous environment, range of growth, temperature, morphology, life history, special properties and pathogenicity. We must thus be content with a provisional and incomplete 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 classification. 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 sub- division 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. 115). 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 represented in every cell. They present three distinct type forms, the coccus, the bacillus, and the spirillum ; endogenous sporulation may occur. They may also be motile. 1. The Cocci. — In this group the cells range in different species from '5 //, to 2 //, in diameter, but most measure about 1 p. Before division they may increase in size in all directions. The species are usually classified according to the method of division. 1 For the illustration of this aud the succeeding systematic paragraphs, vide Fig. 1. 12 GENERAL MORPHOLOGY AND BIOLOGY 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 strepto- coccus. If division takes place irregularly the resultant mass may be compared to a bunch of grapes, and the species is often called a staphylococcus. 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 mfcrbcocci 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 phenomenon 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 staphylococci probably exceeds 150. Usually included in this group are coccus-like organisms which divide in three axes at right angles to one another. These are usually referred to as sarcinm. 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 //. 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 con- sequence of the different artilicial meanings assigned to the essentially synonymous terms bacterium and bacillus. Migula, for instance, applies THE LOWER BACTERIA 13 15& IS , FIG. 1. — 1. Coccus. 2. Streptococcus. 3. Staghylococcus. 4. Capsulated cliplococcus. 5. " Biscuit "-shaped coccus.. 6. Tetrads.. ?. Sarcina form. 8. Types of bacilli (1-8 are diagrammatic). 9. Non-septate spirillum xlOOO. 10. Ordinary spirillum — (a) comma-shaped element; (b) formation of spiral by comma-shaped elements XlOOO. 11. Types of spore formation. 12. Flagellated bacteria. 13. Changes in bacteria produced by plasmolysis (after Fischer). 14. Bacilli with terminal proto- plasm (Biitschli). 15. (a) Bacillus composed of five protoplasmic meshes ; (b) proto- plasmic network in npcrococcus (Biitschli). 16. Bacteria containing nietachromatic granules (Ernst, Neisser) — some contain polar granules. 17. Beggiatoa alba. Both filaments contain sulphur granules — one is septate. 18. Thiothrix tenuis (Wino- gradski). 19. Leptothrix innominata (Miller). 20. Cladothiix dichotoma (Zopt). 21. Streptothrixactinomyces'(Bostrom), (a) colony under low power ; (b) filament showing true branching ; (c) filament containing coccus-like bodies ; (d) filament with club at end. 14 GENERAL MORPHOLOGY AND BIOLOGY the former term to non-motile species, the latter to the motile. Hueppe, on the other hand, calls those in which endogenous sporulation 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 (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 alternating, then a wavy or spiral thread results. An example of this is the chojej^-microbe (Fig. 1, No. 10). This latter type is of much more frequent occurrence, and contains the more important species. Among the first group motility is often not associated, as far as is known, with the possession of flagella. The cells here apparently move by an undulating 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, spirochaete, vibrio. Migula makes " vibjyo " 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 " spirochfete " is reserved for the long unflagellated spiral cells. Hueppe applies the term " spirochfete " to forms without endospores, "vibrio" to those with endospores in which during sporula- tion the organism changes 'its form, and "spirillum" to the latter when no change of form takes place in sporulation. Flugge, another systematist, applies " spirochsete " and "spirillum" indiscriminately to any wavy or corkscrew form, and "vibrio" to forms where the undula- tions 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. Quite recently great doubt has arisen as to whether many of the non-septate spirillary forms are to be looked on as bacteria at all, — the view being taken that in, it may be, many cases they represent a stage in the life history of what are really protozoa of the nature of trypanosomes. The ultimate classifica- tion of the spirilla must thus be left an open question. II. The Higher Bacteria. — These show advance on the lower in consisting of definite filaments branched or unbranched. In THE HIGHER BACTERIA 15 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 interdependent on one another, and special staining methods are often necessary to demonstrate the septa which demarcate 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 advance, however, consists in the setting apart among most of the higher bacteria of the free terminations of the filaments for the produc- tion of new individuals, as has been described (p. 2). 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 hggcizatoa 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 re- sembles 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 thiothrix 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, and on dividing again, it 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, and several other important pathogenic agents. Here the organism consists of a felted mass of non-septate filaments, in which true dichotomous branching occurs. Under certain circumstances 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 16 GENERAL MORPHOLOGY AND BIOLOGY spores. Sometimes too the protoplasm of the filaments breaks up into bacillus-like elements, which may also have the capacity of originating new individuals. In the streptothrix 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, though its morphology and relationships are much disputed, may be looked on as 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 bacteria are chiefly found living on 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. 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 complexity of 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 the natural conditions. 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, 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 pro- duces pigment, e.g. b. pyocyaneus growing on gelatin. In supplying artificial food for bacterial growth, the general principle ought to be to imitate as nearly as possible the natural surround- RELATION TO GASEOUS ENVIRONMENT 17 ings, though it is found that there exists a considerable adapta- bility among organisms. With the pathogenic 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, infusions of the plants on which they grow are frequently used. Some bacteria can exist on inorganic food, but most require organic material to be supplied. Of the latter, some require for their proper nourishment proteid to be present, while others can derive their nitrogen from such a non-proteid as asparagin. All bacteria require nitrogen to be present in some form, and many require to derive their carbon from carbohydrates. Mineral salts, especially sulphates, chlorides, and phosphates, and also salts of iron are necessary. Occasionally special substances are needed 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 spirillum, will not grow in the presence of the smallest amount of free acid. Moisture. — The presence of water is necessary for the con- tinued 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 staphylococcus pyogenes aureus will survive ten days' drying, and the bacillus diphtherise still more. In the case of spores 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. Relation to Gaseous Environment. — The relation of 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 2 18 GENERAL MORPHOLOGY AND BIOLOGY called obligatory anaerobes. 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 can be anaerobes, and those which are preferably anaerobes, but can be aerobes. As a matter of fact such (littVivnces are manifested to a slight degree, but all such organisms are usually grouped as facultative anaerobes, i.e. pre- ferably aerobic but capable of existing without oxygen. Examples of obligatory aerobes are b. proteus vulgaris, b. subtilis ; of obligatory anaerobes, b. tetani, b. oedematis 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 atmosphere 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 or 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 tempera- ture above which growth does not take place, and a minimum temperature below which growth does not take place. As a general rule the optimum temperature 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 (20° to 24° C.) may be taken as the average optimum, while for organisms normally inhabiting animal tissues 35° to 39° C is a fair average. The lowest limit of ordinary growth is from 12° to 14° C., and the upper is from 42° to 44° C. In exceptional cases growth may take place as low as 5° C., and as high as 70° C. Some organisms which grow best at a temperature of from 60° to 70° C. have been isolated from dung, the intestinal tract, etc. These have been called thermophilic bacteria. 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 16° C., a culture might CONDITIONS AFFECTING BACTERIAL MOTILITY 19 be cooled to -32° C. without being killed. With regard to the upper limit, few ordinary organisms in a spore-free condition will survive a temperature of 57° 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 tempera- tures, lose their capacity of producing pigment, e.g. spirillum rubrum. 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. It has been found that an exposure of dry anthrax spores for one and a half hours to sunlight kills them. When they are moist, a much longer exposure is necessary. Typhoid bacilli are 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 ex- cluded, 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 sun- light. 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. Some bacteria especially occurring on the dead bodies of fresh fish are phosphorescent. 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, movement 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 the fact that bacilli may be 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 20 • GENERAL MORPHOLOGY AND BIOLOGY occur when *the bacteria were dead and therefore only subject to physical conditions. 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. Pfeffer 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 this into a drop of fluid containing the bacteria under a cover-glass, and to watch the effect through the microscope. 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 concentration 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 chemio- tactic 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 chief effect of bacterial action in nature is to break up into more simple combinations the complex molecules of the organic substances which form the bodies of plants and animals, or which are excreted by them. In some cases we kno*v 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, and that from urea ammonium carbonate is produced by the micrococcus ureae. That the very compli- cated process of putrefaction is due to bacteria is absolutely proved, for any organic substance can be preserved indefinitely from ordinary putrefaction by the adoption of some method of killing all bacteria present in it, as will be afterwards described. This statement, however, does not exclude the fact that molecular ACTION OF BACTERIAL FERMENTS 21 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 circum- stances, 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 saprophytes. They are normally engaged in breaking up dead animal and vegetable matter. Others normally live on or in the bodies of plants and animals and produce disease. _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 distinction. 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 saprophytes. 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 undergo in being split up by bacteria 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. 22 GENERAL MORPHOLOGY AND BIOLOGY 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 destruc- tion always goes further, and still simpler substances, e.g. indol, and, it may be, crystalline bodies of an alkaloidal nature, 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 produced. During bacterial growth there is not infrequently the abundant production of such gases as sulphuretted 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 substances, 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 production by them of ferments of a very varied nature and complicated action. Thus the digestive action on albumins probably 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, also occur. 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 ferment diffused out into the sur- rounding medium probably break down the constituents of the latter to some extent, and prepare them for a further, probably intracellular, disintegration. Thus in certain putre- factions 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 pro- VARIABILITY AMONG BACTERIA 23 duction 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 ureae, which from urea forms ammonium carbonate by adding water to the urea molecule. Here, if after the action has commenced, 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. In considering the effects of bacteria in nature it must be recognised that some species are capable of building up complex substances out of simple chemical compounds. Examples of these are found in the bacteria which in the soil make nitrogen more available for plant nutri- tion by converting ammonia into nitrites and nitrates. Winogradski, by using media containing non-nitrogenous salts of magnesium, potassium, and ammonium, and free of organic matter, has demonstrated the existence of forms which convert, by oxidation, ammonia into nitrites, and of other forms which convert these nitrites into nitrates. Both can derive their necessary carbon from alkaline carbonates. Other bacteria, or organisms allied to the bacteria, exist which can actually take up and combine into new compounds the free nitrogen of the air. These are found in the tubercles which develop on the rootlets of the leguminosae. Without such organisms the tubercles do not develop, and without the development of the tubercles the plants are poor and stunted. Bacteria thus play an important part in the enrichment and fertilisation of the soil. 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, and at one time very divergent views were held. Some even thought that the same species might at one time give rise to one disease, — at another time to another. There is, however, now practical unanimity that bacteria show as distinct species as the other lower plants and animals, though, of course, the difficulty of defining the concept of a species is as great in them as it is in the latter. Still, we can say that among the bacteria we see exhibited (to use the words of De Bary) "the same periodically repeated course of development within certain empirically determined limits of variation " which justifies, among higher forms of life, a species to be recognised. What at first raised doubts as to the occurrence of species among the bacteria was the observation in certain cases of what is known as pleomorphism. By this is meant that one species may assume at different times different forms, e.g. appear as a coccus, a bacillus, or a leptothrix. Undoubtedly, many of the cases where this was alleged to have been observed occurred before the elaboration of the modern technique for the obtaining of pure cultures, but at the present day there are cases where evidence appears to exist 24 GENERAL MORPHOLOGY AND BIOLOGY of the occurrence of pleomorphisra. This is especially the case with certain bacilli, and it may lead to such forms being classed among the higher bacteria. Pleomorphism is, however, a rare condition, and with regard to the bacteria as a whole we may say that each variety tends to conform- to a definite type of structure and function which is peculiar to it and to it alone. 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 have forty-eight or more generations under obser- vation within twenty- four hours. CHAPTEE 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. 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. The recognition of different species of bacteria depends, in fact, far more on the characters 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. Again, in inquiring as to the possible possession of pathogenic properties by a bacterium, the obtaining of pure cultures is absolutely essential. 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 used 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 preparation 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 the latter have been obtained. We shall here find that different methods 25 26 METHODS OF CULTIVATION OF BACTERIA are necessary according as we are dealing with aerobes or atiaerobes. 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, but underlying these methods is the general principle that all bacteria are destroyed by heat. 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 100° 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 100° 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 will be found, are so constantly in use. • A dull heat is used for cauteries, the points of forceps, 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 in the top 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 STERILISATION BY MOIST HEAT 27 one hour to a temperature of 170° C., is sufficient to kill all the organisms which usually pollute articles in a bacteriological laboratory, though circum- stances might arise where this would be insufficient. This means of sterilisation is used for the glass flasks, 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 heat- ing it, and to allow them to stand in it after sterilisation till the temperature falls. Sud- den heating or cooling is apt to cause glass to crack. The method is manifestly unsuitable for food media. FIG. 2. — Hot-air steriliser. B. Sterilisation by Moist Heat. B. (1) By Boiling. — The boiling of a liquid for five minutes is sufficient to kill ordinary germs if no spores be present, and this method is useful for sterilising distilled or tap water which may be required in various manipulations. It is best to sterilise knives and instruments used in autopsies by boiling in water to which a little sodium carbonate has been added t9 prevent rusting. ' Twenty minutes' boiling will here be sufficient. The boiling of any fluid at 100° C. for one and a half hours will ensure sterilisation under almost any circumstances. B. (2) By Steam at 100° C.— This is by far the most useful means of sterilisation. It may be accomplished in an ordinary potato steamer placed on a kitchen pot. The apparatus ordinarily used is " Koch's steam steriliser" (Fig. 3). This consists FIG. 3.— Koch's steam of a tal1 metal cylinder on legs, provided steriliser. with a lid, and covered externally by 28 METHODS OF CULTIVATION OF BACTERIA some bad conductor of heat, such as felt or asbestos. A perforated tin diaphragm is fitted in the interior at a little distance above the bottom, and there is a tap at the bottom by which water may be supplied or withdrawn. If water to the depth of 3 inches be placed in the interior and heat applied, it will quickly boil, and the steam streaming up will surround any flask or other object standing on the diaphragm. Here no evaporation takes place from any medium as it is surrounded during sterilisation by an atmosphere saturated with water vapour. It is convenient to have the cylinder tall enough to hold a litre flask with a funnel 7 inches in diameter standing in its neck. The funnel may be supported by passing its tube through a second perforated diaphragm placed in the upper part of the steam chamber. With such a " Koch " in the laboratory a hot-water filter is not needed. As has been said, one and a half hour's steaming will sterilise any medium, but in the case of media containing gelatin such an exposure is not practic- able, as with long boiling, gelatin tends to lose its physical property of solidification. The method adopted in this case is to steam for a quarter of an hour on each of three succeeding days. This is a modification of what is known as " Tyndall's intermittent sterilisation." The fundamental principle of this method is that all bacteria in a non-spored form are killed by the temperature of boiling water, while if in a spored form they may not be thus killed. Thus by the sterilisation on the first day all the non-spored forms are destroyed — the spores remaining alive. During the twenty-four hours which intervene before the next heating, these spores, being in a favourable medium, are likely to assume the non-spored form. The next heating kills these. In case any may still not have changed their spored form, the process is repeated on a third day. Experience shows that usually the medium can now be kept indefinitely in a sterile condition. .Steam at 100° C. is therefore available for the sterilisation of all^ ordinary media. In using the Koch's steriliser, especially when a large bulk of medium is to be sterilised, it is best to put the media in while the apparatus is cold, in order to make certain that the whole of the food mass reaches the temperature of 100° C. The period of exposure is reckoned from the time boiling commences in the water in the steriliser. At any rate allowance must always be made for the time required to raise the temperature of the medium to that of the steam surrounding it. If we wish to use such a substance as blood serum as a medium, the albumin would be coagulated by a temperature of 100° C. Therefore other means have to be adopted in this case. STERILISATION" BY HIGH-PRESSURE STEAM 29 °o o o o o o B. (3) Sterilisation by Steam at High Pressure. — This is the most rapid and effective means of sterilisation. It is effected in an autoclave (Fig. 4). This is a gun-metal cylinder supported in a cylindrical sheet-iron case ; its top is fastened down with screws and nuts and is furnished with a safety valve, pressure- gauge, and a hole for thermometer. As in the Koch's steriliser, the contents are supported on a perforated diaphragm. The source of heat is a large Bunsen beneath. The temperature employed is usually 115° C. or 120° C. To boil at 115° C., water requires a pres- sure of about 23 Ibs. to the square inch (i.e. 8 Ibs. plus the 15 Ibs. of ordinary atmo- spheric pressure). To boil at 120° C., a pressure of about 30 Ibs. (i.e. 15 Ibs. plus the usual pressure) is necessary. In such an apparatus the desired temperature is main- tained by adjusting the safety valve so as to blow off at the corresponding pressure. One exposure of media to such temperatures for a quarter of an hour is amply sufficient to kill all organisms or spores. Here, again, care must be taken when gelatin is to be sterilised. It must not be exposed to a temperature above 105° C., and is best sterilised by the intermittent method. Cer- tain precautions are necessary in Using the autoclave. In all cases it is necessary to Fia 4 Autoclave. allow the apparatus to cool well below 1 00° C., before opening it or allowing steam to blow off, otherwise there will be a sudden development of steam when the pressure is removed, and fluid media will be blown out of the flasks. Sometimes the instrument is not fitted with a thermometer. In this case care must be taken to expel all the air initially present, otherwise a mixture of air and steam being present, the pressure read off the gauge cannot be accepted as an indication of the temperature. Further, care must be taken to ensure the presence of a residuum of water when steam is fully up, otherwise the steam is super- heated, and the pressure on the gauge again does not indicate the temperature correctly. B. (4) Sterilisation at Low Temperatures. — Most organisms in a non-spored form are killed, by a prolonged exposure to a temperature of 57° C. This fact has been taken advantage of for the sterilisation of blood serum, which will coagulate if exposed to a rt. Safety-valve. 6. Blow-off pipe, c. Gauge. 30 METHODS OF CULTIVATION OF BACTERIA FIG. o. — Steriliser for blood serum. temperature above that point. Such a medium is sterilised on Tyndall's prin- ciple by exposing it for an hour at 57° C. for eight consecutive days, it being allowed to cool in the interval to the room temperature. The apparatus shown in Fig. 5 is a small hot- water jacket heated by a Bunsen placed beneath it, the temperature being controlled by a gas regulator. To ensure that the temperature all around shall be the same, the lid also is hollow and filled with water, and there is a special gas burner at the side to heat it. This is the form originally used, but serum sterilisers are now constructed in which the test-tubes are placed in the sloped position, and in which inspissation (vide p. 40) can after- wards be performed at a higher temperature. THE PREPARATION OF CULTURE MEDIA. The general principle to be observed in the artificial culture of bacteria is that the medium used should approximate as closely as possible to that on which the bacterium grows naturally. In the case of pathogenic bacteria the medium therefore should resemble the juices of the body. The serum of the blood satisfies this condition and is often used, but its application is limited by the difficulties in its preparation and preservation. Other media have been found which can support the life of all the pathogenic bacteria isolated. These consist of proteids or carbohydrates in a fluid, semi-solid, or solid form, in a trans- parent or opaque condition. The advantage of having a variety of media lies in the fact that growth characters on particular media, non-growth on some and growth on others, etc., constitute specific differences which are valuable in the identification of bacteria. The most commonly used media have as their basis a watery extract of meat. Most bacteria in growing in such an extract cause only a grey turbidity. A great advance resulted when Koch, by adding to it gelatin, provided a transparent solid medium in which growth characteristics of particular bacteria PREPARATION OF MEAT EXTRACt 31 become evident. Many organisms, however, grow best at a temperature at which this nutrient gelatin is fluid, and there- fore another gelatinous substance called agar, which does not melt below 98° C., was substituted. Bouillon made from meat extract, gelatin, 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 containing less fat than the others ; though generally quite 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 1000 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 gathering up the edges of the latter in the left hand, squeeze out the juice still held back in the con- tained 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, other- wise in the later stages it will become opalescent. A large quantity of the extract may be made at a time, and what is not FIG. 6. — Meat press. 32 METHODS OF CULTIVATION OF BACTERIA 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 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. 1. Bouillon Media. — These consist of meat extract with the addition of certain substances to render them suitable for the growth of bacteria. (1) (a). Peptone Broth or Bouillon. — This has the com- position : — Meat extract .... 1000 c.c. Sodium chloride ... 5 grms. Peptone albumin . . . 10 „ Boil till the ingredients are quite dissolved, and neutralise with a saturated 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. Neutralisation 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. 27, 29). This method of neutralisation is to be recommended 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. 165) 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, will- not grow at all on even a slightly acid medium. STANDARDISING THE REACTION OF MEDIA 33 Standardisation of Reaction of Media. — While the above procedure of dealing with the reaction of a medium is sufficient for ordinary work, it has been thought advisable 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 undertaken by the beginner. It entails the preparation of solutions of acid and alkali which may be used for determining the original reaction of the medium, and for accurately making it of a definite degree of alkalinity. Normal J and decinormal solutions of sodium hydrate and hydrochloric acid are used. Preparation of Standard Solutions. — The first requisites here are normal solutions of acid and alkali. The latter is prepared as follows : — 85 grammes of pure sodium bicarbonate are heated to dull redness for ten minutes in a platinum vessel and allowed to cool in an exsiccator ; just over 54 grammes of sodium carbonate should now be present. Any excess is quickly removed, and the rest being dissolved in one litre of distilled water, abnormal solution is obtained. A measured quantity is placed in a porcelain dish, and a few drops of a '5 per cent solution of phenol- phthaleine in neutral methylated spirit is added to act as indicator. The alkali produces in the latter a brilliant rose pink which, however, disappears on the least excess of acid being present. The mixture is boiled and a solution of hydrochloric acid of unknown strength is run into the dish from a burette till the colour goes and does not return after very thorough stirring. The strength of the acid can then be calculated, and a normal solution can be obtained. From these two solutions any strength of acid or alkali (such as the decinormal solution of NaOH mentioned below) may be derived. As Eyre has suggested, the reaction "of a medium may be conveniently expressed by the sign + or - to indicate acid or alkaline respectively, and a number to indicate the number of cubic centimetres of normal acid or alkaline solution necessary to make a litre of the medium neutral to phenol-phthaleine. Thus, for example, "reaction = - 15," will mean that the medium is alkaline, and requires 15 c.c. of normal HC1 to make a litre 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 compound by one atom of hydrogen (e.g. sodium), an equivalent is the molecular weight in grammes. In the case of NaCl, it would be 58'5 grammes (atomic weight of Na = 23, of Cl = 35'5). If the metal be bivalent, i.e. requiring two atoms of H for its replacement in a compound (e.g. calcium), an equivalent is the molecular weight in grammes divided by two. Thus iu the case of CaClo an equivalent would be 55 '5 grammes (atomic weight of Ca — 40, of Clo — 71). 3 34 METHODS OF CULTIVATION OF BACTERIA neutral. It has been found that when a medium such as bouillon reacts neutral to litmus, its reaction to phenol-phthaleine, accord- ing to the above standard, is on the average + 25. 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. It is probably safe to say, however, that when a medium has been rendered neutral to phenol-phthaleine by the addition of NaOH, the optimum degree is generally attained by the addition of from 10 to 15 c.c. of normal HC1 per litre, i.e. the optimum reaction is •+ 10 to + 15. In other words, the optimum reaction for bacterial growth lies, as Fuller has pointed out, about midway between the neutral point indicated by phenol-phthaleine and the neutral point indicated by litmus. The only objection to the use of phenol-phthaleine is that its action is somewhat vitiated if free CO2 be present. This can be completely obviated as follows. Before testing 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. 44, 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 following procedure includes most of the improvements introduced by Eyre. The medium with all its constituents dissolved is filtered and then heated for about 45 minutes in the steamer, the maximum acidity being reached after this time. Of the warm medium take 25 c.c. and put in a porcelain dish, add 25 c.c. distilled water, and 1 c.c. phenol- phthaleine solution. Run in decinormal soda till neutral point is reached, indicated by the first trace of pink colour, the mixture being kept hot.1 Repeat process thrice, and take the mean ; this divided by 10 will give the amount (x) of normal soda required to neutralise 25 c.c. of medium ; then 40 x = amount necessary to neutralise a litre; and 40 #-10 = amount of normal soda necessary to give a litre its optimum reaction. Then measure the amount of medium to be dealt with, and add the requisite amount of soda solution. 1 The beginner may find considerable difficulty in recognising the first tint of pink in the yellow bouillon. A good way of getting over this is to take two samples of the medium, adding the indicator to one only ; then to run the soda into these from separate burettes ; for each few drops run into the medium containing the indicator the same amount is run into the other. Thus the recognition of the first permanent change in tint will be at once recognised by comparing the two lots of solution. GELATIN MEDIA 35 Eyre uses a soda solution of ten times normal strength, which is delivered out of a 1 c.c. pipette divided into hundred ths ; this obviates, to a large extent, the error introduced by increasing the bulk of the medium on the addition of the neutralising solution. 1 (b). 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 there- fore glucose broth is used as a culture fluid for anaerobic organisms. 1 (c). Glycerin Broth. — The initial steps are the same as in 1 (a), but after filtration 6 to 8 per cent of glycerin (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. Gelatin Media. — These are simply the above broths, with gelatin added as a solidifying body. 2 (a). Peptone Gelatin : — Meat extract Sodium chloride Peptone albumin Gelatin . 1000 c.c. 5 grms. 10 „ 100-150 (the "gold label" gelatin of Coignet et Cie, Paris, is the best). The gelatin 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 or diaphragm, 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 funnel with water- jacket which is heated, as shown in Fig. 7. Whichever instrument be used, before filtering shake up the melted medium, as it is apt FIG. 7. — Hot-water funnel. 36 METHODS OF CULTIVATION OF BACTERIA 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 cir- cumstances is quite clear. A litre flask of the finished product ought to be quite transparent. If, however, it is partially opaque, add the white of an egg, shake up well, 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. 27. If the autoclave be used the temperature employed must not be above 105° 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 gelatin medium to lose its property of solidification. The exact percentage of gelatin 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 gelatin be too stiff, it will split on the perforation of its substance by the platinum needle used in inoculating it with a bacterial growth ; 15 per cent gelatin melts at about 24° C. 2 (&). Glucose Gelatin. — 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 gelatin 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 gelatin. Unlike the latter, which is a proteid, agar is a carbohydrate. It is derived from the stems of various sea-weeds growing in the Chinese seas, popularly classed together as " Ceylon Moss." For bacteriological purposes the dried stems of the seaweed may be used, but there is in the market a purified product in the form of a powder ; this is preferable. 3 (a). " Ordinary " Agar. — This has the following composi- tion : — Meat extract . . . . . . 1000 c.c. Sodium chloride . . . . * 5 grms. Peptone albumin . l. . . 10 ,, Agar 15 „ AGAR MEDIA 37 Cut up the agar into very fine fragments (in fact till it is as nearly as possible dust), add to the meat extract with the other ingredients, and preferably allow to stand all night. Then boil gently in a water bath for two or three hours, till the agar is thoroughly melted. The process of melting may be hastened by boiling the medium in a sand bath and passing through it a stream of steam generated in another flask ; the steam is led from the second flask by a bent glass tube passing from just beneath the cork to beneath the surface of the medium (Eyre). After melting render slightly alkaline with sodium hydrate solution, make up to original volume with distilled water, and filter. Filtration here is a very slow process and is best 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 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 minute's or in Koch's steriliser for one and a half hours. Agar melts just below 100° C., and on cooling solidifies about 39° C. 3 (6). Glycerin Agar.— To 3 (a) after filtration add 6 to 8 per cent of glycerin 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 gelatin. It is also a superior culture medium for some aerobes, e,g. the b. diphtherise. These bouillon, gelatin, and agar preparations constitute the most frequently used media. Growths in 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 gelatin media. These have, however, the disadvantage 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 gelatin. It is, for instance, never liquefied, whereas some organisms, by their growth, liquefy gelatin and others do not — a fact of prime importance. Litmus Media. — To any of the above media litmus (French, 38 METHODS OF CULTIVATION OF BACTERIA tournesol) may be added to show change in reaction during bacterial growth. The litmus is added, before sterilisation, as a strong watery solution (e.g. the Kubel-Tiemann solution, vide p. 42) 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. Use of neutral red. — This dye has been introduced as an aid in determining the presence or absence of members of the b. coli group, especially in the examination of water. The media found most suitable are agar or bouillon containing '5 per cent of glucose, to which *5 per cent of a one per cent watery solution of neutral red is added. The use of these media and their probable value are described below (vide Typhoid Fever). Blood Agar : Serum Agar. — The former medium was intro- duced 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 pneumococcus. Human blood or the blood of animals may be used. " Sloped tubes " (vide p. 48) of agar are employed (glycerin agar is' not so suitable). Purify a finger first with 1-1000 corrosive sublimate, dry, and then wash with 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. 49), 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 or two days at 38° C. before 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. A medium composed of one part of fresh blood (drawn aseptically) and two parts of fluid agar at 40° C., has been used for the cultivation of the bacillus of soft sore. Serum agar is prepared in a similar way by smearing the surface of the agar with blood serum, or by adding a few drops of serum to the tube and then allowing it to flow over the surface. Peptone Solution. V A simple solution of peptone (Witte) constitutes a suitable culture medium for many bacteria, The peptone in the propor- tion of 1 to 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 reaction is usually distinctly alkaline, which condition is suitable for most purposes. For BLOOD SERUM 39 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 (p. 75). Litmus may be added to show any change in reaction. Blood Serum. Koch introduced this medium, and it is prepared as follows : Plug the mouth of a tall cylindrical glass vessel (say of 1000 c.c. capacity) 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 subsequently 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. If a centrifuge is available, a large yield of serum may be obtained by centri- fugalising the freshly drawn blood. If coagulation has occurred, the clot must first be thoroughly broken up. 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 con- taminated 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 on each of eight successive days. It is always well to incubate it for a day at 37° C. before use, to see that the result is successful. After sterilisation it is "inspissated," by which process a clear solid medium is obtained. " Inspissa- tion " is probably an initial stage of coagulation, and is effected by keeping the serum at 65° 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 for the purpose is one of the various forms of serum steriliser (e.g. Fig. 8), generally a chamber with water- jacket heated with a Bunsen below. The temperature is con- trolled by a gas regulator, and such an apparatus can, by altering the temperature, be used either for sterilisation or inspissation. As is evident, the preparation of this medium is tedious, but its 40 METHODS OF CULTIVATION OF BACTERIA use is necessary for the observation of particular characteristics in several pathogenic 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 precau- tions, then sterilisation of the serum is unnecessary. To this end the mouth of the cylinder used for col- lecting 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 conveni- ent 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. In every case the serum must be incubated before use, to make sure that it is sterile. Coagulated Blood Serum. — If fresh serum be placed in sterile tubes and be steamed in the sloped position for an hour it coagulates, and there is thus obtained a solid medium very useful for the growth of the diphtheria bacillus for diagnostic purposes. Loffler's Blood Serum. — This is the best medium for the FIG. 8. — Blood serum inspissator. BLOOD SERUM 41 growth of the b. diphtheriae and may be used for other organisms. It has the following composition. Three parts of calf's 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 (p. 29). Alkaline Blood Serum (Lorrain Smith's Method). — To each 100 c.c. of the serum obtained as before, add 1 to 1*5 c.c. of a 10 per cent solution of sodium hydrate and shake 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. Lorrain 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. diphtherise. 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 diffi- culty 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 usual aseptic precautions" being taken. In the case of these media, sterilisation is effected by method B (4), and they are used fluid. Hiss's Serum Water Media. — These are composed of one part of ox's serum and three parts of distilled water with 1 per cent litmus ; various sugars in a pure condition are added in the proportion of 1 per cent. The development of acid by fermentation is shown by the alteration of the colour and by 42 METHODS OF CULTIVATION OF BACTERIA coagulation of the medium. These media do not coagulate at 100° C. and thus can be sterilised in the steam steriliser. They have been extensively used by American workers in studying the fermentative properties of the b. dysenteriae, b. coli, etc. Drigalski and Conradi's Medium. — This is one of the media used for the study of intestinal bacteria and especially for the isolation of the typhoid group of organisms, (a) Three pounds of meat are treated with two litres of water overnight ; the fluid is separated as usual, boiled for an hour, filtered, and there are added 20 grammes Witte's peptone, 20 grammes nutrose,1 10 grammes sodium chloride ; the mixture is then boiled for an hour, 60 grammes finest agar are added, and it is placed in the autoclave till melted (usually one hour) ; it is then rendered slightly alkaline to litmus, filtered, and boiled for half an hour, (b) 260 c.c. Kubel- Tiemann litmus 2 solution is boiled for ten minutes, 30 grammes milk sugar (chemically pure) are added, and the mixture is boiled for fifteen minutes ; (a) and (b) are then mixed hot, well shaken, and, if necessary, the slightly alkaline reaction restored. There are then added 4 c.c. of a 10 per cent sterile solution of water-free sodium hydrate and 20 c.c. of a freshly prepared solution made by dissolving '1 gramme crystal- violet B, Hoechst, in 100 c.c. hot sterile distilled water. This is the finished medium, and great care must be taken not to overheat it or to heat it too long, "as changes in the lactose may be originated. It is convenient to distribute the medium in 80 c.c. flasks. The principle of the medium is that while there is a food supply very favourable to the b. typhosus and the b. coli the antiseptic action of the crystal-violet tends to inhibit the growth of other bacteria likely to occur in material which has been subjected to intestinal contamination. In examining faeces a little is rubbed up in from ten to twenty times its volume of sterile normal salt solution ; in the case of urine or water the fluid is centrifugalised and the deposit or lower portion is used for the inoculation procedures. For use the medium is distributed in Petri capsules in a rather thicker layer than is customary in an ordinary plate. The sheet of medium must be transparent, but must not be less than 2 mm. in thickness — in fact, ought to be about 4 mhi. After being poured, the capsules are left with the covers off for an hour or so, to allow the superficial layers of the medium to become set hard. The effect of this is that during incubation no water of condensation forms on the lid of the capsule, and thus the danger of this fluid dropping on to the developing colonies is avoided. The antiseptic nature of the crystal- violet is sufficient to prevent the growth of any aerial organisms falling 1 Nutrose is an alkaline preparation of casein. 2 The litmus solution is made as follows. Solid commercial litmus is digested with pure spirit at 30° C. till on adding fresh alcohol the latter becomes only of a light violet colour. A saturated solution of the residue is then made in distilled water and filtered. When this is diluted with a little distilled water it is of a violet colour, which further dilution turns to a pure blue. To such a blue solution very weak -sulphuric acid (made by adding two drops of dilute sulphuric acid to 200 c.c. water) is added till the blue colour is turned to a wine-red. Then the saturated solution of the dye is added till the blue colour returns. MACCONKEY'S BILE-SALT MEDIA 43 on the agar during its exposure to the air. The plates are usually inoculated by means of a glass spatula made by bending three inches of a piece of glass rod at right angles to the rest of the rod. This part is dipped in the infective material and smeared in all directions over the surfaces of three or four plates successively without any intervening sterilisation. The plates are again exposed to the air after inoculation for half an hour and then incubated for twenty-four hours. At the end of such a period b. coli colonies are 2 to 6 mm. in diameter, stained distinctly red, and are non-transparent. Colonies of the b. typhosus are seldom larger "than 2 mm., they are blue or bluish-violet in colour, are glassy and dew-like in character, and have a single contour. Sometimes in the plates b. subtilis and its congeners appear, and colonies of these organisms have a blue colour. Their growth is, however, more exuberant than that of the typhoid bacillus, — being often heaped up in the centre, — and the contour of the colony is often double. MacConkey's Bile-Salt Media. — These media were introduced for the purpose of differentiating the intestinal bacteria, and have been exten- sively used for the study of the b. coli, b. typhosus, b. dysenterise, etc. The characteristic ingredients are bile salts and various sugars. The stock solution is the following : — Commercial sodium taurocholate, 0 '5 gramme ; Witte's peptone, 2'0 grammes ; distilled water, 100 c.c. For a liquid medium there is added to this "5 per cent of a freshly prepared 1 per cent solution of neutral red l and the sugar, — when glucose is used 0'5 per cent is added, in the case of other sugars 1 per cent. The fluid is distributed in Durham's fermentation tubes and sterilised in the steamer for ten minutes on two successive days, care being taken not to overheat the medium. For bile-salt agar 1*5 to 2 per cent agar is dissolved in the stock solution in the autoclave, if necessary cleared with white of egg and filtered. Neutral red and a sugar are added, as in the case of the liquid medium. As with Drigalski's medium, it is well to sterilise it in flasks containing 80 c.c., this being an amount sufficient for three Petri capsules. When this medium is used for examining urine or feces, plates are inoculated as with Drigalski's medium (supra} ; for its use in water examinations see p. 136. In the bile-salt bouillon the formation of both acid and gas is observed if such formation occurs, and in the bile-salt agar acid produc- tion is recognised by the red colour of the colonies of the acid-producing organisms. MacConkey's original medium was a 1 per cent bile-salt lactose agar with no indicator, and was used for the detection of intestinal bacteria in water. Such a medium is unfavourable to all the common spore- bearing organisms found in water, and by incubating at 42° C. tubes, in which there is probably a mixed infection from such a source, the growth of most other water bacteria is inhibited. B. coli and b. typhosus, on the other hand, grow readily. "With the former the surface colonies are broad, irregular, and flat, of opaque colour, and with a small spot of yellow or orange in the centre, and the colony is surrounded by a haze ; the deep colonies are lens-shaped, of orange colour, and are likewise surrounded by a haze. With the typhoid organism at the end of forty-eight hours the surface colonies are small, round, raised, and semi-transparent, while the deep colonies are lens-shaped, white, and have no surrounding 1 Neutral red gives a deep crimson with acids and a yellow -red with alkalies. 44 METHODS OF CULTIVATION OF BACTERIA haze. The haze in the case of b. coli is due to the ready production of acid from the lactose causing a precipitate of the taurocholate. Any other organism capable of producing acid from lactose will give a similar reaction, and the haze can be readily cleared up by floating a drop of ammonia on the surface of the medium. MacConkey also used with a similar object a 5 per cent glucose bile-salt bouillon tinted with neutral litmus as in Drigalski's medium. With reference to MacConkey's fluid media, organisms are divided into (1) those which produce both acid and gas ; (2) those producing acid only ; (3) those growing but not producing either acid or gas ; (4) those incapable of growing. B. coli belongs to the first group and b. typhosus to the second, and to these groups also belong most ordinary organisms growing in faeces, practically none of which are found in the third and fourth classes. Thus if any growth takes place on this medium when inoculated with, say, water, the probability is that the bacteria have been derived from faeces, but of course their identification might present some difficulty. With the neutral-red solid media the colonies of any organism giving rise to acid will be of a beautiful rose red colour. Petruschky's Litmus Whey. — 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 precipita- tion 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 sufficient Kubel-Tiemann solution of litmus is added, the medium is put into tubes and then sterilised. After growth has taken place, the amount of acid formed can be estimated by dropping in standardised soda solution till the tint of an uninoculated tube is reached. Media for growing Trichophyta, Moulds, etc. — 1. Beer Wort Agar. Take beer wort as obtainable from the brewery and dilute it till it has an s.g. of 1100. Add 1'5 per cent of powdered agar and heat in the Koch till it is dissolved (usually about two hours are necessary). Filter rapidly and fill into tubes. Sterilise in the Koch for twenty minutes on three successive days. If the medium is heated too long it loses the capacity of solidifying. 2. Sabouraud's medium (modified). Take 40 grammes maltose and 10 grammes Witte's peptone and dissolve these in one litre of water, then add 13 grammes of powdered agar. Heat in the Koch till the agar is dissolved, filter and fill into tubes, sterilise in the autoclave for twenty minutes at 120° C. To use these for isolating, say, the Tinea tonsurans, pick out an infected hair, wash in absolute alcohol for a few seconds, then wash in changes of sterile water and stab the hair into the surface of the medium in a number of places ; incubate at 24° C. Usually it is sufficient to stab the hair as it is picked from the skin into the medium. Potatoes as Culture Material. (a) In Potato Jars. — The jar consists of a round, shallow, glass vessel with a similar cover (vide Fig. 9). It is washed POTATOES AS CULTURE MATERIAL 45 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 1-1000 corrosive sublimate. They are steamed in the Koch's steriliser for thirty minutes or longer, or in the auto- clave for a quarter of an hour. When cold, each is grasped between the FIG. 9. — Potato jar. FIG. 10.— Cylinder of potato cut obliquely. left thumb and forefinger (which have been sterilised with sub- limate) and cut through the middle with 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 upper- m* most. Smaller jars, each of which holds half of a potato, are also used in the same way and are very convenient. (6) By Slices in Tubes. — -This method, intro- duced by Ehrlich, is the best means of utilising potatoes as a medium. A large, long potato is well washed and scrubbed, and peeled with a clean knife. A cylinder is then bored from its interior with an apple corer or a large cork borer, and is cut obliquely, as in Fig. 10. Two wedges are thus obtained, each of which is placed broad end downward in a test-tube of special form (see Fig. 11). In the wide part at the bottom 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 potato. introduced. After the latter are inserted, the tubes are sterilised in the Koch steam steriliser for one hour, or in the autoclave for fifteen minutes, at 115° C. An ordinary test- FIG. 11. — Ehrlich's tube contain- ing piece of 46 METHODS OF CULTIVATION OF BACTERIA tube may be used with a piece of sterile absorbent wool in its bottom, on which the potato may rest. Glycerin potato, suitable for the growth of the tubercle bacillus, may be prepared by covering the slices in the tubes with 6 per cent solution of glycerin in water and steaming for half an hour. The fluid is then poured off and the sterilisation continued for another half hour. 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-phthaleine. The required degree of acidity or alkalinity is obtained by adding the necessary quantity of HC1 or NaOH solution (p. 34) and steaming for other fifteen minutes. The water is then poured off and sterilisation continued for another half hour. Potatoes before being inoculated ought always to be incubated at 37° C. for a night, to make sure that their sterilisation has been successful. Eisner's Medium. — This is one of the media introduced in the study of the comparative reactions of the typhoid bacillus and the B. coli. The preparation is as follows : 500 grammes potato are grated up in a litre of water, allowed to stand over night, then strained, and added to an equal quantity ol' ordinary 15 per cent peptone gelatin which has not been neutralised. Normal sodium hydrate solution is added till the reaction is feebly acid to litmus, the whole boiled together, filtered, and sterilised. Just before use potassium iodide is added so as to constitute one per cent of the medium. Moore has used a similar agar preparation. Here 500 grammes potato are scraped up in one litre of water, allowed to stand for three hours, strained, and put aside over night. The clear fluid is poured off, made up to one litre, rendered slightly alkaline, 20 grammes agar are added, and the whole is treated as in making ordinary agar. The medium is distributed in test-tubes — 10 c.c. to each — and immediately before use, to each is added '5 c.c. of a solution of 10 grammes potassium iodide to 50 c.c. water. 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 THE USE OF THE CULTURE MEDIA 47 facilitate further separation of 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 obtained 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 torulse, 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 f in. These ought to be very thoroughly washed and dripped, and their mouths plugged with plain cotton wool. They are then sterilised for one hour at 170° C. If the tubes be new, the glass, being usually packed in straw, may be contaminated with the extremely resisting spores of the b. subtilis. Cotton -wool plugs are universally used for protecting the sterile contents of flasks and tubes from con- tamination with the bacteria of the air. 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 place the bouillon, gelatin, 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 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. 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 gelatin media, tubes filled one -third full and allowed to solidify while standing upright, are those commonly used. With 48 METHODS OF CULTIVATION OF BACTERIA 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 gelatin 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 contents extend 3 b c FIG. 13.— Tubes of media. - tube- maybe used for filling tubes. c< « Deep " tube for cultures of anaerobes. The apparatus explains itself. The india-rubber stopper with its tubes ought to be sterilised before use. to 4 inches up, giving an oblique surface after solidification. Thus agar is commonly used in such tubes (less frequently gelatin 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 one in which only one orgc\nism is present. The methods of USE OF CULTURE MEDIA 49 \ obtaining pure cultures will presently be described. When a fresh tube of medium is inoculated from an already 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 examination, 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.1 Every worker should have three such wires. Two are 2^ inches long, one of these being straight (Fig. 14, a), and the other having a loop turned upon it (Fig. 14, 6). The latter is referred to as the platinum "loop." or platinum " eyelet," and is used for many purposes. i FIG. 14. — Platinum wires in glass handles. die for ordinary puncture inoculations, b. " I c. Long needle for inoculating " deep " tubes. a. Straight needle for ordinary puncture inoculations, b. " Platinum loop." c. Long needle for " Taking a loopf ul " is a phrase constantly used. The third wire (Fig. 14, c) ought to be 4| inches long and straight. It is used for making anaerobic cultures. It is also very useful to have at hand a platinum-indium spud. This- consists of a piece of platinum-iridium about 1^ inches long, 2 mm. broad, and of sufficient thickness to give it a firm consistence ; its distal end is expanded into a diamond shape, and its proximal is screwed into an aluminium rod. It is very useful for making scrapings from organs and for disintegrating felted bacterial cultures ; in such manipulations the ordinary platinum wire is awkward to work with as it bends so easily. Cultures on a solid medium are referred to (1) as "puncture" or "stab" cultures (German, Stichkultur), or (2) as "stroke" or "slant" cultures (Strichkultur), according as they are made (1) on tubes solidified in the upright position, or (2) on sloped tubes. 1 Aluminium rods are made which are very convenient. The end is split with a knife, the platinum wire is inserted and fixed by pinching the aluminium on it in a vice. 50 METHODS OF CULTIVATION OF BACTERIA FIG. 15. — Another method of inoculating solid tubes. To inoculate, say, one ordinary upright gelatin tube from another, the two tubes are held in an inverted position between the forefinger and thumb of the left hand with their mouths towards the person holding them ; the plugs are twisted round once or twice, to make sure they are not adher- ing to the glass. The short, straight platinum wire is then heated to redness from point to insertion, and 2 to 3 inches of 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 cul- ture tube 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 gelatin 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 FJG- 16.— Rack for platinum needles, other between the middle and ring fingers, then to make the inoculation (Fig. 15). If a tube contain a liquid medium, it must be held in a sloping position between the same fingers, as above. For a stroke culture 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 removing the plugs, to SEPARATION OF BACTERIA 51 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 contaminated, they must be removed by heat- ing the inoculating needle red-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 Fig. 16. To prevent contamination of cultures by bacteria falling on the plugs while these are exposed to the air during inoculation manipulations, some 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. PLATE CULTURES. The general principle underlying the methods of separation is the distribution of the bacteria in one of the solid media liquefied by heat and the dilution of the mixture so that the growths produced by the individual bacteria — called colonies — shall be suitably apart. 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 — hence the term "plate cultures." As the optimum temperature varies with different bacteria, it is necessary to use both gelatin and agar media. Many pathogenic organisms, e.g. pneumococcus, b. diphtheriae, etc., grow too slowly on gelatin to allow its ready use. On the other hand, many organisms, e.g. some occurring in water, do not develop on agar incubated at 37° C. Separation by Gelatin Media. — As the naked-eye and micro- scopic appearances of colonies are often very characteristic, plate cultures, besides use in separ- ation, are often taken advantage of in the description of individual organisms. The plate - culture method can also be used to test whether a tube culture is or is not pure. The suspected culture is plated (three plates being pre- pared, as will be described). If FIG. 17.— Petri's capsule, all the colonies are the same, then (Cover shown partially raised.) the cultures may be held to be pure. Either simple plates of glass 4 inches by 3 inches are used, 52 METHODS OF CULTIVATION OF BACTERIA or, what are more convenient, circular glass cells 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 level by a special apparatus while the medium is solidifying, 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 170° 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 of glass ; in the usual laboratory routine Petri's capsules are to be preferred for the above reasons. The contents of three gelatin tubes, marked a, &, c,1 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, and can only be regulated by experience. 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 gelatin from a to b. Shake b and transfer five 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 organ- isms on its rim are thus killed. The capsules are labelled and set aside till growth takes place. For accurate work it will be found convenient to carry out the dilutions in definite proportions. The following is the pro- cedure which we have found very serviceable. In a number of small sterile test-tubes '95 c.c. sterile water is put. To the first tube we add '05 c.c. of the bacterial mixture. The contents of the tube are well shaken up, and the pipette is sterilised by being washed out with boiling water. It is allowed to cool, and •05 c.c. of fluid is transferred from the first tube to the second. By a similar procedure '05 c.c. is transferred from the second to 1 For marking glass vessels it is convenient to use the red, blue, or yellow oil pencils made for the purpose by Faber. KOCH'S METHOD OF PLATE CULTURES 53 the third and so on. There is thus effected a twenty-fold dilution in each successive tube. After these steps have been carried out a definite amount, say, '05 c.c. is transferred from each tube 'to a tube of melted gelatine, — the gelatine being after- wards plated and the colonies counted when growth occurs. The number of tubes required will vary according to the number of bacteria in the original mixture, but usually four or five will be sufficient. It is quite evident that this method not only enables us to separate bacteria, but if necessary gives us a means of estimating exactly the number in the original mixture. The colonies appear as minute rounded points, whitish or variously coloured. Their characters can be more 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 characters, colour, shape, contour, appearance of surface, liquefaction or non-liquefaction of the gelatin, 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. Gelatin or agar tubes may then be inoculated from a colony of each variety, 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. 115). 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 solidi- fying is, as has been said, necessary. An apparatus devised by Koch is used (Figs. 18, 19). 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 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 out- side and inside with perchloride of mercury 1-1000, and a circle of filter- paper moistened with the same is 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 inocu- lated as in using Petri's capsules (p. 52). The hands having been washed in perchloride of mercury 1-1000 and dried, the plate box is opened, and a plate lifted by its opposite edges and transferred to the 54 METHODS OF CULTIVATION OF BACTERIA levelled ground glass (as in Figs. 18, 19). The bell jar of the levelle being now lifted a little, the gelatin in tube a is poured out on thi surface of the sterile plate, and while still fluid, is spread by stroking FIG. 18. — Koch's levelling apparatus for use in preparing 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. with the rim of the tube. After the medium solidifies, the plate is transferred to the moist chamber as rapidly as possible, so as to avoid 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 con- taminate it. atmospheric contamination. In doing this, it is advisable to have an assistant to raise the glass covers. Tubes b and c are similarly treated, SEPARATION BY AGAR MEDIA 55 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 on the gelatin plates. The further procedure is of the same nature as with Petri's capsules. 3. EsmarcKs Roll Tubes. — Here the principle is that of dilution as before. In each of three test-tubes 1J or 1J inch in diameter, gelatin to the depth of f of an inch is placed. These are sterilised. The gelatin is melted and in- oculated in series 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 sheet of gelatin instead of a flat one. A con- venient 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 liquefying the gelatin be present, the liquefied gelatin contamin- ates the rest of the medium. Separation by Agar Media. — 1. Agar Plates. — The only difference between the technique here and that with gelatin 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 40° C. As it is dangerous to expose organisms to a temperature much above 42° C., it is necessary 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 42° 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 gelatin. 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 FIG. 20. Esmarch's tube for roll culture. 56 METHODS OF CULTIVATION OF BACTERIA 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 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 may 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 incubator in the upright position and must be handled carefully 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. 123) inoculate tubes of suitable media from characteristic lesions situated away from the seat of inoculation, e.g. from spleen in the case of b. tuberculosis, spleen or liver in the case of b. mallei, and heart blood in the case of pneumococcus. 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 SEPARATION OF ANAEROBES 57 be boiled for a few minutes all the vegetative 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. (1) For the growth of anaerobes this oxygen may be expelled by the prolonged passing of an inert gasT 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 interfere 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 employed. The preparation of such media has already been described (pp. 36, 37). In this case the medium ought to be of considerable thickness. The Supply of Hydrogen for Anaerobic Cultures. — The gas is generated in a large Kipp's apparatus from pure sulphuric acid and pure zinc. It is passed through three wash-bottles, as in Fig. 21. In the first is placed a solution 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 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) By Roll-tubes. — A 1J inch test-tube has as much gelatin 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, 58 METHODS OF CULTIVATION OF BACTERIA and covered with plugs of cotton wool. Three such test-tubes are prepared, and they are sterilised in the steam steriliser (p. 27). After sterilisation the gelatin is melted and one tube inoculated with the mixture containing the anaerobes ; the second is inocu- lated from the first, and the third from the second, as in making ordinary gelatin plates. After inoculation the gelatin 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 it and each test-tube is rolled as in Esmarch's method till the gelatin solidifies as a thin layer on the internal FIG. 21. — Apparatus for supplying hydrogen for anaerobic cultures. a. Kipp's apparatus for manufacture of hydrogen. 6. Wash-bottle containing 1-10 solution of lead acetate, c. Wash-bottle containing 1-10 solution of silver nitrate, d. Wash-bottle containing 1-10 solution of pyrogallic acid. (&, c, and d are intentionally drawn to a larger scale than a to show details.) surface. A little hard paraffin may be run between the rim of the test-tube and the stopper, and round the perforations for the gas- supply tubes, to ensure that the apparatus is air-tight. The gelatin 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 admirably suited for all anaerobes which grow at the ordinary temperature. (b) Bulloch's Apparatus for Anaerobic Culture. — This can be recommended for plating out mixtures containing anaerobes, and for obtaining growths (especially surface growths) of the latter. It consists (Fig. 23) of a glass plate as base on which a bell jar can be firmly luted down with unguentum resinae. In CULTURE OF ANAEROBES 59 22.— Esmarch's roll- tube adapted for culture containing anaerobes. the upper part of the bell jar are two apertures furnished with ground stoppers, and through each of the latter passes a glass tube on which is a stop-cock. One tube, bent slightly just after passing through the stopper, extends nearly to the bottom of the chamber; x L the other terminates immediately below the stopper. In using the apparatus there is set on the base-plate a shallow dish, of slightly less diameter than that of the bell jar, and having a little heap of from two to four grammes of dry pyrogallic acid placed in it towards one side. Culture plates made in the usual way can be stacked on a frame of glass rods resting on the edges of the dish, or a beaker containing culture tubes can be placed in it. The bell jar is then placed in position so that the longer glass tube is situated over that part of the bottom of the shallow dish farthest away from the pyrogallic acid, and the bottom and stoppers are luted. The air in the bell jar is now expelled by passing a current of hydrogen through the short glass-tube, and both stoppers are.closed. A partial vacuum is then effected in the jar by connecting up the short tube with an air-pump, opening the tap, and giving a few strokes of the latter. A solution of 109 grms. solid caustic potash dissolved in 145 c.c. water is made, and into the vessel containing it a rubber tube connected with the long glass tube is made to dip, and the stopper of the latter being opened, the fluid is forced into the chamber and spreads over the bottom of the shallow dish ; potassium pyrogallate is thus formed, which absorbs any free oxygen still present. Before the whole of the fluid is forced in, FIG. 23. — Bulloch's apparatus for anaerobic plate cultures. the rubber tube is placed in a little boiled water, and this, passing through the glass tubes, washes out the potash and prevents 60 METHODS OF CULTIVATION OF BACTERIA erosion of the glass. The whole apparatus may be placed in the incubator till growth occurs. 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, 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. From such " deep " cultures growths may be maintained indefinitely by successive sub-cultures in similar tubes. Even ordinary gelatin and agar can be used in the same way if the medium is heated to boiling-point before use to expel any absorbed oxygen. Carroll's Method for Anaerobic Cultures. — This may be used with culture tubes containing any of the media suitable for anaerobes, with Esmarch's roll-tubes, or with fermentation tubes. There is required a dry tube of the same diameter as the culture tube, a short U-shaped glass tube, and two pieces of rubber tub- ing all of like diameter. The culture tube having been inoculated, the plug is pushed home below the lip of the tube. The ends of the U-tube are smeared with vaseline and a rubber tube slipped over each ; the end of the culture tube being similarly treated, the free end of one of the rubber tubes is pushed over it till the glass of the U-tube is in contact with the glass of the culture tube. In the dry tube one or two grammes of pyrogallic acid are placed and the powder is packed down with a layer of filter paper. Ten or twenty cubic centimetres of a ten per cent solution of sodium hydrate are then poured in and the tube is quickly connected up by the rubber tubing with the other end of the U-tube. In this apparatus the oxygen is absorbed by the sodium pyrogallate and the conditions for anaerobic growth are fulfilled. Cultures of Anaerobes in Liquid Media. — It is necessary to employ such in order to obtain the toxic products of the growth CULTURE OF ANAEROBES IN LIQUID MEDIA 61 of anaerobes. Glucose broth is most convenient. It is placed either (1) in a conical flask with a lateral opening and a perfor- ated india-rubber stopper, through which a bent glass tube passes, as in Fig. 24, a, by which hydrogen may be delivered, or (2) in a conical flask with a rubber stopper furnished with two holes, as in Fig. 24, 6, 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 FIG. 24. a. Flask for anaerobes in liquid media. Lateral nozzle and stopper fitted for hydrogen supply, b. A stopper arranged for a flask without lateral nozzle. 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 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 organism 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 62 METHODS OF CULTIVATION OF BACTERIA 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 dis- connected from the hydrogen apparatus. In the case of flask (2), first the exit tube and then the entrance tube are sealed off in the flame before the flask is discon- nected from the hydrogen apparatus. It is well in the case of both flasks to run some melted paraffin all over the rubber stopper. Some- times much gas is evolved by anaerobes, and in dealing with an organism where this FIG. 25.-Flask arranged for culture of will occur provision must be made for its escape. This is conveniently done by lead- ing down the exit tube, and letting the end just dip into a trough of mercury (Fig. 25), 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. The method of culture in fluid media is used to obtain the soluble products of such anaerobes as the tetanus bacillus. 6 is a trough of mercury into which exit tube dips. FIG. 26. — Tubes for auaerobic cultures on the surface of solid media. When it is desired to grow anaerobes on the surface of a solid medium such as agar, tubes of the form shown in Fig. 26, 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 HANGING-DROP PREPARATIONS 63 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 FIG. 27. A. Hollow-ground slide for hanging-drop cultures shown in plan and section. B. Another form of slide for similar cultures. 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. 27. 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 by 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 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, a loopful 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 64 METHODS OF CULTIVATION OF BACTERIA 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 covered with vaseline, and the preparation is then complete and may be placed under the microscope. If necessary, it may be first in- cubated 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 course adheres. The slide with the cover attached is then quickly 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 85. Anaerobic Hanging-drop Cultures. — The growth and examination of bacteria in hanging-drops under anaerobic conditions involve consider- FIG. 28. — Graham Brown's chamber for anaerobic hanging-drops. (A portion of one edge of upper plate is shown cut away.) able difficulty, but may be carried out in an apparatus devised by Graham Brown (Fig. 28). It consists of two brass plates (a and a') which can be approximated by screws, and which have rounded apertures in their middles f in. in diameter. These support two rubber rings, an upper thinner one (b) and a lower thick one (d), the inner diameters being the same as that of the apertures in the plates. Between THE COUNTING OF COLONIES 65 b and d is placed a stout cover-glass of suitable size (c) ; d is separated from the plate of by a square plate of glass (e) (a portion of an ordinary glass-slide for microscopical purposes does well). Two small metal tubes (/) are inserted through the rubber d. 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 FIG. 29. — Apparatus for counting colonies. of colonies which develop 7vlieii_ that amount is added to a tube of suit- Tttlle medium, and the latter plated and incu- bated. An ordinary plate should be used in such a case, and the medium poured out in as rect- angular a shape as pos- sible. For the counting, an apparatus such as is shown in Fig. 29 is employed. This consists of a sheet of glass ruled into squares as indicated, and supported 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 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 purposes of count- ing. The bottoms of such dishes are, however, 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 5 66 METHODS OF CULTIVATION OF BACTERIA 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. Method of counting Living Bacteria in a Culture. — This is accomplished by putting into practice a dilution method such as that described on p. 52. Measured amounts of high dilutions are plated, and the numbers of colonies which subsequently develop are counted. In applying such a method it is necessary to have pipettes capable of measuring small quantities of fluid. Those discharging -05 and '1 c.c. will be found con- venient, and such pipettes can have sub- divisions which enable them to be used for measuring still smaller fractions of a cubic centimetre. Pipettes of this kind can be obtained at the instrument makers. Wright has described a method by which a pipette (Fig. 30) for measuring small quantities of fluid can be made from ordinary quill tubing. The method is as follows : — A .piece of quill tubing about 15 cm. long is drawn out to a capillary stem. A standard 5 c.mm. pipette (such as that of the Gower's haemocytometer), or the pipette described later on p. 108, is filled with mercury and the metal transferred to the capillary stem and run down to near its extremity ; the upper and lower limits of the mercury are marked with an oil pencil ; Fl°- 30-— w^h*>sthe mercury is then displaced up the tube 250 c.mm. pipette ,.„ ., . , , ,. , , N . fitted with nipple *"* ^s previously distal end is at the proximal of the two marks, and a third mark is made at the new position of the upper end of the droplet ; the mani- pulation is repeated three more times, and finally the tip of the tube beyond the lowest mark is broken off. Thus on the capillary part of the pipette we have five divisions, each cap- able of holding 5 c.mm. of fluid. The rest of the pipette is now calibrated so as to determine that part capable of containing 225 c.mm. and 250 c.mm. This is done by placing a rubber nipple on the wide end of the pipette and sucking up some water tinted with, say, methylene-blue till the 25 c.mm. mark is reached ; a small air-bubble is then allowed to enter the 250 c. mm, 225 20 15 10 2-5 METHOD OF COUNTING BACTERIA 67 pipette, then other 25 c.mm. of fluid, then another bubble, and so on till nine volumes each of 25 c.mm. have been sucked up. A mark is then made on the tube at the upper level of this amount, other 25 c.mm. are sucked up, and another mark made. The fluid is expelled, the tube dried, and that part containing the 225 and 250 marks is drawn out into an almost capillary diameter, the manipulation by which the marks were originally arrived at is repeated, and thus in the new marks made a more accurate calibration for these amounts is attained. In order to form a safety chamber a second bulb is formed by drawing out the tube a little higher up as in the figure, and finally the upper inch or two are bent at right angles to the calibrated limb. In doing this a loop may be thrown on the plastic melted capillary tube exactly in the way in which a similar loop may be thrown on a piece of cord. With such a pipette any required dilution of a culture can be made on the principles already described. Wright's Method of counting the Bacteria in Dead Cultures. — In the making of vaccines for use in Wright's pro- cedures it is necessary to know the total number of bacterial cells, whether dead or living, present in a culture, for the dead as well as the living contain the toxins which may stimulate the therapeutic capacities of the body. The method consists in making a mixture of blood (whose content in red blood corpuscles is known) with the bacterial culture and comparing the number of bacteria with the number of corpuscles. The observer first estimates the red cells in his blood ; a capillary pipette with a rubber nipple and with a mark near its capillary extremity is then taken, blood is sucked up to the mark, then an air-bubble, then (according to the empirical estimate the observer forms of the strength of his bacterial emulsion) either one volume of culture and three volumes of diluting fluid (e.g. !85 per cent sodium chloride) or two of culture and two of fluid, and so' on ; the five volumes are thoroughly mixed by being drawn backwards and forwards in the wide part of the pipette, a drop is then blown out on to a slide, and a blood film is spread which may be stained by Irishman's method. The bacteria and blood-corpuscles are now separately enumerated in a series of fields in different parts of the preparation. If a dilution has been taken in which a large number of bacteria are present, an artificial field may be used, made by drawing with the oil pencil a small square on a circular cover-glass and dropping the latter on to the diaphragm of the microscope eye-piece. Suppose, now, that the observer's blood contained 5,000,000 red cells per c.mm., that one volume of bacterial 68 METHODS OF CULTIVATION OF BACTERIA emulsion and three of diluent had been present in the mixture, and that in the fields examined there were 500 red cells and 600 bacteria. It is evident that in the undiluted culture for 500 red cells there would have been 2400 bacteria. Now 500: 2400:: 5,000,000: 24,000,000, which last figure is the number of bacteria per c.mm. of the emulsion. The Bacteriological Examination of the Blood. — (a) This may be done by taking a small drop from the skin surface, e.g. the lobe of the ear. The part should be thoroughly washed with 1-1000 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. Film preparations for microscopic examination may be made at the same time. It is rare to obtain growths from the blood of the human subject by this method (vide special chapters), and if colonies appear the procedure should be repeated to exclude the possibility of accidental con- tamination. (6) A larger quantity of blood may be obtained by puncture of a vein ; this is the only satisfactory method, and should be the one followed whenever practicable. The skin over a vein in the forearm or on the dorsum of the foot having been sterilised, the vein is made turgid by pressure, and the needle of a syringe of 10-15 c.c. capacity, carefully sterilised, is then plunged obliquely through the skin into the lumen of the vessel. Several cubic centimetres of blood can thus be withdrawn into the syringe. Some of the blood (e.g. 1 c.c.) should be added to small flasks containing 50 c.c. of bouillon ; the rest may be used for smearing the surface of agar tubes or may be added to melted agar at 42° C., which is then plated. The flasks, etc., are then incubated. By this method cultures can often be obtained where the former method fails, especially in severe conditions such as ulcerative endocarditis, streptococcus infection, etc. Part of the blood may be incubated by itself for twenty-four hours and cultures then made. 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. 87.) Bacteriological Examination of the Cerebro-spinal Fluid- Lumbar Puncture. — This diagnostic procedure, which is some- times called for in .cases of meningitis, can be carried out with EXAMINATION OF CEREBRO-SPINAL FLUID 69 a sterilised " antitoxin needle" as follows. The patient should lie on the right side, with knees somewhat drawn up and left shoulder tilted somewhat forward, so that the back is fully exposed. The skin over the lumbar region is then carefully sterilised, as above described, and the hands of the operator should be similarly treated. The spines of the lumbar vertebrae having been counted, the left thumb or forefinger is pressed into the space between the 3rd and 4th spines in the middle line ; the jgfidlft JiLthp.Ti in^rted al>OUt bftlf an inr-h t.n tliA riprht rf thft middle line atthis leveLand pushed through the tissues, its ourse being directed slightlyinwrdad^upwards, ffll it. hP nHVrfll ^nrfL wh"n m* the needle, sometimes actually spurting out, and should be received in a sterile test-tube. Several cubic centimetres of fluid can thus usually be obtained, no suction being required ; thereafter it can be examined bacteriologically by the usual methods. The depth of the subdural space from the surface varies from a little over an inch in children to three inches, or even more, in adults — the length of the needle must be suited accordingly. In making the puncture it is convenient to have either a sterile syringe attached, or to have the thick end of the needle covered with a pad of sterile wool, which is of course removed at once when the fluid begins to flow. It is advisable to use the platinum needles which are specially made for the purpose, ;is a .sudden movement of the patient may snap an 'ordinary steel needle. The Bacteriological Examination of Urine. — In such an examination care must be taken to prevent the contamination of the urine by extraneous organisms. In the male it is usually sufficient to wash thoroughly the glans penis and the meatus with 1-1000 corrosive sublimate — the lips of the meatus being everted for more thorough cleansing. The urine is then passed into a series of s,terile 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 au hour's exposure in a plugged flask to a temperature of 120° 0. 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. Filtration of Cultures. — For many purposes it is necessary 70 METHODS OF CULTIVATION OF BACTERIA to filter all the organisms from fluids in which they may have been growing. This is done especially in obtaining the soluble toxic products of bac- teria. The only filter capable of keeping back such minute bodies as bacteria is that formed from a tube of unglazed porcelain as introduced by Chainberland. The efficiency of such a filter depends on the fineness of the grain of the clay from which it is made ; the finest is the Kitasato filter and the Chamber- land " B " pattern ; the next finest is the Cham- berland "F" pattern, which is quite good enough for ordinary work. There are several filters, differing slightly FIG. 31. — Geissler's vacuum pump arranged with manometer for filtering cultures. (The tap and pump are intentionally drawn to a larger . scale than the manometer board to show 1 *"» a11 P01 details.) the 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, FlG 32._Chamberland's candle they are forced through a porcelain and flask arranged for filtration, cylinder by compressed carbonic acid gas. For ordinary bacteriological work, filters of various kinds are in the market (such as those of Klein and THE FILTEATION OF CULTURES 71 others), but the most generally convenient is that in which the fluid is sucked through the porcelain by exhausting the air in the receptacle into which it is to flow. This is con- veniently done by means of a Geissler's water-exhaust pump (Fig. 31, 0), 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 it with rubber solution strips of the rubbered canvas used for mending punctures FIG. 33. — Chamberland's bougie arranged with lamp funnel for filtering a small quantity of fluid. FIG. 34. — bougie in- serted through rubber stopper for same purpose as in Fig. 33. in the outer case of a bicycle tyre. A manometer tube (6) and a receptacle (c) (the latter to catch any back flow of water from the pump if the filter accidentally breaks) are intercepted between the filter and the pump. These are usually arranged on a board a, as in Fig. 31. Between the tube /and the pump g, and between the tube d and the filter, it is convenient to insert lengths of flexible lead-tubing connected up at each end with short, stout-walled rubber-tubing. Filters are arranged in various ways, (a) An apparatus is arranged as in Fig. 32. The fluid to be filtered is placed in the cylindrical vessel a. Into this a "candle" or "bougie" of porcelain dips. From the upper end of the bougie a glass tube with thick rubber connections, as in Fig. 32, proceeds to flask b 72 METHODS OF CULTIVATION OF BACTERIA 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 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. (6) A very good apparatus can be arranged with a lamp funnel and the porcelain bougie. These may be fitted up in two ways. (1) An india-rubber washer is placed round the bougie c at its glazed end (vide Fig. 33). On this the narrow end of the funnel d, which must, of course, be of an appropriate size, rests. A broad band of sheet rubber is then wrapped round the lower end of the 4 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, FIG. 35. — Muencke's modification of Chamberland's filter. is connected by a glass tube with the lateral tube of the flask b } the tube a is con- nected 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 in Fig. 34. Into the narrow part of the funnel an india-rubber bung is fitted, with a perforation in it sufficiently large to receive the candle, which it should grasp tightly. (c) Muencke's modification of the Chamberland filter is seen in Fig. 35. It consists of a thick-walled flask a, the lower part conical, the upper cylindrical, with a strong flange on the lip. There are two lateral tubes, one horizontal to connect 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 projecting rim on the flange of the flask, an asbestos washer, c, being interposed. The fluid to be filtered is placed THE FILTRATION OF CULTURES 73 in the porcelain cylinder, and the whole top covered, as shown at /, with an india-rubber cap with a central perforation ; the tube d is connected with the exhaust-pump and the tube e plugged with a rubber stopper. When a large quantity of fluid is to be filtered, . a receptacle such as that shown in Fig. 36 may be used. The tap in its bottom enables the filtrate to be removed without the apparatus being unshipped, but it is difficult to get the tap to fit so accurately as not to allow air to pass into the vacuum chamber. For filtering small quantities of fluid the apparatus shown in Fig. 37 may be used. It consists of a small Chamberland bougie fitted by a rubber tube to a funnel, the stem of which has been passed through a rubber cork ; this cork fits into a tri- angular flask with side arm for connection with exhaust. Before any one of the above apparatus is used, it ought to be con- nected up as far as pos- sible and sterilised in the Koch's steriliser. The ends of any im- portant 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 oft7 all organic matter. If the latter is allowed to accumu- late 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 candies 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 FIG. 36.— Flask fitted with porcelain bougie for filtering- large quantities of fluid. FIG. 37. — Flask for filtering small quanti- ties of fluid. 74 METHODS OF CULTIVATION OF BACTERIA thoroughly closed, and if these vessels be kept in a cool place in the dark. A layer of sterile toluol about half an inch thick ought to be run on to the top of the filtered fluid to protect the latter from the atmospheric oxygen. 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 practicable to kill the bacteria, by heat when their soluble products are to be studied, as many of the latter are destroyed by a lower temperature than is required to kill the bacteria themselves. Bacteria can be almost entirely removed from fluid cultures by spinning the latter in a centrifuge of very high speed (e.g. C. J. Martin's turbine centrifuge), and this method is sometimes adopted in practice. The Observation of Bacterial Fermentation of Sugars, etc. — The capacity of certain species of bacteria to originate fermenta- tions in sugars constitutes an important biological factor. It is well to consider this factor in relation to the chemical con- stitution of the sugars. These bodies are now known to be (to use the definition of Holleman) aldehyde or ketone alcohols containing one or more hydroxyl groups, one of which is directly linked to a carbon atom in union with carbonyl. The group characteristic of a sugar is thus - CHOH - CO - . The sugars are divided into monosaccharides or monoses, disaccharides (dioses), and polysaccharides (polyoses). The members of the last two groups may be looked on as derived from the combination of two or more molecules of a rnonosaccharide with the elimination of water (e.g. 2C6H12O6 = C12H22On + H2O). Monosaccharides. — These are classified according to the number of C atoms they contain. The pentoses ordinarily used are arabinose (obtained from gum arable), rhamnose and xylose (from wood). Among the hexoses are glucose (dextrose) with dextro-rotatory properties. Glucose is an aldehyde alcohol (aldose). In fruit there is also a ketone alcohol (ketose) called fructose, which from its laevo-rotatory properties is also known as laevulose. Other hexoses are mannose (from the vegetable ivory nut) and galactose (a hydrolytic derivative of lactose). Disaccharides (C]2H22On). — The ordinary members of this group are maltose (derived from starch), lactose, and cane sugar (sucrose, saccharose). Polysaccharides. — Examples are starch, raffinose, inulin (from dahlia roots), dextrin, arabin, glycogen, cellulose. BACTERIAL FERMENTATION OF SUGARS 75 If we consider sugars generally from the point of view of the capacity of yeast to originate alcoholic fermentation in them, we may say that the simpler the constitution of the sugar the more easily is it fermented. Thus the monosaccharides are more easily acted on by yeast than the di- or polysaccharides. Usually an independent process resulting in the splitting of the higher into the lower is preliminary to the alcoholic fermentation. Thus yeast first inverts cane sugar into glucose and fructose and then acts on these products. From what is known it is probable that similar facts hold with regard to the action of bacteria. Besides sugars other alcohols with large molecules may be broken down by bacterial action, and these bodies have been used for differentiating the properties of allied bacteria. Among these substances may be mentioned the trihydric alcohol glycerol (glycerin), the tetrahydric erythritol and the hexahydric dulcitol (dulcite), mannitol (mannite), and sorbitol (sorbite). Similarly certain glucosides, such as salicin, coniferin, etc., have been used for testing the fermentative properties of bacteria. Other substances allied to sugars (e.g. inosite) have also been used. The end products of bacterial fermentations may be various. They differ according to the sugar employed and according to the species of bacterium under observation, and frequently a species which will ferment one sugar has no effect on another. The sub- stances finally produced, speaking roughly, may be alcohols, acids, or gaseous bodies (chiefly carbon dioxide, hydrogen, and methane). For the estimation of the first groups complicated chemical procedure may be necessary. The tests usually employed for the detection of ordinary fermentative processes depend on two kinds of changes, namely (a) the evolution of gases and (b) the formation of acids. Generally speaking, we may say that such tests are reliable and the methods to be pursued are simple. Besides such gases as those named 'some organisms give rise to sulphuretted hydrogen by breaking up the proteid. The formation of this gas can be detected by the blackening of lead acetate when it is added to the g'as-containing medium. In testing the effect of a bacterium on a given sugar it is essential that this sugar alone be present ; the basis of the medium ought therefore to be either peptone solution (v. p. 38) or a dextrose-free bouillon (v. infra). The sugar or other substance is added in the proportion of from a half to one per cent, and care is taken not to overheat during sterilisation. 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 76 METHODS OF CULTIVATION OF BACTERIA dextrose, and allow the latter to act for forty-eight hours. The bouillon is then filtered and re-sterilised. A sample is tested for another period of forty-eight 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 sugar in solid form be placed in the bouillon and this then sterilised, there is danger that chemical changes may take place in the sugar, in consequence of its bei-ng heated in the presence of substances (such as the alkali) which may act deleteriously upon it ; in any case sterilisation should not be at a temperature above 100° C. For the observation of gas-formation either of the following methods may be employed : — (1) Durham's Tubes (Fig. 38, 6).— The plug of a tube which contains about one-third more than usual of a liquid medium is It a c FIG. 38. — Tubes for demonstrating gas-formation by bacteria. «, tube with "shake" culture. 5, Durham's fermentation tube, c, ordinary form of fermentation tube. removed, and a small test-tube is slipped into the latter mouth downwards. The plug is replaced and the tube sterilised thrice for ten minutes at 100° C. The air remaining in the smaller tube is thereby expelled. The tube is then inoculated with the bacterium to be tested. Any gas developed collects in the upper part of the inner tube. (2) The Fermentation Tube (Fig. 38, c).— This consists of a tube of the form shown, and the figure also indicates the extent BACTERIAL FERMENTATION OF SUGARS 77 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. For the observation of the effect of an organism on glucose the following method may be employed : — Gelatin Shake Cultures (Fig. 38, a). — The gelatin 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 temperature. If the bacterium used is a gas-forming one, then, as growth occurs, little bubbles appear round the colonies. In this method the gas -formation results from fermenta- tion of the glucose naturally present in the medium from transformation of the glycogen of muscle. The amount of glucose naturally present, however, varies much, and therefore glucose should be added to the medium if the effects on this sugar are to be observed with certainty. The shake culture method may be utilised for observing fermentation in other sugars by adding to peptone solution containing the sugar 10-15 per cent of gelatin. The development of an acid reaction is demonstrated by the addition of an indicator to the medium, litmus being generally used. The details of composition of such media have already been given. In Hiss's serum water media the production of acid also leads to coagulation of the medium. Sometimes acid is formed very slowly from sugars, so that it is well to keep the cultures under observation for several days. Acid and gas-formation may be simultaneously tested for, by placing the fluid medium containing the indicator in Durham's tubes. In all tests in which sugars are used a control uninoculated tube ought to be incubated with the bacterial cultures, as changes in reaction sometimes spontaneously occur in media containing unstable sugars. The capacity of an organism to produce acid may be measured by taking a standard amount of a fluid medium and allowing growth to take place for a standard time, and then adding an amount of, say, decinormal soda solution sufficient to bring the litmus back to the tint of the original medium. The Observation of Indol-formation by Bacteria. — The formation of indol from albumin by a bacterium sometimes con- stitutes an important specific characteristic. To observe indol 78 METHODS OF CULTIVATION OF BACTERIA production the bacterium is grown, preferably at incubation temperature, in a fluid medium containing peptone. The latter may either be ordinary bouillon or preferably peptone solution (see p. 38). Indol production is recognised 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 usually in making the test (e.g. in the case of b. coli) the nitrites must be added. This is effected 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. In many instances incubation at 37° C. for several days may be necessary before the presence of indol is demonstrable. 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 peptones 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. The Drying of Substances in vacuo. — As many substances, for example toxins and antitoxins, with which bacteriology is concerned would be destroyed by drying with heat as is done in ordinary chemical work, it is necessary to remove the water at the ordinary room temperature. This is most quickly effected by drying in vacuo in the presence of some substance such as strong sulphuric acid which readily takes up water vapour. The vacuum produced by a water-pump is here not available, as in such a vacuum there must always be water vapour present. An air-pump is therefore to be employed. Here we have found the Geryk pump most efficient, and it has this further advantage, that its internal parts are lubricated with an oil of very low vapour density so that almost a perfect vacuum is obtainable. The apparatus is shown in Fig. 39. The vacuum chamber consists of a bell-jar set on a brass plate. A perforation in the centre of the latter leads into the pipe a, which can be connected by strong- walled rubber-tubing with the air-pump, and which can be cut off from the latter by a stop-cock 6. In using the apparatus the substance to be dried is poured out in flat dishes (one-half of a Petri capsule does very well), and these are stacked alternately with similar dishes of strong sulphuric acid on a STORING AND INCUBATION OF CULTURES 79 stand which rests on the brass plate. The edge of the bell-jar is well luted with unguentum resinse and placed in position and the chamber exhausted. In a few hours, if, as is always advis- able, each dish have contained only a thin layer of fluid, the drying will be complete. The vacuum is then broken by admitting air very slowly through a bye-pass c, and the bell-jar is removed. In such an apparatus it is always advisable, as is shown in the figure, to have interposed between the pump and the vacuum chamber a Wolffs bottle containing sulphuric acid. This protects the oil of the pump from contamination with FIG. 39. — Geryk air-pump for drying in vacuo. water vapour. Whenever the vacuum is produced the rubber- tube should be at once disconnected from a, the cock b being shut. It is advisable when the apparatus is exhausted to cover the vacuum chamber and the Wolff's bottle with wire guards covered with strong cloth, in case, under the external pressure, the glass vessels give way. The Storing and Incubation of Cultures. — Gelatin cultures must be grown at a temperature below their melting-point, i.e. for 10 per cent gelatin, below 22° C. 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 12° C. to 18° C. Agar and serum media are usually employed to grow bacteria at a higher temperature, corresponding to that at which 80 METHODS OF CULTIVATION OF BACTERIA the organisms grow best, usually 37° 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 nuid (water or glycerin and water) is placed, which, when raised to a certain temperature, 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 y design. Sometimes a mechanism devised in .-•--.... Koch's laboratory is affixed, which auto- a matically turns off the gas if the light be accidentally extinguished. Between the tap supplying the gas, and the burner, is inter- posed a gas regulator. Such regulators vary in design, but for ordinary chambers which require to be kept at a constant tem- perature, Reichert's is as good and simple as any and is not expensive. It is shown in Fig. 40. It consists of a long tube /closed at the lower eiid, 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 J 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 perpendicular tube fits accurately FIG. 40. — Reichert's a bent tube g, drawn out below to a comparatively gas regulator. 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 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. When the thermometer in the interior of the chamber indicates that the desired temperature 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 alternation 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 STORING AND INCUBATION OF CULTURES 81 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. 41. — Hearson's incubator for use at 37° C. 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 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 82 METHODS OF CULTIVATION OF BACTERIA 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 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 gelatin at 21° to 22° C. An incubator of this kind fitted with a low-temperature Hearson's regulator is in the market. Method of mounting Bacterial Cultures as Permanent Museum Specimens. (Richard Muir). — (a) Stab or stroke cultures in nutrient gelatin or agar media. — When the culture shows typical characters, further growth is arrested by placing tube in a formol vapour chamber, or by saturating the cotton- wool plug with strong formalin. Then leave for a day or two. Make up following : — (1) Thymol Water (saturated in cold) ... 100 c.c. Glycerin . . . . . . . 20 c.c. Acetate of Potash ...... 5 grams. Coignet's (gold label) Gelatin . . . 10 grams. Render the mixture acid to litmus with acetic acid ; clear with white of egg and filter. Warm to about 40° C., and removing cotton-wool plug from culture take a little of the preserving fluid in a pipette and allow to run gently over surface of medium in tube. Place in such a position that a thin layer of the preserving medium remains completely covering the growth and the surface of culture medium. The gelatin is now allowed to solidify. Add three or four drops of strong formalin to the tube and fill up to within a quarter of an inch of the top of the tube with the following fluid : — (2) Thymol Water (saturated in cold) . . . 100 c.c. Glycerin . 20 c.c. Acetate of Potash ...... 5 grams. Cover top of tube with a small piece of paper so as to keep out dust, allow to stand for a day or two so that small air-bells may rise to the surface. To seal tube, pour melted paraffin gently on to the surface of fluid to near the top of tube ; allow to solidify. Cover paraffin with layer of alcoholic orange shellac cement ; allow this to set and repeat until the cement becomes level with top of test tube. When set, a few drops of black lacquer are put on and a circular cover-glass of about the same diameter as the mouth of tube is placed so as completely to seal it. GENERAL LABORATORY RULES 83 (b) The following method is useful for preserving plate cultures. Instead of making the cultures in Petri's capsules, use ordinary watch-glasses. The watch-glass is sterilised in a Petri's capsule, and the inoculated medium is poured out into the watch-glass, allowed to solidify in the usual way, and left in the Petri's cap- sule until the colonies of growth have developed. The watch- glass is now removed from capsule and a layer of the preserving gelatin medium (1), to which have been added a few drops of strong formalin, is allowed to spread over the surface of the culture medium. When the layer is solidified the watch-glass is filled up with the same, and a clean square or oblong piece of glass (which of course should be of slightly larger diameter than the watch-glass) is now carefully placed over watch-glass, care being taken that no air-bells are formed. The edge of watch-glass should be closely applied to the glass cover and left in position until the gelatin has solidified. The superfluous gelatin is now removed, and the glasses sealed first with the orange shellac cement, then with black lacquer. It is now finished off by using a circular mask of suitable size. The various kinds of solid media used in the cultivation of bacteria, such as blood serum, potato, bread paste, etc., can be treated in the same manner with excellent results. 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 contained 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. When, as in public-health work, a large number of tubes are being daily put out of use, they may be placed in an enamelled slop- pail and this when full is placed in the steam s'teriliser. A white glazed tile on which a bell-jar can be set is very 84 METHODS OF CULTIVATION OF BACTERIA convenient to have on a bench. Infective material in watch- glasses can be placed thus under cover while investigation is going on, and if anything is spilled the whole can be easily disinfected. In making examinations of organs containing virulent bacteria, the hands should be previously dipped in 1-1000 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 1-1000 mercuric chloride and then with yellow soap. In the case of any fluid containing bacteria being accidentally spilt on the bench or floor, 1-1000 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 BACTERIO- LOGICAL 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 (flat 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 corresponding to these. The oil immer-- sion 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 tlofc condenser. When the observer has finished for the time being with the 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, which will dissolve the material by which the lens is fixed in its metal carrier. Microscopic Examination of Bacteria. 1. Hanging -drop Preparations. — Micro-organisms may be examined : (1) 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 85 86 MICROSCOPIC METHODS 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. 63). In examining them microscopically, 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 hanging-drop is made up with a minute quantity of an 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 them. 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 obtaining such. The test of this being accomplished is that, when the drop of fluid containing the bacteria is placed upon the glass, it can be uniformly spread with the platinum needle all over the surface without showing any tendency to retract into droplets. 1 In bacteriological work it is essential that cover-glasses of No. 1 thick- ness (i.e. '14 ram. thick) should he used, as those of greater thickness are not suitable for a ^-in. lens. FILM PREPARATIONS 87 The best method is that recommended by Van Ermengem. The cover-glasses are placed for some time in a mixture of con- centrated sulphuric acid 6 parts, potassium bichromate 6 parts, water 100 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 pre- paration 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 distilled water is placed on the cover-glass and a minute par- ticle of growth rubbed up in it and spread over the glass. The great mistake made by begin- . . \ 4,, FIG. 42. — Cornet s forceps for holding ners is to take too much of the cover-classes, 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 tjie 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 may be smeared directly on the cover-glasses. In the case of blood, a fairly large drop should be allowed to spread itself between two clean 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 may be fixed by one of the following methods : by being placed (a) in a hot-air 88 MICROSCOPIC METHODS chamber at 120° C. for half an hour, (b) in a mixture of equal parts of alcohol and ether for half an hour, then washed ^ and dried, (c) in formol-alcohol (Gulland) (formalin 1 part, absolute alcohol 9 parts) for five minutes, then washed and dried, or (d) in a saturated solution of corrosive sublimate for two or three minutes, then washed well in running water and dried. (Fig. 71 shows a film prepared by the last method.) In using the Romanowsky stains no previous fixation is necessary (vide infra). In the case of urine, the specimen must be 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. Within recent years it has become common to make blood films on ordinary microscopic slides instead of upon cover- glasses. Here the slides must be clean. This can be effected by washing thoroughly first with weak alkali and then with water and storing in alcohol. For use, a slide is taken from the alcohol and the fluid adhering to it set on fire and allowed to burn off, a dry clean slide being thus obtained. To make a film on such, a small drop of blood is placed near one end, the edge of a second clean slide is lowered through the drop on to the surface of the glass on which the blood has been placed. This second slide is held at an angle to the first, on which it rests by its edge. The droplet of blood by capillarity spreads itself in the angle between the two slides. The edge of the second slide is then stroked along the surface of the first slide, and in this procedure the blood is spread out in a film whose thickness can be regulated by the angle formed by the second slide. Large- sized films can thus be obtained, and when these are stained they are often examined without any cover-glass being placed upon them. A drop of cedar oil is placed on the preparation, and after use this can be removed by the careful application of xylol. 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 " wet " films for the " dried " films, the preparation of which has been described. The nuclear structure, mitotic figures, etc., are EXAMINATION OF BACTERIA IN TISSUES 89 by this method well preserved, whereas these are considerably distorted in dried films. The initial stages in the preparation of wet films are the same as above, but instead of being dried in air they are placed, while still wet, film downwards in the fixative. The following are some of the best fixing methods : — (a) A saturated solution of perchloride of mercury in '75 per cent sodium chloride ; fix for five minutes. Then place the films for half an hour, with occasional gentle shaking, in 75 per cent sodium chloride solution to wash out the corrosive sublimate ; they are thereafter washed in successive strengths of methylated spirit. After this treatment the films are stained and treated as if they were sections. (b) Formol-alcohol — formalin 1 part, absolute alcohol 9. Fix films for three minutes ; then wash well in methylated spirit. This is an excellent and very rapid method. (c) Another excellent method of fixing has been 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 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 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 (a) 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 examina- tion 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 permanently maintain, as far as possible, the condition it was in when re- moved 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 (e.g. Cathcart's or one of the newer instru- ments in which the freezing is accomplished by compressed carbonic acid gas), 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 bacterio- logical 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. — The following are amongst the best methods for bacteriological purposes : — (a) 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 90 « MICROSCOPIC METHODS is sufficient to keep it in this reagent for a few hours. 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. 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 preserved in 50 per cent spirit. (b) Formol-alcohol — formalin 1, absolute alcohol 9. Fix for not more than twenty-four hours ; then place in absolute alcohol if the tissue is to be embedded at once, in 50 per cent spirit if it is to be kept for some time. For small pieces of tissue fixation for twelve hours or even less is sufficient. The method is a rapid and very satisfactory one. (c) Corrosive sublimate is an excellent fixing agent. It is best used as a saturated solution in '75 per cent sodium chloride solution. Dis- solve 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. They should then be tied up in a piece of gauze, and placed in a stream of running water for from twelve to twenty-four hours, according to the size of the pieces, to wash out the excess of sublimate. They are then placed for twenty-four hours in each of the following strengths of methylated spirit (free from naphtha l) : 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 afterwards described. (d) Methylated Spirit. — Small pieces of tissue may be placed in methylated spirit, which is to be changed after the first day. In from 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 ttte Freezing Microtome. — Pieces of tissue hardened by any of the above methods must have all the alcohol removed from them by wash- ing 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 on a freezing microtome 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. 1 In Britain ordinary commercial methylated spirit has wood naphtha added to it to discourage its being used as a beverage. The naphtha being insoluble in water a milky fluid results from the dilution of the spirit. By law, chemists can only sell 8 ounces of pure spirit at a time. Most patho- logical laboratories are, however, licensed by the Excise to buy "industrial spirit," which contains only one -nineteenth of wood naphtha. THE CUTTING OF SECTIONS 91 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 impregnation 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 per- meates the tissue more rapidly. The more gradually the tissues are changed from reagent to ceagent 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. Beichert's, being of course necessary. The tissues occurring in pathological work have a tendency to "become brittle if overheated, and therefore the best results are not obtained by using paraffin melting about 58° 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 54° C. This mixture has a melting-point between 52° 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 : — 1. Pieces of tissue, however hardened, are placed in fresh absolute alcohol for twenty-four hours in order to their complete dehydration. 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 or longer. 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, otherwise sufficient chloroform will be present to vitiate the final result and not give the perfectly hard block obtained with pure paraffin. With ex- perience, the persistence of the slightest trace of chloroform can be recognised by smell. 92 MICROSCOPIC METHODS In the case of very small pieces of tissue the time given for eacli 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 de- scribed. When it is advisable to avoid all shrinkage it is well to change the paraffin every few hours during the embedding process. 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 Cutting of Paraffin Sections. — Sections must be cut as thin as possible, the Cambridge rocking microtome being, on the whole, most suitable. They should not exceed 8 /x, in thick- FIG. 43. — Needle with square of paper on end for manipulating paraffin sections. ness, and ought, if possible, to be about 4 p. For their mani- pulation it is best to have two needles on handles, two camel's- hair brushes on handles, and a needle with a rectangle of stiff writing paper fixed on it as in the diagram (Fig. 43). When cut, sections are floated on the surface of a beaker of water kept at a temperature about 10° 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 down- wards, 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 manipulations necessary during staining and mounting. (b) 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 down- DEHYDRATION AND CLEARING 93 wards, 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 of the slide 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 corresponding method of fixing the sections on the slide must be used. Preparation of Paraffin Sections for Staining. — Before stain- ing, 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. These can be removed by placing the sections, before staining, for a few minutes in equal parts of Gram's iodine solution (p. 99) 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. Dehydration and Clearing. — It is convenient, first of all, to indicate the final steps to be taken after a specimen is stained. Dry films after being stained are washed in water, dried and mounted in xylol balsam • ivet Jilms and sections must be dehydrated, 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 placed on a slide is partially dried by draining with fine blotting-paper. Some aniline oil is placed on the section and 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. Balsam as ordinarily supplied has often an acid reaction, and 94 MICROSCOPIC METHODS preparations stained with aniline dyes are apt to fade when mounted in it. It is accordingly a great advantage to use the acid-free balsam supplied by Griibler. Paraffin sections can usually be dehydrated and cleared by the mixture of aniline oil and xylol alone. 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 dehydration 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. With a little experience the progress, not only of these processes but also of staining, can be very accurately judged of by observing the appearances under a low objective. 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 complicated compounds derived from the coal-tar product aniline (C(5H5 . 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 therefore 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 : — Violet Stains. — Methyl- violet, R-5R (synonyms : Hoffmann's violet, dahlia). Gentian-violet (synonyms : benzyl-violet, Pyoktanin). THE STAINING OF BACTERIA 95 Crystal violet. Blue Stains. — Methylene-blue1 (synonym : phenylene-blue). Victoria- blue. Thionin-blue. Red Stains.— -Basic fuchsin (synonyms : basic rubin, magenta). Safranin (synonyms : fuchsia, Girofle). Brown 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 plates. It is most convenient to keep saturated alcoholic solutions 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 1 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 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. 44). The figure explains itself. 1 Tliis is to be distinguished from methyl-blue, which is a different com- pound. 96 MICROSCOPIC METHODS 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. It is sometimes advantageous 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. 100), but with this exception, practically all bacterial films made from cultures can be stained in this way. Some bac- teria, 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 suffi- cient for diagnostic purposes. The blue dyes are 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. The Romanowsky stains (v. p. 105) are here most useful, as by these the preparations are fixed as well as stained. Fixation by heat which is apt to injure delicate cellular structures is thus avoided. 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 speci- men is washed without any violent application of water, and the bacteria are not displaced. FIG. 44. — Syphon wash- bottle for distilled water used in washing prepara- tions. MORDANTS AND DECOLORISING AGENTS 97 For the general staining of films a saturated watery solution of methylene-blue will be found to be the best stain to com- mence with, the Gram method (v. infra) is then applied, and subsequently any special stains which may appear advisable. 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 subse- quent 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. (6) 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 (hydro- chloric, nitric, sulphuric), vegetable acids (especially acetic acid), alcohol (either methylated spirit or absolute alcohol), or a com- bination of spirit and acid, e.g. methylated 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 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 an acid, they should be well washed in tap water, or in distilled water with a little lithium carbonate added. The methods embracing the use of a stain with a mordant, and a decoloriser, are very numerous, and we can only enumerate the best of them. Different organisms take up and retain the stains with various degrees of intensity, and thus duration of staining and decolorising must be modified accordingly. We sometimes have to deal with bacteria which show a special tendency to be decolorised. This tendency can be obviated by adding a little 98 MICROSCOPIC METHODS of the stain to the alcohol, or aniline oil, employed in dehydra- tion. 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 is used, the stain 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 Combinations. 1. Ldfflers Methylene-blue. Saturated solution of methylene-blue in alcohol . . 30 c.c. Solution of potassium hydrate in distilled water (1-10,000) . 100 ,, (This dilute solution may be conveniently made by adding 1 c.c. of a 1 per cent solution to 99 c.c. of water.) Sections may be stained in this mixture for from a quarter of an hour to several hours. They do not readily overstain. The tissue containing the bacteria is then decolorised if necessary with £-1 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 Lbffler'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 ,, 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 are quite sufficient. Wash again thoroughly with water. Dehydrate with absolute alcohol. 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. As a contrast stain, 1 per cent watery solution of eosin may be used before staining with the thionin. 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 GRAM'S STAIN 99 to prevent access of light. (&) Make a saturated solution of gentian- violet in alcohol. When the stain is to be used, 1 part of (6) is added to 10 parts of (a), and the mixture filtered. The mixture should be made nob 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: 5. Carrol- Gentian-Violet. — 1 part of saturated alcoholic solution of gentian-violet is mixed with 10 parts of 5 per cent solution of carbolic acid. It is used as No. 4. 6. Carbol-Fuchsin (see p. 101). — This is a very powerful stain, and, when used in the undiluted condition, ^-1 minute's staining is usually sufficient. It is better, however, to dilute with from 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. Gram's Method and its Modifications. — In the methods already described the tissues, and more especially the nuclei, 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 bacteria, 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. 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 ..... 1 part Potassium iodide ... 2 parts Distilled water . . . 300 „ The following is the method : — 1. Stain in aniline oil gentian- violet or in carbol-gentian- violet (vide supra) 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. 100 MICROSCOPIC METHODS 3. Decolorise with absolute alcohol or methylated spirit till the 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. In stage (3) the process of decolorisation is more satisfactorily performed by using clove oil after sufficient dehydration with alcohol, the clove oil being afterwards removed by xylol. As a contrast stain for the tissues carmalum or lithia carmine is used before staining with gentian-violet (1). As a contrast stain for other bacteria which are decolorised by Gram's method carbol-fuchsin diluted with ten volumes of water or a saturated watery solution of Bismarck - brown may be used before stage (4). The following modifications of Gram's method may be given : — 1. Weigerfs Modification. — The contrast staining of the tissues and stages (1) and (2) are performed as above. (3) After using the iodine solution the preparation is dried by blotting and then decolorised by aniline-xylol (aniline-oil 2, xylol 1). (4) Wash well in xylol and mount in xylol balsam. Film preparations after being washed in xylol may be dried and thereafter dilute carbol- fuchsin may be used to stain bacteria which have been decolorised. This modification probably gives the most uniformly successful results. 2. Nicolles Modification. — Carbol-gentian-violet is used as the stain. Treatment with iodine is carried out as above and decolorisation is effected with a mixture of acetone (1 part) and alcohol ^2 parts). 3. Kuhnes Modification. — (1) Stain for five minutes in a solution made up of equal parts of saturated alcoholic solution of crystal-violet (" Kry stall- vi olet ") 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 Kuhne : — Iodine ...... 2 parts Potassium iodide . . . . 4 ,, Distilled water . ' . . . . 100 ,, 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. There is great variability in the avidity with which organisms stained by Gram retain the dye when washed with alcohol, and sometimes difficulty is experienced in saying whether an organism does or does not stain by this method. Stain for Tubercle and other Acid -fast 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 its action may be aided TUBERCLE STAINS 101 by a short application of heat. When once stained, however, they resist decolorising even with very powerful acids ; they are therefore called " acid-fast." The smegma bacillus also resists decolorising with strong acids (p. 254), and a number of other acid-fast bacilli have recently been discovered (p. 252). 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. t Basic fuchsin . . . 1 part Absolute alcohol . . . .10 parts Solution of carbolic acid (1 : 20) . 100 „ 1. Place the specimen in this fluid, and having heated it till steam rises, allo\v 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 practice, it is best to remove the preparation from the acid every few seconds ani wash in water, replacing the specimen in the acid and re- washing till the proper pale pink tint is obtained. Then wash in alcohol for half a minute and replace in water. 4. Contrast stain with a saturated watery solution of rnethylene-blue for half a minute, or with saturated watery 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. Fraenkel's Modification of the Ziehl-Neelsen 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 : — Distilled water . .. . . i 50 parts Absolute alcohol . . '. . . 30 ,, Nitric acid 20 ,, Methylene-blue in crystals to saturation. 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. 102 MICROSCOPIC METHODS 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 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 considerable tenacity. The following is the simplest 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 methyl- ated spirit. This removes the stain from the bacilli. 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 decolorising agent for use. If sulphuric acid stronger than 1 per cent is used the spores of many bacilli are readily decolorised. Moller's Method. — The following method, recommended by Mbller, is much more satisfactory than the previous. Before being stained, the films are placed in chloroform for 2 minutes, and then in a 5 per cent solution of chromic acid for £-2 minutes, the preparation being well washed after each reagent. Thereafter they are stained and decolorised as above. The Staining of Capsules. — The two following methods may be recommended in the case of capsulated bacteria : — (a) Welch's Method. — This depends on the fact that in many cases the capsules can be fixed with glacial acetic acid. Films when still wet are placed in this acid for a few seconds. The superfluous acid is removed with filter-paper and the preparation is treated with gentian-violet in aniline oil water repeatedly till all the acetic acid is removed. Then wash with 1-2 per cent solution of sodium chloride and examine in the same solution. The capsule appears as a pale violet halo around the deeply stained bacterium. (b) Richard Muirs Method (as recently modified). 1. The film containing the bacteria must be very thin. It is dried and stained in filtered carbol-fuchsin for half a minute, the preparation being gently heated. STAINING OF FLAGELLA 103 2. Wash slightly with spirit and then well in water. 3. Place in following mordant for a few seconds : — Saturated solution of corrosive sublimate . . .2 parts Tannic acid solution — 20 per cent . . . 2 ,, Saturated solution of potash alum . . . . 5 , , 4. Wash well in water. 5. Treat with methylated spirit for about a minute. The preparation has a pale reddish appearance. 6. Wash well in water. 7. Counterstain with watery solution of ordinary methylene-blue for half a minute. 8. Dehydrate in alcohol, clear in xylol, and mount in balsam. The bacteria are a deep crimson and the capsules of a blue tint. The capsules of bacteria in cultures may sometimes be demonstrated by this method. 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 two 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 ten to eighteen hours at 37° 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 87. 1. Pitfield's Method as modified by Richard Muir. Prepare the following solutions : — A. The Mordant. Tannic acid, 10 per cent watery solution, filtered . 10 c.c. Corrosive sublimate, saturated watery solution Alum, saturated watery solution . . . 5 ,, Carbol-fuchsin (vide p. 101) . . . . 5 ,, 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 . . . 10 c.c. Gentian- violet, saturated alcoholic solution . . 2 ,, 104 MICROSCOPIC METHODS 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 of xylol balsam. This method has yielded the best results in our hands. 2. Van Ermengems 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 Solution C. (Bain reducteur et reinforqateur) — Gallic acid 5 grm. Tannin 3 ,, Fused potassium acetate . . . . . 10 ,, Distilled water . . . . . . . 350 c.c. Keep in this for a few seconds. 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 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 bacillus 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 flagella 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 first method. Staining of Spirochsete in Sections. — The following im- pregnation method, which is practically that of Ramon-y-Cajal THE ROMANOWSKY STAINS 105 for nerve fibrillse, has been applied for this purpose by Levaditi and gives excellent results. (1) The tissues, which ought to be in thin slices, about 1 mm. in thickness, are best fixed in 10 per cent formalin solution for twenty-four hours. (2) They are washed for an hour in water and then brought into 96 per cent alcohol for twenty-four hours. (3) They are then placed in 1'5 per cent solution of nitrate of silver in a dark bottle, and are kept in an incubator at 37° C. for three days. (4) They are washed in water for about twenty minutes, and are thereafter placed in the following mixture, namely : — Pyrogallic acid, 4 parts. Formalin, 5 parts. Distilled water up to 100 parts. They are kept in this mixture in a dark bottle for forty-eight hours at room temperature. (5) They are then washed in water for a few minutes, taken through increasing strengths of alcohol, and embedded in paraffin in the usual way. The sections ought to be as thin as possible. In satisfactory preparations the spirochaetes appear of an almost black colour against the pale yellow background of the tissues. The latter can be contrast stained by weak carbol- fuchsin or by toluidin blue. (For the staining of spirocheetes in films see p. 107.) The Romanowsky Stains. — Within recent years the numerous modifications of the Romanowsky stain have been extensively used. The dye concerned is the compound which is formed when watery solutions of medicinal methylene-blue and water- soluble eosin are brought together. This compound is insoluble in water but soluble in alcohol — the alcohol employed being methyl alcohol. The stain was originally used by Romanowsky for the malarial parasite, and its special quality is that it imparts to certain elements, such as the chromatin of this organism, a reddish-purple hue. This was at first thought to be simply due to the combination of the methylene-blue and the eosin, but it is now recognised that certain changes, such as occur in methylene-blue solutions with age, are necessary. In the modern formulae these changes are brought about by treatment with alkalies, especially alkaline carbonates, as was first practised by Unna in the preparation of his polychrome methylene-blue. It is not certainly known to what particular 106 MICROSCOPIC METHODS new body the reddish hue is due, but it may be to methyl-violet or to methyl-azure, both of which result from the action of alkali on methylene-blue. The stains are much used in staining blood- films (in which the characters of both nucleus and cytoplasm are beautifully brought out), in staining bacteria in tissues or exudates, the malaria parasite, trypanosomes, the pathogenic spirochsetes (such as the spirochaete pallida), and protozoa generally. The following are the chief formulae in use : — 1. Jenners Stain. — This is an excellent blood stain, but is not so good for the study of parasites as the others to be mentioned. In its preparation no alkali is used. It is made by mixing equal parts of (a) a 1 '2 to 1 '25 per cent solution of Griibler's water soluble eosin (yellow shade) in distilled water and (b) 1 per cent Griibler's medicinal methy- lene-blue (also a watery solution). The mixture is allowed to stand twenty-four hours, is filtered, and the residue is dried at 55° C. ; the powder is shaken up in distilled water, filtered, washed with distilled water and dried. Of the powder, 5 grms. are dissolved in 100 c.c. Merck's methyl alcohol. For use a few drops are placed on the dried unfixed film for one to three minutes, the dye is poured off, and the preparation washed with distilled water till it presents a pink colour ; it is then dried between filter-paper and mounted in xylol balsam. 2. Leishmans Stain. — The following solutions are prepared : (a) to a 1 per cent solution of medicinal methylene-blue is added '5 per cent sodium carbonate ; the mixture is kept at 65° C. for twelve hours and then for ten days at room temperature ('25 per cent formalin may be added as a preservative) ; (b) 1-1000 solution of eosin, extra B.A., in distilled water. Equal volumes of the two solutions are mixed and allowed to stand for six to twelve hours with occasional stirring, the precipitate is collected, filtered, washed with distilled water and dried. For use '15 per cent is dissolved in Merck's methyl alcohol ("for analysis, acetone free ") as follows : the powder is placed in a clean mortar, a little of the alcohol is added and well rubbed up with a pestle ; the undissolved powder is allowed to settle and the fluid decanted into a dry bottle ; the process is repeated with fresh fractions of the solvent till practically all the stain is dissolved, and the bottle is well stoppered ; the stain will keep for a long period. For the staining of films a few drops of the stain are placed on the unfixed preparation for fifteen to thirty seconds so as to cover it with a shallow layer (the stain may be conveniently spread over the film with a glass rod) and the film is tilted to and fro so as to prevent drying. This treatment efficiently fixes the film by the action of the methyl alcohol. About double the quantity of distilled water is now dropped on the film, and the stain and diluent are quickly mixed with the rod. Five minutes are now allowed for staining, and the stain is then gently washed off with distilled water. A little of the water is kept on the film for half a minute to intensify the colour contrasts in the various cells. For certain special structures such as Schiiffner's dots or Maurer's dots in the malarial parasite a longer staining (up to one hour) may be necessary, and in any case it is well to practise being able to control the depth of the staining effect by observation with a low power objective. If a preparation is to be stained for a long time it THE ROMANOWSKY STAINS 107 must be kept covered, and if in such cases a granular deposit is formed this may be got rid of by a quick wash with absolute alcohol. If in blood films the red corpuscles appear bluish instead of pink the colour may be restored by washing the tilm with acetic acid, 1-1500. The film is dried between filter-paper and mounted. For staining sections a little modification is necessary. A paraffin section is taken into distilled water as usual, the excess of water is drained off, and a mixture of one part of stain' and two parts of distilled water is placed on it. The stain is allowed to act for five to ten minutes till the tissue appears a deep Oxford blue ; it is then decolorised with 1-1500 acetic acid — the effect being watched under a low-power lens. The blue begins to come out, and the process is allowed to go on till only the nuclei remain blue. The section is then washed with distilled water, rapidly dehydrated with alcohol, cleared and mounted. If, as some- times happens, the eosin tint be too well marked it can be lightened by the action of 1-7000 solution of caustic soda, this being washed off whenever the desired colour has been attained. 3. J. H. Wright's Stain. — In this modification 1 per cent methylene- blue (BX or Ehrlich's rectified) and \ per cent sodium carbonate (both in water) are mixed and placed in a Koch's steriliser for an hour. When the fluid is cold, 1-1000 solution of extra B. A. eosin is added till the mixture becomes purplish and a finely granular black precipitate appears in suspension (about 500 c.c. eosin to 100 c.c. methylene-blue solution are required) ; the precipitate is filtered off and dried without being washed. A saturated solution of this is made in the pure methyl alcohol ; this is filtered and diluted by adding to 80 c.c. of the saturated solution 20 c.c. of methyl alcohol. The application of the stain is almost the same as with Leishman's. A few drops are placed on the preparation for a minute for fixation ; water is then dropped on till a green iridescent scum appears on the top of the fluid and staining goes on for about two minutes ; the stain is then washed off with distilled water and a little is allowed to remain on the film till differentiation is complete ; the preparation is carefully dried with filter-paper and mounted. 4. Giemsa's Stain. — Giemsa believes that the reddish-blue hue characteristic of the Romanowsky stain is due to the formation of methyl-azure, and he has prepared this by a method of his own under the name " Azur I." From this, by the addition of equal parts of medicinal methylene-blue, he prepares what he calls "Azur II.," and from this again by the addition of eosin he prepares " Azur II. -eosin." The latest formula for the finished stain is as follows : — Azur II. -eosin 3 gr., Azur II. 8 gr., Glycerin (Merck, chemically pure) 250 gr., Methyl alcohol (Kahlbaum, I.) 250 gr. This stain has been extensively used for demonstrating the Spirochsete pallida, but it can be used for any other purpose to which the Romanowsky stains are applicable. For the spirochsete the following are Giemsa's directions: — (1) Fix films in absolute alcohol for fifteen to twenty minutes, dry with I filter-paper. (2) Dilute stain with distilled water — one drop of stain to 1 c.c. water (the mixture being well shaken). (Sometimes the water is made alkaline by the addition of one drop of 1 per cent potassium carbonate to 10 c.c. water). (3) Stain for fifteen minutes. (4) Wash in brisk stream of distilled water. (5) Drain with filter-paper, dry, and mount in Canada balsam. With regard to the Jenner and Giemsa stains it is best to obtain the solutions from Griibler ready for use ; the powder for Leishman's stain may be obtained from the same source and the solution made up by 108 MICROSCOPIC METHODS the worker himself. Cabot states that Wright's stain can be obtained from the Harvard Co-operative Society, Boylston Street, Boston, U.S.A. Neisser's Stain. — Neisser introduced the following stain as an aid to the diagnosis of the diphtheria bacillus. Two solutions are used as follows : (a) 1 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 grins. 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 (b), 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 after 9-24 hours' incubation at 34-35° C. Satisfactory results are not always obtained in the case of films prepared from membrane, etc., but there is no doubt that here also the method is one of considerable value. SPECIAL BACTEKIOLOGICAL METHODS. Wright's Methods of measuring small amounts of Fluids. — In ordinary work fine calibrated pipettes may be used for measur- ing small quantities of fluids, but such pipettes are not always available, and by Wright's technique if a Gower's 5 c.mm. haemocytometer pipette be at hand any measurements may be undertaken, — in fact, once the pipette now to be described (see Fig. 45) is made we are independent of other means of measurement. A piece of quill tubing is drawn out to capillary dimensions, and the extreme tip of it is heated in a peep flame and then drawn out till it is of the thickness of a hair though FIG 45 —Wright's 5 st^ Possessing a bore. If the point be c» nun. pipGttG, Aj ^ i i • Casing of quill tubing ; into the tube the metal will be caught B, rubber nipple ; C, where the tube narrows and will pass no wax luting ; E to F, f urther — in fact, though air will pass, -E — -A W C — F E, hair capillary. this tube 5 c.mm. of mercury, measured from a Gower's pipette, is run down till it will go no further. A mark is made on the tube at the proximal end of the mercury, which is now allowed to run out, and the tube is carefully cut through at the mark. A piece of ordinary quill tubing is drawrn out and broken off just below where its narrowing has begun, the capillary tube has TESTING OF PROPERTIES OF SERUM 109 some wax moulded round its middle, the hair end is slipped through the broken-off end just mentioned, and the tube is fixed in position as shown in the figure. A rubber nipple placed on the end of the pipette completes the apparatus. If by pressing the nipple the air be expelled from the pipette and the end dipped under mercury, exactly 5 c.mm. will be taken up. Thus when pressure on the nipple is relaxed, other tubes can be very readily calibrated by the mercury being expelled into them and its limits marked on their bores. For measuring equal parts of different fluids the pipette described in connection with agglutination is very useful (see Fig. 46 d). The Testing of Agglutinative and Sedimenting Properties of Serum. liv ti'i'jl lit! nation is meant the aggregation into clumps of uniformly disposed bacteria in a Huid ; by sedimentation the" of n 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 ; these being chiefly met with when the individual is suffering from the disease produced 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 ; sediment- ation 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 : (a) controls should always be made with normal serum, and (b) the serum to be tested should never be brought in the undiluted 110 MICROSCOPIC METHODS condition into contact with the bacteria. The stages of pro- cedure are the following : — 1. Blood is conveniently 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 a Wright's blood capsule (Fig. 47) or into the bulbous portion of a capillary pipette, such as in Fig. 46, a. (These pip- ettes can be readily made by drawing out quill glass tubing in a flame. It is convenient 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 pip- ette. The serum is then to be diluted. 2. The serum may be diluted (a) by means of a grad aated pipette — either a leucocytometer pipette (Fig. 46, b) or some corre- sponding form. In this way successive dilutions of 1 : 10, 1 : 20, 1 : 100, etc., can be rapidly made. This is the best method. , (b) By means of a capillary \ / -§- n pipette with a mark on the tube, the serum is drawn b d up to the mark and then . blown out into a glass FIG. 46.-Tubes used in testing agglutinating and capsule . equal quantities segmenting properties of serum. 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 THE OPSONTC TECHNIQUE 111 is to draw a drop of Hood 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. 46, 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 wrhen 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, inciibated at 37° 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 a 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 microscope. The form of glass slide used for hang-drop cultures (Fig. 27) 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. 46, d. Diluted serum is drawn up to fill the space mn, a small quantity of air 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 Jcl. 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. It may be said that.it is often important to observe not only the •strongest concentration of a serum which will produce agglutination but also the weakest. Method of measuring the Phagocytic Capacity of the Leucocytes — the Opsonic Technique. — This was first done by Leishman by a very simple method as follows : A piece of quill tubing is drawn out to a capillary diameter so as to make a pipette about six inches long. The point is broken off and a rubber nipple adjusted to the wide end, a mark is made with an oil pencil about three-quarters of an inch above the orifice. Blood is drawn from the finger up to the mark, then an air-bubble is allowed to pass in. A thin 112 MICROSCOPIC METHODS emulsion of the bacterium to be tested having been prepared, a quantity of this is also drawn up to the mark. The two fluids are then thoroughly mixed by being first blown out on to a sterile slide and then being drawn back into the pipette and expelled, — this being repeated several times. A cover-glass is placed over the drop, and the slide is placed in the incubator at 37° C. for fifteen minutes. The cover-glass is then slipped off so as to make a film preparation which in the case of ordinary bacteria may be stained by Leishman's method. The number of bacteria present in, say, 50 polymorphonuclear cells succes- sively examined is determined and an average struck. The method was first used for showing that in cases of staphylococcus infection the average number of bacteria taken up was less than in a control in which the same bacterial emulsion was exposed to the blood of a healthy individual. In making such an observation drops from the two mixtures are placed on the same slide under separate cover-glasses and the preparation incubated. One cover is then slipped to one end of the slide and the other to the other, — the two films being then stained as one. According to Wright's view the process of phagocytosis in blood outside the body is not a simple one, and^e^pj^.^, leucocyte takes up a bacterium the latter most be acted on_ in some way by substances present in the serum, which Wrignt 'c&lls^ogsoniiis^ (see Immunity). The technique by which the actions of these opsonins is studied has been elaborated by Wright and his co-workers in connection with his work on bacterial vaccines, especially in relation to infection by the pyogenic cocci and the tubercle bacillus. This technique involves (1) the preparation of the bacterial emulsion, (2) the preparation of the leucocytes, (3) the preparation of samples of (a) serum from a normal person, (6) serum from the infected person. (1) Preparation of bacterial emulsion. In the case of the pyogenic cocci a little of a twenty-four hour living culture off a sloped agar tube is taken and rubbed up in a watch-glass with •85 per cent saline. The mixture is placed in a tube and centri- fugalised so as to deposit any masses of bacteria which may be present. Only by experience can a knowledge be gained of the amount of culture to be used in the first instance, but the resultant emulsion usually should exhibit only the merest trace of cloudiness to the naked eye. Wright states it will then contain from 7000 to 10,000 million bacteria per c.cm. If too strong an emulsion be used the leucocytes may take up so many organisms that these cannot be accurately enumerated. In the case of PREPARATION OF THE SERA 113 the tubercle bacillus as short a variety of the organism as possible should be selected, and a mass of growth off a solid medium is taken (bacilli in mass can be obtained in the market from wholesale chemists) and is well washed with changes of distilled water, dried on filter paper in a Petri dish and thoroughly rubbed up with a little 1*5 per cent saline in an agate mortar so as to disintegrate the bacterial masses and get an emulsion composed as far as possible of individual bacilli. This must be controlled by microscopic examination. A thick cream should be obtained, and this should be sterilised by steaming for half an hour on three successive days. Before sterilisation it is convenient to seal up the stock emulsion in small quantities in a number of pieces of quill tubing so that in the subsequent procedures only small portions of the emulsion are exposed to aerial contamination at one time. For actual use, one of those tubes is opened, a little is withdrawn with a sterile pipette, and a weak emulsion made in the same way as with the staphylococcus except that 1/5 per cent saline is used. The stock tube may be sealed with wax and kept for use again. A fresh emulsion ought to be made up for each day's work. (2) Preparation of leucocytes. Here the observer uses his own blood cells. A 1*5 per cent solution of sodium citrate in •85 per cent sodium chloride is prepared. This is placed in a glass tube three inches long made by drawing out a piece of half -inch tubing to a point, the tube being filled nearly to the brim. A handkerchief being bound round the finger, this is now pricked and the blood allowed to flow directly into the fluid, to the bottom of which it sinks. The tube ought to be inverted between the addition of every few drops of blood so as to bring the blood in contact with the citrate and prevent coagulation. The equivalent of about ten to twenty drops of blood should be obtained. The diluted blood is then centri- fugalised, and when the corpuscles are separated the super- natant fluid is removed, '85 per cent saline is substituted and the centrifugalisation repeated. The fluid is again removed, care being taken not to disturb the layer of white cells lying on the top of the red corpuscles. This layer is then pipetted off into a watch-glass or tube and the leucocytes required are thus obtained. (3) Preparation of the sera. The serum whose sensitising effect on the bacteria it is desired to test is obtained by Wright as follows. A "blood capsule" is made by drawing a piece of No. 3 quill tubing into the shape shown in Fig. 47, the part not drawn out being about one inch in length. It is convenient to 8 114 MICROSCOPIC METHODS make a number of these capsules at one time and to draw off their extremities and seal them in the flame. For use the tips of both extremities are broken off, the finger is pricked, and blood allowed to pass into the capsule through the bent limb till the capsule is about half full. The air remaining in the capsule is rarefied by passing the straight end through a flame and then sealing it off. By this manipulation the blood is sucked over the bend into the straight part of the tube, and the bent end is now also sealed off or closed with wax. It is well to shake the blood down towards the closed straight end, care being taken to previously allow the glass to cool sufficiently. The capsule is now hung by the bend on the edge of a centrifuge tube 'and the serum separated by spinning the instrument. In any particular case a capsule of serum from the infected person and one from a normal individual are prepared. The emulsion, corpuscles, and serum being thus prepared, the next step is to mix them. This is done by taking a piece of quill tubing and drawing it out to a capillary point so as to make a pipette about eight inches long ; on the thick end of this a rubber teat is fixed, and about one inch from the capillary point a mark is made with FTO. 47.-Wright'8 Blood-cap- an oi! Penci}' From the ™tch-glass sule and method of filling containing the separated leucocytes a same. portion is sucked up to the mark and then an air-bubble is allowed to pass in. A similar portion of the serum is drawn up, and then another air-bubble, and finally a similar portion of the bacterial emulsion. The three droplets are carefully blown on to a slide and are thoroughly mixed with one another by being alternately drawn up into the tube and expelled several times. The mixture is then drawn into the tube and the end sealed off in the flame. The rubber nipple is removed and the tube placed in the incubator at 37° for fifteen minutes. A slide is now prepared by rubbing it once or twice with very fine emery paper (No. 000) and thoroughly wiping it. This is a procedure adopted by Wright to cause an evenly distributed film to be made. The tube being removed from the incubator and the end broken off, its contents are again mixed by expelling and drawing up into the tube. A minute droplet is placed on the prepared slide, and by means of the edge of the end GENERAL BACTERIOLOGICAL DIAGNOSIS 115 of another slide a film is made which is then dried and is ready for staining. Films containing staphylococci are stained either by Leishman's stain (q.v.) or with carbol-thionin blue. In the former case no fixation is necessary, in the latter it is usual to fix in saturated perchloride of mercury for 1J minutes, wash in water and then stain. With tubercle films the following is the procedure : the film is fixed for two minutes in perchloride of mercury, washed thoroughly, stained with carbol-fuchsin as usual, decolorised with 2 '5 per cent sulphuric acid, cleared with 4 per cent acetic acid, counterstained with watery solution of methylene-blue, and dried. In applying the technique two preparations are made, in both of which the same emulsion and the same leucocytes are employed, but in one of which the bacteria have been exposed to the serum of the infected individual under observation, and in the other to that of a normal person — usually the observer himself. Each of these is now examined microscopically with a movable stage, the number of bacteria in the protoplasm of at least 50 polymorphonucleated leucocytes is counted and an average per leucocyte struck ; the proportion which this average in the case of the abnormal serum bears to the average in the preparation in which the healthy serum was used, constitutes the opsonic index, — that of the healthy serum being reckoned as unity. The reliability of the method of course depends on the phagocytic activity of the 50 cells counted representing the phagocytic activity of all the cells in the preparation. 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 (\\ of a micro- ^ scopic examination r\\ fH ™"*"™"i .I.T. «.;**„ a . (2) oran attemni^ To isolate tfie organisms present ; and (3) of the identification of , ili ' ii'ganisms isolated.. \Vo 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 116 GENERAL BACTERIOLOGICAL DIAGNOSIS being contaminated with extraneous 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 patho- logical 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 for the incision is preferably to be sterilised by boiling, the first part of the pus which escapes allowed to flow away (as it might be spoiled by containing some of the antiseptics used in the purification) 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 ab- sorbent 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 impregnated 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 ^e secured with sterile pipettes. To make T of *ese> 'ake nine inches °/ ordinary quill glass-tubing, draw out one end to a capillary diameter, and place a little plug of cotton wool 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 replace in its former position in the test-tube (Fig. 48). Another method very convenient for transport is to' make two constrictions on the glass tube at FIG. 48.— Test-tube and pipette ar- ing bacteria. in the other end. ROUTINE EXAMINATION OF MATERIAL 117 suitable distances, according to the amount of fluid to be taken. The fluid is 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. 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 spud 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 faeces 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 : — (1) Several cover-glass preparations should V>P( |pa.rlp ^Onp. ought to HDC stamea with saturated watery methy1pT1p-hl"pi one with a stain containing a mordant such as Ziehl-Neelsen p.a.rhnl-fijfljisjfl. one _by Gram's method ^2} (a} Gelatin plates should be made and^ ^kgpi at room temperature, (b] a series of agar p1a.t,ps or successive ^trolves on agiir tuLcs_(p. 55) should be made and incubated at 37° C. Method (b) 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 plate the first question to be cleared up is; Do all the colonies present consist of the same __ ba^tejium ? flie shape of the colony, its size, the appearance of the margin, the graining of the substance, its colour, etc., are all 118 GENERAL BACTERIOLOGICAL DIAGNOSIS 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 arrangement 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 colonies on a plate having been classified, a microscopic examina- tion of each group may be made by means of cover-glass preparations, and tubes of gelatin 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. The general lines along which observation is to be made in the case of a particular bacterium may be indicated as follows : — 1. Microscopic Appearances. — For ordinary descriptive pur- poses 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 1 How does it behave towards Gram's method 1 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 1 (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 GROWTH CHARACTERISTICS 119 temperature, (2) to oxygen 1 These can be answered from some of the following experiments : — A. Growth on gelatin. (1) Stab culture. Note (a) rate of growth ; (6) 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, (6) deep. (5) Growth in fluid gelatin at 37° C. B. Growth on agar at 37° C. (1) Stab. (2) Streak. Also on glycerin agar, blood agar, etc. Appearances of colonies in agar plates. C. Growth in bouillon, (a) character of growth, (b) smell, (c) reaction. D. Growth on special media. (1) Solidified blood serum. (2) Potatoes. (3) Lactose and other sugar media. Does fermenta- tion occur and is gas formed ? (4) Milk. Is it curdled or turned sour 1 (5) Litmus media. Note changes in colour. (6) Peptone solution. Is indol formed 1 E. What is the viability of organism on artificial media 1 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, how- ever, 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 characteristics 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 combinations, 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 microphoto- graphs 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 120 INOCULATION OF ANIMALS often vary with the age. It is suggested that in the case of cultures grown at from 36° to 37° 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 22° C. the appearances between 48 and 72 hours should be employed. The culture fluids 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. l 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 laboratory purposes. In the case of the mouse and rat the variety must be carefully noted, as there are differences in susceptibility 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 from 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 susceptible 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 platmum-iridium needles. Before use the syringe and the needle are sterilised by boiling for five minutes. The materials used for inoculation are cultures, animal exudations, or the juice of organs. If the bacteria already exist in a fluid there is no difficulty. The syringe 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 off and shaken up in sterile distilled water or '85 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 f in. glass-tubing 3 in. long, drawing 1 Experiments on animals, of course, caunot be performed in this country without a license granted by the Home Secretary. INOCULATION OF ANIMALS 121 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 ; (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 charged with the fluid to be inoculated. The hair is cut off the part to be inoculated, and the skin purified with 1 to 1000 corrosive sublimate. The skin is then pinched up and, the needle being inserted, the requisite dose is administered. The wound is then sealed with a little collodion. 3. Intraperitoneal Injection. — This may be 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. 49). The hair over the lower part of the abdomen is cut, and the skin purified with an antiseptic. The whole thickness of the ab- dominal 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 inoculation can also be practised with an FIG. 49.— Hollow needle with lateral aperture (at a) for intra- peritoneal in- oculations. 122 INOCULATION OF ANIMALS ordinary needle. The mode of procedure is similar, but after the needle is plunged through the abdominal fold, it is partially •withdrawn till the point is felt to be free in the peritoneal cavity when the injection 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 pressing on it between the point of inoculation and the heart. The needle is then plunged into the vein arid the fluid injected. That it has perforated the vessel will be shown by the escape of a little blood; and that the injection has taken place into the lumen of the vessel will be known by the absence of the small swelling which occurs in subcutaneous injections. If preferred, the vein may be first laid bare by snipping the skin over it. The needle is then introduced. 5. Inoculation into the Anterior Chamber of the Eye. — Local anaesthesia is established by applying a few drops of 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 accomplished. Sometimes inoculations are made by planting small pieces of pathological tissues in the subcutaneous tissue. This is especially done 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 comfortable cages, which must be capable of easy and thorough disinfection subsequently. 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 COLLODION CAPSULES 123 from above. Otherwise 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 vaselin, and the bulb inserted just within the sphincter, where it is allowed 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. Collodion Capsules.— These have been used to allow the sojourn of bacteria within the animal body without their coming into contact with the cells of the tissues. Various substances in solution can pass in either direction through the wall by diffusion, but the wall is impermeable alike to bacteria and leucocytes. The following method of preparing such capsules is that of M'Rae modified by Harris. A gelatine capsule, such as is used by veterinary surgeons, is taken, and in one end there is fixed a small piece of thin glass tubing by gently heating the glass and inserting it. The tube becomes fixed when quite cold, and the junction is then painted round with collodion, which is allowed to dry thoroughly. The bore of the tubing is cleared of any obstructing gelatine, and the whole capsule is dipped into a solution of collodion so as to coat it completely. The collodion is allowed to dry and the coating is repeated ; it is also advis- able to strengthen the layer by further painting it at the extremity and at the junction. The interior of the capsule is then filled with water by a fine capillary pipette, and the capsule is placed in hot water in order to liquefy the gelatine, which can be removed from the interior by means of the fine pipette. The sac is filled with bouillon and is placed in a tube of bouillon. It is then sterilised in the autoclave. A small quantity of the bouillon is removed, and the contents are inoculated with the particular bacterium to be studied or an emulsion of the bacterium is added. The glass tubing is seized in sterile forceps and is sealed off in a small flame a short distance above the junction. The closed sac ought then to be placed in a tube of sterile bouillon to test its impermeability. The result is satisfactory if no growth occurs in the surrounding medium. The sac with its contents can now be transferred to the peritoneal cavity of an animal. Autopsies on Animals dead or killed after Inoculation.— These should be made as soon as possible after death. It is 124 INOCULATION OF ANIMALS necessary to have some shallow troughs, constructed either of metal or of wood covered with metal, convenientfy 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 in 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 important, prevents hairs which might be affected with pathogenic products from getting into the air of the laboratory. The instruments 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 by surgeons. Two sets at least ought to be used in an autopsy, and they may be placed, after boiling, on a sterile glass plate covered by a bell -jar. It is also necessary to have a medium- sized hatchet-shaped cautery, or other similar piece of metal. It is well to have prepared a few freshly-drawn-out capillary tubes stored in a sterile cylindrical glass vessel, and also some larger sterile glass pipettes. The hair of the abdomen of the animal is removed. If some of the peritoneal fluid is wanted, a band should be cauterised down the linea alba from the sternum to the pubes, and another at right angles to the upper end of this ; an incision should be made in the middle of these bands, and the abdominal walls thrown to each side. One or more capillary tubes should then be filled with the fluid collected in the flanks, the fluid being allowed to run up the tube and 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 instruments, and it is convenient to place them pending examination 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, introducing a platinum eyelet, inoculate tubes and make cover-glass preparations at once. To examine any organ, sear the surface with a cautery, cut into it, and inoculate tubes and make film preparations with a platinum loop. For removing small parts of organs for making inoculations AUTOPSIES ON ANIMALS 125 on tubes, a small platinum spud is very useful, as the ordinary wires are apt to become bent. Place pieces of the organs in some preservative fluid for microscopic examination. The organs ought not to be touched with the fingers. When the examination is concluded the body should have corrosive sublimate or carbolic acid solution poured over it, and be forthwith burned. The dissecting trough and all the instruments ought to be boiled for half an hour. The amount of precaution to be taken will, of course, depend on the character of the bacterium under investiga- tion, but as a general rule every care should be used. CHAPTER IV. BACTERIA IN AIR, SOIL, AND WATER. ANTISEPTICS. IT is impossible here to do more than indicate the chief methods which are employed by bacteriologists in the investigation of the bacteria present in air, soil, and water, and to add an outline of the chief results obtained. In dealing with the latter the subject has been approached mainly from the standpoint of the bearings which the results have towards human pathology. In dealing with antiseptics, so far as possible the effects of the various agents on the chief pathogenic bacteria have been given, though in many cases our information is very imperfect. AIR. Very little information of value can be obtained from the examination of the air, but the following are the chief methods used, along with the results obtained. More can be learned from the examination of atmospheres experimentally contamin- ated than by the investigation of the air as it exists under natural conditions. Methods of Examination. — The methods employed vary with the objects in view. If it be sought to compare the relative richness of different atmospheres in organisms, and if the atmospheres in question be fairly quiescent, then it is sufficient to expose gelatin plates for definite times in the rooms to be examined. Bacteria, or the particles of dust carrying them, fall on the plates, and from the number of colonies which develop a rough idea of the richness of the air in bacteria can be obtained. Petri states that in five minutes the bacteria present in 10 litres of air are deposited on 100 square centimetres of a gelatin plate. 126 METHODS OF EXAMINATION 127 More complete results are available when some method is employed by which the bacteria in a given quantity of air are examined. The oldest method employed, and one which is still used, is that of Hesse. The apparatus is shown in Fig. 50. It consists of a cylindrical tube a about 20 inches long and 2 inches in diameter. At one end this is closed by a rubber cork having a piece of quill tubing, /, passing through it and projecting some distance into the interior. For use the tube is sterilised in a tall "Koch," and then a quantity of peptone gelatin, sufficient to cover the whole interior to the thickness of an ordinary gelatin plate, is poured in. This gelatin is kept from escaping by ./ the projection of the quill tubing into the lumen of the large tube. A plug of cotton wool is now placed in the outer end of the quill tubing. Over the other end of the large tube is tied a sheet of rubber having a hole about a quarter of an inch in diameter in its centre, and over this again is tied a piece of similar but un- perforated sheet rubber. The tube is then steri- lised in the tall " Koch.' On removal from this it is rolled, after the manner of an Esmarch's tube (q.v.), till the gelatin is set as a layer over its interior, and it is then placed horizontally on the tripod as shown. The other part of the appar- atus is an aspirator by means of which a known quantity • of air can be brought in contact with the gelatin. It consists FIG. 50. — Hesse's tube, mounted for use. of two conical glass flasks connected by means of a tube which passes through the cork of each down to the bottom of the flask. When this tube is filled with water, it, of course, can act as a syphon tube between volumes of water in the flasks. Such a syphon system being established, the levels of the water are marked on the flasks, and to one a litre of water is added, and by depressing flask b the whole litre can be got into it and the connecting tube c is then clamped. The two flasks are then connected by a rubber tube with the tube/, the clamp on c is opened, and the passing of a litre of water into d will draw a litre of air through the gelatin tube, when the outer rubber sheet is removed from the end and the clamp li opened. By disconnecting at g and reversing the syphon flasks, another litre can be sucked through, and so any desired quantity of air can be brought in contact with the gelatin. The speed ought not to be more than one litre in two minutes, and in such a case practically 128 BACTERIA IN AIR all the organisms will be found to have fallen out of the air on to the gelatin in the course of their transit. This fact can be tested by interposing between the tube a and the aspirator a second tube prepared in the same way, which ought, of course, to show no growth. When forty-eight hours at 20° C. or four days at lower temperature have elapsed, the colonies which develop in a may be counted. The dis- advantage of the method is that if particles of dust carrying more than one bacterium alight on the gelatin, these bacteria develop in one colony, and thus the enumeration results may be too low ; difficulties may also arise from liquefying colonies developing in the upper parts of the tubS and running over the gelatin. Petri's Sand-Filter Method. — A glass tube open at both ends, and about 3| inches long and half an inch wide, is taken, and in its centre is placed a transverse diaphragm of very fine iron gauze (Fig. 51, e) ; on each side of this is placed some fine quartz sand which has been well washed, dried, and burned to remove all impurities, and this is kept in position by cotton plugs. The whole is sterilised by dry heat. One plug is removed and a sterile rubber cork, c, inserted, through which a tube, d, passes to an exhausting apparatus. The tube is then clamped in an upright position in the atmo- sphere to be examined, with the remaining plug, /, uppermost. The latter is removed and the air sucked through. Difficulty may be experienced from the resistance of the sand if quick filtration be attempted. The best means to adopt is to use an air-pump — the amount of air drawn per stroke of which is accurately known — and have a manometer (as in Fig. 31) interposed between the tube and the pump. Between each two strokes of the air-pump the mercury is allowed to return to zero. After the required amount of air has passed, the sand a is removed, and is distributed among a number of sterile gelatin tubes which are well shaken ; plate cultures are then made, and when growth has occurred the colonies are enumerated ; the sand b is similarly treated and acts as a control. When it is necessary to examine air for particular organisms, special methods must often be adopted. Thus in the case of the suspected presence of tubercle bacilli a given quantity of air is drawn through a small quantity of water and then injected" into a guinea-pig. It must be admitted that comparatively little information bearing on the harmlessness or harmfulness of the air is obtain- able by the mere enumeration of the living organisms present, for under certain conditions the number may be increased by the presence of many individuals of a purely non-pathogenic character. The organisms found in the air belong to two groups — firstly, a great variety of bacteria ; secondly, yeasts and the spores of moulds and of the lower fungi. With regard to the spores, the organisms from which they are derived often FIG. 51. — Petri's sand filter. PETRI'S SAND-FILTER METHOD 129 consist of felted masses of threads, from which are thrust into the air special filaments, and in connection with these the spores are formed. By currents of air these latter can easily be detached, and may float about in a free condition. With the bacteria, on the other hand, the case is different. Usually these are growing together in little masses on organic materials, or in fluids, and it is very much by the detachment of minute particles of the substratum that the organisms become free. The entrance of bacteria into the air, therefore, is associated with conditions which favour the presence of dust, minute droplets of fluid, etc. The presence of dust in particular would specially favour a large number of bacteria being observed, and this is the case with the air in many industrial conditions, where the bacteria, though numerous, may be quite innocuous. Great numbers of bacteria thus may not indicate any condition likely to injure health, and this may be true also even when the bacteria come from the crowding together of a number of healthy human beings. On the other hand, there is no doubt that disease germs can be disseminated by means of the air. The possibility of this has been shown experimentally by infecting the mouth with the b. prodigiosus, which is easily recognised by its brilliantly coloured colonies, and then studying its subsequent distribution. Most important here is the infection of the air from sick persons. The actions of coughing, sneezing, speaking, and even of deep breathing, distribute, often to a considerable distance, minute droplets of secretions from the mouth, throat, and nose, and these may float in the air for a considerable time. Even five hours after an atmosphere has been thus infected evidence may be found of bacteria still floating free. Before this time, however, most of the bacteria have settled upon various objects, where they rapidly dry, and are no longer displaceable by ordinary air currents. The diseases of known etiology where infection can thus take place are diphtheria, influenza, pneumonia, and phthisis ; and here also probably whooping-cough, typhus fever, and measles are to be added, though the morbific agents are unknown. In the case of phthisis, the alighting of tubercle bacilli has been demonstrated on cover-glasses held before the mouths of patients while talking, and animals made to breathe directly in front of the mouths of such patients have become infected with tuberculosis. Apart from direct infection from individuals, however, pathogenic bacteria may be spread in some cases from the splashing of infected water, as from a sewage outfall. This possibility has to be recognised especially in the cases of typhoid and cholera. Besides infection through fluid particles, 130 BACTERIA IN AIR infection can be caused in the air by 'dust coming, say, from infected skin or clothes, etc. Fliigge, in dealing with this subject in an experimental inquiry, distinguishes between large particles of dust which require an air current moving at the rate of 1 centimetre per second to keep them suspended, and the finer dust which can be kept in suspension by currents moving at from 1 to 4 millimetres per second. In the former case, when once the particles alight they cannot be displaced by currents of air except when these are moving at, at least, 5 metres per second, but the brushing, shaking, or beating of objects may, of course, distribute them. In the case of the finer dust the particles will remain for long suspended, and when they have settled can be more easily displaced, as by the waving of an arm, breathing, etc. With regard to infection by dust, a most important factor, however, is whether or not the infecting agent can preserve its vitality in a dry condition. In the case of a sporing organism such as anthrax, vitality is preserved for long periods of time, and great resistance to drying is also possessed by the tubercle and diphtheria bacilli ; but apart from such cases there is little doubt that infection is usually necessarily associated with the transport of moist particles, and is thus confined to a limited area around a sick person. Among diseases which may occasionally be thus spread cholera and typhoid have been classed. Considerable controversy has arisen with regard to certain out- breaks of the latter disease, which have apparently been spread by dusty winds, although we have the fact that the typhoid bacillus does not survive being dried even for a short time. It appears, however, that in such epidemics the transport of infection by means of insects carried by the wind has not been entirely excluded. As in the cases of the soil and of water, presently to be described, attempts have been made to obtain indirect evidence of the contamination of the air by man. Thus Gordon has shown that certain streptococci are common in the saliva ; these resemble the streptococcus pyogenes, but are relatively non-pathogenic, grow well at 37° C. and under anaerobic conditions, cause clotting and acid-formation in litmus milk at 37°, and in neutral-red media have an action resembling that of b. coli. These characters serve, according to Gordon, to differentiate organisms of human origin from ordinary streptococci occurring in the air and which he states grow better at about 22° C., are facultative anaerobes and do not produce the changes in milk and in neutral-red media. Thus the finding of streptococci of the first group in plates exposed to air would indicate that a human source was probable, and, if the observation were made on air from the neighbourhood of a sick person, that risk of the dissemination of disease germs was present. The value of this as a practical method has yet to be determined. BACTERIA IN SOIL 131* SOIL. The investigation of the bacteria which may be found in the soil is undertaken from various points of view. Information may be desired as to the change its composition undergoes by a bacterial action, the result of which may be an increase in fertility and thus in economic value. Under this head may be grouped inquiries relating to the bacteria which convert ammonia and its salts into nitrates and nitrites, and to the organisms concerned in the fixation of the free nitrogen of the air. The discussion of the questions involved in such inquiries is outside the scope of the present chapter, which is more con- cerned with the relation of the bacteriology of the soil to questions of public health. So far as this narrower view is concerned, soil bacteria are chiefly of importance in so far as they can be washed out of the soils into potable water supplies. An important aspect of this question thus is as to the significance of certain bacterio- logical appearances in a water in relation to the soil from which it has come or over which it has flowed. In this country these questions have been chiefly investigated by Houston, and it is from his papers that the following account is largely taken. Methods of Examination. — For examination of soil on surface or not far from surface, Houston recommends tin troughs 10 in. by 3 in. and pointed at one extremity, to be wrapped in layers of paper and sterilised by dry heat. If several of these be provided, then the soil can be well rubbed up and a sample secured and placed in a sterile test-tube for examination as soon as convenient after collection. If samples are to be taken at some depth beneath the surface, then a special instrument of which many varieties have been devised must be used. The general form of these is that of a gigantic gimlet stoutly made of steel. Just above the point of the instrument the shaft has in it a hollow chamber, and a sliding lateral door in this can be opened and shut by a mechanism controlled at the handle. The chamber being sterilised and closed, the instrument is bored to the required depth, the door is slid back, and by varying devices it is effected that the chamber is filled with earth ; the door is reclosed and the instrument withdrawn. In any soil the two important lines of inquiry are first as to the total number of organisms (usually reckoned per gramme of the fresh sample), and secondly as to the varieties of organisms present. The number of organisms present in a soil is often, however, so enormous that it is con- venient to submit only a fraction of a gramme to examination. The method employed is to weigh the tube containing the soil, shake out an amount of about the size of a bean into a litre of distilled water, and re- weigh the tube. The amount placed in the water is distributed as thoroughly as possible by shaking, and, if necessary, by rubbing down with a sterile glass rod, and small quantities measured from a graduated pipette are used for the investigation. For estimating the total number of organisms present in the portion of soil used, small quantities, say *1 c.c. and 1 c.c., of the fluid are added to melted tubes of ordinary alkaline peptone gelatin ; after being shaken, the gelatin is plated, incubated at 132 BACTERIA IN SOIL 22° C., and the colonies are counted as late'as the liquefaction, which always occurs round some of them, will allow. From these numbers the total number of organisms present in the amount of soil originally present can be calculated. The numbers of bacteria in the soil vary very much. Accord- ing to Houston's results, fewest occur in uncultivated sandy soils, these containing on an average 100,000 per gramme. Peaty soils, though rich in organic matter, also give low results, it being possible that the acidity of such soils inhibits free bacterial growth. Garden soils yield usually about 1,500,000 bacteria per gramme, but the greatest numbers are found in soils which have been polluted by sewage, when the figures may rise to 115,000,000. In addition to the enumeration of the numbers of bacteria present, it is a question whether something may not be gained from a knowledge of the number of spores present in a soil relative to the total number of bacteria. This is a point which demands further inquiry, especially by the periodic investi- gation of examples of different classes of soils. The method is to take 1 c.c. of such a soil emulsion as that just described, add it to 10 c.c. of gelatin, heat for ten minutes at 80° C. to destroy the non-spored bacteria, plate, incubate, and count as before. Besides the enumeration of the numbers of bacteria present in a soil, an important question in its bacteriological examination lies in inquiring what kinds of bacteria are present in any par- ticular case. Practically this resolves itself into studying the most common bacteria present, for the complete examination of the bacterial flora of any one sample would occupy far too much time. Of these common bacteria the most important are those from whose presence indications can be gathered of the con- tamination of the soil -by sewage, for from the public health standpoint this is by far the most important question on which bacteriology can shed light. Bacillus mycoides. — This bacillus is 1'6 to 2*4 /j, in length and about *9 in breadth. It grows in long threads which often show motility. It can be readily stained by such a combination as carbol-thionin, and re- tains the dye in Gram's method. All ordinary media will support its growth, and, in surface growths on agar or potato spore, formation is readily produced. Its optimum temperature is about 18° C. On gelatin plates it shows a very characteristic appearance. At first under a low power it shows a felted mass of filaments throwing out irregular shoots from the centre, and later to the naked eye these appear to be in the form of thick threads like the growth of a mould. They rapidly spread over the surface of the medium, and the whole resembles a piece of wet teased-out cotton wool. The gelatin is liquefied. Cladothrices. — Of these several kinds are common in the soil. The ordinary dadothrix dichotoma is among them. This organism appears BACTERIA IN SOIL 133 as colourless flocculent growth with an opaque centre, and can be seen under the microscope to send out into the medium apparently branched threads which vary in thickness, being sometimes 2 p across. They consist of rods enclosed in a sheath. These rods may divide at any point, and thus the terminal elements may be pushed along the sheath. Sometimes the sheath ruptures, and thus by the extrusion of these dividing cells and their further division the branching appearance is originated. Reproduction takes place by the formation of gonidia in the interior of the terminal cells. These gonidia acquire at one end a bundle of flagella, and for some time swim free before becoming attached and forming a new colony. Houston describes as occurring in the soil another variety, which with similar microscopic characters appears as a brownish growth with a pitted surface, and diffusing a bismarck-brown pigment into the gelatin which it liquefies. A few experiments made with an ordinary field soil will, however, familiarise the worker with the non-pathogenic bacteria usually present. We have referred to these two because of their importance. In regard to pathogenic organisms, especially in relation to possible sewage con- tamination, attention is to be directed to three groups of organisms, those resembling the b. coli, the bacillus enteritidis sporogenes, and the streptococcus pyogenes. The characters of the first two of these will be found in the chapter on Typhoid Fever ; of the third in Chap. VI. For the detection of these bacteria Houston recommends the following procedure. (a) The B. coli group. A third of a gramme of soil is added to 10 c.c. phenol broth (vide chapter on Typhoid Fever) and incubated at 37° C. In this medium very few if any other bacteria except those of the b. coli group will grow, so that if after twenty-four hours a turbidity appears, some of the latter may be suspected to be present. In such a case a loopful of the broth is shaken up in 5 c.c. sterile distilled water, and of this one or two loopfuls are spread over the surface of a solid plate of phenol gelatin in a Petri capsule either by means of the loop or of a small platinum spatula, and the plate is incubated at 20° C. Any colonies which resemble b. coli are then examined by the culture methods detailed under that organism. Further, all organisms having the microscopic appearances of b. coli, and which generally conform to its culture reactions, are to be reckoned in the coli group. The media of MacConkey and Drigalski are very useful in connection with the separa- tion of such soil organisms (v. pp. 42, 43). (b) The Bacillus enteritidis sporogenes. To search for this organism 1 gramme of the soil is thoroughly distributed in 100 c.c. sterile distilled water, and of this 1 c.c., "1 c.c., and '01 c.c. is added to each of three sterile milk tubes. These are heated to 80° C. for ten minutes and then cultivated anaerobically at 37° C. for twenty-four hours. If the characteristic appear- ances seen in such cultures of the b. enteritidis (q.v.) are developed, then it may fairly safely be deduced that it is this organism which has produced them. (c) Fcecal Streptococci. The method here is to pour out a tube of agar into a Petri capsule, and when it has solidified to spread out "1 c.c. of the emulsion of soil over it and incubate at 37° C. for twenty-four hours. At this temperature many of the non-pathogenic bacteria grow with difficulty, and thus the number of colonies which develop is relatively small. Colonies having appearances resembling those of the streptococcus pyogenes (q.v.} can thus be investigated. Much work has been devoted to the question of these feecal streptococci presenting specific characters by 134 BACTERIA IN SOIL which they could be differentiated from other streptococci. No definite results have as yet been obtained. Houston gives as the general characters of these organisms that they usually grow in short chains, that they produce uniform turbidity in broth, that they give rise to acid and clot in litmus milk at 37° C., and that they are non-pathogenic to mice. The important point is to recognise that streptococci of fairly ordinary types exist in great numbers in human fseces, and that when in any circumstances faecal contamination is suspected the isolation of streptococci strengthens the suspicion. We may now give in brief the results at which Houston has arrived by the application of these methods. First of all, un- cultivated soils contain very few, if any, representatives of the b. mycoides, and this is also true to a less extent of the cladothrices. Cultivated soils, on the other hand, do practically always contain these organisms. With regard to the b. coli its presence in a soil must be looked on as indicative of recent pollution with excremental matter. The presence of b. enteritidis is also evidence of such pollution, but from the fact that it is a sporing organism this pollution may not have been recent. With regard to the streptococci, on the other hand, the opinion is advanced that their presence is, on account of their want of viability outside the animal body, to be looked on as evidence of extremely recent excremental pollution. The very great importance of these results in relation to the bacteriological examination of water supplies will be at once apparent, and will be referred to again in connection with the subject of water. While such means have been advanced for the obtaining of indirect evidence of excremental pollution of soil, and therefore of a pollution dangerous to health from the possible presence of pathogenic organisms in excreta, investigations have also been conducted with regard to the viability in the soil of pathogenic bacteria, especially of those likely to be present in excreta, namely, the typhoid and cholera organisms. The solution of this problem is attended with difficulty, as it is not easy to identify these organisms when they are present in such bacterial mixtures as naturally occur in the soil. Now there is evidence that bacteria when growing together often influence each other's growth in an unfavourable way, so that it is only by studying the organisms in question when growing in unsterilised soils that information can be obtained as to what occurs in nature. For instance, it has been found that the b. typhosus, when grown in an organically polluted soil which has been sterilised, can maintain its vitality for fifteen weeks, but if the conditions occurring naturally be so far imitated by growing it in soil in the presence of a pure culture BACTERIA IN WATER 135 of one or other certain soil bacteria, it is found that sometimes the typhoid bacillus, sometimes the soil bacterium in the course of a few weeks, or even in a few days, disappears. Further, the char- acter of the soil exercises an important effect on what happens ; for instance, the typhoid bacillus soon dies out in a virgin sandy soil, even when it is the only organism present. In experiments made by sowing cultures of cholera and diphtheria in plots in a field it was found that after, at the longest, forty days they were no longer recognisable. Further, it is a question whether ordinary disease organisms, even if they remain alive, can multiply to any great extent in soil under natural conditions. If we are dealing with a sporing organism such as the b. anthracis, the capacity for remaining in a quiescent condition of potential pathogenicity is, of course, much greater. The most important principle to be deduced from these experiments is that the ordinary conditions of soil rather tend to be unfavourable to the continued existence of pathogenic bacteria, so that by natural processes soil tends to purify itself. It must, however, be noted that such an organism as the typhoid bacillus can exist long enough in soil to be a serious source of danger. WATER. In the bacteriological jexamination of water three lines of inquiry may have to be followed. First, the number of bacteria per cubic centimetre may be estimated. Second, the kinds of bacteria present may be investigated. Third, it may be necessary to ask if a particular organism is present, and, if so, in what number per c.c. it occurs. Methods. — In the two first cases a small quantity ("5-1 c.c.) is taken in a sterile pipette and added to a tube of gelatin, which is then plated and incubated at the room temperature. In the case of water taken from a house tap the water 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 precaution be not adopted an altogether erroneous idea of the number present may be obtained. In the case of the examination of river water the gelatin plates ought to be prepared on the spot ; at any rate, the time clasping between the sample being taken and the plates being prepared 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 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 136 BACTERIA IN WATEK 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. The bottles ought to be packed in ice and sawdust, and plates must be prepared from the samples as soon as possible. When the object in view is to determine the number of bacteria per cubic centimetre, it is important to note that water bacteria grow at very varied rates, and therefore it is well that the same time should always elapse before the colonies are counted. The period of growing usually allowed is forty- eight hours at 20° C. Several points may be here noted. It has been found, for instance, that slight variations in the reaction of the medium affect the number of colonies which develop. A slightly greater degree of alkalinity than peptone gelatin, as ordinarily prepared, possesses — such an increased degree as that caused by the addition of '01 grm. Na2C03 to 10 c.c. peptone gelatin — will give a greater yield of colonies than the ordinary gelatin. Again, the natural temperature of the growth of water bacteria in temperate climates is comparatively low, being not often above 18° C., and, on account of this, gelatin suggests itself as the most suitable medium. This can be seen by comparing the growth on an agar plate inoculated with a given quantity of water, and incubated at 37° C., with the growth on a precisely similar gelatin plate incubated at 20° C. , as it will be found that many more colonies have developed on the latter. This fact may be taken advantage of when pathogenic bacteria are being sought for in a water. The latter usually grow well at 37° C., and thus if agar plates be used the search may be facilitated. Apart from the difference of incubation temperatures, however, in such a case as that cited, it is probable that agar is a less suitable medium than gelatin for the growth of water bacteria, for in plates incubated at the same temperature the colonies which grow on the agar are often fewer than those on the gelatin. Probably no one medium will support the growth of all the organisms present in a given sample of water, and under certain circumstances special media must therefore be used. Thus Hansen found that in testing waters to be used in brewing it was advisable to have in the medium employed some sterile wort or beer, so that the organisms in the test experiments should be provided with the food materials which would be present in the commercial use of the water. Manifestly this principle applies generally in the bacteriological examina- tion of waters to be used for industrial purposes. In ordinary public health work it may be taken that the most frequent and important inquiry is directed towards the presence or absence of the b. coli and its congeners. Many methods are here used but we consider that in which MacConkey's bile-salt media are employed the most convenient. For small quantities of water, — up to 1 c.c., — the sample is simply added to a Durham's tube of bile-salt glucose neutral- red broth and incubated for 48 hours. When it is necessary to examine larger samples it is convenient, as Savage recommends, to have the bile- salt broth made of double, treble, or quadruple its usual strength. The water to be examined is used as the diluent by which the medium is brought down to the ordinary concentration. If gas forms, some of the coli group are almost certainly present. The organisms may be plated out by smearing a little of the broth on bile-salt agar for further isolation and examination. With regard to the objects with which the bacteriological BACTERIA IN WATER 137 examination of water may be undertaken, though these may be of a purely scientific character, they usually aim at contribut- ing to the settlement of questions relating to the potability of waters, to their use in commerce, and to the efficiency of pro- cesses undertaken for the purification of waters which have undergone pollution. The last of these objects is often closely associated with the first two, as the question so often arises whether a purification process is so efficient as to make the water again fit for use. Water derived from any natural source contains bacteria, though, as in the case of some artesian wells and some springs, the numbers may be very small, e.g. 4 to 100 per c.c. In rain, snow, and ice there are often great numbers, those in the first two being derived from the air. Great attention has been paid to the bacterial content of wells and rivers. With regard to the former, precautions are necessary in arriving at a judgment. If the water in a well has been standing for some time, multiplication of bacteria may give a high value. To meet this difficulty, if practicable,- the well ought to be pumped dry and then allowed to fill, in order to get at what is really the im- portant point, namely, the bacterial content of the water entering the well. Again, if the sediment of the well has been stirred up a high value is obtained. Ordinary wells of medium depth contain from 100 to 2000 per c.c. With regard to rivers very varied results are obtained. Moorland streams are usually very pure. In an ordinary river the numbers present vary at different seasons of the year, whilst the prevailing temperature, the presence or absence of decaying vegetation, or of washings from land, and dilution with large quantities of pure spring water, are other important features. Thus the Franklands found the rivers Thames and Lea purest in summer, and this they attributed to the fact that in this season there is most spring water entering, and very little water as washings off land. In the case of other rivers the bacteria have been found to be fewest in winter. A great many circumstances must therefore be taken into account in dealing with mere enumerations of water bacteria, and such enumerations are only useful w7hen they are taken simultaneously over a stretch of river, with special reference to the sources of the water entering the river. Thus it is usually found that immediately below a sewage effluent the bacterial content rises, though in a comparatively short distance the numbers may markedly decrease, and it may be that the river as far as numbers are concerned may appear to return to its previous bacterial content. The numbers of 138 BACTERIA IN WATER bacteria present in rivers vary so greatly that there is little use in quoting figures, most information being obtainable by comparative enumerations before and after a given event has occurred to a particular water. Such a method is thus of great use in estimating the efficacy of the filter beds of a town water supply. These usually remove from 95 to 98 per cent of the bacteria present, and a town supply as it issues from the filter beds should not contain more than 100 bacteria per c.c. Again, is it found that the storage of water diminishes the number .of bacteria present. The highest counts of bacteria per c.c. are observed with sewage ; for example, in the London sewage the numbers range from six to twelve millions. Much more important than the mere enumeration of the bacteria present in a water is the question whether these include forms pathogenic to man. The chief interest here, so far as Europe is concerned, lies in the fact that typhoid fever is so frequently water-borne, but cholera and certain other intestinal diseases have a similar source. The search in waters for the organisms concerned in these diseases is a matter of the greatest difficulty, for each belongs to a group of organisms morpho- logically similar, very widespread in nature, and many of which have little or no pathogenic action. The biological characters of these organisms will be given in the chapters devoted to the diseases in question, but here it may be said that from the public health standpoint the making of their being found a criterion for the condemning of a water is impractic- able. There is no doubt that the typhoid and cholera bacteria can exist for some time in water — at least this has been found to be the case when sterile water has been inoculated with these bacteria. But to what extent the same is true when they are placed in natural conditions, which involve their living in the presence of other organisms, is unknown, for it may be safely said that by no known method can the presence of either be demonstrated in the complex mixtures which occur in nature. With regard to the typhoid bacillus, of late the tendency has been to seek for the presence of indirect bacterio- logical evidence which might point in the direction of the possibility of the presence of this organism. The methods employed and the lines along which such investigations have gone have already been alluded to in connection with soil. The whole question turns on the possibility of recognising bacteriologically the contamination of water with sewage. Klein and Houston here insist on the fact that in crude sewage the b. coli or the members of the coli group are practically BACTERIAL TREATMENT OF SEWAGE 139 never fewer than 100,000 per c.c., and therefore if in a water this organism forms a considerable proportion of the total number of organisms present, then there is grave reason for suspecting sewage pollution. Houston holds that, the nearer the majority of the coli organisms in a water approach to the typical reactions of coli the more likely is sewage contamination to be present. The reactions regarded by him as typical are, gas production in gelatin shake culture, production of indol, clotting of milk, production of fluorescence in neutral-red broth, acid and gas production in lactose peptone solution (v. b. coli). The presence of b. coli in 100 c.c. of deep well water or in 10 c.c. of river or shallow well water is sufficient to condemn that water. As the b. coli is fairly widespread in nature, Klein and Houston hold that valuable supporting evidence is found in the presence of the b. enteritidis sporogenes and of the streptococci, both of which are probably constant inhabit- ants of the human intestine. The spores of the former usually number 100 per c.c. in sewage, and the presence of the latter can always be recognised in *001 grm. of human faeces. The deductions to be drawn from the presence of these in water are the same as those to be drawn from their presence in soil. A further point here is that it is well, wherever practicable, that the indirect evidence as to the potability of a water which is usually derived from chemical analysis should be supplemented by a bacteriological search for the three groups of organisms mentioned. It has been found that in water artificially polluted with sewage containing them, they can be detected by bacterio- logical methods in mixtures from ten to a hundred times more dilute than those in which the pollution can be detected by purely chemical methods. Bacterial Treatment of Sewage. — Of late years the opinion has been growing that the most appropriate method of dealing with the disposal of sewage is to imitate as far as possible the processes which occur in nature for the breaking up of organic material. These practically depend entirely on bacterial action. Hence the rationale of the most approved methods of sewage disposal is to encourage the growth of bacteria which naturally exist in sewage, and which are capable of breaking up organic compounds and of converting the nitrogen into nitrates and nitrites. The technique by which this is accomplished is very varied and sometimes rather empirical, but probably the general principles underlying the different methods are comparatively simple. It is probable that for the complete destruction of the organic matter of sewage both aerobic and anaerobic bacteria 140 BACTERIA IN WATER are required, though on this point there may be some difference of opinion. Certainly very fair results are obtained when apparently the conditions chiefly favour aerobic organisms alone. This is usually effected by running the sewage on to beds of sand, or preferably of coke, allowing it to stand for some hours, slowly running the effluent out through the bottom of the bed, and leaving the bed to rest for some hours before recharging. The final result is better if the effluent be afterwards run over another similar coke-bed. According to some authorities the sewage, as it runs into the first bed, takes up from the air considerable free oxygen, which, however, soon disappears during the stationary period, so that on leaving the first bed the sewage contains little oxygen. In the latter part of its stay it has thus been submitted to anaerobic conditions. Further, while by the passage of the effluent out of the first bed oxygen is sucked in, this rapidly dis- appears, and during the greater part of the resting stage the interstices of the bed are filled with carbonic acid gas, with nitrogen partly derived from the air, partly from putrefactive processes, and thus in the filter anaerobic conditions prevail, under which the bacteria can act on the deposit left on the coke. On this latter point there is difference of opinion, for, in examin- ing London sewage, Clowes has found oxygen present in abundance from four to forty hours after the sewage has been run off. Sometimes the treatment of the sewage consists in allowing it continuously to trickle through sand or gravel or coke beds. Probably the best results in sewage treatment are obtained when it is practicable to introduce a step where there can be no doubt that the conditions are anaerobic. This involves as a pre- liminary stage the treatment of the sewage in what is called a septic tank, and the method has been adopted at Exeter, Button, and Yeovil in this country, and very fully worked at in America by the State Board of Health of Massachusetts. In the explana- tion given of the rationale of this process, sewage is looked on as existing in three stages. (1) First of all, fresh sewage — the newly mixed and very varied material as it enters the main sewers. (2) Secondly, stale sewage — the ordinary contents of the main sewers. Here there is abundant oxygen, and as the sewage flows along there occurs by bacterial action a certain formation of carbon dioxide and ammonia which combine to form ammonium carbonate. This is the sewage as it reaches the purification works. Here a preliminary mechanical screening may be adopted, after which it is run into an air-tight tank — the septic tank. (3) It remains there for from twenty-four to thirty-six hours, and becomes a foul-smelling fluid — the septic sewage. The chemical ANTISEPTICS 141 changes which take place in the septic tank are of a most complex nature. The sewage entering it contains little free oxygen, and therefore the bacteria in the tank are probably largely anaerobic, and the changes which they originate consist of the formation of comparatively simple compounds of hydrogen with carbon, sulphur, and phosphorus. As a result there is a great reduction in the amount of organic nitrogen, of albuminoid ammonia, and of carbonaceous matter. The latter fact is important, as the clogging of ordinary filter beds is largely due to the accumulation of such material, and of matters generally consisting of cellulose. One further important effect is that the size of the deposited matter is decreased, and therefore it is more easily broken up in the next stage of the process. This consists of running the effluent from the septic tank on to filter beds, preferably of coke, where a further purification process takes place. By this method there is first an anaerobic treatment succeeded by an aerobic ; in the latter the process of nitrification occurs by means of the special bacteria concerned. The results are of a satisfactory nature, there being often a marked diminution in the number of coli organisms present. Often the effluent from a sewage purification system contains as many bacteria as the sewage entering, but, especially by means of the septic tank method, there is often a marked diminution. It is said by some that pathogenic bacteria do not live in sewage. The typhoid bacillus has been found to die out when placed in sewage, but it certainly can live in this fluid for a much longer period than that embraced by any purification method. Thus the constant presence of b. coli, b. enteritidis, and streptococci which has been observed in sewage effluents must here still be looked on as indicating a possible infection with the typhoid bacillus, and it is only by great dilution and prolonged exposure to the conditions present in running water that such an effluent can be again a part of a potable water. ANTISEPTICS. The death of bacteria is judged of by the fact that when they are placed on a suitable food medium no development takes place. Microscopically it would be observed that division no longer occurred, and that in the case of motile species move- ment would have ceased, but such an observation has only scientific interest. From the importance of being able to kill bacteria an enormous amount of work has been done in the way of investigating the means of doing so by chemical means, 142 ANTISEPTICS and the bodies having such a capacity are called antiseptics. It is now known that the activity of these agents is limited to the killing of bacteria outside the animal body, but still even this is of high importance. Methods. — These vary very much. In early inquiries a great point was made of the prevention of putrefaction, and work was done in the way of finding how much of an agent must be added to a given solution such as beef extract, urine, etc., in order that the bacteria accidentally present might not develop ; but as bacteria vary in their powers of re- sistance, the method was unsatisfactory, and now an antiseptic is usually judged of by its effects on pure cultures of definite pathogenic microbes, and in the case of a sporing bacterium the effect on both the vegetative and spore forms is investigated. The organisms most used are the staphylococcus pyogenes, streptococcus pyogenes, and the organisms of typhoid, cholera, diphtheria, and anthrax — the latter being most used for testing the action on spores. The best method to employ is to take sloped agar cultures of the test organism, scrape off the growth, and mix it up with a small amount of distilled water and filter this emulsion through a plug of sterile glass wool held in a small sterile glass funnel, add a measured quantity of this fluid to a given quantity of a solution of the antiseptic in distilled water, then alter the lapse of the period of observation to remove one or two loopfuls of the mixture and place them in a great excess of culture medium. Here it is preferable to use fluid agar, which is then plated and incubated ; such a procedure is prefer- able to the use of bouillon tubes, as any colonies developing can easily be recognised as belonging to the species of bacterium used. In dealing with strong solutions of chemical agents it is necessary to be sure that the culture fluid is in great excess, so that the small amount of the antisej.tic which is transferred with the bacteria may be diluted far beyond the strength at which it still can have any noxious influence. Sometimes it is possible at the end of the period of observation to change the antiseptic into inert bodies by the addition of some other substance and then test the condition of the bacteria, and if the inert substances are fluid there is no objection to this proceeding, but if in the process a precipitate results, then it is better not to have recourse to such a method, as sometimes the bacteria are carried down with the precipitate and may escape the culture test. The advisability of, when possible, thus chemically changing the antiseptic was first brought to notice by the criticism of Koch's statements as to the efficacy of mercuric chloride in killing the spores of the b. anthracis. The method he employed in his experiments was to soak silk threads in an emulsion of anthrax spores and dry them. These were then subjected to the action of the antiseptic, well washed in water, and laid on the surface of agar. It was found, however, that with threads exposed to a far higher concentration of the corrosive sublimate than Koch had stated was sufficient to prevent growth, if the salt were broken up by the action of ammonium sulphide and this washed off, growth of anthrax still occurred when the threads were laid on agar. The explanation given was that the antiseptic had formed an albuminate with the case of each spore, and that this prevented the antiseptic from acting upon the contained protoplasm. Such an occurrence only takes place with spores, and the method given above, in which the small amount of antiseptic adhering THE ACTION OF ANTISEPTICS 143 to the bacteria is swamped in an excess of culture fluid, can safely be followed, especially when a series of antiseptics is being compared. Much attention has been paid to the standardisation of antiseptics, and a watery solution of carbolic acid is now generally taken as the standard with which other antiseptics are compared. Rideal and Walker point out that 110 parts by weight of B. P. carbolic acid equal 100 parts by weight of phenol, and they recommend the following method of standardising. To 5 c.c. of a particular dilution of the di.-infectant add 5 drops of a 24-hour-old bouillon culture of the organism (usually b. typhosus) which has been incubated at 87° C. Shake the mixture and make subcultures every 2^ minutes to 15 minutes. Perform a parallel series of experiments with carbolic acid and express the comparative result iu multiples of the carbolic acid doing the same work. The Action of Antiseptics. — In inquiries into the actions of antiseptic^ attention to a great variety of factors is necessary, especially when the object is not to compare different antiseptics with one another, but when the absolute value of any body is being investigated. Thus the medium in which the bacteria to be killed are situated, is important ; the more albuminous the surroundings are, the greater degree of concentration is required. Again, the higher the temperature at which the action is to take place, the more dilute may the antiseptic be, or the shorter the exposure necessary for a given effect to take place. The most important factor, however, to be considered is the chemical nature of the substances employed. Though nearly every sub- stance which is not a food to the animal or vegetable body is more or less harmful to bacterial life, yet certain bodies have a more marked action than others. Thus it may be said that the most important antiseptics are the salts of the heavy metals, certain acids, especially mineral acids, certain oxidising and re- ducing agents, a great variety of substances belonging to the aromatic series, and volatile oils generally. In comparing different bodies belonging to any one of these groups the chemical composition or constitution is very important, and if such comparisons are to be made, the solutions compared must be equimolecular ; in other words, the action of a molecule of one body must be compared with the action of a molecule of another body. This can be done by dissolving the molecular weight in grammes in say a litre of water (see p. 33). When this is done important facts emerge. Thus, generally speaking, the compounds of a metal of high atomic weight are more powerful antiseptics than those of one belonging to the same series, but of a lower atomic weight. Among organic bodies again substances with high molecular weight are more powerful than those of low molecular weight — thus butyric alcohol is more powerful than e thy lie alcohol — and important differences among 144 ANTISEPTICS the aromatic bodies are associated with their chemical constitu- tion. Thus among the cresols the ortho- and para-bodies re- semble each other in general chemical properties, and stand apart from metacresol ; they also are similar in antiseptic action, and are much stronger than the meta-body. The same may be observed in the other groups of ortho-, meta-, and para-bodies. Again, such a property as acidity is important in the action of a substance, and, generally speaking, the greater the avidity of an acid to combine with an alkali, the more powerful an antiseptic it is. With regard to oxidising agents and reducing agents, probably the possession of such properties has been overrated as increasing bactericidal potency. Thus in the case of such re- ducers as sulphurous acid and formic acid, the effect is apparently chiefly due to the fact that these substances are acids. Formic acid is much more efficient than formate of sodium. In the case of permanganate of potassium, which is usually taken as the type of oxidising agents in this connection, it can be shown that the greater amount of the oxidation which takes place when this agent is brought into contact with bacteria occurs after the organisms are killed. Such an observation is, however, not conclusive as to the non-efficiency of the oxidation process, for the death of the bacteria might be due to the oxidation of a very small part of the bacterial protoplasm. Apart from the chemical nature of antiseptic agents, the physical factors con- cerned in their solution, especially when they are electrolytes, probably play a part in their action. The part played by such factors is exemplified in the important fact that a strong solution acting for a short time will have the same effect as a weaker solution acting for a longer time. From what has been said it will be realised that the real causes of a material being an antiseptic are very obscure, and at present we can only have a remote idea of the factors at work. The Actions of certain Antiseptics. — Here we can only briefly indicate certain results obtained with the more common members of the group. Chlorine. — All the halogens have been found to be powerful antiseptics, but from the cheapness with which it can be produced chlorine has been most used ; not only is it the chief active agent in the somewhat complex action of bleaching powder, but it is also the chief constituent of several proprietary substances, of which " Electrozone " is a good example. This last substance is made from electrolysing sea -water, when magnesia and chlorine being liberated, magnesium hypochlorite and magnesium chloride are formed. In the action of this substance free hypo- ACTIONS OF CERTAIN ANTISEPTICS 145 chlorous acid is formed, and the effect produced is thus similar to that of bleaching powder. Nissen, investigating the action of the latter, found that 1^ per cent killed typhoid bacilli in faeces ; and Rideal found that 1 part to 400-500 disinfected sewage in fourteen minutes, and Delepine's results show that 1 part to 50 (equal to '66 per cent of chlorine) rapidly kills the tubercle bacillus, and 1 part to 10 (equal to 3 '3 per cent) killed anthrax spores. Klein found that *05 per cent of chlorine killed most bacterial spores in five minutes. Iodine Terchloride. — This is a very unstable compound of iodine and chlorine, and though it has been much used as an antiseptic, seeing that the- substance only remains as IC13 in an atmosphere of chlorine gas, it is open to doubt whether the effects described are not due to a very complicated action of free hydrochloric acid, hydriodic acid, of oxyacids of chlorine and iodine produced by its decomposition, and also, in certain cases, of organic iodine compounds formed from its contact with albuminous material. It is stated that the action is very potent : a 1 per cent solution is said instantly to kill even anthrax spores, but if the spores be in bouillon, death occurs after from ten to twelve minutes. In serum the necessary exposure is from thirty to forty minutes. A solution of 1-1000 will kill the typhoid, cholera, and diphtheria organisms in five minutes. Nascent Oxygen. — This is chiefly available in two ways — firstly, when in the breaking up of ozone the free third atom of the ozone molecule is seeking to unite with another similar atom ; secondly, when peroxide of hydrogen is broken up into water and an oxygen atom is thereby liberated. In commerce the activity of " Sanitas " compounds is due to the formation of ozone by the slow oxidation of the resin, camphor, and thymol they contain. Per chloride of Mercury. — Of all the salts of the heavy metals this has been most widely employed, and must be regarded as one of the most powerful and useful of known antiseptics. In testing its action on anthrax spores there is no doubt that in the earlier results its potency was overrated from a neglect of the fact already alluded to, that in the spore-case an albuminate of mercury was formed which prevented the contained protoplasm from developing, while not depriving it of life. It has been found, however, that this salt in a strength of 1-100 will kill the spores in twenty minutes, although an hour's exposure to 1-1000 has no effect. The best results are obtained by the addition to the corrosive sublimate solution of -5 per cent of sulphuric acid or hydrochloric acid ; the spores will then be killed by a seventy- 10 146 ANTISEPTICS' minute exposure to a 1-200 solution. When, however, organisms in the vegetative condition are being dealt with, much weaker solutions are sufficient; thus anthrax bacilli in blood will be killed in a few minutes by 1-2000, in bouillon by 1-40,000, and in water by 1-500,000. Plague bacilli are killed by one to two minutes' exposure to 1-3000. Generally speaking, it may be said that a 1-2000 solution must be used for the practically instan- taneous killing of vegetative organisms. Perchloride of mercury is one of the substances which have been used for disinfecting rooms by distributing it from a spray producer, of which the Equifex may be taken as a type. With such a machine it is calculated that 1 oz. of perchloride of mercury used in a solution of 1-1000 will probably disinfect 3000 square feet of surface. Such a procedure has been extensively used in the. disinfection of plague houses, but the use of a stronger solution (1-500 acidulated) is probably preferable. Formalin as a commercial article is a 40 per cent solution of formaldehyde in water. This is a substance which of late years has come much into vogue, and it is undoubtedly a valuable antiseptic. A disadvantage, however, to its use is that, when diluted and exposed to air, amongst other changes which it undergoes it may be transformed, under little understood conditions, into trioxymethylene and paraform aldehyde, these being polymers of formaldehyde. The bactericidal values of these mixtures are thus indefinite. Formalin may be used either by applying it in its liquid form or as a spray, or the gas which evaporates at ordinary temperatures from the solution may be utilised. To disinfect such an organic mixture as pus containing pyogenic organisms a 10 per cent solution acting for half an hour is necessary. In the case of pure cultures, a 5 per cent solution will kill the cholera organism in three minutes, anthrax bacilli in a quarter of an hour, and the spores in five hours. When such organisms as pyogenic cocci, cholera spirillum, and anthrax bacillus infect clothing, an exposure to the full strength of formalin for two hours is necessary, and in the case of anthrax spores, for twenty-four hours. Silk threads impregnated with the plague bacillus were found to be sterile after two minutes' exposure to formalin. The action of formalin vapour has been much studied, as its use constitutes a cheap method of treating infected rooms, in which case some spray-producing machine is employed. It is stated that a mixture of 8 c.c. of formalin with 48 c.c. of water is sufficient when vapourised to disinfect one cubic metre, so far as non-sporing organisms are concerned. It is stated that 1 part ACTIONS OF CERTAIN ANTISEPTICS 147 formalin in 10,000 of air will kill the cholera vibrio in one hour, diphtheria bacillus in three hours, the staphylococcus pyogenes in six hours, and anthrax spores in thirteen hours. In the case of organisms which have become dry it is probable, however, that much longer exposures are necessary, but on this point we have not definite information. Formalin gas has only a limited application ; it has little effect on dry organisms, and in the case of wet organisms, in order to be effective, probably must become dissolved so as to give the moisture a proportion analogous to the strengths stated above with regard to the vapour. Sulphurous Acid. — This substance has long been in use, largely from the cheapness with which it can be produced by burning sulphur in the air. An atmosphere containing "98 per cent will kill the pyogenic cocci in two minutes if they are wet, and in twenty minutes if they are dry ; and anthrax bacilli are killed by thirty minutes' exposure, but to kill anthrax spores an exposure of from one to two hours to an atmosphere containing 1 1 per cent is necessary. For a small room the burning of about a pound and a half (most easily accomplished by moistening the sulphur with methylated spirit) is usually considered sufficient. It has been found that if bacteria are protected, e.g. when they, are in the middle of small bundles of clothes, no effect is produced even by an atmosphere containing a large proportion of the sulphurous acid gas. The practical applications of this agent are therefore limited. Potassium Permanganate. — The action of this agent very much depends on whether it can obtain free access to the bacteria to be killed or whether these are present in a solution containing much organic matter. In the latter case the oxidation of the organic material throws so much of the salt out of action that there may be little left to attack the organisms. Koch found that to kill anthrax spores a 5 per cent solution required to act for about a day ; for most organisms a similar solution acting for shorter periods has been found sufficient, and in the case of the pyogenic cocci a 1 per cent solution will kill in ten minutes. There is little doubt that such weaker solutions are of value in disinfecting the throat on account of their non-irritating properties, and good results in this connection have been obtained in cases of diphtheria. A solution of 1 in 10,000 has been found to kill plague bacilli in five minutes. Carbolic Acid. — Of all the aromatic series this is the most extensively employed antiseptic. All ordinary bacteria in the vegetative condition, and of these the staphylococcus pyogenes 148 ANTISEPTICS is the most resistant, are killed in less than five minutes by a 2-3 per cent solution in water, so that the 5 per cent solution usually employed in surgery leaves a margin of safety. But for the killing of such organisms as anthrax spores a very much longer exposure is necessary ; thus Koch found it necessary to expose these spores for four days to ensure disinfection. The risk of such spores being present in ordinary surgical procedure may be overlooked, but there might be risk of tetanus spores not being killed, as these will withstand fifteen hours' exposure to a 5 per cent solution. In the products of the distillation of coal there occur, besides carbolic acid, many bodies of a similar chemical constitution, and many mixtures of these are in the market — the chief being creolin, izal, and lysol, all of which are agents of value. Of these lysol is perhaps the most noticeable, as from its nature it acts as a soap and thus can remove fat and dirt from the hands. A one-third per cent solution is said to destroy the typhoid and cholera organisms in twenty minutes. A 1 per cent solution is sufficient for ordinary surgical procedures. lodoform. — This is an agent regarding the efficacy of which there has been much dispute. There is little doubt that it owes its efficiency to its capacity for being broken up by bacterial action in such a way as to set free iodine, which acts as a powerful disinfectant. . The substance is therefore of value in the treatment of foul wounds, such as those of the mouth and Tectum, where reducing bacteria are abundantly present. It acts more slightly where there are only pyogenic cocci, and it seems to have a specially beneficial effect in tubercular affections. In certain cases its action may apparently be aided by the presence of the products of tissue degeneration. From the results which have been given it will easily be recognised that the choice of an antiseptic and the precise manner in which it is to be employed depend entirely on the environment of the bacteria which are to be killed. In many cases it will be quite impossible, without original inquiry, to say what course is likely to be attended with most success. CHAPTER V. RELATIONS OF BACTERIA TO DISEASE— THE PRODUCTION OF TOXINS 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 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 experiment. Now we know of no organ- isms which will in all circumstances produce disease in all animals, and, on the other hand, many bacteria described as harmless saprophytes will produce pathological changes if intro- duced 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 149 150 RELATIONS OF BACTERIA TO DISEASE 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, consequently, the diseased condition produced. 1. The Infecting Agent. — In the case of a particular species of bacterium its effect will depend chiefly upon (a) its virulence, and (6) 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 increased 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 that 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 rapidly fatal septicaemia when its virulence is raised. Virulence also has a relation to the animal employed, as occasionally on being in- creased 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 theoretical consideration of virulence must be reserved for a later chapter (see Immunity). 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 any serious result, but if a larger dose be CONDITIONS MODIFYING PATHOGENICITY 151 introduced, a fatal peritonitis may follow. Again, a certain quantity of a particular organism injected subcutaneously 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 ; a dose, moreover, which is modified by various circumstances difficult to control. The path of infection may alter the result, serious effects often following a direct entrance into the blood stream. Staphylococci 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 inflam- matory 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 injection of the pneumococcus produces death more readily than intra- venous injection. 2. The Subject of Infection. — Amongst healthy individuals 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, gonorrhoea, 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 suppura- tive affections. 152 RELATIONS OF BACTERIA TO DISEASE In increasing the susceptibility of a given individual, condi- tions 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 injection 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 injuring or diminishing the vitality of a part. If, for example, previous to an intravenous injection of staphylococci, 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 appears to favour 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 pro- duction 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 destroyed. 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 production of diseases of which the direct cause is a bacterium, may be understood. It is important to keep in view in this con- nection 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 MODES OF BACTERIAL ACTION 153 specific organism may produce lesions through which the other organisms gain entrance, e.g. in typhoid, diphtheria, etc. A notable example of diminished resistance to bacterial infection is seen in the case of diabetes7; tuberculosis and infection with pyogenic organisms are prone to occur in this disease and are of a severe character. It is not uncommon to find in the bodies of those who have died from chronic wasting disease, collections of micrococci 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 specific- ally increased belong to the subject of immunity, and are decribed in the chapter on that subject. Modes of Bacterial Action. — In the production of disease by micro-organisms there are two main factors involved, namely, (a) the multiplication of the living organisms after they have entered the body, and (b) 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. 1 . 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 exten- sive (for example, the septicaemia produced by the pneumococcus in rabbits) ; but in septicaemia 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 is rare, and even culture methods may give negative results unless a large amount of blood is used. 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 septicaemia produced by strepto- cocci. In the human subject more frequently one of two things happens. In the first place, the organisms may remain local, producing little reaction around them, as in tetanus, or a well- marked lesion, as in diphtheria, pneumonia, etc. Or in the second place, they may pass by the lymph or blood stream to 154 RELATIONS OF BACTERIA TO DISEASE 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 formation 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 after- wards 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 tuber- culosis. 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, continuously, and in a certain degree of concentration, and these conditions cannot be exactly reproduced by experiment. It is also to be noted that more than one poison may be produced by a given bacterium, e.g. the tetanus bacillus (p. 380). 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. The separated toxin of diphtheria, like various vegetable and animal toxins (vide infra), however, possesses a local toxic action of very intense character, evidenced often by extensive necrotic change. The injection of large quantities of many different pathogenic organisms in the dead conditions results in the production of a local inflammatory change which may be followed by suppura- tion, this effect being possibly brought about by certain sub- stances in the bacterial protoplasm common to various species, or at least possessing a common physiological action (Buchner and others). When dead tubercle bacilli, however, are intro- duced 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 resistant and TISSUE CHANGES PRODUCED BY BACTERIA 155 slowly acting substance which gradually diffuses around and produces effects (vide Tuberculosis). Summary. — We may say then that the action of bacteria as disease-producers, as in fact their power to exist and multiply in the living body, depends upon the chemical products formed directly or indirectly by them. This action is shown by tissue changes produced in the vicinity of the bacteria or throughout the system, and by toxic symptoms of great variety of degree and character. We shall first consider the effects of bacteria on the body generally, and afterwards the nature of the chemical products. EFFECTS OF BACTERIAL ACTION. These may be for convenience arranged in a tabular form as follows : — A. Tissue Changes. (1) Local changes, i.e. changes produced in the neigh- bourhood of the bacteria. Position (a) At primary lesion. (6) At secondary foci. Character (a) Tissue reactions ) Acute or (b) Degeneration and necrosis j Chronic. (2) Produced at a distance from the bacteria, directly or indirectly, by the absorption of toxins. (a) In special tissues — (a) as the result of damage, e.g. nerve cells and fibres, secreting cells, vessel walls, or (/3) changes of a reactive nature in the blood- forming organs. (&) General anatomical changes, the effects of mal- nutrition or of increased waste. B. Changes in Metabolism. The occurrence of fever, of errors of assimilation and elimination, etc. A. Tissue Changes produced by Bacteria. — The effects of bacterial action are so various as to include almost all known pathological changes. However varied in character, they may be classified under two main headings : — (a) those of a degenera- tive or necrotic nature, the direct result of damage, and (6) those 156 RELATIONS OF BACTERIA TO DISEASE of reactive nature, defensive or reparative. The former are the expression of the necessary vulnerability of the tissues, the latter of protective powers evolved for the benefit of the organism. In the means of defence both leucocytes and the fixed cells of the tissues are concerned. Both show phagocytic properties, i.e. have the power of taking up bacteria into their protoplasm. The cells are guided towards the focus of infection by chemiotaxis, and thus we find that different bacteria attract different cells. The most rapid and abundant supply of phagocytes is seen in the case of suppurative conditions where the neutrophile leuco- cytes of the blood are chiefly concerned. When the local lesion is of some extent there is usually an increase of these cells in the blood — a neutrophile leucocytosis. And further, recent observations have shown that associated with this there is in the bone-marrow an increased number of the mother-cells of these leucocytes — the neutrophile myelocytes. The passage of the neutrophile leucocytes from the marrow into the blood, with the resulting leucocytosis, is also apparently due to the absorbed bacterial toxins acting chemiotactically on the marrow. These facts abundantly show that the means of defence is not a mere local mechanism, but that increased proliferative activity in distant tissues is called into play. In addition to direct phagocyt- osis by these leucocytes, there is now abundant evidence that an important function is the production in the body of bactericidal and other antagonistic substances. In other cases the cells chiefly involved are the mononuclear hyaline leucocytes, and with them the endothelial cells, e.g. of serous membranes, often play an important part in the defence ; this is well seen in typhoid fever, where the specific bacillus appears to have little or no action on the neutrophile leucocytes. In other cases, again, the reaction is chiefly on the part of the connective cells, though their proliferation is always associated with some variety of leucocytic infiltration and usually also with the forma- tion of new blood vessels. Such a connective tissue reaction occurs especially in slow infections or in the later stages of an acute infection. The tissue changes resulting from cellular activity in the presence of bacterial invasion are naturally very varied — examples of this will be found in subsequent chapters — but they may be said to be manifestations of the two funda- mental processes of (a) increased functional activity — movement, phagocytosis, secretion, etc. — and (b) increased formative activity — cell growth and division. The exudation from the blood vessels has been variously interpreted. There is no doubt that the exudate has bactericidal properties and also acts as a diluting LOCAL LESIONS 157 agent, but it must still be held as uncertain whether the process of exudation ought to be regarded as primarily defensive or as the direct result of damage to the endothelium of the vessels. It may also be pointed out that the various changes referred to are none of them peculiar to bacterial invasion ; they are examples of the general laws of tissue change under abnormal conditions, and they can all be reproduced by chemical substances in solution or in a particulate state. What constitutes their special feature is their progressive or spreading nature, due to the bacterial multiplication. (1) Local Lesions. — In some diseases the lesion has a special site ; for example, the lesion of typhoid fever and, to a less extent, that of diphtheria. In other cases it depends entirely upon the point of entrance, e.g. malignant pustule 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 in tubercle. In some cases the site has a mechanical explanation. When organisms gain an entrance to the blood from a primary lesion, the organs specially liable to be affected vary greatly in different diseases. Pyogenic cocci show a special tendency to settle in the capillaries of the kidneys and produce miliary abscesses, whilst these lesions rarely occur in the spleen. 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 susceptibility 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 character is observed. 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 oedema ; it may be localised or spreading in character ; it may be followed by suppuration, and may be accompanied or lead up to necrosis of the tissues of the part, a good example of the latter event being found in a boil. Examples will be given in subsequent chapters. The necrotic or degenerative changes 158 RELATIONS OF BACTEKIA TO DISEASE affecting especially the more highly developed elements of tissues are chiefly produced by the direct action of the bacterial poisons, though aided by the disturbances of nutrition involved in the vascular phenomena. It may here be pointed out that a well- marked inflammatory reaction 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 vhere is less vascular disturbance and a greater preponderance of the proliferative processes, leading to new formation of connective tissue or a modified connective 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 lin£, however, cannot be drawn between such conditions and those described above as acute. In glanders, for example, especially in the human subject, the lesion 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, bnt on the other hand, this is often impossible. These changes often include the occurren3e of degenerations or of actual necrosis in the newly formed tissue. In the granulomata, 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, intestine, etc. (2) General Lesions produced by Toxins. — In the various in- fective conditions produced by bactaria, changes commonly occur in certain organs unassociated vrith the presence of the bacteria ; these are produced by the 'action of bacterial pro- ducts circulating in the blood. Many such lesions can be pro- duced 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 DISTURBANCES OF METABOLISM, ETC. 159 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 toxins, e.g. of diphtheria, and also of vegetable poisons, e.g. ricin and abrin. Skin eruptions occurring in the exanthemata 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 corresponding 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 reproduced experimentally by the products of the diphtheria bacilli. There is also experi- mental evidence that the bacillus coli communis and the strepto- coccus pyogenes may, by means of their products, produce areas of softening in the spinal cord, and this may furnish an explana- tion of some of the lesions found clinically. It is also possible that some serous inflammations may be produced in the same way. B. Disturbances of Metabolism, etc. — It will easily be realised that such profound tissue changes as have been detailed cannot occur without great interference with the normal bodily metabolism. General malnutrition and cachexia are 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 pro- gressive, leading to the death of the animal. In bacterial disease assimilation is often imperfect, for the digestive glands are affected, it may be, by actual poisoning by bacterial products, it may be by the occurrence of fever. The fatty degenerations which are so common are indicative of a breaking down of the proteid molecules, and are associated with increased urea produc- tion, while the degeneration of the kidney epithelium renders the excretion of waste products deficient or impossible, and this is not infrequently the immediate cause of death. But of all the changes in metabolism the most difficult to understand is the occurrence of that interference with the heat-regulating mechanism which results in fever. The degree and course of the latter vary, sometimes conforming to a more or less definite type, 160 RELATIONS OF BACTERIA TO DISEASE where the bacilli are selective in their field of operation, as in croupous pneumonia or typhoid, sometimes being of a very ir- regular kind, especially when the bacteria from time to time invade fresh areas of the body, as in pysemic affections. The main point of interest regarding the development of fever is as to whether it is a direct effect of the circulation of bacterial toxins, or if it is to be looked on as part of the reaction of the body against the irritant. This question has still to be settled, and all that we can do is to adduce certain facts bearing on it. Thus in diphtheria and tetanus, where toxic action leading to degeneration plays such an important part, fever may be a very subsidiary feature, except in the terminal stage of the latter disease ; and in fact in diphtheria profoundly toxic effects may be produced with little or no interference with heat regulation. On the other hand, in bacterial disease, where defensive and re- parative processes predominate, fever is rarely absent, and it is nearly always present when an active leucocytosis is going on. In this connection it may be remarked that several observers have found that, when a relatively small amount of the dead bodies of certain bacteria are injected into an animal, fever occurs; while the injection of a large amount of the same is followed by subnormal temperatures and rapidly fatal collapse. It might appear as if this indicated that the occurrence of fever had a beneficial effect, but this is one of the points at issue. Certainly such an effect is not due to the bacteria being unable to multiply at the higher degrees of temperature occurring in fever, for this has been shown not to be the case. Whether the increase of bodily temperature indicates the occurrence of changes resulting in the production of bactericidal bodies, etc., is very doubtful ; a production of antagonistic- substances may be effected without the occurrence of fever or of any appar- ent disturbance of health. If we consider the site of the heat production in fever we again are in difficulties. It might appear as if the tissue destruction, indicated by the occurrence of fatty degeneration, would lead to heat development, but frequently excessive heat production with increased proteid metabolism occurs without any discoverable changes in the tissues ; and further, in phosphorus poisoning there is little fever with great tissue destruction. The increased work performed by the heart in most bacterial infections no doubt contributes to the rise of bodily temperature. But we must bear in mind that in fever there is more than mere increase of heat production — there is also a diminished loss of heat from interference with the nervous mechanism of the sweat apparatus. The known facts would THE- TOXINS PRODUCED BY BACTERIA 161 indicate that in fever there is a factor involving the nervous system to be taken into account. The whole subject is thus very obscure. 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, corre- sponding changes have not yet been discovered. Of the latter those associated with fever, with its disturbances of metabolism and manifold affections of the various systems, are the most important. The nervous system is especially liable to be affected — convulsions, spasms, coma, paralysis, etc., being common. The symptoms due to disturbance or abolition of the functions of secretory glands also constitute an important group, forming, as they do, a striking analogy to what is found in the action of various drugs. These tissue changes and symptoms are given only as illus- trative examples, and the list might easily be greatly amplified. The important fact, however, is that necwly all, if not quite all, the changes found throughout the organs {without the actuaLl presence of bacteria), and also the symptoms occurring in infectvti£[ diseases, can either be exyerimeu tally revrnduMd by tfi* *.i ~~bf bacterial /><>is<>n.* or ka.v<- an, analogy in the action of drugs. THE TOXINS PKODUCED BY BACTERIA. Early Work on Toxins. — We know that bacteria are capable of giving rise to poisonous bodies within the animal body and also in artificial media. We know, however, comparatively little of the actual nature of such bodies, and therefore we apply to them as a class the general term toxins. The necessity for accounting for the general pathogenic effects of certain bacteria, which in the corresponding diseases were not distributed throughout the body, directed attention to the probable exist- ence of such toxins ; and the first to systematically study the production of such poisonous bodies was Brieger. This observer isolated from putrefying substances, and also from bacterial cultures, nitrogen-containing bodies, which he called ptomaines. Similar bodies occurring in the ordinary metabolic processes of the body had previously been described and called leucomaines. Ptomaines isolated from pathogenic bacteria in no case repro- duced the symptoms of the disease, except perhaps in tetanus and this only owing to their impurity. The methods by which they were isolated were faulty, and they have therefore only a historic interest. 11 162 THE TOXINS PRODUCED BY BACTERIA The introduction of the principle of rendering fluid cultures bacteria-free by nitration through unglazed porcelain, and its application by Roux and Yersin to obtain, in the case of the b. diphtherias, a solution containing a toxin which reproduced the symptoms of this disease (vide Chap. XV.), encouraged the further inquiry as to the nature of this toxin. An attempt on the part of Brieger and Fraenkel to obtain a purified diphtheria toxin by precipitating bouillon cultures by alcohol (the product being denominated a toxalbumin) did not greatly advance know- ledge on the subject, and further investigation soon showed that characteristic toxins can be isolated from but few bacteria. General Facts regarding Bacterial Toxins. — The following may be regarded as the chief facts regarding bacterial toxins which have been revealed by the study, partly of the bodily tissues of animals infected by the bacteria concerned, partly of artificial cultures of : these bacteria. The dead bodies of certain bacteria have been found to be very toxic. When, for instance, tubercle bacilli are killed by heat and injected into the body tissues of 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 toxins, seeing that characteristic lesions result. The bodies of the cholera vibrio are likewise toxic. Such intracellular toxins, as they have been called, may appear in the nuids in which the bacteria are living (1) by excretion in an un- or alto/rod condition, (2) by the disintegration of the which we know are always in any bacterial growth/ The death of bacteria occurs also rn the body of an infected animal, and the disintegration of these dead bacteria constitutes an important means by which the poisons they contain are absorbed. There is some evidence that often bacteria produce during growth poisons which are hurtful to their own vitality, and also that ferments are produced by them which have a solvent effect on the poisoned members of the colony. Such a process of autolysis^ as it has been called, may have an important effect in liberating intracellular toxins. We do not, however, under- stand all that takes place under such circumstances ; for the dead bodies of many bacteria, such as those of anthrax and diphtheria, are relatively non-toxic. As it is impossible, at present, to obtain intracellular toxins apart from other deriva- tives of the bacterial protoplasm, all our knowledge concerning their effects is derived from the study of what happens when the bodies of bacteria killed by chloroform vapour or by heat are FACTS REGARDING BACTERIAL TOXINS 163 injected into animals. When effects are produced by such injections they do not present in any particular case specific characters. They are of the nature of general disturbances of metabolism, as manifested by fever, loss of weight, etc., often of such serious degree as to result in death. It is important to note that when pathogenic effects are produced these usually appear very soon, it may be in a few hours after injection of the toxic material; there is not the definite period of incubation which with other toxins often elapses before symptoms appear. Sometimes the media in which bacteria are growing become extremely toxic. This is more marked in some cases than in others. The two best examples of bacteria thus producing soluble toxins are the diphtheria and tetanus bacilli. In these and similar cases when bouillon cultures are filtered bacterium- free by means of a porcelain filter, toxic fluids are obtained, which on injection into animals reproduce the highly character- istic symptoms of the corresponding diseases. In the case of the b. anthracis and of many others, at any rate when grow- ing in artificial media, such toxin production is much less marked, a filtered bouillon culture being relatively non-toxic. Pnianna nppftan'ncr in nnltnrq mftHio. m cellular toxins, but we cannot as yet say whether they are excreted by the bacteria or whether they are produced by the bacteria acting on the constituents of the media. We therefore cannot as yet draw a hard and fast line between intra- and extracellular toxins, but the terms are convenient, and may apply to two actually different sets of bodies. That the poisonous capacities of a bacterium may be very compli- cated is shown by what is known in the case of the cholera vibrio, where the poisons which dissolve out into the culture fluid are probably different in their nature from those which act when the dead bacteria are injected into an animal. The extra- cellular toxins are the more easily obtainable in large quantities, and it is their nature and effects which are best known. No method, however, has been discovered of obtaining them in a pure form, and our knowledge of their properties is exclusively derived from the study of the toxic filtrates of bouillon cultures — these filtrates being usually referred to simply as the toxins. These toxins differ in their effects from the intracellular poisons in that specific actions on certain tissues are often manifested. Thus the toxins of the diphtheria, the tetanus, and the botu- lismus bacilli all act on the nervous system ; with some of the pyogenic bacteria, on the other hand, poisons, probably of similar nature, produce solution of red blood corpuscles (this 164 THE TOXINS PRODUCED BY BACTERIA last may explain, in part at least, the anaemias so common in the associated diseases). In the action of many of these toxins the occurrence of a period of incubation between the introduction of the poison into the animal tissues and the appearance of symptoms is often a feature. We have seen that in certain cases there is difficulty in under- standing the action of bacteria which do not form toxins in fluid media, especially as in the cases of some of these the bacterial protoplasm does not seem very toxic. Yet we often see effects produced at a distance from the focus of infection, e.g. in anthrax. To explain such occurrences it has long been put forward as a possibility that some bacteria are only capable of producing toxins within the animal tissues, and it has further been thought possible that bacteria, such as, for example, the typhoid bacillus, which do in media give rise to intracellular toxins, might either produce these toxins more readily in the tissues or might produce in addition other toxins of a different nature. Recently such toxins have been much studied, and the name aggressins has been given to them. The evidence adduced for the existence of these aggressins as a separate group of bacterial poisons is of the following kind. An animal is killed by a dose of the typhoid, dysentery, cholera, or tubercle bacillus, or by a staphylococcus, the organism being introduced into one of the serous cavities. After death the serous exudation, which in all these cases is present, is removed, and centrifugalised to remove the bacteria so far as this can be done by such a procedure ; the bacteria which are left are killed by shaking the fluid up with toluol and leaving it to stand for some days. It is stated that such a fluid is of itself without pathogenic effect, but has the property of transforming a non-lethal dose of the bacterium used into one having fatal effect. Further, the effects of the combined actions of the bacteria and aggressins are often of a much more acute character than can be obtained with toxic products developed in vitro. Thus, in the case of the action of a non- lethal dose of the tubercle bacillus plus its aggressin, death may occur in twenty hours, a result never obtained with artificial cultures of the organism. The results obtained are attributed to. a paralysing action which the aggressin is supposed to have on the phagocytic functions of the leucocytes. The subject is full of difficulties, and in the case of certain of the organisms employed, it is stated that results similar to those attributed to aggressin action have been observed with macerated cultures, — the deduction being that in the aggressins we are merely deal- ing with concentrated intracellular toxins. On the other hand, THE NATURE OF TOXINS 165 as evidence of the existence of a special group of toxins, it has been stated that a special type of immunity against the aggressins can be originated. Perhaps the most important aspect of the controversy is the recognition of the existence of toxins having an action on the leucocytes. A poison causing death of these cells in connection with the pus-forming action of the pyogenic cocci has been described under the name of leucocidin. The investigation of such poisons must be of the highest importance in view of the part played by the blood- cells in the protection of the body against infection, and it is possible that toxins having a fatal effect in strong con- centrations, may, when dilute, be responsible for the phenomena of attraction or repulsion of leucocytes which we know occur round a focus of bacterial growth in the body. It is to be noted that in the case of any particular bacterium several different toxins may be at work, and it is also possible that one toxin may have different effects on different tissues of the body. Intracellular toxins of an organism may cause general metabolic disturbances, and its special toxins may act on special tissues. Thus the staphylococcus pyogenes aureus may cause fever, wasting, etc., by its intracellular poisons, a special action on the leucocytes by a leucocidin toxin, and anaemia by its hsemolytic properties. The phenomena of any bacterial disease may thus in reality be due to very different and complex causes. The Nature of Toxins. — There is still comparatively little known regarding this subject, and it chiefly relates to the extra- cellular toxins. The earlier investigations upon toxins suggested that analogies exist between the modes of bacterial action and what takes place in ordinary gastric digestion, and the idea was worked out for anthrax, diphtheria, tetanus, and ulcerative endocarditis by Sidney Martin. This observer took, not solutions artificially made up with albumoses,1 but the natural fluids of the body or definite 1 In the digestion of albumins by the gastric and pancreatic juices the albumoses are a group of bodies formed preliminarily to the production of peptone. Like the latter they differ from the albumins in their not being coagulated by heat, and in being slightly dialysable. They differ from the peptones in being precipitated by dilute acetic acid in presence of much sodium chloride, and also by neutral saturated sulphate of ammonia. Both are precipitated by alcohol. The first albumoses formed in digestion are proto-albumose and hetero-albumose, which 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 secondary 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 166 THE TOXINS PEODUCED BY BACTERIA solutions of albumins, and, further, never subjected the results of the bacterial growth to heat above 40° C., or to any stronger agent than absolute alcohol. He found that albumoses and sometimes peptones were formed by the action of the patho- genic bacteria studied, and further, that the precipitate contain- ing these albumoses was toxic. In certain cases the process of splitting up of the albumins went further than in peptic diges- tion, and organic bases or acids might be formed. According to Martin, the characteristic symptoms of the diseases could be explained by compound actions, in which the albumoses were responsible for some of the effects, the remaining bodies for others. A similar digestive action has been traced in the case of the tubercle bacillus by Kiihne. Further evidence that bacterial toxins are either albumoses or bodies having a still smaller molecule is furnished by C. J. Martin. This worker, by filling the pores of a Chamberland bougie with gelatin, has obtained what is practically a strongly supported colloid membrane through which dialysis can be made to take place under great pressure, say, of compressed oxygen. He finds that in such an apparatus toxins, — at least 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 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 toxins when growing in proteid-free fluids. In the case of diphtheria when the toxin is produced in such a fluid a proteid reaction appears. Of course this need not necessarily be caused by the toxin. Further investigation is here required, for Uschinsky, applying Brieger and Boer's method to a toxin so produced, states that the toxic body is not precipitated by zinc salts, but remains free in the medium. If the toxins 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 toxic albumoses of Sidney Martin may be due to the precipitation of the true toxins along with these other bodies. From the chemical standpoint this is quite possible. When we take into account the extraordinary potency of these poisons (in trace of acetic acid be present. Dysalbumose is probably merely a temporary modification of hetero-albumose. Further digestion of deutero-albumose results in the formation of peptone. THE NATURE OF TOXINS 167 the case of tetanus the fatal dose of the pure poison for a guinea-pig must often be less than '000001 gr.), we can under- stand how attempts by present chemical methods to isolate them in a pure condition are not likely to be successful, and of their real nature we know nothing. In a recent research Friedberger and Moreschi have shown that the intravenous injection in the human subject of a fraction of a loopful of a dead typhoid culture gives rise to toxic symptoms, including marked febrile reaction. Such injections are followed by the appearance of agglutinating and bacteriolytic substances in the serum. These results show that intracellular toxins may be comparable with extra-cellular toxins so far as concerns the extremely small dose sufficient to produce toxic effects. Amongst the properties of the extracellular toxins are the following. They are certainly all uncrvstallisable • they are soluble in water and they are dialysable ; they are pre- • •ipitated along \yith proteids by concentrated alcohol, and als<3 by ammonium sulphate; if they are proteids they are either alEumoses or allied to the albumoses ; they are often relatively un- stable, having their toxicity HiminiiAed or destroyed bv heat (the 'degree of heat wmcn is destructive varies much in different cases), light, and by certain chemical agents. Their potency is often altered in the precipitations practised to obtain them in a pure or concentrated condition, but among the precipitants ammonium sulphate has little if any harmful effect. Regarding the toxins which are more intimately associated with the bacterial proto- plasm 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. those of the tubercle bacillus, already mentioned. In the case of all toxins the fatal dose for an animaJLjsajdfia-jdtlL, species, body weight, age, a.nrl 'prevTona' p.n™titinnff y trT food, temperature, etc. In estimating the minimal lethal dose of a toxin tiiese factors must be carefully considered. The following is the best method of obtaining concentrated extra- cellular toxins. The toxic fluid is placed in a shallow dish, and ammonium sulphate crystals are well stirred in till no more dissolve. Fresh crystals to form a bulk nearly equal to that of the whole fluid are added, and the dish set in an incubator at 37° C. over night. Next day a brown scum of precipitate will be found floating on the surface. This contains the toxin. It is skimmed off with a spoon, placed in watch glasses, and these are dried in vacuo and stored in the dark, also in vacuo, or in an exsiccator containing strong sulphuric acid. For use the contents of one are dissolved up in a little normal saline solution. The comparison of the action of bacteria in the tissues in the production of these toxins to what takes place in the gastric 168 THE TOXINS PRODUCED BY BACTERIA digestion, has raised the question of the possibility of the elabora- tion by these bacteria of ferments by which the process may be started. Thus Sidney Martin puts forward the view that ferments may be produced, which we may look on as the primary toxic agents, and which act by digesting surrounding material and producing albumoses, — these poisons being, as it were, secondary poisons. Hitherto all attempts at the isolation of bacterial ferments of such a nature have failed. But apart from the fact that with such bacteria as these of tetanus and diphtheria, a digestive action may occur, analogies have been drawn between ferment and toxic action. The chief facts upon which such analogies have been founded are as follows. Thus the toxic products of these and other bacteria lose their toxicity by exposure to a temperature which puts an end to the activity of such an undoubted ferment as that of the gastric juice. If a bouillon containing diphtheria toxin be heated at 65° C. for one hour, it is found to have lost much of its toxic effect, and in the case of b. tetani all the toxicity is lost by exposure at this temperature. In both diseases there is a still further fact which is adduced in favour of the toxic substances being of the nature of ferments, namely, the existence of a definite period of incubation between the injection of the toxic bodies and the appearance of symptoms. This may be inter- preted as showing that after the introduction of, say, a filtered bouillon culture, further chemical substances are formed in the body before the actual toxic effect is produced. Too much reliance must not be placed on such an argument, for in the case of tetanus, at least, the delay may be explained by the fact that the poison apparently has to travel up the nerve trunks before the real poisonous action is developed. Further, with some poisons presently to be mentioned which are closely allied to the bacterial toxins an incubation period may not exist. It would not be prudent to dogmatise as to whether the toxins do or do not belong to such an ill-defined group of substances as the ferments. It may be pointed out, however, that the essential concept of a ferment is that of a body which can originate change without itself being changed, and no evidence has been adduced that toxins fulfil this condition. Another property of ferments is that so long as the products of fermenta- tion are removed, the action of a given amount of ferment is indefinite. Again, in the case of toxins no evidence of such an occurrence has been found. A certain amount of a toxin is always associated with a given amount of disease effect, though a process of elimination of waste products must be all the time VEGETABLE AND ANIMAL POISONS 169 going on in the animal's body. Again, too much importance must not be attached to loss of toxicity by toxins at relatively low temperatures. This is not true of all toxins, and further- more many proteids show a tendency to change at such temperatures ; for instance, if egg albumin be kept long enough at 55° C. nearly the whole of it will be coagulated. We must therefore maintain an open mind on this subject. 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 precatorius (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 toxins. They are soluble in water, they are precipitable by alcohol, but being less easily dialysable than the alburnoses 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 active poisons known — ricin being the more powerful. When they are 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 hsemorrhagic 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 venins, and a very representative group of such venins derived from different species has been studied. To speak generally there is derivable from the natural secretions of the poison glands a series of venins which have all the reactions of the bodies previously considered. Like ricin and abrin, they are not so easily dialysable as bacterial toxins, 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, and some will stand boiling. There is also evidence that in a crude venin there may be several poisons differently sensitive to heat. All the venins are very powerful poisons, but here there is practically no period of incubation — the effects are almost immediate. An outstanding feature of the venins is the complexity of the crude poison secreted by any particular species of snake. C. J. Martin in summing up the results of many observers has pointed out that different venoms have been found to contain one or more of the following poisons : a neurotoxin acting on the respiratory centre, a neurotoxiu acting on the nerve-endings in muscle, a toxin causing haemolysis, toxins acting on other cells, e.g. the endothelium of blood-vessels (this from its effects has been named haemerrhagin), leucocytes, nerve-cells, a toxin causing thrombosis, a toxin haying an opposite effect and preventing coagulation, a toxin neutralising the bactericidal qualities of the body fluids and thus favouring putrefaction, a toxin causing agglutination of the red blood corpuscles, a proteolytic 170 THE TOXINS PRODUCED BY BACTERIA ferment, a toxin causing systolic standstill of the excised heart. Any particular venom contains a mixture in varying proportions of such toxins, and the different effects produced by the bites of different snakes largely depend on this variability of composition. The neurotoxic, the thrombotic, and the hsemolytic toxins are very important constituents of any venom. The toxicity of different venoms varies much, and no general statement can be made with regard to the toxicity of different poisons towards man. Lamb has calculated that the fatal dose of crude cobra venom for man is probably about -015 of a gramme, and that if such a snake bites with full glands many times this dose would probably be injected, but, of course, the amount emitted depends largely on the period which has elapsed since the animal last emptied its glands. When a dose of a venom not sufficient to cause immediate death from general effects be given, very rapid and widespread necrosis often may occur in a few hours round the site of inoculation. An extremely important fact was discovered by Flexner and Noguchi, namely, that the hsemolytic toxin of cobra venom in certain cases has no action by itself, but produces rapid solution of red corpuscles when some normal serum is added, the latter containing a labile complement-like body, which activates the venom. In this there is a close analogy to what holds in the case of a hsemolytic serum deprived of complement by heat at 55° C. (p. 479). Kyes and Sachs further showed that in addition to serum-complement a substance with definitely known constitution, namely lecithin, had the property of activating the hsemoly tic substance in cobra venom, the two apparently uniting to form an actively toxic substance. Later still, Kyes succeeded in demonstrating the union of the two substances to form a cobra-lecithid, and in separating the latter as a practically pure compound, which is, unlike lecithin, insoluble in ether, but soluble in chloroform. So far no example of activating a bacterial toxin is known, but the results mentioned point to the possibility of this occurring in some cases in the tissues of the body. The Theory of Toxic Action. — While we know little of the chemical nature of any toxins 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 afterwards described, when introduced into the bodies of susceptible animals they stimulate the production of substances called antitoxins. The nature of the antagonism between toxin and antitoxin will be discussed later. Here, to explain what follows it may be stated (1) that the molecule of toxin most probably forms a chemical combination with the molecule of antitoxin, and (2) that it has been shown that toxin molecules may lose much of their toxic power and still be capable of uniting with exactly the same proportion of antitoxin molecules. From these and other circumstances Ehrlich has advanced the view that the toxin molecule has a very com- plicated structure, and contains two atom groups. One of these, the haptophorous (aTrmj/, to bind to), is that by which com- THE THEORY OF TOXIC ACTION 171 bination takes place with the antitoxin 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 haptophorous group. Ehrlich explains the loss of toxicity which with time occurs in, say, diphtheria toxin, on the theory that the toxophorous group undergoes disintegration. And if we suppose that the haptophorous group remains unaffected we can then understand how a toxin may have its toxicity diminished and still require the same proportion of antitoxin molecules for its neutralisation. To the bodies whose toxophorous atom groups have become degenerated, Ehrlich gives the name toxoids. The theory may afford an explanation of what has been suspected, namely, that in some instances toxins 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 antitoxin which corresponds almost completely with that produced by another vegetable poison, robin (vide supra), though ricin and robin are certainly different. This may be ex- plained according to the view 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. With regard to the intracellular toxins we shall see -it is difficult to determine whether or not they share with the extra- cellular poisons the property of stimulating antitoxin formation, — if they do not, then they may belong to an entirely different class of substances. It is certain that a tolerance against such poisons is difficult to establish and is not of a lasting character. We thus cannot say what the mechanism is by which these poisons act. It may be said that Macfadyen by grinding up typhoid bacilli frozen by liquid air claimed that on thawing he obtained the intracellular toxins in liquid form, and he further stated that by using this fluid he could immunise animals not only against the toxins but also against the living bacteria. We have already pointed out that those who claim for the aggressins a special character hold that the activity of these bodies has as its effect the interference with the phagocytic functions of the leucocytes. They .also hold that a special type of immunity can be developed against the aggressive bodies. CHAPTER VI. INFLAMMATORY AND SUPPURATIVE CONDITIONS. THIS subject is an exceedingly wide one, and embraces a great many pathological conditions which in their general characters and results are widely different. Thus in addition to suppuration, various inflammations, ulcerative endocarditis, septicaemia and pyaemia, will come up for consideration. With regard to these the two following general statements, established by bacteriological research, may be made in introducing the subject. In the first place, there is no one specific organism for any one of these conditions ; various organisms may produce them, and not in- frequently more than one organism may be present together. In the second place, the same organism may produce widely varying results under different circumstances, — at one time a local inflammation or abscess, at another multiple suppurations or a general septicaemia. The principles on which this diversity in results depends have already been explained (p. 151). Furthermore, there are conditions like acute pneumonia, epidemic meningitis, acute rheumatism, etc., which have practically the character of specific diseases and yet which as regards their essential pathology belong to the same class. The arrangement followed is to a certain extent one of convenience. It may be well to emphasise some of the chief points in- the pathology of these conditions. In. suppuration the two main phenomena are — (a) a. prn^rpsaive immigration of leucocytes, chiefly of the polymorpho-nuclear (neutrophile) variety, and (6) a liquefaction oT digestion pt 1M JJUppoTlllUr eleineut§ of the tissue along with necrosis of the cells 01 tne part. The result" is that the tissue aflectecT Le< -nines replaced by the cream-like fluid called pus. A suppurative inflammation is thus to be distinguished on the one hand from an inflammation without destruction of tissue, and on the other from necrosis or death 172 NATURE OF SUPPURATION 173 en masse, where the tissue is not liquefied, and leucocyte accumulation may be slight. 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 in 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. Many experiments have been performed to determine whether suppuration can be produced in the absence of micro-organisms by various chemical substances, such as croton oil, nitrate of silver, turpentine, etc. — care, of course, being taken to ensure the absence of bacteria. The general result obtained by in- dependent observers is that as a rule suppuration does not follow, but that in certain animals and with certain substances it may, the pus being free from bacteria. It is still, however, questioned by some whether the pus thus produced really corresponds histologically and chemically with that due to bacterial action. Buchner showed that suppuration may be produced by the injection of dead bacteria, e.g. sterilised cultures of bacillus pyocyaneus, etc. The subject has now more a scientific than a practical interest, and the general statement may be made that practically all cases of true suppuration met with clinically are due to the action of living micro-organisms. The term septicaemia is applied to conditions in which the ^organisms multiply within the blood and give rise to symptoms j)f genera, i poisoning ragout, however, producing abscesses in the .organs.^ In all cases of septicaemia the organisms are more numerous in the capillaries of internal organs than in the peripheral circulation, and, in the case of the human subject, it may be impossible to detect any in the blood during life, though they may be seen in large numbers in the capillaries of the kidneys, liver, etc., post mortem. The essential fact in_pycemia, on the Other hand, is the op.r-nrrenfp. of nmltiplft a.bsp.ftsaftsin 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. 186). If the term "pyaemia" be used to embrace all such conditions, their method of production should always be distinguished. 174 INFLAMMATION AND SUPPURATION BACTEKIA AS CAUSES OF INFLAMMATION AND SUPPURATION. A considerable number of species of bacteria have been found in acute inflammatory and suppurative conditions, and of these many have been proved to be causally related, 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 1881), 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. Rosen- bach shortly afterwards (1884), by means of cultures, differentiated several varieties of micrococci, to which he gave the following special names : stayhylococcus pyogenes aureus. pyogenes albus, streptococcus pi/o•, micrococcus pyogenes ti-imls. ^ OllM' oTgaliislns are met with in suppuration, such as staphylo- coccus pyogenes citreus. stavhvlococcus cereus albus. staphylococcus cereus flavus neumococcus, pneumobacillus '(ITriedl&nder ba^ly* pvodenes foetidus (assetj&aci^f/.s- c) streptococcus brevis, which is common in the mouth in normal conditions, and is usually non-pathogenic ; and (c) streptococcus conglomeratus, so called from its forming in bouillon minute granules composed of very long chains. It may be stated that pathogenic streptococci obtained from the human subject usually form fairly long chains on agar, whilst the short STREPTOCOCCUS PYOGENES 179 streptococci obtained from the mouth and intestine are usually devoid of virulence. But to these statements exceptions occur, as short streptococci may be associated with grave lesions ; it has also been found that the length of the chains is not a constant feature. As in the case of other organisms attempts have also been made to differentiate streptococci by means of their fermentative properties. Mervyn Gordon introduced for this purpose nine tests, namely : — (1) The clotting of milk, (2) the reduction of neutral red, (3-9) the fermentation with acid production of saccharose, lactose, raffinose, inulin, salicin, coniferin, and mannite. Andrewes and Horder by means of these have differentiated six varieties, of which five occur in the human subject. These are : — (a) A short-chained form called streptococcus mitis, which occurs chiefly in the saliva and faeces as a saprophyte. (6) The streptococcus pyogenes, which is the most important pathogenic variety, and has the characters described above, (c) The streptococcus salivarius, which corre- sponds to the streptococcus brevis of the mouth, and which, as regards fermentative action, seems to bear the same relation to the next variety as the streptococcus mitis does to the strepto- coccus pyogenes. It has more active fermentative properties and clots milk, (d) The streptococcus anginosus, which corre- sponds with the so-called streptococcus scarlatinae and the strepto- coccus conglomeratus. It usually clots milk and does not grow on gelatin at 20° C. (e) The streptococcus fcecalis, a short- chained form, which abounds in the intestine and which has great fermentative activity. It forms sulphuretted hydrogen, and is devoid of haemolytic action. (/) The sixth variety is the streptococcus equinus, which is common in the air and dust of towns, and appears to be derived from horse dung.1 Schottmuller has employed the appearance of the colonies of ^streptococci on blood agar as a means of separating varieties, the medium used consisting of two parts human blood and five parts melted agar. He distinguishes the streptococcus lonVA 180 INFLAMMATION AND SUPPURATION It will be thus seen from this account that the streptococcus pyogenes as described above is the organism most frequently associated with the pathogenic processes, and that short-chained forms are common saprophytes in the human body, although they may be associated with conditions of disease ; these may be subdivided according to their fermentative activity as detailed. And lastly, there is the streptococcus conglomeratus (anginosus), which is specially abundant in the throat in scarlet fever, though it also occurs in other acute catarrhal states. No definite statement can yet be made as to the etiological relation of streptococci to scarlet fever ; we can only say that streptococci are almost invariably present in the fauces, and that to them many of the complications of the disease are due. Bacillus coli communis. — The microscopic and cultural characters are described in the chapter on typhoid fever. The bacillus lactis cerogenes and the bacillus pyogenes f&tidus 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 gelatin, etc., being thicker and whiter than that of the bacillus coli. Bacillus aerogenes encapsulatus sometimes invades the tissues before death, and is characterised by the formation of bubbles of gas in the infected parts. Its characters are described in Chapter XVI. Bacillus pyocyaneus. — This organism occurs in the form of minute rods 1'5 to 3 fji. in length and less than '5 At in thickness (Fig. 56). Occasionally 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-gelatin 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 gelatin. 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 gelatin. Ultimately liquefaction reaches the wall of the tube. In plate cultures the colonies appear as minute whitish points, those on 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 liquefaction. Around the colonies a greenish tint appears. On agarthe 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 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 EXPERIMENTAL INOCULATION 181 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 haemorrhagic oedema results, which may be attended by septicaemia. Intravenous injection may produce, according to the dose, rapid septicaemia with nephritis, or sometimes a more chronic condition of wasting attended by albuminuria. Micrococcus tetragenus. — This organism, first de- scribed by Gaffky, is char- acterised by the fact that it divides in two planes at right angles to one another (Fig. 57), and is thus generally found in the tissues in groups of four or tetrads, which are often seen to be surrounded by a capsule. The cocci measure 1 /* in diameter. They stain readily with all the ordinary stains, and also retain the stain in Gram's method. It grows readily on all the media at the room tem- perature. In a puncture cul- ture on peptone - gelatin a F^alo'fthet^of "hee *»• 57 -Micrococcus tet.ag^s ; young SS&SS& ^Jtewsteart -co. of whitish colour. The gela- tin is not liquefied. 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 viscid or tenacious character, owing to the gelatinous character of the sheaths of the cocci. White mice are exceedingly susceptible to this organism. Subcutaneous 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 arbscess with a good deal of necrotic change results ; sometimes there is also septicaemia. 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 suppura- tion depends upon the number of organisms introduced into the tissues, the number necessary varying not only in different animals, but also in different parts of the same animal, — a smaller number producing suppuration in the anterior chamber of the eye, for example, than in the peritoneum. The virulence of the 182 IMFLAMMATION AND SUPPURATION organism also may vary, and corresponding results may be pro- duced. 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. 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. intravenous injection). Intravenous injection in rabbits, for example, produces interest- ing results which vary according to the quantity used. If a con- siderable 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 circulating 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 haemorrhage. Similar small abscesses may be produced in the heart wrall, 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 carbuncular condition which healed only after some weeks. Confirmatory 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 also one which loses its virulence rapidly in cultures. Even highly virulent cultures, if grown under ordinary conditions, in the course of time lose practically all BACILLUS COLI COMMUNIS 183 pathogenic power. By passage from animal to animal, however, the virulence may be much increased, andparipassu the effects of inoculation are correspondingly varied. Marmorek, for example, 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 41), and (6) in the body of a rabbit ; ultimately, after several passages it possesses a super- virulent character, so that even an extremely minute dose intro- duced into the tissues of a rabbit produces rapid septicaemia, with death in a few hours. It has been proved by Marmorek's experi- ments, 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 erysi- pelatous condition, or again a general septicaemic infection, according as its virulence is artificially increased. Such experi- ments are of extreme importance as explaining to some extent the great diversity of lesions in the human subject with which strep- tococci are associated. 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 septica3mic 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 septica3mia with scattered hemorrhages in various organs. Other Effects.— It has been found by independent observers that in cases where rabbits recover after intravenous injection of bacillus coli communis, a certain proportion suffer from paralysis and sometimes 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 proportion 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 improbable 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, consider 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. 1 - r CsTLAMMATKHT AKD SUPPUEATHW in rnanT of tfe CMK off LESIONS IX THE HUMAN SUBJECT 1-5 In pyaemia they are frequently 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 coli communis is found in a great many inflam- matory and suppurative conditions in connection with the ali- mentary tract — for example, in suppuration in the peritoneum, or in the extraperitoneal tissue with or without 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, — endocarditis, pleurisy, etc., which in some cases are associated with lesions of the intestine, though in others such cannot be found. It is also frequently present in inflammation of the urinary passages, cystitis, pyelitis, abscesses in the kidneys, etc., these lesions being in fact most frequently caused by this or closely allied organisms. 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 eruptions on the skin, and general marasmus. 186 INFLAMMATION AND SUPPURATION Suppurative and inflammatory 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 on the skin and mucous membranes of healthy individuals. Staphylococci are commonly present on the skin, FIG. 59. — Minute focus of commencing suppuration in brain — case of acute ulcerative endocarditis. In the centre a small haemorrhage ; to right side dark masses of staphylococci ; zone of leucocytes at periphery. Alum carmine and Gram's method, x 50. and also occur in the throat and other parts, and streptococci can often be cultivated from the secretions of the mouth in normal conditions. The pneumococcus of Fraenkel and the pneumobacillus of Friedlander have also been found in the mouth and in the nasal cavity, 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 readily understood. Their action will, of course, be favoured by any condition of depressed vitality. Though in normal conditions the blood is bacterium-free, we must suppose ENTRANCE AND SPREAD OF BACTERIA 187 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 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. If, however, there be a local weakness, they may settle in that part FIG. 60. — Secondary infection of a glomerulus of kidney by the staphylo- coccus aureus, 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. and produce suppuration, and from this other parts of the body may be infected. Such a supposition as this is necessary to explain many inflammatory and suppurative conditions met with clinically. In some cases of multiple suppurations due to staphylococcus infection, which we have had the opportunity to examine, only an apparently unimportant surface lesion was present ; whilst in others no lesion could be found to explain the origin of the infection. The term cryptogenetic has been applied by some writers to such cases in which the original 188 INFLAMMATION AND SUPPURATION point of infection cannot be found, but its use is scarcely necessary. The paths of secondary infection may be conveniently sum- marised 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 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 em- bolism ; and we may add (c) by a direct extension along a vein, producing a spreading thrombosis and suppuration 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 pylephlebitis suppurativa. Although many of the lesions produced by the bacteria under consideration have already been mentioned, certain con- ditions may be selected for further consideration on account of their clinical importance or bacteriological interest. Endocarditis. — There is now strong presumptive evidence that all cases of endocarditis are due to bacterial infection. In the simple or vegetative form, so often the result of acute rheumatism, the micrococcus rheumaticus (p. 193) has been cultivated from the valves in a certain number of cases, and is probably the causal agent in most instances. Endocarditis of the nlcerative various organisms, chiefly pyogenic._ Of these the staphylococci *anct streptococci are most frequently found. In some cases ol ulcerative endocarditis following pneumonia, t.hp. pnftnmor;op.p.ns- (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 endocarditis encap- sulatus, bacillus endocarditidis griseus (Weichselbaum), and others. In some cases the bacillus coli cominunishas_beenjojiiid, and occasionally in endocarcTillij following^ typhoid the typhoid bacillus has been described as the organism present, but further observations on this point are desirable. The^onococcr[s also has been shown to affect the heart valves (p. 22o]7tnough this is a very rare occurrence. TnT-raj-nlp. no^jplpa on the ENDOCARDITIS 189 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 organisms may attack healthy valves, producing a primary ulcerative endo- carditis, but more frequently the valves have been the seat of previous endocarditis, secondary ulcerative endocarditis being FIG. 61. — Section of a vegetation jn ulcerative endocarditis showing numerous staphylococci lying in the spaces. The lower portion is a fragment in process of separation. Stained by Gram's method and Bismarck -brown. x 600. thus produced. In some cases, 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 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 surface, or occurring in large masses or clusters in spaces in the vegetation (Fig. 61). By their action a certain amount 190 INFLAMMATION AND SUPPURATION of softening or breaking down of the vegetations occurs, and the emboli thus produced act as the carriers of infection to other organs, and give rise to secondary suppurations. 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. Orth and Wyssokowitsch at a comparatively early date produced the condition by damaging the aortic cusps by a glass rod introduced through the carotid, and after- wards 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 in salt solution so as to form an emulsion, and then injected into the circulation, some minute fragments became arrested at the attachment of the chordae tendinese 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. The great majority of cases are caused by the ^vogenic cocci, of which one or two varieties may be present, the staphylococcus aureus, 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 the same organisms or in the course of infective fevers, but in a great many cases the path of entrance cannot be determined. In the course of this disease serious secondary infections are always very liable to follow, such as small abscesses in the kidneys, 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 arrange- ment of the veins in the bone and marrow. Experimental. — Multiple abscesses in the bones and under the peri- osteum 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 CONJUNCTIVITIS 191 few organisms which have reached the part by the blood to settle and multiply. Moreover, if a bone be experimentally 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 may be produced by a variety of organisms, but the disease in the human subject in its characteristic form is almost in- variably 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 inocu- lations on the human subject as a therapeutic measure in malignant disease, he was able to reproduce erysipelas. 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 con- ditions due to streptococci do not appear liable to be followed by erysipelas. On the other hand, the connection between erysipelas and puerperal septicaemia is well established clinically. 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. The streptococci may extend to serous and synovial cavities and set up inflammatory or suppurative change, — peritonitis, meningitis, and synovitis may thus be produced. Conjunctivitis. — A considerable number of organisms are concerned in the production of conjunctivitis and its associated lesions. Of these a number appear to be specially associated with this region. Thus a small organism, generally known as the Koch- Weeks bacillus, is the most common- cause of acute contagious conjunctivitis, especially prevalent in Egypt, but also common in this country. This organism is very minute, being little more than 1 //, in length, and morphologically resembles the influenza bacillus : its conditions of growth are jsven more restricted, as it rarely ^rows on blood agar, the best "medium being serum ap^ar. un tnls medium it produces minute transparent colonies like drops of dew. The obtaining of • pure cultures is a matter of considerable difficulty, and it is nearly always accompanied by the xerosis bacillus. It can readily be 192 INFLAMMATION- AND SUPPURATION found in the muco-purulent secretion by staining films with ^^^^^^_ weak (1 : 10) carbol- fuchsin, and is often to be seen in the interior of leucocytes (Fig. 62). Another organism ex- ceedingly like the prev- ious, apparently differing from it only in the rather wider conditions of growth, is Muller's bacil- lus. It has been culti- vated by^him in a con- siderable proportion of cases of trachoma, but its relation to this con- FIG. 62. -Film preparation from a case of dition is sti11 matter of acute conjunctivitis, showing Koch-Weeks dispute. Another bacil- bacilli, chiefly contained within a leucocyte, lus which is now well (From a preparation by Dr. Inglis Pollock.) recognised is the diplo- bacillus of conjunctivitis first described by Morax. It is especially common in the more subacute cases of conjunctivitis. Eyre found it in 2 '5 per cent of all cases of conjunc- tivitis. Its cultural char- acters are given below. The xerosis bacillus, which is a small diph- theroid organism (Fig. 123), has been found in r xerosis of the conjunc- tiva, in follicular con- junctivitis, and in other conditions ; it appears to occur sometimes also in the normal conjunc- tiva. It is doubtful whether it has any pathogenic action of im- portance. Acute con- junctivitis is also pro duced by the pneu mo- coccus, epidemics of the disease being sometimes due to this organism, and also by FIG. 63. — Film preparation of conjunctival secretion showing the Morax diplo-bacillus of conjunctivitis. x 1000. ACUTE RHEUMATISM 193 streptococci and staphylococci. True diphtheria of the con- junctiva caused by the Klebs-LorHer bacillus also occurs, whilst in gonorrha'al conjunctivitis, often of an acute purulent type, the gonococcus is present (p. 225). Diplo - bacillus of Conjunctivitis. — This organism, discovered by Morax, is a small plump bacillus, measuring 1 x 2 /*, and usually occur- ring in pairs, or in short chains of pairs (Fig. 63). It is non-motile, does not form spores, and is decolorised by Gram's method. It does not grow on the ordinary gelatin and agar media, the addition of blood or serum being necessary. On serum it forms small rounded colonies which produce small pits of liquefaction ; hence it has been called the bacillus lacunatus. In cultures it is distinctly pleomorphous, and involution forms also occur. It is non-pathogenic to the lower animals. Acute Rheumatism. — There are many facts which point to the infective nature of this disease, and investigations from this point of view have yielded important results. Of the organism isolated, the one which appears to have strongest claims is a small coccus observed by Triboulet, and by Westphal and Wassermann, the characters and action of which were first investigated in this country by Poynton and Paine. It is now usually spoken of as iho^nicrococcus rheumaticus. The organism is sometimes spoken of as a diplococcus, but it is best described as a streptococcus growing in short chains ; in the tissues, how- ever, it usually occurs in pairs. It is rather smaller than the streptococcus pyogenes, and although it can be stained by Gram's method, it loses the colour more readily than the streptococcus. In the various media it produces a large amount of acid, and usually clots milk after incubation for two days ; on blood agar it alters the haemoglobin to a brownish colour. Its growth on media generally is more luxuriant than that of the streptococcus, and it grows well on gelatin at 20° C. Intravenous injection of pure cultures in rabbits often produces polyarthritis and synovitis, valvulitis and pericarditis, without any suppurative change — lesions which it is stated are not produced by the ordinary streptococci (Beattie). In one or two instances choreiform movements have been observed after injection. The organism is most easily obtained from the substance of inflamed synovial membrane where it is invading the tissues ; a part where there is special congestion should be selected as being most likely to give positive results. It is only occasionally to be obtained from the fluid in joints. It has also been cultivated from the blood in rheumatic fever, from the vegetations on the heart valves, and from other acute lesions ; in many cases, how- ever, cultures from the blood give negative results. Poynton 13 194 INFLAMMATION AND SUPPURATION and Paine cultivated it from the cerebro- spinal fluid in three cases where chorea was present, and also detected it in the membranes of the brain. They consider that this disease is probably of the nature of a slight meningo-myelitis produced by this organism. The facts already accumulated speak strongly in favour of this organism being causally related to rheumatic fever, though this cannot be considered completely proved. Andrewes finds that the organism has the same cultural characters and fermentative effects as the streptococcus fsecalis, a common inhabitant of the intestine. Even, however, if the two organisms were the same, it might well be possible that rheumatic fever is due to an infection of the tissues by this variety of streptococcus. The clinical data, in fact, rather point to rheumatic fever being due to an infection by some organism frequently present in the body, brought about by some state of predisposition or acquired susceptibility. Vaccination Treatment of Infections by the Pyogenic Cocci. — From his study of the part played by phagocytosis in the successful combat of the pyogenic bacteria by the body, Wright was led to advocate the treatment of such infections by the origination during their course of an active immunisation by dead cultures of the infecting agent. The treatment is applicable when the infection is practically local as in acne pustules, in boils, etc. (For the theoretical questions raised see Immunity.) It is best to attempt to isolate the causal organism from the lesion and to test the opsoriic index of the patient against it. To prepare the vaccine an agar slope culture is taken and the growth washed off with normal saline. The organism is then killed by steaming for an appropriate time, and the efficacy of the sterilisation tested by inoculating fresh agar tubes. The strength of the emulsion is estimated by the method of counting dead bacteria described on p. 67. The number of bacteria used for an injection is from 250,000,000 to 500,000,000, and in the details of the measurement of this quantity and in its injection every aseptic precaution must, of course, be adopted. If repeated injections are necessary Wright recommends that the opsonic index should be observed every few days and the injections only practised during a positive phase. If it is not practicable to use the infecting strain for the preparation of the vaccine, then laboratory cultures must be used, and in such cases it is well to use a mixture of strains ; in skin infections a mixture of staphylococcus aureus and albus may be employed. Such means have been extensively used in the treatment of acne, boils, sycosis, infections of the genito-urinary tract by the METHODS -OF EXAMINATION 195 b. coli, infections of joints by the gonococcus, and in many cases considerable success has followed the treatment. Methods of Examination in Inflammatory and Suppurative Conditions. — These are usually of a comparatively simple nature, and include (1) microscopic examination, (2) the making of cultures. (1) The pus or other fluids should be examined microscopi- cally, 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. 98), 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 gelatin media at ordinary temperatures, pure cultures can be readily obtained by the ordinary plate methods. But in many cases the separation can be effected much more rapidly by the method of successive streaks on agar tubes, which are then incubated at 37° 0. 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 streptococci. Inoculation experiments may be carried out as occasion arises. In cases of suspected blood infection the examination of the blood is to be carried out by the methods already described (p. 68). CHAPTER VII. INFLAMMATOKY AND SUPPURATIVE CONDITIONS, CONTINUED: THE ACUTE PNEUMONIAS, EPI- DEMIC CEREBRO-SPINAL MENINGITIS. Introductory. — The term Pneumonia is applied to several con- ditions which present differences in pathological anatomy and in origin. All of these, however, must be looked on as varieties of inflammation in which the process is modified in different ways depending on the special structure of the lung or of the parts which compose it. There is, first of all, and, in adults, the com- monest type, the^acute croupous or lobar pneumonia, in which an inflammatory process attended by abundant fibrjnous exuda- J^ion affects, by continuity~jtlie entire tissue of a jobe or' of a^ large portion of the lun^'iT^Teparts trom ike course of an" ordinary inflammation in that the reaction of the connective tissue of the lung is relatively slight, and there is usually no tendency for organisation of the inflammatory exudation to take place. Secondly, there is the acute catarrhal or lobular pneumonia, where a catarrhal inflammatory process spreads from the capillary bronchi to the air vesicles, and in these a change, consisting largely of proliferation of the endothelium of the alveoli, takes place which leads to consolidation 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, however, influenza in an epidemic form has become frequent, catarrhal pneumonia has been of much more frequent occurrence in adults, has assumed a very fatal tendency, and has presented the formerly quite unusual feature of being sometimes the precursor of gangrene of the lung. Besides these two definite types other forms also occur. Thus instead of a fibrinous material the exudation may 196 TYPES OF PNEUMONIA 197 be of a serous, haemorrhagic, or purulent character. Cases of mixed fibrinous and catarrhal pneumonia also occur, and in the catarrhal there may be great leucocytic emigration. Hsemorrhages also may occur here. Besides the two chief 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 ; these often set up a purulent capillary bronchitis and lead to infection of the air cells and also of the interstitial 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.) In these septic pneumonias various changes, resembling those found in the other types, are often seen round the septic foci. In pneumonias, therefore, there may be present a great variety of types of inflammatory reaction. We shall see that with all of them bacteria have been found associated. 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 was supposed to be an effect of exposure to cold ; but many observers were dissatisfied with this view of its etiology. Not only did 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 con- formed to the type of an acute infective fever ; it was thus suspected by some to be due to a specific infection. This view of its etiology was promulgated in 1882-83 by Friedlander, whose results were briefly as follows. In pneumonic lungs there were cocci, adherent usually in pairs, and possessed of a definitely contoured capsule. These cocci could be isolated and grown on gelatin, and on inoculation in mice they produced a kind of septicaemia with inflammation of the serous membranes. The blood and the exudation in serous cavities contained numerous capsulated diplococci. There is little doubt that many of the organisms seen by Friedlander were really Fraenkel's pneumococcus, to be presently described. By many observers it had been 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 capsulated diplococci were found. A. Fraenkel 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 septicsemia," as it was called, differed from Friedlander's cocci in several respects, to be presently studied. Fraenkel further 198 THE ACUTE PNEUMONIAS investigated a few cases of pneumonia, and isolated from them cocci identical in microscopic appearances, cultures, and pathogenic effects, with those isolated in sputum septicaemia. The most extensive investi- gations on the whole question were those of Weichselbaum, published in 1886. This author examined 129 cases of the disease, including cases not only of acute croupous pneumonia, but of lobular and septic pneumonia. From them he isolated four groups of organisms. (1) Diplococcus pneumonice. This he described as an oval or lancet- formed coccus, corresponding in appearance and growth characters to Fraenkel's coccus. (2) Streptococcus pneumonice. This on the whole presented similar characters to the last, but it was more vigorous in its growth, and could grow below 20° C., though it preferred a temperature of 37° C. (3) Staphylococcus pyogenes aureus. (4) Bacillus pneumonice. This was a rod-shaped organism, and was identical . with Friedlander's pneumo- coccus. Of these organisms the diplococcus pneumonias was by far the most frequent. It also occurred in all forms of pneumonia. Next in frequency was the streptococcus pneumonias, and lastly the bacillus pneumoniae. Inoculation experiments were also performed by Weichsel- baum with each of the three characteristic cocci he isolated. The diplococcus pneumonias and the streptococcus pneumonias 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 nneumncnc,r.u.^ whir».h is recognised to be identical with the coccus of "sputum septicaemia," with Weichselbaum 's diplococcus pneumoniae, and with his streptococcus pneumonise. 2. Friedldnder's ^^(^^^^,e (-n^™ known as Friedlander's pneumobacillus), which is almost certainly the bacillus pneu- monias of Weichselbaum. We shall use the terms " Fraenkel's pneuinococcus " and ".Friedlander's pTiftiimobar'.illns." 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 pneumonic lung (preferably from a part in a stage of acute congestion or early hepatisation), or from the gelatino*us 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 Bismarck-brown or Ziehl-Neelsen carbol-fuchsin (one part to ten of water) as a contrast stain ; with the latter it is best either to stain for only a few seconds, or to overstain and then decolorise with alcohol till the ground of the preparation is just tinted. The capsules can also be BACTERIA IN PNEUMONIA 199 stained by the methods already described (p. 102). 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) FraenkeVs Pneumococcus. — -This organism occurs in the form of a small oval coccus, about 1 p in longest diameter, arranged generally in pairs (diplo- cocci), but also in chains / of four to ten (Fig. 64). t ,/; * The free ends are often >•;_ pointed like a lancet, hence * » <"'* ^ the term diplococcus lance- Ltlajtus has also been ap- plied to it. These cocci have round them a capsule, which, in films stained by ordinary methods, usually appears as an unstained halo, but is sometimes stained more deeply than the ground of the pre- paration. This difference in Staining depends, in nartfltlpfl^t on thpamount partat least, on tneamount of decolorisation to which the preparation 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 (Ira/ml*. m.?thnd_ It is the organism of by far the most frequent occurrence in true croupous pneumonia, and in fact may be said to be rarely absent. (2) Friedldnder's Pneumobacillus. — As seen in the sputum and tissues, this organism, both in its appearance and arrange- ment, 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 classed amongst the bacilli, especially in view of the fact that elongated FIG. 64.— Film preparation of pneumonic sputum, showing numerous pneumococci (Praenkel.s) with unstamed capsules; some are arranged in short chains. Stained with carbol-fuchsiu. x 1000. 200 THE ACUTE PNEUMONIAS rod forms may occur (Fig. 65). The capsule has the same general characters as that of FraenkePs organ- ism. JFriedlanderV pneumobacillus stains readily with the basic aniline stains, but loses the stain in Gram's method, and is accord- ingly coloured with the contrast stain, — fuchsin or Bismarck -brown, as above recommended. A valuable means is thus afforded of distinguish- ing it from Fraenkel's pneumococcus in inicro- Fio. 65. — Friedlander's pneumobacillus, showing scopic preparations, the variations in length, also capsules. Film preparation from exudate in a case of pneu- monia, x 1000. sometimes it Friedlander's organ- ism is much less fre- quently present in pneu- is associated with the inonia than Fraenkel's; latter; very rarely it occurs alone. In sputum prepara- tions the capsule of both pneumococci 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. Some- times in preparations stained by ordinary methods 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. FIG. 66. — Fraenkel's pneumococcus in serous exudation at site of inoculation in a rabbit, showing capsules stained. Stained by Rd. Muir's method. x 1000. This CULTIVATION OF PNEUMOCOCCUS 201 difficulty can always be overcome by having the groundwork 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 post 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 commenc- ing red hepatisation, and incubating them at 37° C. The colonies of the pneumo- coccus appear as almost transparent small discs which have been compared to drops of dew (Fig. 67). This method is also sometimes successful in the case of sputum. The appearances presented in cultures FK} 67>_stroke cnlture of 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 glycerin agar. In a stroke culture on blood serum growth appears as an almost transparent pellicle along the track, with isolated colonies at the margin. On agar 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 vigorous, and is more delicate in appearance. A similar statement also applies to cultures in gelatin 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 transparent periphery. In bouillon, growth forms a slight turbidity, which settles to the Fraenkel's pneumococcus on blood agar. The colonies are large and un- usually distinct. Twenty- four hours' growth at 37° C. Natural size. 202 THE ACUTE PNEUMONIAS bottom of the vessel as a slight dust-like deposit. On potatoes^ 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 ultimately 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 Washbourn, 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 rab- bits' blood. The agar \ t must be prepared with X ' p% "'• A s Witte's peptone, must \ \ % n°t be neated over 100° * i \ C., and after neutralisa- tion (rosolic acid being s** V | > used as the indicator) i,,, \ '^jj must have '5 per cent of ^ ^ normal sodium hydrate added. The tubes when . inoculated are to be kept ^ ^ \ at 37' 5° C. and sealed to prevent evaporation. In %«^. none of the ordinary arti- ^ ficial media do pneumo- cocci develop a capsule. FIG. 68. — P raeukel s pneumococcus from a pure m, -,, culture on blood agar of twenty-four hours' Thev Dually appear as growth, some iu pairs, some in short chains, diplococci, but in pre- Stained with weak carbol-fuchsin. x 1000. parations made from the surface of agar or from bouillon, shorter or longer chains may be observed (Fig. 68). After a few days' growth they lose their regular shape and size, and involution forms appear. Usually the pneumococcus does not grow below 22° C., but forms in which the virulence has disappeared often grow well at 20° C. Its optimum temperature is 37° C., its maximum 42° C. It is preferably an aerobe, but can exist without oxygen. It prefers a slightly alkaline medium to a neutral, and does not grow on an acid medium. These facts show that when growing outside the body on artificial media, the pneumococcus is a comparatively delicate organism. There has been described by Eyre and Washbourn a non- pathogenic type of the pneumococcus which may be found in the healthy mouth, and which may also be produced during the saprophytic growth of the virulent form. From the latter it CULTIVATION OF PNEUMOBACILLUS 203 differs generally in its more vigorous growth, in producing a uniform cloud in bouillon, in slowly liquefying gelatin, and in growing on potato. The Cultivation of Friedlander's Pneumobacillus. — This organism, when present in sputum or in a pneumonic lung, can be readily separated by making ordinary gelatin 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 ap- pearance of a stab culture in gelatin 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 I of an inch above the level of the gelatin ; UHfeM along the needle track there is a white ^5 FIG. 69.— Stab culture of Friedlander's pneumobacillus in peptone gelatin, showing the nail- like appearance ; ten days' growth. Natural size. •«.•>* .<«&. ••X. ^ &'* FIG. 70. — Friedlander's pneumobacillus,1 from a young culture on agar, showing some rod-shaped forms. Stained with thionin-blue. x 1000. granular appearance, so that the whole resembles a white round- headed nail driven into the gelatin (Fig. 69). Hence the name 1 The apparent size of this organism, on account of the nature of its sheath, varies much according to the stain used. If stained with a strong stain, e.g. carbol-fuchsin, its thickness appears nearly twice as great as is shown in the figure. 204 THE ACUTE PNEUMONIAS "nail-like" which has been applied. Occasionally bubbles of gas develop along the line of growth. There is no liquefaction of the medium. On sloped agar it forms a 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. 70). On the surface 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 on glycerin. The substances produced by the fermentation vary with the sugar fermented, but include ethylic alcohol, acetic acid, laevolactic acid, succinic acid, along with hydrogen and carbonic acid gas. The amount of acid produced from lactose seems only exceptionally sufficient to cause coagula- tion of milk. It is said by some that this bacillus is identical with an organism common in sour milk, and also a normal inhabitant of the human intestine, viz. the bacterium lactis aerogenes of Escherich. The Occurrence of the Pneumobacteria in Pneumonia and other Conditions. — 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 microscopic- ally 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 most recent, e.g. in an area of acute congestion in a case of croupous pneumonia, and therefore such parts are preferably to be selected for microscopic examination, and as the source of cultures. Some- times there occur in pneumonic consolidation areas of suppura- tive 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 destruction of large portions of the lung. In these a great variety of bacteria, both aerobes and anaerobes, are to be found. DISTRIBUTION OF PNEUMOBACTERIA 205 In ordinary broncho -pneumonias also Fraenkel's pneumo- coccus is usually present, sometimes along with pyogenic 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 accompanied 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 associated with the presence of the pneumococcus. By direct extension to neigh- bouring 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, peritoneum, joints, kidneys, liver, etc.), in ptitis media, ulcerative endocarditis (p. 188), and meningitis. Tnese conditions may take place either as complications of pneumonia, or they may constitute the primary disease. The occurrence of meningitis is of special importance, 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 1 IK QK Capillary bronchitis/ • • • Meningitis . 13 '00 Empyema Otitis Endocarditis Liver abscess 8-53 2-44 1-22 1-22 (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. 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. As bearing on the occurrence of pneumococcal infections 206 THE ACUTE PNEUMONIAS secondary to such a local lesion as pneumonia, it is important to note that in a large proportion of cases of the latter disease the pneumococcus can be isolated from the blood. Experimental Inoculation. — The pneumococcus of Fraenkel is pathogenic to various animals, though the effects vary somewhat with the virulence of the race used. The susceptibility of different species, as Gamaleia has shown, varies to a considerable FIG. 71. — 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 1000. extent. The rabbit, and especially the mouse, are very sus- ceptible ; the guinea-pig, the rat, the dog, and the sheep occupy an intermediate position ; the pigeon is immune. In the more susceptible animals the general type of the disease produced is not pneumonia, but a general septiccemia. 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 EXPERIMENTAL INOCULATION 207 often enlarged and firm, and the blood contains capsulated pneumococci in large numbers (Fig. 71). If the seat of inocula- tion 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- pulmonary injection in sheep is followed by a typical pneumonia, 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. Inoculation by inhalation appears only to have been performed in the susceptible mouse and rabbit ; here also septicaemia resulted. The general conclusion to be drawn from these experiments thus is that in highly susceptible animals virulent pneumococci produce a general septicaemia ; whereas in more 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 sufficient to kill a rabbit is injected sub- cutaneously in the human subject, it gives rise to a local inflam- matory 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. In this con- nection the occurrence of manifestations of general infection associated with pneumonia in man is of the highest import- ance. We have seen that meningitis and other inflammations are not very rare complications of the disease, and such cases form a link connecting the local disease in the human subject with the general septicsemic processes which may be produced 208 THE ACUTE PNEUMONIAS artificially in the more susceptible representatives of the lower animals. A fact which at first appeared rather to militate against the pneumococcus being the cause of pneumonia was 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 confirmed 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, only indicates 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 possibility 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 effects produced by such bacteria as the b. typhosus and the b. diphtherise 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 to the catarrhal patches in broncho-pneumonia. It is quite likely that in the former condition the organism is possessed of a different order of virulence, though of this we have no direct proof. We have, however, a closely analogous fact in the case of erysipelas ; this disease, 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 re- garding the relation of Fraenkel's pneumococcus to the disease PNEUMOCOCCUS INFECTION 209 by saying that it can be isolated from nearly all cases of acute croupous pneumonia, and also from a considerable 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 con- ditions 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 there- fore 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 septicaemic complications of pneumonia, such as empyema and meningitis, render it possible that it may be the causal agent in a few cases of the disease. In the septic pneumonias the different pyogenic organisms already described are found, and sometimes in ordinary pneumonias, especially the catarrhal forms, other organisms, such as the b. coli or its allies, may be the causal agents. The Pathology of Pneumococcus Infection. — The effects of the action of the pneumococcus, at any rate in a relatively insusceptible animal such as man, seem to indicate that toxins must play an important part. 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 general nervous depression that death usually results. These considerations, taken in connection with the fact that in man the organisms are found in the greatest numbers in the lung, suggest that a toxic action is at work. Various attempts have been made to isolate toxins from cultures of the pneumococcus, e.g. by precipitating 14 210 THE ACUTE PNEUMONIAS bouillon cultures with alcohol or ammonium sulphate, and poisonous effects have been produced by certain substances thus derived ; but the effects produced are, as in so many other similar cases, of a non-specific character and to be classed as interferences with general metabolism. The general conclusion has been that the toxins at work in pneumonia are intracellular ; but no special light has been thrown on the common effects of the members of this group of bacterial poisons. • Immunisation against the Pneumococcus. — Animals can be immunised against the pneumococcus by inoculation with cultures which have become attenuated by growth on artificial media, or with the naturally attenuated cocci which occur in the sputum after the crisis of the disease. Netter effected immun- isation by injecting an emulsion of the dried spleen of an animal dead of pneumococcus septicaemia. Virulent cultures killed by heating at 62° C., rusty sputum kept at 60° C. for one to two hours and then filtered, and filtered or unfiltered bouillon cultures similarly treated have also been used. In all cases one or two injections, at intervals of several days, are sufficient for immunisation, but the immunity has often been observed to be of a fleeting character and may not last more than a few weeks. The serum of such immunised animals protects rabbits against subsequent inoculation with pneumococci, and if injected within twenty-four hours after inoculation, prevents death. A protective serum was obtained by Washbourn, who employed pneumococcus cultures of constant virulence. This observer immunised a pony by using successively (1) broth cultures killed by one hour's exposure to 60° C. ; (2) living agar cultures ; (3) living broth cultures. From this animal there was obtained a serum which protected ' susceptible animals against many times an otherwise fatal dose, and which also had a limited curative action. It is stated 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, similar to that exhibited by the serum of immune animals. The Klemperers treated a certain number of cases of human pneumonia by serum derived from immune animals, and appar- ently with a certain measure of success, and sera prepared by Washbourn and by others have also been used. The results obtained by different observers have, however, been rather con- tradictory. The use of these sera apparently causes the tempera- ture in some cases to fall, and even may hasten a crisis, but further experience is necessary before their value in therapeutics can be properly estimated. PNEUMOCOCCUS INFECTION 211 There has been considerable difference of opinion as to the explanations to be given of the facts observed regarding immunisation against the pneumococcus and especially regarding the properties of immune sera. At first these sera were supposed to possess antitoxic qualities — largely on the ground that no bactericidal effect was produced by them on the bacterium in vitro. As no specific toxin has been proved to be concerned in the action of the organism the development of an antitoxin during immunisation must, in the present state of knowledge, be looked on as not yet proved. To explain the action of a serum in preventing and curing pneumococcal infections, it has been thought to have the complex character seen in anti- typhoid sera in which two substances — immune body and com- plement (see Immunity) — are concerned, and the variability in the therapeutic results obtained has been accounted for on the view that there might be a deficiency of complement, such as occurs in other similar cases. The absence of bactericidal effect, however, raises several difficult points. It is stated that no such effect is observable either in immune sera, or in the serum of patients who have successfully come through an attack of the natural disease. Some effect of the kind would be ex- pected to be present if the anti-pneumonic serum were quite comparable to the antityphoid serum. Within recent times many have turned to the opsonic property of sera to account for the facts observed. In this connection Mennes observed that normal leucocytes only become phagocytic towards pneumo- cocci when they are lying in the serum of an animal immunised against this bacterium. Wright had in his early papers looked to the phagocytosis of sensitised bacteria to explain their destruction in the absence of bactericidal qualities in the serum alone, and Neufeld and Rimpau have described the occurrence of an opsonic effect in the action of an anti-pneumococcic serum. Further work may show that along these lines lies the explanation of the facts observed. In studying further the relationship of the opsonic effect to pneumococcal infection, inquiry has been directed to the opsonic qualities of the blood of pneumonic patients, especially with a view to throwing light on the nature of the febrile crisis. According to some results the opsonic index as compared with that of a healthy person is not above normal, but if the possible phagocytic capacities of the whole blood of the sick person be taken into account these will probably be much above normal in consequence of the leucocytosis which usually accompanies a successful resistance to this infection. It has been observed, 212 THE ACUTE PNEUMONIAS however, that as the crisis approaches in a case which is to recover the opsonic index rises, and after defervescence gradually falls to normal. And further, as bearing on the factors in- volved in the successful resistance of the organism to the pneumococcus, it has been noted that avirulent pneumococci are more readily opsonised than more virulent strains. Further observations along such lines are to be looked for with interest, and it may be said that Wright's vaccination methods have been applied to the treatment of pneumonia cases, and in certain instances are said to have been followed by favourable result. It may be noted here, in conclusion, that in man it is probable that immunity against pneumonia may be short-lived, as in a good many cases of pneumonia a history of a previous attack is elicited. Agglutination of the Pneumococcus. — If a small amount of a culture of Fraenkel's pneumococcus be placed in an anti-pneumo- coccic serum, an aggregation of the bacteria into clumps occurs. Such an agglutination, as it is called, is frequently observed under similar circumstances with other bacteria. The pheno- menon is not invariably associated with the presence of protective bodies in a serum, but it has been used for diagnostic purposes in the differentiation of sore throats due to pneumococcus infection from those due to other bacteria. Whether the method is reliable has still to be proved. 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. (pp. 99, 101), in the latter case without decolorising the groundwork of the preparation. (2) By cultures, (a) Fraenkel's pneumococcus. With similar material make successive strokes on agar, blood agar, or blood serum. The most certain method, however, 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. In some cases of severe pneumococcic infection the organism may be cultivated from the blood obtained by venesection (p. 68). (b) Friedldnder's pneumobacillus can be readily isolated either by ordinary gelatin plates or by successive strokes on agar media. EPIDEMIC CEREBRO-SPINAL MENINGITIS 213 EPIDEMIC CEKEBE.O-SPINAL MENINGITIS. As the result of observations on this disease in different parts of the world, it has been now established that the causal agent is the dijdococcns intracdlidaris meningitidis first described by WeicTiselbaum. This organism is a small coccus measuring about 1 /x in diameter and usually occurs in pairs, the adjacent sides being somewhat flattened against each other. In most cases the cocci are chiefly contained within polymorphonuclear leucocytes in the exudation (Fig. 72) ; in some cases, however, the majority may be lying free. It stains readily with basic aniline dyes, but loses the stain Gram's metJtmcL tne readiness with which the organism decolorises varying with different strains. Both in appear- ance and in its staining reactions it is superfici- ally similar to the gono- coccus (vide infra). The organism can readily be cultivated outside the body, but the conditions of growth are somewhat restricted — agar with an admixture of serum or blood (preferably human) is most suitable.1 Strains separated in different epidemics appear to present slight in- dividual variations, but the following description may be taken as summing up the common characters. Growth takes place best at the temperature of the body, and practically ceases at 25° C. On serum agar the colonies are circular discs of almost transparent appearance and possessing a smooth shining surface ; they have little tendency to become confluent. When examined under a low magnification the colour is seen to be somewhat yellowish and the margins usually are smooth and 1 A very good medium is one composed of 1 part of ascitic fluid and 6 parts of 1 per cent glucose agar ; the serum obtained aseptically^is added to the agar in the melted state at 45° C. and the tubes are tested as regards sterility by incxibation. Fia. 72. — Film preparation of exudation from a case of meningitis, showing the diplococci within leucocytes. Stained with carbol-thionin-blue. x 1000. 214 THE ACUTE PNEUMONIAS regular, though on some media slight crenation may appear. The colonies may be of considerable size, reaching sometimes a diameter of 2 to 3 mm. on the third day. On plain agar the colonies are very much smaller, and sometimes no growth occurs ; sub-cultures especially often fail to give any growth on this medium. In serum bouillon the organism produces a general turbidity with formation of some deposit after a day or two. It ferments maltose, galactose, and dextrose with acid production, a property which distinguishes it from the micro- coccus catarrhalis (vide infra}. Buchanan has pointed out that this may be demonstrated by making up lots of Loifler's medium (p. 40) in Petri dishes . • with each of these sugars * • •• * added. In all cases / *• *** ., * 5 „ . growth occurs best when »*' *.%*** /V the medium has a neutral v •» *"* ? • V * %^\ or very slightly alkaline + ^* -*, „, **; **** '-/ %*t*»* reaction. In cultures the », .** •*%• "* '*.!V*.' *„ *•' « organism presents the . *%**** •• V * same appearance as in the **• - ** . • . , - . **, body and often shows •' *«•%«*, * % * tetrad formation. There . "' •* ** ***** T' is also a great tendency i • » t^, ^ t to the production of in- *-.+ .'• • f . * volution forms (Fig. 73), ,%«X,» \+* many of the cocci be- * coming much swollen, FIG. 73,-Pure culture of diplococcus intra- staining badly. and after' cellularis, showing involution forms. wards undergoing disin- tegration. This change, according to Flexner's observations, would appear to be due to the production of an autolytic enzyne, and he has also found that this substance has the property of producing dissolution of the bodies of other bacteria. The life of the organism in cultures is a comparatively short one ; after a few days cultures will often be found to be dead, but, by making sub-cultures every three or four days strains can be maintained alive for considerable periods. The organism is readily killed by heat at 60° C., and it is also very sensitive to weak antiseptics ; drying for a period of a day has been found to be fatal to it. The facts established accord- ingly show it to be a somewhat delicate parasite. As stated above, the organism occurs in the exudate in the meninges and in the cerebro-spinal fluid, and it can usually be obtained by lumbar puncture. In acute cases, especially in the EPIDEMIC CEREBRO-SPINAL MENINGITIS 215 earlier stages, it is usually abundant ; but in the later stages of cases of more sub-acute character, its detection may be a matter of difficulty, and only a few examples may be found after a prolonged search ; in extremely acute cases also the organism may be difficult to demonstrate. In most cases the disease is practically restricted to the nervous system, but occasionally complications occur, and in these the organism may sometimes be found. It has been observed, for example, in arthritis, peri- carditis, pneumonic patches in the lung, and in other inflam- matory conditions associated with the disease. In a small proportion of cases it has been obtained from the blood during life, but cultures in most instances give negative results. Experimental inoculation shows that the ordinary laboratory animals are relatively insusceptible to this organism. An inflammatory condition may be produced in guinea-pigs by intra-peritoneal injection, but large quantities of cultures must be used, and none of the characteristic lesions found in the human subject are reproduced. The intra-peritoneal injection of the cerebro-spinal fluid or of cultures in mice is frequently followed by death, the cocci being found in the exudate and even in the blood. Flexner has shown that cerebro-spinal meningitis may be produced in monkeys by injections of the organism into the spinal canal. In such experiments the organism extends upwards to the brain, and produces meningitis within a very short time. The resulting lesions, both as regards their dis- tribution and general characters, and also as regards the histological changes, resemble the disease in the human subject. Even these animals, however, are, in comparison with man, relatively insusceptible, as a considerable amount of culture has to be injected. Many questions of great importance Math regard to the spread of the disease still require further investigation. The organism has been obtained by culture from the throat and nasal cavities of those suffering from the disease in a consider- able number of instances. It has also been obtained from the same positions in healthy individuals, during an epidemic of the disease. In some epidemics also a pharyngitis has been found to occur, and the organism has been obtained from the affected fauces. The general opinion is that the organism spreads by means of the lymphatics from the pharynx or nose to the base of the brain, but a spread by means of the blood stream cannot be excluded, and infection by the alimentary canal has also been suggested. Flexner in his experiments found that when the organism was injected into the spinal 216 THE ACUTE PNEUMONIAS canal marked congestion and inflammatory change in the nasal mucous membrane followed, and in this position he was able to find a Gram-negative diplococcus ; he was, however, unable to recover the diplococcus intracellularis in culture from this situation. These results would seem to indicate that the organism might spread from the brain to the nasal cavity, but if this be so, it also follows that an extension may take place in the reverse direction. On the whole the evidence at present tends to show that the entrance of the organism into the body is by the naso-pharynx, and that this usually results by inhalation of the organism distributed in fine particles of expectoration, etc. In fact, as regards the mode and conditions of infection, an analogy would appear to hold between this disease and influenza. Apart from the epidemic form of the disease, cases of sporadic nature also occur, in which the lesions are of the same nature, and in which the diplococcus intracellularis is present. The facts stated would indicate that the origin and spread of the disease in the epidemic form depend on certain conditions which produce an increased virulence of the organism. We are, however, as yet entirely ignorant as to what these conditions may be. In simple posterior basal meningitis in children, a diplococcus is present, as described by Still, which has the same microscopic and cultural characters as the diplococcus intracellularis ; it has been regarded as probably an attenuated variety of the latter. Recently, however, Houston and Rankin have found that the serum of a patient suffering from epidemic meningitis does not exert the same opsonic and agglutinative effects on the diplococcus of basal meningitis as on the diplococcus intracellularis; and this result points to the two organisms being distinct, though closely allied, species. An agglutination reaction towards the diplococcus intracellu- laris is given by the serum of patients suffering from the disease, where life is prolonged for a sufficient length of time, but the degree of the reaction does not possess much clinical significance. It usually appears about the fourth day, when the serum may give a positive reaction in a dilution of 1 : 50 ; at a later stage it has been observed in so great a dilution as 1 : 1000. There is thus no doubt that anti-substances are produced in epidemic meningitis as in other diseases, and this is also found to be the case on inoculation of animals with pure cultures. Attempts had been made to obtain an anti-serum, and a certain measure of success has been obtained so far as experimental results are concerned. Flexner obtained such a serum from a goat, and EPIDEMIC CEREBRO-SPINAL MENINGITIS 217 found that it had a certain protective effect in guinea-pigs and monkeys against infection by the organism, but, on the whole, better results were obtained with the serum of inoculated monkeys. As yet no important applications towards the treatment of the disease have been effected. In the nasal cavity there occur other diplococci which have a close resemblance to the diplococcus intracellularis. These occur in the healthy state but are especially abundant in catarrhal conditions ; of these the diplococcus catarrhalis has the closest resemblance to the diplococcus p intracellularis. In addition to occurring in health this organism has also been found in "numbers in epidemic catarrh. Its microscopic appearances are "practically similar to those described above, and it also occurs within leucocytes. _Its colonies on pfiir™ QFQr Qro ™p™> ripQqnf__ than those of the diplococcus intracellularis, and they have a tnan tnose 01 tne diplococcus intracellularis, ana tney u tough consistence^so that they afe1 JjOInetmiejLJcejjQOvedLgft bv the platinum needle. The organism grows on gela The organism grows on gelatin at iv C. without liquefying the medium, and it has none of the . fermentative properties described itfjnvft s\^ Kplrmgmg- t^ the Ljdiplococcus intracellularis. _.. Other species of Gram - negative micrococci have also been isolated, and a Gram-positive diplo- coccus called the diplococcus crassus is of common occurrence ; this organism is rather larger than the diplococcus intracellularis, and especially in sub-cultures may tend to assume staphylo- coccal forms. It is thus evident that the nasal cavity is the common habitat for a number of closely allied diplococci, and that the identification of any suspected organism as the diplo- coccus intracellularis can only be effected by cultivation tests. Apart from the epidemic form of the disease, meningitis may be produced by almost any of the organisms described in the previous chapter, as associated with inflammatory conditions. A considerable number of cases, especially in children, are due to the pneumococcus. In many instances where no other lesions are present the extension is by the Eustachian tube to the middle ear. In other cases the path of infection is from some other lesion by means of the blood stream. This organism also infects the meninges not infrequently in lobar pneumonia, and in some cases with head symptoms we have found it present where there was merely a condition of congestion. The pneumobacillus also has been found in a few cases. Meningitis is not infrequently produced by streptococci, especially when middle ear disease is present, less frequently by one of the staphylococci ; occasionally more than one organism may be concerned. In meningitis following influenza the influenza bacillus has been found in a 218 THE ACUTE PNEUMONIAS few instances, but sometimes the pneumococcus is the causal agent ; and in tubercular meningitis the tubercle bacillus of course is present, especially in the nodules along the sheaths of the vessels. An invasion of the meninges by the anthrax bacillus occurs, but is a rare condition ; it is attended by diffuse haemorrhage in the subarachnoid space. In conclusion here it may be stated that mixed infections may occur in meningitis. Thus the pneumococcus has been found associated with the tubercle bacillus and also with the diplococcus intracellularis. Further, in infection with the latter, Gram-negative bacilli of a diphtheroid appearance have also been observed ; the significance of these is unknown. CHAPTER VIII. GONORRHCEA, SOFT SORE, SYPHILIS. GONORRHOEA. Introductory. — The micrococcus now known to be the cause of gonorrhoea, and now called the gonococcus, was first described by Neisser, who in 1879 gave an account of its microscopical characters as seen in the pus of gonorrhoeal affections, both of the urethra and of the conjunctiva. He considered that this organism was peculiar to the disease, and that its .characters were distinctive. 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 usually is seen 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. 74). When division takes place in the two members of a diplococcus, a tetrad is formed, which, however, soon separates into two sets of diplococci — that is to say, arrangement as diplococci is much commoner than as tetrads. Cocci in process of degeneration are seen as spherical elements of varying size, some being considerably swollen. 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 219 220 GONORRHOEA, SOFT SORE, SYPHILIS \ 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 leuco- 5B|^^ cytes appear to be filled with them, and their nuclei are obscured. As the dis- ease 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 gon- orrhoeal conjunctivitis, also in various parts of the female genital organs when FIG. 74.— Portion of film of gonorrhceal pus, tkege partg are t^e geat Qf showing the characteristic arrangement , i i • £ ,• of the gonococci within leucocytes true gonorrhoeal infection, Stained with fuchsin. x 1000. and they have been found in some cases in the second- ary infections of the joints in the disease, as will be described below. Staining. — The gonococcus stains readily and deeply with a watery solution of any of the basic aniline dyes — methylene-blue, fuchsin, etc. It is, however, easily decolorised, 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 40° 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 25° 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 CULTIVATION OF GONOCOCCUS 221 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 usu'ally 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 some- what 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 prac- tically pure condition, and if the meatus of the urethra be sterilised by washing with weak solution of cor- rosive sublimate and then with absolute alcohol, and the material for inoculation be expressed from the deeper part of the urethra, cultures may often be ob- tained which are pure from the first. By successive sub-cultures at short intervals, growth may be maintained indefinitely, and the organism gradually nourishes more luxuriantly. In culture the organisms have similar microscopic characters to those described (Fig. 75), but show a remarkable tendency to undergo degeneration, becoming swollen and of various sizes, and staining very irregularly. Degenerated forms are seen even on the second day, whilst in a culture four or five days old comparatively few normal cocci may be found. The less suitable the medium the more rapidly does degeneration take place. On ordinary agar and on glycerin agar growth does not take place, or is so slight that these media are quite unsuitable for FIG. 75. — Gonococci, from a pure culture on blood agar of twenty - four hours' growth. Some already are beginning to show the swollen appearance common in older cultures. Stained with carbol-thionin-blue. x 1000. 222 GONORRHOEA, SOFT SORE, SYPHILIS purposes of culture. The organism does not grow on gelatin,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 temperatuft of 40° C. These are then successively inoculated with the pus or other material in the same manner as gelatin tubes for ordinary plates (vide p. 52). To each tube is added an equal part of ordinary agar which has been thoroughly liquefied by heating and allowed to cool also to 40° 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 a"nd 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 invariably present in the urethral discharge in gonorrlwea, and also in other parts of the genital tract when these are the seat of true gonorrhoeal infection. Its presence in these different 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 gonorrhoeal pus itself. In fact, hitherto it has been found impossible to reproduce the disease by any means in the lower animals. On a considerable number of occasions inoculations of pure cultures have been made on the human urethra, both in the male and female, and the disease, with all its characteristic symptoms, has resulted. (Such experiments have been performed independently by Bumm, Steinschneider, Wertheim, and others.) The causal relationship of the organism to the disease has therefore been completely 1 Turro has announced that he has cultivated the gonococcus on acid gelatin, i.e. ordinary peptone gelatin 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. DISTRIBUTION OF GONOCOCCUS 223 established, and it is interesting to note how the conditions of growth and the pathogenic effects of the organism agree with the characters of the natural disease. Intraperitoneal injections of pure cultures of the gonococcus 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 subsides, 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. Toxin of the Gonococcus.— De Christmas 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 ; such a fluid constitutes the "toxin." The toxic substances are precipitated along with the proteids by alcohol, and the precipitate after being desiccated possesses the toxic action. In young rabbits injection of the toxin 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 toxin resulted after five successive injections at intervals. In a more recent publication he points out that the toxin on intracerebral injec- tion has marked effects ; he also claims to have produced an antitoxin. He states that the toxin diffuses out in the culture medium, and does not merely result from disintegration of the organisms. This has, how- ever, been called in question by other investigators. 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 with great increase of secretion. There occurs also a gradually increasing emigration of leucocytes, which take up a large number of the organisms. 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. Even, however, when the gonococci have disappeared from the urethral dis- charge, they may still be present in the deeper part of the 224 GONORRHCEA, SOFT SORE, SYPHILIS mucous membrane of .the urethra, possibly also in the prostate, and may thus be capable of producing infection. The prostatic secretion may sometimes be examinecLJby 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 considerable degree of inflammatory affection of the prostate and vesiculae seminales, but whether these conditions are always 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. Gonococci have, however, been obtained in pure culture from peri-urethral abscess and from epididymitis : it is likely that the latter condition, when occurring in gonorrhoea, is usually due to the actual presence of gonococci. During the more chronic stages other organisms may 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 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 invariably 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 gonorrhoeal vulvo- vaginitis of young subjects. They have also been found in suppurations in connection with Bartholini's glands, and some- times produce an inflammatory 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 mem- brane there. From the pus in cases of pyosalpinx they have been cultivated 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 DISTRIBUTION OF GONOCOCCUS 225 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 gonorrhceal 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 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. Relations to Joint Affections, etc. — The relations of the gono- coccus to the sequelae of gonorrhoea form a subject of great interest and importance, and the application of recent methods of examination shows that the organism is much more frequently present in such conditions than the earlier results indicated. The following statements may be made with regard to them. First, in a considerable 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 many others. A similar statement applies to inflammation of the sheaths of tendons following gonorrhoea. Secondly, in a large proportion of cases no organ- isms have been found. It is, however, possible that in a number of these the gonococci may have been present in the synovial membrane, as it has been observed that they may be much more numerous in that situation than in the fluid. Thirdly, in some cases, especially in those associated with extensive suppuration, occasionally of a pyaemic nature, various pyogenic cocci have been found to be present. In the instances in which the gono- coccus has been found in the joints, the fluid present has usually been described as being of a whitish-yellow tint, somewhat turbid, and containing shreds of fibrin-like material, sometimes purulent in appearance. In one case Bordoni-Uffreduzzi culti- vated the gonococcus from a joint-affection, and afterwards produced gonorrhoea in the human subject by inoculating with the cultures 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 gonorrhoeal endocarditis has been established by recent observations. Cases apparently of this nature occurring in the course of gonorrhoea had been previously described, but the complete bacteriological test has now been satisfied in several instances. In one case 15 226 GONORRHCEA, SOFT SOEE, SYPHILIS Lenhartz produced gonorrhoea in the human subject by inoculation with the organisms obtained from the vegetations. That a true gonorrhoeal septicaemia may also occur has also been established, cultures of the gonococcus having been obtained from the blood during life on more than one occasion (Thayer and Blumer, Thayer and Lazear, Ahmann). 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 methy- lene- or thionin-blue, as they do not overstain, and the films do not need to be decolorised. Staining 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 examina- tion 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 consider 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 examina- tion alone may give a definite positive result. When other organ- isms 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 examination, therefore, though often giving positive results, will sometimes be inconclusive. As regards lesions in other parts of the body microscopic examination alone is quite insufficient ; it is practi- cally impossible, for example, to distinguish by this means the gonococcus from the diplococcus intracellularis of meningitis. 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 227 SOFT SORE. The bacillus of soft sore was first described by Ducrey in 1889, who found it in the purulent discharge from the ulcerated surface; and later, in 1892, Unna 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. Microscopical Characters. — The organism occurs in the form of minute oval rods measuring about 1 '5 /x in length, and '5 //, in thickness (Fig. 76). 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 pur- ity amongst the leucocytic infiltration. In this posi- tion it is usually arranged in chains which may be of considerable length, and which are often seen FIG. 76. — Film preparation of pus from soft lying; in parallel rows be- chancre, showing Ducrey's bacillus, chiefly ,, mi arranged in pairs ; stained with carbol- tween the cells. Ine fuchsin and slightly decolorised, x 1500. bacilli chiefly occur in the free condition, but occasionally a few may be contained within leucocytes. There is no doubt that in many cases the organism is present in the buboes in a state of purity ; it has been found there by microscopic examination, and cultures have also been obtained from this source. The negative results of some observers are probably due to the organism having died off. On the whole the evidence goes to show that the ordinary bubo associated with soft sore is to be regarded as another lesion produced by Ducrey's bacillus. Sometimes the ordinary pyogenic organisms become superadded. This bacillus takes up the basic aniline stains fairly readily, 228 GONORRHCEA, SOFT SORE, SYPHILIS -'£» h*->&v but loses the colour very rapidly when a decolorising 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 Kiihne'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. 93) 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. Cultivation. — Although for a long period of time attempts to obtain cultures were unsuccessful, success has been attained within recent years. Benzangon, Griffon, and Le Sourd obtained pure cultures in four cases, the medium used being a mixture of rabbit's blood and agar, in the proportion of one part of the former to two of the latter. The blood is added to the agar in the melted condition at 45° C., and the tubes are then sloped. Davis con- firms these results, and finds that another good medium is freshly-drawn human blood distributed in small tubes; this method is specially suitable, as the blood inhibits the growth of various extraneous organisms. On the solid medium (blood- agar) the growth appears in the form of small round globules, wjiich attain their complete development in forty-eight hours, having then a diameter of 1 to 2 mm.; the colonies do not become confluent. Microscopic examination of these colonies, which are dissociated with some difficulty, shows similar appear- ances to those observed when the organism is in the tissues (Fig. 77), but occasionally long undivided filaments are observed which Davis regards as degenerative forms. Within a comparatively short period cultures undergo marked degenerative changes, and great irregularities of form and shape are to be founct It We are indebted to Dr. Davis for the use of Figs. 76 and 77. FIG. 77. — Ducrey's bacillus from a 24-hour culture in blood-bouillon. x 1500.1 SYPHILIS 229 would appear that a comparatively large amount of blood is necessary for the growth of this organism, and even sub-cultures on the ordinary media, including blood -serum media, give negative results. Inoculation of the ordinary laboratory animals is not attended by any result, but it has been found that some monkeys are susceptible, small ulcerations being produced by superficial inoculation, and in these the organism can be demon- strated. Tomasczewski cultivated the organism for several generations, and reproduced the disease by inoculation of the human subject. The causal relationship of this bacillus must therefore be considered as completely established, and the con- ditions under which it grows show it to be a strict parasite — a fact which is in conformity with the known facts as to the transmission of the disease. SYPHILIS. Up till quite recent times practically nothing of a definite nature was known regarding the etiology of syphilis. Most interest for a long time centred around the observations of Lustgarten, who in 1884 described a characteristic bacillus, both in the primary sore and in the lesions in internal organs. This organism occurred in the form of slender rods, straight, or slightly bent, 3 to 4 /x in length, often forming little clusters either within cells or lying free in the lymphatic spaces ; it took up basic aniline dyes with some difficulty, but was much more easily decolorised by acids than the tubercle bacillus. 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. Much controversy arose regarding the significance of this bacillus. Some considered it to be the tubercle bacillus, whilst others supposed that it was the smegma bacillus which had invaded the tissues. The etiological relationship of the organism to the disease was, however, not generally accepted, and in view of the recent work on syphilis, the organism cannot be regarded as having any pathological importance. Spirochsete pallida. — An entirely new light has been thrown on the etiology of the disease by the work of Schaudinn and Hoffmann which appeared in 1905. Since their first publication a great amount of work has been undertaken in order to test 230 GONORRHOEA, SOFT SORE, SYPHILIS their conclusions, and the general result may be said to be of a confirmatory nature. These observers found in certain cases an organism to which they gave the name spirochcete pallida ; it now also goes by the name spironema pallidum. As described by them, it is a minute spiral-shaped organism, showing usually from six to eight curves, though longer forms are met with ; the curves are small, comparatively sharp, and regular (Figs. 78, 79). It may be said to measure 4-14 fj. in length, while it is extremely thin, its thickness being only '25 p. In a fresh specimen, say a scraping from a chancre suspended in a little salt solution, the organism shows active movements, which are of three kinds — rotation about the long axis, gliding movements to and fro, FIGS. 78 and 79. — Film preparations from juice of hard chancre showing spirochaete pallida,— Giemsa's stain, x 1000. (From preparations by Dr. A. MacLennan.) and movements of flexion of the whole body. The ends are pointed and tapering. Its detection is comparatively difficult, as the organism is feebly refractile, and more difficult to see than most other organisms; the movement of small particles in the vicinity, however, is of assistance in finding it. In ulcerated syphilitic lesions other organisms are, of course, present, and not infrequently another spiral organism, to which the name spirochcete refringens has been given. This organism is usually somewhat longer, and is distinctly thicker than the spirochaete pallida. As the name implies, it is more highly refractile, and is much more easily detected than the latter organism ; its curves also are opener and much less regular, and they vary in their appearance during the movements. In stained films (see p. 107) the differences between the organisms come out more distinctly, as can be gathered from the accom- panying photograph (Fig. 81). The spirochaete pallida by the SYPHILIS 231 Giemsa stain is coloured somewhat faintly, and of reddish tint, whilst the regular spiral twistings are preserved ; the spirochsete refringens shows natter, wave-like bends, and, like other organ- isms, is stained of a bluish tint. By using Loffler's stain for the flagella of bacteria, Schaudinn was able to demonstrate a single deli- cate flagellum at each pole of the spirochsete pallida, while no undu- lating membrane could be detected ; on the other hand, several other species, including the spirochaete refringens, showed a distinct un- dulating membrane. Two flagella at one pole of the spirochaete pallida were also seen, an appear- FIG. 80. — Section of spleen from ance which Schaudinn thought a case of congenital syphilis, might represent the commencement showi^ .8ev*£l, examples of PI ., T i ^ - spirochsete pallida ; Levaditi s of longitudinal fission. mJetho(L x 1000. The number of publications with regard to the distribution of the spirochsete pallida is already very large, and a summary of the results may be given. In the primary sore and in the related lymphatic glands, the juice of which can be conveniently obtained by means of a hypodermic syringe, the organism has been found in a very large majority of cases. It has been also obtained in the papular and roseolar erup- tions, in condylomata and mucous patches — in fact, one may say generally, in all the primary and secondary lesions. It has been ob- tained from the spleen during life, and on a few occasions, e.g. by Schaudinn. also from the blood during life in secondary syphilis. In the congenital form of the disease FIG. 81. — Spirochsete refringens ,, , , • in film preparation from a case the organism may be present in of balanitis. x 1000. large numbers, as was first shown by Buschke and Fischer, and by Levaditi. In the pemphigoid bullse, in the blood, in the in- ternal organs, the liver, lungs, spleen, supra-renals, and even in the heart its detection may be comparatively easy, owing to the large numbers present (Fig. 80). It can readily be demonstrated 232 GONORRHCEA, SOFT SORE, SYPHILIS in sections of the organs by the method described on p. 104. In such preparations large numbers of spirochaetes, chiefly extra- vascular in position, can be seen, and many may occur in the interior of the more highly specialised cells, for example, liver- cells ; in many cases examination has been made within so short a period after the death of the child as to practically exclude the possibility of contamination from without. It also abounds sometimes on mucous surfaces, e.g. of the bladder and intestine in cases of congenital syphilis. Shortly after the discovery of the organism, Metchnikoff was able to detect it in the lesions produced in monkeys by inoculation with material derived from syphilitic sores, and his observations have since been confirmed. Although various organisms may be associated with it in the lesions of the skin or mucous membranes, there is a comparative agreement amongst observers that this organism occurs alone in syphilitic lesions where the entrance of bacteria, etc., from outside is excluded. The high percentage of cases in which it is found would, in view of the difficulty in detecting it, almost point to its invariable presence, and, as a matter of fact, Schaudinn in his last series of cases, numbering over seventy, found it in all. Jn gummata and other tertiary lesions, however, the spirochaete Vhas rarely, if ever, been detected, and it is probable, as Schaudinn /suggests, that it has passed into some resting condition which 'has not yet been found. Another question of considerable importance is, as to whether this organism has. been found in other conditions. Observations show that in various conditions, such as ulcerated carcinomata, balanitis, etc., spirochaetes are of comparatively common occurrence. There is no doubt what- ever that the great majority of these are readily distinguishable by their appearance from the spirochaete pallida, but others resemble it closely. Hoffmann, however, who has seen many of these spirochaetes from other sources, considers that even by their microscopic appearance they are capable of being dis- tinguished, though with considerable difficulty. It must, of course, be borne in mind that the finding of an organism in non-syphilitic lesions with exactly the same microscopical char- acters does not show that it is the same organism as the spiro- chaete pallida. It cannot be claimed that the pathological relation of this organism to the disease is absolutely demon- strated ; but the facts stated are sufficient to form very strong presumptive evidence that in the spirochaete pallida we have the true cause of syphilis. Transmission of the Disease to Animals. — Although various experiments had previously been from time to time SYPHILIS 233 made by different observers, in some cases with reported successful result, it is to the papers of Metchnikoff and Roux (1903-5) that we owe most of our knowledge. These observers have carried on a large series of observations, and have shown that the disease can be transmitted to various species of monkeys. Of these the anthropoid apes are most susceptible, the chimpanzee being the most suitable for experimental purposes. Their results have been confirmed by Lassar, Neisser, Kraus, and others. Inoculations made by scarification resulted in the production of typical primary lesions in all of more than twenty animals used. The primary lesion is in the form of an indurated papule or of papules, in every respect resembling the human lesion. Along with this there is a marked enlargement and induration of the corresponding lymphatic glands. The primary lesion appeared on an average about thirty days after inocula- tion, and secondary symptoms appeared in rather more than half of the cases after a further period of rather longer duration. These were of the nature of squamous papules on the skin, mucous patches in the mouth, and sometimes palmar psoriasis. As a rule, the secondary manifestations were of a somewhat mild degree, and in no instance up to the present has any tertiary lesion been observed. By inoculation from the secondary lesions, the primary manifestations with their typical characters have been reproduced. The orang-outang has been found to be less susceptible, whilst Roux's experiments on the gorilla have been too few to admit of any conclusion. The disease may also be produced in baboons and macaques (macacus sinicus is one of the most susceptible), but these animals are less susceptible. In the case of many of them no result follows, and when a lesion is produced it is only of the nature of a primary papule, secondary manifestations never appearing. There is thus no doubt that the disease may be produced in apes, and, to speak generally, the severity of the affection increases according to the nearness of the relationship of the animal to the human subject. The production of the disease, experimentally, has supplied us with some further facts regarding the nature of the virus. It has been shown repeatedly that the passage of fluid con- taining the virus through a Berkefeld filter deprives it completely of its infectivity. In other words, the virus does not belong to the ultra-microscopic group of organisms. The virus is also readily destroyed by heat, a temperature of 51° C. being fatal. With regard to the production of immunity, very little of a satisfactory nature has so far been established. It has been 234 GONORRHCEA, SOFT SORE, SYPHILIS found that the virus from a macaque monkey produces a less severe disease in the chimpanzee than the virus from the human subject, inasmuch as secondary lesions do not follow ; the virus would thus appear to have undergone a certain amount of attenuation in the tissues of that monkey. Recently corneal ulcers in rabbits have been produced by Bertarelli and by Hoff- mann by inoculation with syphilitic material ; they appear after a long period of incubation, and the spirochaBte can be demon- strated in the lesions. The effects of injecting emulsions of tertiary lesions or of serum from syphilitic patients, at the time of inoculation with the virus, appear to be practically nil ; so also the employment of the virus rendered inactive by heating has apparently no influence in acting as a vaccine. There is some evidence that the serum from a patient suffering from the disease when mixed with the virus before inoculation modifies the disease to a certain extent, but further evidence on this point is necessary. As mentioned above, the spirochsete pallida has been found in the lesions in monkeys, Metchnikoff and Roux obtaining positive results in more than 75 per cent of the cases, and it is to be noted that here also the organism has been found deep in the substance of the papules, unaccompanied by any other organisms. Hoffmann failed to find any spirochsetes in monkeys which had not been inoculated with syphilitic material. This observer produced a lesion on the upper eyelid of a macacus by inoculation with the blood of a man who had suffered from the disease for six months, and a papule appeared which contained spirochaetes. This result is in conformity with that given by microscopic examination, and shows that the organism is sometimes present in the circulating blood in severe cases of the disease, and that the blood is accordingly infective. Castellani has described in yaws or frambcesia the occurrence of a spirochsete closely resembling the spirochsete pallida in appearance, and to this organism he has given the name spiro- chcete pertenuis. He has found it not only in the skin lesions but also in the spleen and lymphatic glands of patients suffering from the disease. He has produced the disease in monkeys by direct inoculation and has found the spirochsete in the resulting lesions. He finds that the immunity reactions of the two organ- isms— spirochaete pallida and spirochsete pertenuis — are quite distinct ; hence we have probably to deal with two distinct species. 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 important 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 infection. A definite answer has in this way been supplied to many questions which were previously the subject of endless discussion. Historical. — By the work of Armanni and of Cohnheim and Salomonsen (1870-80) it had been demonstrated that tubercle was an infective disease. The latter observers found on inoculation of the anterior chamber of the eye of rabbits with tubercular material 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 disorgan- isation of the globe of the eye. Later still, 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 in 1884 (Mitth. a. d. K. Gsndhtsamte., Berlin). Koch's work on this subject will remain as a classical masterpiece 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, 235 236 TUBERCULOSIS 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 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 im- portance in relation to the infection of the human subject. Amongst the domestic animals the disease is commonest in cattle (bovine tuberculosis), in which animals the lesions are very various, both in character and distribution. In most cases the lungs are affected, and contain numerous rounded nodules, many being of considerable 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 pneumonia, 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 pedun- culated, the condition being known in Germany as Perlsucht, in France as pommeliere. Lesions similar to the last may be chiefly confined to the peritoneum and pleurae. In other cases, again, the abdominal organs are principally involved. The udder becomes affected in a certain pro- portion of cases of tuberculosis in cows — in 3 per cent according to Bang — but primary affection of this gland is very rare. Tuberculosis is also a comparatively common disease in pigs, in which animals it in many cases affects the abdominal organs, in other cases produces a sort of caseous pneumonia, and sometimes 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 seat 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 of rare occurrence, especially in the former animals. It may occur spontaneously 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. THE TUBERCLE BACILLUS 237 Tuberculosis in fowls (avian tuberculosis) is a common and very infectious disease, nearly all the birds in a poultry-yard being sometimes affected. 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 relation of the different forms of tuberculosis is discussed below. Tubercle Bacillus — Microscopical Characters. — Tubercle bacilli are minute rods which usually measure 2 '5 to 3 -5 ^ in length, and '3 /A in thickness, i.e. in proportion to their length they are comparatively thin organisms (Figs. 82 and 83). Some- times, however, longer forms, up to 5 /A or more in length, are met with, both in cultures and in 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 uni- formly coloured, or may present small uncoloured spots along their course, with darkly -stained parts between. In such a min- ute organism it is ex- tremely difficult to deter^ FJG 82._Tubercle bacilli, from a pure mine the exact natures. of culture on glycerin agar. the unstained points. Ac- Stained with carbol-fuchsin. x 1000. cordingly, we find that some observers consider these to be spores, while others find that it is impossible to stain them by any means whatever, and con- sider 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 condensed and highly-stained particles are spores. It is impossible to speak definitely 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 ; this latter condition is, however, not associated with greater powers of resistance. 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 238 TUBERCULOSIS 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 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 larger elements V FIG. 83. — Tubercle bacilli in phthisical sputum ; they are longer than is often the case. Film preparation, stained with carbol-fuchsin and rnethylene-blue. x 1000. may occur. These may be in the form of long filaments, some- times swollen or clubbed at their extremities, may be irregularly beaded, and may even show the appearance of branching. Such forms have been studied by Metclmikoff, Maffucci, Klein, and others. Their significance has been variously interpreted, for while some look upon them as degenerated or involution forms, others regard them as indicating a special phase in the life history of the organism, allying it with the higher bacteria. Recent observations, however, go to establish the latter view, and this is now generally accepted by authorities. It has also CULTIVATION OF TUBERCLE BACILLUS 239 been found that under certain circumstances tubercle bacilli in the tissues produce a radiating structure closely similar to that of the actinomyces. This was found by Babes and also by Lubarsch to be the case when the bacilli were injected under the dura mater and directly into certain solid organs, such as the kidneys in the rabbit. Club-like structures may be present at the periphery ; these are usually not acid-fast, but they retain the stain in the Weigert-Gram method. Similar results obtained with other acid-fast bacilli will be mentioned below, and these organisms would appear to form a group closely allied to the streptothriceae, the bacillary parasitic form being one stage of the life history of the organism. This group is often spoken of as the mycobacteria. Staining Reactions. — The tubercle bacillus takes up the ordinary stains very slowly and 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. One of the best and most convenient methods is the Ziehl-Neelsen method (see p. 100). 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. Within recent years certain other bacilli have been discovered which present the same staining reactions as tubercle bacilli ; they are therefore called " acid-fast " (vide infra). The spores of many bacilli become decolorised more readily than tubercle bacilli, though some retain the colour with equal tenacity. Bulloch and Macleod, by treating tubercle bacilli with hot alcohol and ether, extracted a wax which gave the characteristic staining reactions of the bacilli themselves. The remains of the bacilli, further, when extracted witli caustic potash, yielded a body which was probably a chitin, and which was acid-fast when stained for twenty-four hours with carbol- fuchsin. Cultivation. — The medium first used by Koch was inspissated blood serum (vide p. 39). If inoculations are made on this medium with tubercular material free from other organisms, 240 TUBERCULOSIS there appear in from ten to fourteen days minute points of growth of dull whitish colour, rather irregular, and slightly raised above the surface (it is advisable to plant on the medium an actual piece of the tubercular tissue and to fix it in a wound of the surface of the serum). Koch compared the appearance of these to that of small dry scales. In such cultures the growths usually reach only a compara- tively small size and re- main separate, becoming confluent only when many occur close together. In sub - cultures, 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 surf ace of the serum and at the bottom of the tube may grow over the surface of the condensa- tion water on to the glass (Fig. 84, A). The growth is always of a dull ap- pearance and has a con- siderable degree of con- sistence, so that it is diffi- cult to dissociate a portion thoroughly in a drop of water. In older cultures th° %™^ may acquire a slightly brownish or buff colour. When the Small colonies are examined •, ,. , , Under a lOW power OI 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 glycerin 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 pro- FIG. 84.-Cultures of tubercle bacilli on glycerin 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. POWERS OF RESISTANCE 241 gresses more rapidly than on serum, but 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. In glycerin 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 super- ficially 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 tuberculin (vide infra}. The culture has a peculiar fruity and not unpleasant odour. On ordinary agar and on gelatin media no growth takes place. It Avas 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 glycerin is added. He also found that cultures from tubercular lesions could be obtained on glycerin potato (p. 46). The optimum temperature for growth is 37° to 38° C. Growth ceases above 42° and usually below 28°, but on long- continued cultivation outside the body and in special circum- stances, growth may take place at a lower temperature, e.g. Sander found that growth took place in glycerin-potato broth even at 22° to 23° C. Powers of Resistance. — Tubercle bacilli have considerable powers of resistance to external influences, and can retain their vitality for a long time outside the body in various conditions ; in fact, in this respect they may be said to occupy an inter- mediate position between spores and spore-free 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 - 3° C. for three hours, even when this is repeated several times. It has been found that 16 242 TUBERCULOSIS when completely dried they can resist a temperature of 100° C. for an hour, but, on the other hand, exposure in the moist condition to 70° 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 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, the structure of which varies in different situations and according to the intensity of the action of the bacilli. 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 — the so-called epithelioid cells. These proliferative changes may be well seen on the fifth day after inoculation 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 epithelioid cells become swollen 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 quickly, then giant-cell formation may occur in the centre of the follicle, this constituting one of the characteristic features of the tuber- cular lesion ; or after the occurrence of caseation giant-cells may be formed in the cellular tissue around. 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 discussion 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 pro- liferation without the protoplasm dividing. These epithelioid cells may sometimes be the lining cells of capillaries. Some con- sider that the giant-cells result from a fusion of the epithelioid ACTION ON THE TISSUES 243 cells ; but, though there are occasionally appearances which indicate 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 may be seen round a small central thrombus. Such an occurrence gives rise to an appearance closely resembling a typical giant -cell. 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. There can be no doubt that the cell necrosis and subsequent 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, perspiration, wasting, etc., are to be ascribed to the absorption and distribution throughout the system of the toxic products of the bacilli ; in the case of phthisical cavities and like conditions where other bacteria are present, the toxins of the latter also play an im- portant part. 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 tuber- cular lesions. 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 these are tubercle nodules with much connective 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 244 TUBERCULOSIS not, the important fact has been established that tubercular material in which no bacilli can be found microscopically, may be proved, on experimental 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. FIG. 85. — Tubercle bacilli in section of human lung in acute phthisis. The bacilli are seen lying singly, and also in large masses to left of field. The pale background is formed by caseous material. Stained with carbol-fuchsin and Bismarck -brown, x 1000. They are most numerous in acute lesions, especially where caseation is rapidly spreading, for example, in such conditions as caseous catarrhal pneumonia (Fig. 85), 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, but retains its form and staining power for a much ACTION ON THE TISSUES 245 longer period than most organisms. As a rule the bacilli are extra-cellular in position. Occasionally they occur within the giant-cells, in which they may be arranged in a somewhat radiate manner at the periphery, occasionally also in epithelioid cells and in leucocytes. The above statements, however, apply only to tuberculosis in the human subject, and even in this case there are exceptions. » FIG. 86. — Tubercle bacilli in giant-cells, showing the radiate arrangement at the periphery of the cells. Section of tubercular udder of cow. Stained with carbol-fuchsin and Bismarck-brown. x 1000. In the ox, on the other hand, the presence of tubercle bacilli within giant-cells is a very common occurrence ; and it is also common to find them in considerable numbers scattered irregularly throughout the cellular connective tissue of the lesions, even when there is little or no caseation present (Fig. 86). 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 both in their number and in their site is met with in tuberculosis of other animals. 246 TUBERCULOSIS 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 demonstrated almost invariably at some period, and sometimes their numbers are very large (for method of staining see p. 101). 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 usually present in the && ^ urine; but as they are |^ much diluted it is difficult to find them unless a de- posit is obtained by means of the centrifuge. This deposit is examined in the same way as the sputum. The bacilli often occur in little clumps, as shown in Fig. 87. In tubercular ulceration of the intestine their presence in the faeces may be demonstrated^ as FIG. 87. — Tubercle bacilli in urine ; showing was first shown by Koch '} one of the characteristic clumps, in which ^ut jn t^g cage fa^ s Timotll s bacillus L 20 per cent sulphuric acid. x 1000. J6)' The Petri-Rabinowitch butter bacillus. (c) Bacillus of fish tuberculosis. bacillus I. but are less marked. Moeller also obtained a similar organism from milk. He also discovered a third acid-fast bacillus which he obtained from manure and therefore called the " Mistbacillus " (dung bacillus). This organism has analogous characters, though presenting minor differences. It also has pathogenic effects. Petri and Rabinowitch independently cultivated an acid- fast bacillus from butter ("butter bacillus") in which it occurs with comparative frequency. The organism resembles the tubercle bacillus, although it is on the whole shorter and thicker. Its lesions closely resemble tuber- culosis, especially when injection of the organism is made into the peritoneal cavity of guinea-pigs, along with butter, — the method usually adopted in searching for tubercle bacilli in butter. This organism produces pretty rapidly a wrinkled growth (Fig. 89, b) not unlike that of Moeller's grass bacillus II. Korn has also obtained other two bacilli from butter which he holds to be distinct from one another and 254 TUBERCULOSIS \ t V from Rabinowitch's bacillus. The points of distinction are of a minor character. Other more or less similar bacilli have been cultivated by Tobler, Coggi, and others.1 Another bacillus of considerable interest is Johne's bacillus or the bacillus of "chronic bovine pseudo-tuberculous enteritis," the lesions produced by it being corrugated thickenings of the mucous membrane, especially of the small intestine. The disease has now been observed in various countries, and several cases in Britain have been recorded by M'Fadyean. The bacilli occur in large numbers in the lesions, and can readily be found in scrapings from the surface. They resemble the tubercle bacillus in appear- ance, but on the whole are rather shorter ; they are equally acid-fast. The organ- ism has not yet been culti- vated outside the body. Smegma Bacillus. —This organism is of importance, as in form and staining re- action it somewhat resembles the tubercle bacillus and may be mistaken for it. It occurs often in large numbers in the smegma prseputiale and in the region of the external genitals, especially where there is an accumulation of fatty matter from the secre- tions. Morphologically it is a slender slightly curved organism, like the tubercle bacillus but usually dis- tinctly shorter (Fig. 90). Like the tubercle bacillus it stains with some difficulty and resists decolorisation with strong mineral acids. Most observers ascribe the latter fact to the fatty matter with which it is surrounded, and find that if the specimen is treated with alcohol the organism is easily decolorised. Czaplewski, however, who claims to have cultivated it on various media, finds that in culture it shows resistance to decolor- isation both with alcohol and with acids, and considers, therefore, that the reaction is not due to 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 readily distinguished from the tubercle bacillus. We, moreover, believe that minor points of difference in the microscopic appearances of the two organisms are quite sufficient to make the experienced observer suspicious if he should meet with the smegma bacillus in urine, and lead him to apply the decolorising test. Difficulty will only occur when a few scattered bacilli retaining the fuchsin occur. Its cultivation, which is attended with some difficulty, was first FIG. 90. — Smegma bacilli. Film preparation of smegma. Ziehl-Neelsen stain. x 1000. 1 For further details on this subject, vide Potet, Etudes sur les bacilles dites acidophiles. Paris, 1902. ACTION OF DEAD TUBERCLE BACILLI 255 effected by Czaplewski. 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 glycerin agar and in bouillon. It is non-pathogenic to various animals which have been tested. Cowie has recently found that acid-fast bacilli are of common occur- rence in the secretions of the external genitals, mammse, etc., in certain of the lower animals, and that these organisms vary in appearance. He considers that the term "smegma bacillus" probably represents a number of allied species. The question may be asked — do these results modify the validity of the staining reaction of tubercle bacilli as a means of diagnosis'? The source of any acid-fast bacilli in question is manifestly of importance, and it may be stated that when these have been obtained from some source outside the body, or where contamination from without has been possible, their recognition as tubercle bacilli cannot be established by microscopic examina- tion alone. In the case of material coming from the interior of the body, however, — sputum, etc., — the condition must be looked on as different, and although an acid-fast bacillus (not tubercle) has been found by Rabinowitch in a case of pulmonary gangrene we have no sufficient data for saying that acid-fast bacilli other than the tubercle bacillus nourish within the tissues of the human body except in such rare instances as to be practically negligible. (To this statement the case of the leprosy bacillus is of course an exception.) Accordingly, up till now, the microscopic ex- amination of sputum, etc., cannot be said to have its validity shaken, and we have the results of enormous clinical experience that such examination is practically of unvarying value. Never- theless the facts established with regard to other acid-fast bacilli must be kept carefully in view, and great care must be exercised when only one or two bacilli are found, especially if they deviate in their morphological characters from the tubercle bacillus. 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, but no caseation, were occasionally present, and which were characterised by more growth of fibrous tissue than in ordinary tubercle. The subject was very fully investigated with confirmatory results by Straus and Gamaleia, who found that, if the number of bacilli introduced into the circulation were large, there resulted very numerous tubercle nodules with well- 256 TUBERCULOSIS formed giant -cells, and occasionally traces of caseation. The bacilli can be well recognised in the nodules by the ordinary staining method. In these experiments the bacilli were killed by exposure to a temperature of 115° C. for ten minutes before being injected. Similar nodules can be produced by intra- peritoneal 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 experiments, 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. S. Stockman has found that an animal inoculated with large numbers of dead tubercle bacilli afterwards gives the tuberculin reaction. 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 experiments 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 ventilating 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 ACTION OF DEAD TUBERCLE BACILLI 257 inoculating them with dust collected from the walls of a con- sumptive ward. Tubercle bacilli are also discharged in consider- able 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 phthisical 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 should only expectorate on to pieces of rag which are afterwards to be burnt, or into special receptacles which are to be then sterilised either by boiling or by the addition of a 5 per cent solution of carbolic acid. Another great source of infection is in all probability 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 subjects. 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 infection, 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, in the first place, tuberculosis 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 tubercular 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 inhaled tubercle bacilli may also be swallowed and contamination of food by tubercular material from the human subject may occur. Alike when inhaled and when ingested, tubercle bacilli may lodge about the pharynx and thus come to 17 258 TUBERCULOSIS 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. The Toxins of the Tubercle Bacillus. — Two outstanding features of the action of the tubercle bacillus are the occurrence of necrosis in the cells of tubercle nodules and the production of general disturbances of metabolism accompanied by fever. It is natural to refer these phenomena to the effects of toxins formed by the organism. The study of such toxins centres round the substance known as tuberculin which Koch brought forward in 1890-1 as a curative agent for tubercular affections. Koch's Tuberculin. — Koch stated that if in a guinea-pig suffering from the effects of a subcutaneous inoculation with tubercle bacilli, a second subcutaneous inoculation of tubercle bacilli 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 tuberculosis. This reaction was further studied by means of tuberculin, which consisted of a concentrated glycerin bouillon culture of tubercle in which the bacilli had been killed by heat. Its essential components probably were the dead and often macerated bacilli and the substances indestructible by boiling which existed in these bacilli, or which were formed during their growth. The injection of '25 c.c. of tuberculin into a healthy man causes, in from three to four hours, malaise, tendency 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), however, of '01 c.c. into a tubercular person gives rise to similar symptoms, but in a much more aggravated form, and in addition there occurs around any tubercular focus great inflammatory reaction, resulting in necrosis and a casting off of the tubercular mass, when this is possible, as for instance in the case of lupus. The bacilli are, it was shown, not killed in the process. Koch's theory of the action of the 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, inflammatory reaction takes place around, and necrosis of the spreading margin occurs very rapidly, the material containing the living or dead bacilli being thrown off en masse instead of being disintegrated piecemeal. It appears, however, that this explanation may not be the true one ; for, on the one hand, other substances besides products of the tubercle bacillus may TOXINS OF THE TUBERCLE BACILLUS 259 give rise to similar effects in tubercular animals, and, on the other, a similar reaction can take place in other diseases where there is locally in the body a deposit of new tissue. Matthes has, for instance, found that albumoses and peptones isolated from the ordinary peptic 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 toxins of the vibrio Metchnikovi ; and later Metchnikoff found that a similar susceptibility existed towards the toxins of the bacillus of fowl cholera. How complicated the tuberculin reaction is is shown by the fact that a similar reaction has taken place when tuberculin has been injected into persons suffering from diseases other than tubercle, e.g. cancer, sarcoma, syphilis. The hopes which the introduction of tuberculin raised, that a curative agent against tuberculosis had been discovered, were soon found not to be justified. It was very difficult to see how the necrosed material which was produced and which contained the still living bacilli, could be got rid 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, arid in a few months the treatment was practically abandoned. 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 : — Tiie animals are kept twenty-four hours in their stalls, and the temperature is taken every three hours, from four hours before the injection till twenty-four after. The average tempera- ture in cattle is 102 '2° F. ; 30 to 40 centigrammes of tuberculin are injected, and if the animal be tubercular the temperature rises 2° or 3° 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 104° 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 has been largely practised in all parts of the world, and is of great value. While it is undoubted that tuberculin contains toxic products formed by the bacilli, we know nothing of the nature of the toxins present. From the fact that filtered cultures cause little 260 TUBERCULOSIS toxic effect, and that trituration of the bacilli increases the poisonous content of a culture, it is inferred that we have to deal with endotoxins, but beyond this statement we cannot go. Hitherto no success has attended attempts to gain a closer knowledge of the nature of such substances. It has been stated that albumoses of a special kind are present in tuberculin, but nothing definite has emerged from the investigation of these bodies. Active Immunisation against the Tubercle Bacillus. — Koch's Tuber culin-R. Our knowledge here centres round the substance introduced by Koch in 1897 under the name of " Tuberculin-R," or the new tuberculin. Koch's new researches consisted (1) of an attempt to immunise animals against the tubercle bacillus by employing its intracellular toxins ; (2) of trying to utilise such an immunisation to aid the tissues of an animal already attacked with tubercle the better to combat the effects of the bacilli. The method of obtaining the intracellular toxins was as follows. Bacilli from young virulent cultures were dried in vacuo, and disintegrated in an agate mill, treated with distilled water and centrifugalised. The clear fluid was decanted,, and is called by Koch " Tuberculin-O." The remaining deposit was again dried, ground, treated with w^ater and centrifugalised, the clear fluid being again decanted, and this process was repeated with successive residues till no residue remained. These fluids put together constitute the " Tuberculin-R." From the fact that tuberculin-O gave no cloudiness when glycerin was added, Koch concluded that it contained the sub- stances present in the glycerin-bouillon extracts originally used by him, and he held this was borne out by the readiness with which a tuberculin reaction could be caused by it. Similarly, as tuberculin-R gave a cloudiness with glycerin and did not readily originate a reaction, he considered that it contained different products of the bacillus. When injected into animals in repeated and increasing doses, TJy- mgrm. being the initial dose, tuberculin-R is said to produce immunity against the original extract, against tuberculin-O, and against living and virulent tubercle bacilli. Another preparation has also been introduced known as " Koch's new tuberculin " (Baiillenemulsion). This is an emulsion of ground tubercle bacilli in water containing 50 per cent of glycerin ; it thus really contains both tuberculin-O and tuberculin-R. Both, especially tuberculin-R, have been used for the treatment of tuberculosis in man, especially for early localised lesions. In the case of both substances commencing with from to yj^ mgrm, gradually increasing doses were given every OPSONINS IN TUBERCULOSIS 261 second day, and the rule originally laid down for the regulation of the dosage was that no amount should be given which raised the temperature more than *5° F. Very various opinions have been expressed as to the efficacy of such treatment. There is little doubt that in certain cases of lojcal conditions, such as lupus, tubercular joints, glands and genito-urinary tuberculosis, improve- ment has followed its application ; but where febrile conditions indicate that general disturbances are in existence, there is little or no justification for its being applied, and even in many local conditions the absence of benefit is so marked that by many physicians the method has been abandoned. Active Immunisation associated with Opsonic Observations. — Within recent years attention has been directed to the possibility of controlling the use of tuberculin-R by observations of its effect on the opsonic qualities of the serum. Wright, early in his work, showed that tubercle bacilli when sensitised by an appropriate serum, were readily phagocyted by the polymorpho- nucleate leucocytes, and the relative sensitising capacities of serum from tubercular and non-tubercular cases has been widely studied. According to Wright, in strictly localised tuberculosis the opsonic index is persistently low, varying from '1 to '9, while in tubercle with general disturbances it fluctuates greatly from day to day, being sometimes below, sometimes above unity. To take the former and simpler case, he holds that if the treat- ment with injections of tuberculin-R be controlled by noting the effect produced on the opsonic index, great improvement in the patient's condition may result. Wright's interpretation of what occurs is bound up with his views on the nature of the effects produced. These views are briefly as follows. For reasons unknown the opsonic qualities of the body fluids may become abnormally low, and the tubercle bacilli, if they gain admission to the body, can multiply locally. This multiplication is associated with a still further local diminution of the opsonins. By the introduction of such a substance as Koch's tuberculin-R the bodily mechanism, whatever it is, which produces the opsonins is stimulated, and a rise in the general opsonic index occurs. Naturally this is accompanied by a passing to the site of infec- tion of fluids more rich in opsonins than previously, the activity of the phagocytes comes into play and the tubercle bacilli are destroyed. But any such vaccination process must be controlled by constant observations of the opsonic index, and it is only by this means that not only good results can be obtained, but that the production of harmful effects can be prevented. The reason of this is that in a great many cases the injection of a bacterial 262 TUBEECULOSIS vaccine is followed by a decrease in the opsonic qualities of the serum, — the occurrence of a negative phase. J)uring such a period of depression there is probably an increased susceptibility to the action of the bacilli. Now, in order to get permanent benefit from the vaccination process, repeated injections of the tuberculin must be practised, and if an injection be given during a negative phase, actual harm may be done. The course of a successful vaccination is that, after the passing off of the negative phase, the opsonic index should rise to above its original level, — the occurrence of a positive phase. It is when this positive phase is fully developed that a fresh inoculation can be practised with success. The new negative phase which will now occur may not cause a drop to below the level of the original state of the serum, and the hope is that its succeeding positive phase will carry the opsonic index still higher and ensure a still greater resistance to the bacterium. The importance of the observations of the opsonic index lies in this that in antibacterial vaccina- tions the degree of active immunisation which can be attained is always much less than is the case with immunisation against such a substance as the diphtheria toxin, although in the latter there also occur negative and positive phases of a precisely similar character. If an injection be practised during a negative phase, then a still further drop in the opsonic content of the serum will occur and a fresh growth of the invading bacilli is likely. There are very great variations in the capacities shown by tubercular patients to react to a vaccination process. In certain cases good positive phases are readily and quickly produced, while in others after an inoculation the negative phase is long con- tinued and may even show no tendency to pass into a positive phase. The irregularities in the opsonic index in cases where there is a general disturbance of metabolism Wright explains by supposing that they result from very irregular auto-infections of the patient's body by tubercular products from the local lesions, — positive and negative phases being produced without the pur- posive quality which ought to characterise a successful therapeutic vaccination. Such auto-infections may come about in various ways, and Wright is of opinion that exercise, for instance, may disseminate both tubercular products and tubercle bacilli, — he having noticed in tubercle patients a fall in the opsonic index after muscular exertion. With regard to the details of the immunisation, Wright's chief point is that the repeated, uncontrolled injections of tuber- culin such as were originally given may very likely have a harmful result, and that when an injection is practised it is not necessary OPSONINS IN TUBERCULOSIS 263 for constitutional effects to occur in order that a beneficial result may follow. Hence much smaller doses of tuberculin than hitherto are given by him. For ordinary cases with low opsonic index and no evidence of constitutional disturbance, an amount of tuberculin corresponding to from one-thousandth to a six- hundredth of a millegramme of tubercle powder is a sufficient dose, and if any dose seems to produce a pronounced negative phase then a smaller dose ought to be tried at the next inocula- tion. For cases clinically tubercular where the index is about normal, then smaller doses, say, the equivalent of a two- thousandth of a millegramme or less ought to be used, — the effect on the index being carefully watched. In any case, the dose which is found to give the highest positive phase is the optimum dose and one which need not necessarily be increased. Cases where there is constitutional disturbance should be as a rule left untreated. The general position of Wright and his school is, that it is only by the observation of the opsonic index that the application of the tuberculin treatment can be effectually controlled, — deductions based on clinical data, such as absence of interference with pulse rate, temperature, etc., or increase of body weight after an inoculation being unreliable, and further evidence of the unreliability of such tests is brought forward in the fact that, in cases of apparent benefit from sanatorium treatment, the opsonic index may still be very low. With regard to the results obtained, many cases have been brought forward by Wright and others where benefit has followed the putting into practice of the principles enunciated, and there is little doubt that the work done has given a fresh start to the active immunisation method in the treatment of tuberculosis. An outstanding event of Wright's work in this field has been his insistance on the good effects produced by extremely small doses of tuberculin (down to the four-thousandth of a millegramme) given at fairly long inter- vals (say 10 days or more). With regard to the efficacy of the opsonic method as affording an index to the progress of a case it must be recognised that the method is still on its trial, and it is doubtful if even in the work of the most careful observers the limits of the experimental error of the opsonic method have been sufficiently denned. The whole question of immunisation against the tubercle bacillus presents many difficulties, and it is the merit of Wright's work that it has shed fresh light on some of these. One great difficulty arises from the great chronicity of the results of the infection in the majority of human cases. It is probably 264 TUBERCULOSIS true not only of man but of many species of animals used in experimental inquiries, that many individuals are on the border- line between immunity and susceptibility. From the wide spread of the bacilli in centres of human population, it is certain that the opportunity for infection arises in a very large propor- tion of the race ; in many cases no results follow infection, and in many others small lesions occur which do not develop further ; this has actually been shown by morbid anatomists to be the case. The disease being thus so often characterised by transient local effects without constitutional disturbance, the course of an immunisation may be expected to be 'rather different from that obtaining in an ordinary acute affection, though the underlying processes may be of the same nature. It is difficult, for instance, on account of the slowness of tubercular processes, to define recovery from an attack of the disease, or to speak of an animal recovering from the effect of an inoculation during an immunisa- tion. It follows that little is known regarding an attenuation of the tubercle bacillus analogous to what is an important feature in immunisations against other organisms. It has been thought by some that 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 injec- tion of bacilli from avian tuberculosis could protect the dog against bacilli derived from man. But these are not yet conclusive. Agglutinative Phenomena. — The serum of tubercular patients has been found to exert an agglutinating action on the tubercle bacillus. A convenient 'method is to add different amounts of serum, commencing with, say, '1 c.c., to quantities of a dilution of the new tuberculin (Bazillenemulsion) equivalent to 1 part of the bacterial bodies to 10,000 of diluent, and leave the mixture for 24 hours before observing. As with other agglutina- tive observations, it is difficult to correlate the degree of agglu- tinating power of the serum with the degree of immunisation possessed by the individual from which it was derived. Antitubercular Sera. — Several attempts have been made to treat tuberculosis with the serum of animals immunised by the tubercle bacillus or its products. The most successful is perhaps that of Maragliano. This author distinguishes between the toxic materials contained in the bodies of the bacilli (which withstand, unchanged, a temperature of 100° C.) and those secreted into the culture fluid (which are destroyed by heat). METHODS OF EXAMINATION 265 The substance used by him for immunising his animals consists of three parts of the former and one of the latter. 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, but very little importance can be attached to this result. Maragliano does not appear to have studied the effects of this serum on tubercular animals, but it has been tried in a great number of cases of human tuberculosis, 2 c.c. being injected subcutaneously every two days. Improve- ment is said to have taken place in a certain proportion, especi- ally of mild non-febrile cases. An antitubercular serum has also been introduced by Marmorek. This observer considers that the tubercle bacillus cannot produce in ordinary media the toxins which it originates when exposed to the antagonism of the bodily cells. He tries to make good this defect by first growing it in a serum antago- nistic to some of the phagocytic cells of the body ; for this a leucotoxic serum is used. When the bacillus has grown in this presumably favourable soil it is transferred to a medium con- taining a substance which may be unfavourable : and for this there is employed a medium containing liver extract, the liver being an organ in which in man tubercular lesions are comparatively rare. The bacilli being thus accustomed to an unfavourable surrounding are used for immunising animals, the serum of which is now suitable for the treatment of human tuber- culosis. It is too soon to speak of th« effects of this line of treatment. Methods of Examination. — (1) Microscopic Examination. Tuberculosis is one of the comparatively few diseases in which a diagnosis can usually be definitely made by microscopic examina- tion 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. 101). In the 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 negative result is obtained in a suspected case, repeated examination 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 266 TUBERCULOSIS 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 or into the peritoneum ; 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 un- necessary. 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 four or five weeks, to inoculate tubes of solidified blood serum, under strict aseptic precautions, with portions of a tubercular organ, e.g. the spleen. The portions of tissue should be fairly large, and should be well rubbed into the broken surface of the medium. 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 characteristic 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 w^ords, the local effects of the bacilli are well marked, often extreme, whilst the toxic phenomena are proportionately at a minimum. There are two chief forms of leprosy. The one, usually called the tubercular form — lepra tuberosa or tuberculosa — is character- ised 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 resulting. In the other form, the anaesthetic, — maculo- anaesthetic of Hansen and Looft — the outstanding 267 268 LEPROSY 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 face, of the backs of hands and feet, and of the extensor aspects of arms and ££ m FIG. 91. — Sections through leprous skin, showing the masses of cellular granulation tissue in the cutis ; the dark points are clumps of bacilli deeply stained. Paraffin section ; Ziehl-Neelsen stain, x 80. legs. These nodules enlarge and produce 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 (Fig. 91), to a less extent in the subcutaneous tissue. The epithelium often becomes stretched over them, and an oozing surface becomes developed, or actual ulceration 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 growths. Internal organs, especially the spleen, liver, and testicles, may become secondarily affected. In BACILLUS OF LEPROSY 269 all situations the change is of the same nature, — a chronic inflammatory condition attended by abundant formation of granulation tissue, nodular or 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 compara- tively 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 frequently 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 ancesthetic form the lesion of the nerves is the out- standing feature. These are the seat of diffuse infiltrations which lead to the destruction of the nerve fibres. In the earlier stages, in which the chief symptoms are pains along the nerves, there occur patches on the skin, often of 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, parch- ment-like, and anaesthetic ; frequently pemphigoid bullee or other skin eruptions occur. Partly owing to injury to which the feet and arms are liable from their anaesthetic condition, and partly owing to trophic disturbances, 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 scantier, 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 1871, 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 270 LEPROSY 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 fe- * • ***••** * ^ 4 -. FIG. 92. — 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. 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 up the basic aniline stains 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 POSITION OF THE BACILLI 271 bacilli, but to use a weaker solution of sulphuric acid, say 5 per cent, in decolorising ; in the case of films and thin sections, decolorising 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 (vide p. 101). The bacilli are also readily stained by Gram's method. Regarding the presence of spores practically nothing is known, though some FIG. 93. — 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 1100. 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 numbers 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 numerous that the structure of the cells is quite 272 LEPROSY obscured (Fig. 92). They are often arranged in bundles which contain several bacilli lying parallel to one another, though the bundles lie in various directions (Fig. 93). The appearance thus presented by the cells filled with bacilli is very characteristic. 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 connective-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 smaller numbers. In the nerves in the anaesthetic form they are com- paratively 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 distribution 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, 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 arid spinal cord are almost exempt, but in some cases bacilli have been found • even within nerve cells. Relations to the Disease. — Attempts to cultivate the leprosy bacilli outside the body have so far been unsuccessful. From time to time announcements of successful cultivations 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 appearance weeks or even months afterwards, but no multi- plication of the organisms occurs. The only exception to this statement is afforded by the experiments of Melcher andOrthmann, who inoculated the anterior chamber of the eye of rabbits with RELATIONS TO THE DISEASE 273 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 wre cannot say that there is any satisfactory proof that the disease can be transmitted to any of the lower animals. Diphtheroid bacilli of more than one variety have been cultivated from the blood and tissues of leprous patients by Babes and others. Their presence would appear to be by no means infrequent, but it is not possible to say at present what their significance is. It is interesting to note that a disease occurs under natural conditions in rats which presents many points of close similarity to leprosy. It has been observed in Russia, Germany, and England, and an excellent description has recently been given by Dean. In this affection there are lesions in the skin which resemble those in leprosy, and the cells contain enormous numbers of an acid -fast bacillus. The disease can be trans- mitted to rats by inoculation with the tissue juices containing the bacilli, but riot to animals of other species. All attempts to cultivate the characteristic organism outside the body have failed, but Dean has obtained a diphtheroid bacillus— a result of interest in relation to what has been found in leprosy. Whether this disease has any relation to leprosy in the human subject is very doubtful, but the facts which have been ascertained may prove of high importance in connection with the pathology of the latter disease. 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 the objection that 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. There is also the fact that from time to time 18 274 LEPROSY in leprous subjects there occur febrile attacks, 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 inoculation 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 all the facts there can be little doubt that leprosy in certain conditions may be transmitted by direct contact, though its contagiousness is not of a high order. 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 arrange- ment 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 distinguishing the two conditions. CHAPTER XL GLANDERS AND EHINOSCLEROMA. 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 conclusively established its causal relationship. These have been fully confirmed. The same organism has also been cultivated 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, mallein, 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 tuberculin 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 inter- mediate position, the former being rather 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 inocula- tion 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 275 276 GLANDERS into contact with horses ; even amongst them it is a comparatively rare disease. 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 which are at first firm and of somewhat translucent grey appearance. The growth of these is attended usually by inflammatory swelling and profuse catarrhal dis- charge. Afterwards the nodules soften in the centre, break down, and give rise to irregular ulcerations. 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, media- stinum, 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 by a congested zone. The term "farcy" is applied to the affection of the superficial 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 associated lymphatic glands 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 nodules may occur in internal organs and the nasal mucous membrane. The disease is often present in a "latent form," and its presence can only be detected by the mallein test (vide infra). 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 usually with spread- ing 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 inflammatory 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 THE GLANDERS BACILLUS 277 addition to the lesions mentioned there may be foci, usually suppurative, in the lungs (attended of ten. with pneumonic con- solidation),, 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 also have a great tendency to ulcerate, though the lymphatic system is not so prominently affected as in the horse. Deposits may form in the subcutaneous 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, on the other hand, the disease may take on a more acute character and rapidly become fatal. The Glanders Bacil- lus. — Microscopical Characters. — The glan- ders bacilli are minute rods, straight or slightly curved, with rounded ends, and about the same length as tubercle bacilli, but distinctly thicker. Fia 94._Glanders bacim among8t broken- (Flg. 94 ). They show, down cells. Film preparation from a however, considerable glanders nodule in a guinea-pig, variations in size and in staiued with weak carbol-fuchsin. x 1000. appearance, and their pro- toplasm is often broken up into a number of deeply-stained portions with unstained intervals between. These characters are seen both in the tissues and in cultures, but, as in the case of many organisms, irregularities in form and size are more pro- nounced in cultures (Fig 95) ; short filamentous forms 8 to 12 ju in length are sometimes met with, but these are on the whole rare. The organism is non-motile. In the tissues the bacilli 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 considerable numbers ; but in the chronic nodules, especially when softening has taken place, they 278 GLANDERS 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 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 (Rosen thai). But it is very doubtful that such is the case ; the appearances correspond rather with mere breaks in the proto- plasm, such as are met with in many other bacilli which do not contain spores, and the compara- tively low powers of resist- ance of glanders bacilli containing these so-called from 0 a spores, is strongly against FIG. 95.— Glanders bacilli, culture on glycerin agar. carbol-fuchsinand partially decolorised to show segmentation of protoplasm, x 1000. Stained with tneir being of that nature. The power of resistance is after all the important practical point. Staining. — The glanders bacillus differs widely froin 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. We have obtained the best results by carbol-thionin-blue (p. 98), and we prefer to dehydrate by the aniline-oil method. In film preparations of fresh glanders nodules the bacilli can be readily found by staining with any of the ordinary combinations, e.g. carbol-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. M'Fadyean recommends that after sections have been stained in Loffler's methylene blue and slightly decolorised in weak acetic acid, they should be treated for fifteen minutes with a saturated solution of tannic acid ; thereafter they are washed thoroughly in water, and as a contrast stain a 1 per cent solution of acid fuchsin may be applied for CULTIVATION OF GLANDERS BACILLUS 279 half a minute ; they are then dehydrated, cleared, 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° to 37° C. Though a certain amount of growth occurs down to 21° C., a temperature above 25° C. is always desirable. On agar and glycerin 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 sub-cultures on these media at the body temperature growth is visible within twenty-four hours, but when fresh cultures are made from the tissues it may not be visible till the second day. Serum or potato, however, is much more suitable for cultivating from the tissues than the agar media ; on the latter it is some- times 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 fiocculent deposit of slimy and somewhat tenacious consistence. On potato at 30° to 37° C. the glanders bacillus flourishes well and produces a characteristic appearance ; incubation at a high temperature, however, being necessary. Growth proceeds rapidly, and on the third day has usually formed a transparent layer of slightly yellowish tint, like clear honey in appearance. On sub- sequent days, the growth still extends and becomes darker in colour and more opaque, till about the eighth day it has a reddish-brown or chocolate tint, 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). 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 280 GLANDERS 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 are kept at the temperature of the room; on the other hand, they are often found to be dead at the end of two weeks when kept constantly at the body temperature. They have comparatively feeble resistance to heat and antiseptics. Loftier found that they were killed in ten minutes in a fluid kept at 55° C., and in from 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 some- what the tubercle bacillus, but is thicker, and differs widely from it in its staining reactions. For its cultivation the higher temperatures are necessary, and the 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 Lofner and 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. One of the animals died ; after a few weeks the other, showing symptoms of caohexia, 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, ACTION ON THE TISSUES 281 lungs, and liver. In the guinea-pig the disease is less acute, though secondary nodules in internal organs are usually present in considerable numbers. At the site of inoculation an inflam- matory swelling forms, which soon softens and breaks down, leading to the formation of an irregular crateriform ulcer with indurated margins. The lymphatic vessels become infiltrated, 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 a longer period. Secondary nodules, in varying numbers in different cases, may be present 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 great swelling and redness of the testicles, which changes may be noticeable in two or three days. By this method there occur also numerous small nodules on the surface of the peritoneum. Eabbits are less susceptible than guinea- pigs, and the effect of subcutaneous 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. Mayer has found that when the glanders bacillus is injected along with melted butter into the peritoneum of a guinea-pig, it shows filamentous, branching, and club-shaped forms; in other words, it presents the characters of a streptothrix. Lubarsch, on the other hand, in a comparative study of the results of inoculation with acid-fast and other bacilli, found none of the above characters in the case of the glanders bacillus (cf. Tubercle). Action on the Tissues. — From the above facts it will be seen tl;at in many respects glanders presents an analogy to tubercle as regards the general characters of the lesions and the mode of spread. 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 infiltration 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, most of which are polymorpho-nuclear, whilst in the central parts many show fragmentation of nuclei with the formation of a deeply staining granular detritus. And further, the inflammatory change may be followed by suppurative softening of the tissue, especially in certain situations, such as 282 GLANDERS the subcutaneous tissue and lymphatic glands. The nodules, therefore, in glanders, as Baumgarten puts it, occupy an intermediate position between miliary abscesses and tubercles. The diffuse coagulative necrosis 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 ; there may also be fibrinous exudation, whilst at the periphery of the nodules con- nective-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-stream — 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 produced 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 cultures of the bacillus had been pulverised. 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. 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 1 in 50 dilution of the serum produces marked agglutination 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 iu both directions, i.e. negative results by glandered anmials and positive results by non-glandered animals. He finds that a more delicate METHODS OF EXAMINATION 283 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 sedimenting 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. — Mallein is obtained from cultures of the glanders bacillus grown for a suitable length of time, and, like tuber- culin, 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 glycerin or water, but is now usually prepared from cultures in glycerin bouillon. Such a culture, after being allowed to grow for three or four weeks, is sterilised by heat either in the autoclave at 115° C. or by steaming at 100° C. on successive days. It is then filtered 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 throwing the 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. The use of Mallein as a means of Diagnosis. — In 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'Fadyean at the sixth, tenth, fourteenth, and eighteenth hours afterwards, and on the next day. Here both the local reaction and the temperature are of importance. In a glandered animal, at the site of inoculation there is a somewhat painful local swelling, which reaches a diameter of five inches at least, the maximum size not being attained until twenty-four hours afterwards. The temperature rises 1 '5° to 2° C. , or more, the maximum generally occurring in from eight to sixteen hours. If the temperature never rises as much as 1 '5°, the reaction is considered doubtful. In the negative reaction given by an animal free from glanders, the rise of temperature does not usually exceed 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. 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 glycerin agar (preferably the former), and incubating at 37° C. The colonies of the glanders bacillus do not appear till two days after. This method often fails unless a considerable number 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 284 GLANDERS 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 injection. By the latter method, as above described, lesions are much more rapidly produced, and are more characteristic. 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. It is extremely doubtful whether the application of mallein to diagnosis of the disease in the human subject is justifiable. There is a certain risk, that it may lead to the lesions assuming a more acute character ; moreover, culture and inoculation tests are generally available. In the case of horses, etc., a diagnosis will, however, 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 granulomata. It is characterised by the occurrence of chronic nodular thickenings in the skin or mucous membrane of the nose, or in the mucous membrane of the pharynx, larynx, or upper part of the trachea. The nodules are of considerable size, sometimes as large as a pea ; in the earlier stages they are comparatively smooth on the surface, but later they become shrunken and the centre is often retracted. - The disease is scarcely ever met with in this country, but is of not very uncommon occurrence on the Continent, especially in Austria and Poland. 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 gelatinous material which may fill the cell and push the nucleus to the side. Within these cells there is present a characteristic bacillus, occurring in little clumps or masses 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 pneumobacillus. They are usually present in the lesions in a state of purity. It was at first stated RHINOSCLEROMA 285 that they could be stained by Gram's method, but more recent observations 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 or gelatin is said to be less distinct, and the growth on potatoes is more transparent and may show small bubbles of gas ; but 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. From these facts alone it would appear probable that they are the active agents in the production of the lesions. Experimental inoculation has thrown little light on the subject, though one observer has described the production of nodules on the conjunctivas 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 frequently present in ozcena, and is often known as the bacillus ozwnce. 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. There is no doubt that rhinoscleroma is a specific disease with well-marked characters and it is quite possible that one member of this group of organisms may be the causal agent, though indistinguishable from others by culture tests. There is, however, a tendency on the part of recent investigators to consider the "bacillus of rhinoscleroma " to be identical with the pneumobacillus and its presence in the affected tissues to represent merely a secondary invasion. The subject is one on which more light is still required. CHAPTER XII. ACTINOMYCOSIS AND ALLIED DISEASES. ACTINOMYCOSIS is the most important example of a group of diseases produced by streptothrix organisms. 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 Bol linger, 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. It is, however, to be noted that the term " actinomyces," as originally used, does not' represent one parasite but a number of closely allied species, as cultures obtained from various sources have presented considerable differences ; and, further, it is noted that other distinct species of streptothrix have been cultivated from isolated cases of disease in the human subject where the lesions resembled more or less closely those of actinomycosis. In one or two instances the organism has been found to be "acid-fast," and there is no doubt that the actinomyces group is closely related through intermediate forms with the tubercle group (vide p. 239). Naked-eye Characters of the Parasite. — The actinomyces 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, 286 CHARACTERS OF THE ACTINOMYCES 287 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 issue. 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 pigment and by degenerative change, which is usually accompanied 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 some- times described as being always of a distinctly yellow colour, but this is only occasionally the case ; in fact, in the human subject they occur much more frequently as small specks of semi-translucent appearance, and of greenish-grey tint. Microscopical Characters. — The parasite, which is now generally regarded as belonging to the streptothrix group of the higher bacteria (p. 14), presents pleomorphous characters. 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 •6 IJL 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 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 true branching, a character which at once distinguishes 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 of which filaments radiate for a short distance (Fig. 96). The filaments usually stain uniformly in the younger colonies, but some, especially in the older colonies, may be segmented so 288 ACTINOMYCOSIS AND ALLIED DISEASES 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. Spores or Gonidia. — Like other species of streptothrix 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 FIG. 96. — Actinomycosis of 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. • and act as new centres by growing out into filaments. They have somewhat higher powers of resistance than the filaments, though less than the spores of most of the lower bacteria. An exposure to 75° C. for half an hour is sufficient to kill most streptothrices or their spores ; cultures containing spores can resist a temperature from five to ten degrees higher than spore- free cultures (Foulerton). 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. Both the CHARACTERS OF THE ACTINOMYCES 289 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 (Figs. 97, 98). They are usually homogeneous and structureless in appearance. In the human subject the clubs are FIG. 97. — Actinomyces in human kidney, showing clubs radially arranged and surrounded by pus. The filaments had practically disappeared. Paraffin section ; stained with hsematoxylin and rubin. x 500. often comparatively fragile structures, which are easily broken down, and may sometimes 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 usually not coloured by the violet, but take readily a contrast stain, such as picric acid, 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, 19 290 ACTINOMYCOSIS AND ALLIED DISEASES much older colonies, the clubs constitute the most prominent feature, whilst in most colonies the filaments are more or less degenerated, and it may sometimes be impossible to find any. They often form a dense fringe around the colony, and when stained by Gram's method retain the violet stain. They have, in fact, undergone some further chemical change which produces the altered staining reaction. Occasionally in very chronic FIG. 98. — Colonies of actinomyces, showing general structural arrangement and clubs at periphery. From pus in human subject. Stained Gram and safranin. x 60. lesions in the human subject the clubs stain with Gram's method. Clubs showing intermediate staining reaction have been described in the ox by M'Fadyean. The club-formation probably represents a means of defence on the part of the parasite against the phagocytes of the tissue : the view, formerly held, that the clubs are organs of fructification has now been generally abandoned. Tissue Lesions. — In the human subject the parasite pro- duces by its growth a chronic inflammatory change, usually ending in a suppuration which slowly spreads. In some cases TISSUE LESIONS 291 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 associated with abundant growth of the parasite in the filamentous form presenting a honeycomb appearance. In an organ such as the liver, multiple foci of suppuration are seen at the spreading margin of the disease, 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, and often consisting of well-developed fibrous tissue containing areas of younger formation in which irregular abscess formation is usually present. The cells immedi- ately 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, 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 oesophagus, 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 purulent material. Thence it may spread to the peritoneum or to the extraperitoneal tissue, 292 ACTINOMYCOSIS AND ALLIED DISEASES the retrocaecal 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 composed almost exclusively of masses of the actinomyces along with epithelial cells. This, however, is a 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 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 to find leucocytes in the neighbourhood of a colony containing 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 latter producing enlargement and induration, with nodular thickening on the surface — the condition known as " woody tongue." Source of the Parasite. — There is a considerable 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 em- bedded in the tissues. There are besides, in the case of the human subject, a certain number of cases in which 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. Cultivation (for methods of isolation see later). — The de- scriptions of the cultures obtained by various investigators differ in essential particulars, and there is no doubt that the organisms described are different. The following is the account of the organism as cultivated by Bostrom : — On agar or glycerin agar at 37° C., growth is generally CULTIVATION OF ACTINOMYCES 293 visible on the third or fourth day in the form of little transparent drops which gradually enlarge and form rounded projections of a reddish-yellow tint and somewhat transparent appearance, like drops of amber. The growths tend to remain separate, and even when they become confluent, the nodular character is maintained. They have a tough con- sistence, being with diffi- culty broken up, and ad- here firmly to the surface of theagar. Older growths often show on the surface a sort of corrugated aspect, and may sometimes pre- sent the appearance of having been dusted with a brownish-yellow powder (Fig. 99). " In the cultures at an early stage the growth is composed of branching filaments, which stain uniformly (Fig. 100), but later some of the super- ficial filaments may show segmentation into gonidia. Slight bulbous thicken- ings may be seen at the end of some of the fila- ments, but true clubs have not been observed. On gelatin the FIG. 99. — Cultures of the actinomyces on glycerin agar, of about three weeks' growth, showing the appearances which occur. The growth in A is at places somewhat corru- gated on the surface. Natural size. same tendency to grow in little spherical 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. The organism obtained in culture by Wolff and Israel (vide infra} is probably the same as the one which has been recently described in detail by J. H. Wright, who obtained it in pure con- dition from fifteen different cases of the disease. It differs markedly from Bostrom's organism in being almost a strict anaerobe and in 294 ACTINOMYCOSIS AND ALLIED DISEASES ceasing to grow at a temperature a little below that of the body. Under ordinary aerobic conditions either no growth occurs or it is of a very slight character. On the surface of agar under anaerobic conditions the organism produces dense rounded colonies of greyish -white colour, which sometimes assume a rosette form. A somewhat curious feature of growth is described by Wright, namely, that in a shake culture in glucose agar the colonies are most numerous, and form a dense zone about half an inch from the surface of the medium, that is, at a level where there is presum- ably a mere trace of oxygen obtainable (Fig. 101). In bouillon, growth takes place at the bottom of the medium in rounded masses which afterwards undergo disintegration. Wright found when the organism was grown in the presence of serum or other animal fluids that the formation of true clubs occurred at the ex- tremity of some of the fila- ments (Fig. 102). From the conditions under FIG 100.- Actinomyces, from a culture on which th QC he glycerin agar, showing the branching of . . S , the filaments. 1S inclined to regard Stained with fuchsin. . x 1000. it as a true parasite, and doubts whether it can have a saprophytic existence outside the body, e.g. on grain. He is also of opinion that all cases of true actinomycosis, i.e. cases where colonies visible to the naked eye are present, are probably produced by one species, and that the aerobic organisms obtained by Bostrom and others are probably accidental contaminations. It is quite evident that further investigations are required in the light of the results detailed. Certainly the parasite in many cases of actinomycosis in the human subject does not grow on ordinary media under aerobic conditions as Bostrom's organism does. Varieties of Actinomyces and Allied Forms. — It is probable that in the cases of the disease described in the human subject there is more than one variety or species of parasite belonging to the same group. Gasperini has described several varieties of actinomyces bovis according VARIETIES OF ACTINOMYCES 295 to the colour of the growths, and a similar condition may obtain in the case of the human subject. Further- more a considerable number of streptothrices have been found in cases of disease in the human subject, the as- sociated lesions varying in character from tubercle-like nodules on the one hand to suppurative processes on the other. The organisms culti- vated from such sources differ according to their microscopic characters (for example, some form "clubs" whilst others do not) ac- cording to their conditions of growth, staining reac- tions, etc. Of these only a few examples may here be mentioned, but it may be noted that the importance of the streptothrices as causes of disease is con- stantly being extended. Wolff and Israel cultivated from two cases of ' ' actino- mycosis" in man a strep- tothrix which differs in so many important points from the actinomyces of Bostrom FIG. 102. — Section of a colony of actinomyces from a culture in blood serum, showing the formation of clubs at the periphery. x 1500. FIG. 10 1.1 — Shake cultures of actinomyces in glucose agar, showing the maximum growth at some distance from the sur- face of the medium. that it is now regarded as a distinct species. An- other species was culti- vated by Eppinger from a brain abscess, and called by him "cladothrixaster- oides," from the appear- ance of its colonies on culture media. A case of general streptothrix infection in the human subject described by MacDonald was probably due to the same organism as Eppinger's. In the tissues it grows in a somewhat diffuse manner and does not form clubs ; 1 For Figs. 101 and 102 we are indebted to Dr. J. Homer Wright of Boston, U.S.A. 296 ACTINOMYCOSIS AttD ALLIED DISEASES in rabbits and guinea-pigs it produces tubercle-like lesions. Flexner observed a streptothrix in the lungs associated witb lesions somewhat like a rapid phthisis, and applied the name "pseudo-tuberculosis hominis streptothricea " ; an apparently similar condition has been described by Buchholz. Berestnew cultivated two species of streptothrix from suppurative lesions, one of which is acid -fast and grows only in anaerobic conditions. Birt and Leishman have described another acid- fast streptothrix obtained from cirrhotic nodules in the lungs of a man. This organism grows readily on ordinary media, forming a white powdery growth which afterwards assumes a pinkish colour ; it is pathogenic for guinea-pigs, in which it causes caseous lesions. There is, further, the streptothrix madurse described below. 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. Dean has cultivated from a nodule in a horse another streptothrix, which produces tubercle-like nodules in the rabbit with club-formation ; it has close resemblances to the organism of Israel and Wolff. The so-called diphtheria of calves and "bacillary necrosis" in the ox are probably both produced by another streptothrix or leptothrix, which grows diffusely in the tissues in the form of fine felted filaments. Further investigation may show that some of these or other species may occur in the human subject in conditions which are not yet differentiated. Experimental Inoculation. — Inoculation of smaller animals, such as rabbits and guinea-pigs, has usually failed to give positive results. This was the case, for example, in the important series of experiments by Bostrom, and it may be assumed that these animals are little susceptible to the actinomyces. The disease has, however, been experimentally produced in the bovine species both by cultures from the ox and from the human subject. Inoculation with the organism of Israel and Wolff produces nodular lesions both in rabbits and in guinea-pigs, while Wright found that characteristic colonies and lesions resulted although the parasite did not grow to any great extent. Several of the other species of streptothrix have been found to possess active pathogenic properties. Methods of Examination and Diagnosis. — As actinomycosis 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 recognised 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 glycerin or Farrant's solution. To study the filaments, a colony should be broken down on a MADURA DISEASE 297 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 prepara- tions 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 acid fuchsin being afterwards used to stain the clubs. By this method, very striking prepara- tions may be obtained. Cultures should be made both under aerobic and anaerobic conditions. Tubes of agar or glycerin agar should be inoculated and incubated at 37° C. ; deep tubes of melted glucose agar should also be used, the inoculated material being diffused through the medium, separate colonies may thus be obtained. 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. MADUKA DISEASE. Madura disease or mycetoma resembles actinomycosis both as regards the general characters of the lesions and the occurrence of the parasite in the form of colonies. or "granules." There is no doubt, however, that the two conditions are distinct, and it also appears established that the two varieties of Madura disease (vide infra) are produced by different organisms. This disease is comparatively common in India and in various other parts of the tropics : it has also been met with in Algiers and in America. Madura disease differs from actinomyces not only in its geo- graphical 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 operation, the parasite never produces secondary lesions in internal organs. Vincent also found that iodide of potassium, which has a high value as a therapeutic agent in many cases of actinomycosis, had no effect in the case of Madura disease studied by him. 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 298 ACTINOMYCOSIS AND ALLIED DISEASES formation of fistulous openings and ulcers. There are great en- largement 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, com- pared 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 pale variety and a black variety of the disease have been distinguished ; in both varieties the granules mentioned reach a rather larger size than in actinomycosis. These two conditions will be considered separately. Pale Variety. — When the roe-like granules are examined microscopic- ally, they are found, like the actinomyces, to show in their interior an abun- dant mass of branching filaments with mycelial arrangement. There may also be present at the periphery club-like struc- tures, as in actinomyces ; sometimes they are ab- sent. These structures FIG. 103. — Streptothrix Maclurse, showing £, i i . j branching filaments. From a culture of °ften have «* elongated agar. Stained with carbol-thionin-blue. wedge-shape, tormmg an x 1000. outer zone to the colony, and in some cases the filaments can be found to be connected with them. Vincent obtained cultures of the parasite from a case in Algiers, and found it to be a distinct species : it is now known as the streptothrix Maduroe. Morphologically it closely resembles the actinomyces, but it presents certain differences in cultural characters. In gelatin it forms raised colonies of a yellowish colour, with umbilication of the centre, and there is no liquefaction of the medium. On agar the growth assumes a reddish colour; the organism flourishes well in various vegetable infusions in which the actinomyces does not grow. On all the media growth only takes place in aerobic conditions. Experimental inoculation of various animals has failed to reproduce the disease. There is MADUKA DISEASE 299 therefore no doubt that the streptothrix madurae and the actinomyces are distinct species. Black Variety. — The observations of J. H. Wright, who obtained pure cultures of a hyphomycete, show that this variety is a distinct affection from the pale variety. The pigment may be dissolved by soaking the granules for a few minutes in hypochlorite of sodium solution, and the granules may then be crushed out beneath a cover-glass and examined microscopically. The black granules are composed of a somewhat homogeneous ground- substance impregnated with pigment, and in this there is a mycelium of thick filaments or hyphse, many of the segments of which are swollen ; at the periphery the hyphae form a zone with radiate arrangement. In many of the older granules the parasite is largely degenerated and presents an amorphous appear- ance. Wright planted over sixty of the black granules in various culture media, and obtained cultures of a hyphomycete from about a third of these. The organism grows well on agar, bouillon, potato, etc. ; on agar it forms a felted mass of greyish colour, and in old cultures black granules appear amongst the mycelium. Microscopically the parasite appears as a mycelium of thick branching filaments with delicate transverse septa ; in the older threads the segments become swollen, so that strings of oval-shaped bodies result. No signs of spore-formation were noted. Inoculation of animals with cultures gave negative results, as did also direct inoculation with the black granules from the tissues. CHAPTER XIII ANTHRAX.1 OTHER NAMES. — SPLENIC FEVER, MALIGNANT PUSTULE, WOOL- SORTER'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 throughout 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 " 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 now the first part to give rise to symptoms. In all these forms of the affection in the human subject, the bacilli are in their distribution much more re- stricted to the local lesions than is the case in the T>X, 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. 1 In 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." a This must be distinguished from "charbon symptomatique, " which is quite a different disease. 300 BACILLUS ANTHRACIS 301 During the early part of last 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 contribution 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 observa- tions were, shortly afterwards, confirmed in the main 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 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 arrEhfax, 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 ^ thick or a little thicker, and 6 to 8 p 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 302 ANTHRAX protoplasm is very finely granular, and sometimes appears sur- rounded by a thin unstained capsule. When several bacilli lie end to end in a thread, the capsule seems common to the whole thread (Fig. 108). 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 gelatin 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 ex- amination with a higher power is observed to be a filament which turns upon itself (Fig. 104). The whole colony is, in fact, probably one long thread. Such colonies are very suitable for making impression pre- parations (vide p. 118) which preserve perman- ently the appearances de- scribed. On examining such with a high power, the wreaths are seen to be made up of bundles of FIG. 104.— Surface colony of the anthrax l°ng filaments lying par- bacillus on an agar plate, showing the allel with one another, each characteristic appearances, x 30. filament consisting of a chain of bacilli lying end to end, and similar to those observed in the blood (Fig. 105). On gelatin plates, after from twenty-four to thirty-six hours at 20° C., the same appearances manifest themselves, and later they are accompanied by liquefaction of the gelatin. In gelatin plates, however, instead of the characteristically wreathed appear- ance at the margin, the colonies sometimes give off radiating spikelets irregularly nodulated, which produce a star-like form. These spikelets are composed of spirally twisted threads. From such plates the bacilli can be easily isolated, and the appearances of pure cultures on various media studied. In bouillon, after twenty-four hours' incubation at 37° C., there is usually the appearance of irregularly spiral threads suspended in the liquid. These, on being examined, are seen BACILLUS ANTHRACIS 303 to be made up of bundles of parallel chains of bacilli. Later, growth is more abundant, and forms a flocculent mass at the bottom of the fluid. In gelatin stab cultures, the characteristic appearance can be best observed when a low proportion, say 7J per cent, of gelatin FIG. 105. — Anthrax bacilli, arranged in chains, from a twenty-four hours' culture on agar at 37° C. Stained with fuchsin. x 1000. 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 s pikelets which enable the cultures to be easily recog- Plo.106._stab culture of msed. Inese spikelets are longest at the upper part of the needle track (Fig. 106). Not much spread takes place on the surface of the gelatin, but here liquefaction commences, and gradually spreads down the stab and out into the medium, till the whole of the gelatin may be liquefied. Gelatin slope cultures exhibit a thick felted growth, the edges of which show the wreathed appearance seen in plate cultures. Liquefaction here soon ploughs a trough in the surface of the medium. Sometimes " spiking " does not take place in gelatin stab cultures, only the anthrax bacillus in peptone-gelatin ; seven days' growth. It shows the "spiking" and also, at the surface, com- mencing liquefaction. Natural size. 304 ANTHRAX little round particles of growth occurring 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 sloped cultures have the appearance of similar cultures in gelatin, though; of course, no liquefaction takes place. Blood serum sloped cultures present the same appearances 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 recognised by its microscopic appearance, by its growth on agar or gelatin plates, and by its growth in gelatin stab cultures. The growth on plates is specially characteristic, and is simulated by no other pathogenic organism. The Biology of the B. Anthracis. — Koch found that the bacillus anthracis grows best at a temperature of 35° C. Growth, i.e. multiplication, does not take place below 12° C. or above 45° C. In the spore-free condition the bacilli have comparatively low powers of resistance. They do not stand long exposure to 60° C., and if kept at ordinary temperature 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 accord- ingly destroyed in the stomachs of healthy animals. They are also soon killed in the process of putrefaction. They can, how- ever, 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 gelatin always commences at the surface and spreads downwards. Growth is more rapid in the presence of oxygen, and spore formation 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; this gradually increases in size until it forms an oval body about the same thickness as the bacillus lying in the bacillary protoplasm (Fig. 107). The latter gradually loses its staining capacities and finally disappears. The spore thus lies free as*1 an oval highly BIOLOGY OF THE B. ANTHRACIS 305 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. 102). When the spore is 'again about to assume 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 occurrence of spores in cultures outside the body. Many, however, 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 suit- able temperature. The optimum temperature for spore production is 30° C. Koch found that spore formation did not occur below 18° C. Above 42° C. not only does sporula- tion cease, but Pasteur found that if bacilli were kept at this temperature FIG. 107. — Anthrax bacilli containing spores (the darkly coloured bodies) ; from a .. . , , three days' culture on agar at 37° C. tor eight days they did not stained with carbol-fuchsin and methylene- regain the capacity when blue, x 1000. again grown at a lower temperature. 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 140° 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 scraping off a surface culture and rubbing it up in a little sterile water. Into this 20 306 ANTHRAX sterile silk threads are dipped, which, after being dried over strong sulphuric acid in a desiccator, 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 (see Chap. IV.). 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 pre- cautions 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, however, have been largely modified by the system of preventive 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 apparently out of sorts, and does not feed j its pulse and respiration 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 enlarge- ment 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 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 examina- tion it will be found to contain enormous numbers of bacilli ANTHEAX IN ANIMALS 307 mixed with red corpuscles and leucocytes, chiefly lymphocytes and the large mononucleated variety (Fig. 108). 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 FIG. 108. — Scraping from spleen of guinea-pig dead of anthrax, showing the bacilli mixed with leucocytes, etc. (Same appearance as in the ox.) "Corrosive film" stained with carbol-thionin-blue. x 1000. 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 as the result of rupture of the capillaries. The blood throughout the body is usually fluid and of dark colour. 308 ANTHRAX The lymphatic system generally is much affected. The glands, especially the mediastinal, mesenteric, and cervical glands, are enlarged and surrounded by oedematous tissue, the lymphatic vessels are swollen, and both glands and 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. 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 susceptible than this group are the horse, deer, goat, in which the disease occurs from time to time in nature. Anthrax also occurs epidemically in the pig, often from the ingestion of the organs of other animals dead of the disease. It is, however, doubtful if all cases of disease in the pig described on clinical grounds as anthrax are really such, and a careful bacteriological examination is always advisable. The human subject may be said to occupy 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 confined 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. Neverthe- less 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 in laboratory work, white mice and guinea-pigs are the most susceptible to anthrax, and are generally used for test inocula- ANTHRAX IN MAN 309 tions. If a small quantity of anthrax bacilli be 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 appearance, small haemorrhages are often present, and on microscopic examination numerous bacilli are seen. The internal organs show congestion and cloudy swelling, with FIG. 109. — Portion of kidney of a guinea-pig dead of anthrax, showing the bacilli in the capillaries, especially of the glomerulus. Paraffin section ; stained by Gram's method and Bismarck-brown. x 300. sometimes small haemorrhages, and their capillaries contain enormous numbers of bacilli, as has already been described in the case of the ox (Fig. 109) ; the spleen also shows a corre- sponding condition. Highly susceptible animals may be infected by being made to inhale the bacilli or their spores, and also by being fed with spores, a general infection rapidly occurring by both methods. Anthrax in the Human Subject. — As we have noted, man occupies a middle position in the scale of susceptibility to anthrax. It is always communicated to him from animals, and 310 ANTHRAX 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 due to 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, hair, or bristles, which have been taken from animals dead of the disease, and which have been contaminated with blood or secretions con- taining the bacilli, these having afterwards formed spores. This variety is often referred to as " woolsorter's disease," from its occurring in the centres of the woolstapling trade (in England, chiefly in Yorkshire), but it also is found in places where there are hair and brush factories. (1) Malignant Pustule. — This usually occurs on the exposed surfaces — the face, hands, fore-arms, 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 an irregular ring of vesicles, these in turn being surrounded by a congested area. From this pustule as a centre subcutaneous oedema spreads, especially in the direction of the lymphatics ; the neighbouring 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 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 neigh- ANTHRAX IN MAN 311 bouring lymphatics, and, to a certain extent, in the vesicles. It is very important to note that widespread oedema of a limb, enlargement of neighbouring glands, and fever may occur while the bacilli are still confined to the immediate neighbourhood 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 pathological 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. The actual cause of death is therefore the absorption of toxins. 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) Woolsorter's Disease. — The pathology of this affection was worked out in this country especially by Greenfield. The local lesion is usually situated in the lower part of the trachea or in the large bronchi, and is in the form of swollen patches in the mucous membrane often with haemorrhage into them, The tissues are cedematous, and the cellular elements are separated, but there is usually little or no necrosis. There is enormous enlargement of the mediastinal and bronchial glands, and hsemorrhagic infiltration of the cellular tissue in the region. There are pleural and pericardial effusions, and hsemorrhagic spots occur beneath the serous membranes. The lungs show collapse and oedema. There may be cutaneous oedema 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 im- possible to find any. (3) Infection occasionally takes place through the intestine, probably by ingestion of spores as in the case of animals ; but this condition is rare. In such cases there is a local lesion in the intestinal mucous membrane, of similar nature to that in the bronchial form, the central parts of the hsemorrhagic areas being, however, sometimes necrotic and yellowish, and there is a corresponding affection of the mesenteric glands. In a case of this kind, recently recorded by Teacher hsemorrhagic meningitis, 312 ANTHRAX associated with the presence of the bacilli in large numbers, occurred as a complication. The Toxins 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 blocking 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. The discovery of definite toxins which accounted for the pathogenic effects of certain bacteria led to such bodies being sought for in connection with the anthrax bacillus. Among other workers, Sidney Martin investigated this subject. This observer used alkali-albumin on which to grow the bacillus, this medium approaching most closely to the environment of the latter when growing in the animal body. From cultures in this medium, concentrated by evaporation either at 100° C. or in vacuo at 35° to 45° 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 albumoses and the alkaloid, pathogenic effects were produced in animals, closely similar to those produced by the bacilli themselves. Martin, to account for the symptoms of the disease, considered that the fever was mostly due to the albumoses, while the oedema and congestion were 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 similar to those produced by the bacilli growing in this artificial medium. Hankin and Wesbrook arrived at the conclusion that the bacillus anthracis produces a ferment which, diffusing 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. Marmier, after cultivating the b. anthracis in peptone solution containing certain salts, removed all the albumoses from the resultant liquid, and from them, either by dialysis or extraction with glycerin, isolated a body which gave no reactions of albuminoid matter, peptone, propeptone, or alkaloid. This he considered the SPREAD OF THE DISEASE IN NATURE 313 toxin. It killed animals susceptible to anthrax by a sort of cachexia, and in suitably small doses could be used to immunise them against subsequent inoculation with virulent bacilli. It was chiefly retained within the bacilli when these were growing in the most favourable conditions.. Unlike the toxins of tetanus and diphtheria, and unlike ferments, it was not destroyed by heating to 110° C. The toxin 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. It cannot be said that great light has been thrown on the pathology of the disease by these researches. The effects of infection by the b. anthracis are those shared by all other organisms producing inflammation, the tendency to oedema production of an unwonted degree being the chief special feature and one with reference to which Martin's work may be important. That toxic effects do occur in anthrax is undoubted, for frequently, while the bacilli are still locally confined, there may occur pyrexia and oedema spreading widely beyond the pustule, but we have no definite information as to how these effects are produced. In this disease we are probably dealing with another example of the action of intracellular toxins, regarding which, as in other cases, little is known. 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 carcases of animals dying of the disease, 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, and bowel, myriads of bacilli which may rapidly spore, and thus arrive at a very re- sistant 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 un- doubtedly 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 314 ANTHRAX 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 bringing to the surface anthrax spores. Koch made direct experiments on this point, and could get no evidence that such 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 ex- tremely important that anthrax carcases should be disposed of in such a way as to prevent their becoming future sources of infection. If anthrax be suspected as the cause of death no post mortem examination should be made, but only a small quantity of blood 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 sporulation 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 be here adopted. In some countries anthrax carcases are burned, and this, if practicable, is of course the best means of treating them. The chief source of danger to cattle subsequently, however, proceeds from the infection of fields, yards, and byres with the offal and the discharge from the mouths of anthrax animals. All material that can 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 their 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° to 43° C. caused them to lose their capacity of producing spores, and also gradually to lose their virulence, so that after twenty- four days they could no longer kill either guinea-pigs, rabbits, or sheep. Such cultures constituted his premier vaccin, and IMMUNISATION AGAINST ANTHRAX 315 protected against the subsequent inoculation with bacilli which had been grown for twelve days at the same temperature, and the attenuation of which had therefore not been carried so far. The latter constituted the deuxieme vaccin. It was further found that sheep thus twice vaccinated now resisted inoculation 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 deuxieme 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 difficult to arrive at a certain conclusion as to its merits. Undoubtedly a certain number of animals die of anthrax either 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 vaccinated ; and thus to be permanently efficacious the process 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. During the twelve years 1882-93 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. The immunisation of animals against anthrax Jhas always been found to be a difficult proceeding. The most usual technique has been to commence with Pasteur's vaccines, and to follow these by careful dosage with virulent cultures. Marchoux in this way produced immunity, and found that the serum of immune animals had a certain degree of protective and curative action. The most successful attempts in this direction have been those of Sclavo and of Sobernheim. The former observer, after trying various animals, came to the conclusion that the 316 ANTHKAX ass was the most suitable. He first employed a method similar to that of Marchoux; later, however, after noting the effects of the serum of an animal so immunised, he commenced the immunisation by injecting 5 to 15 c.c. of this serum along with a slightly attentuated culture of the bacilli. A few days later this was followed up with injections of virulent cultures which could now be periodically introduced for many months, and a high degree of immunity resulted. What was even more important, the serum of such an animal had strongly protective and curative properties. It has been extensively used in the treatment of anthrax in man. In a case of malignant pustule 30 to 40 c.c. are injected in quantities of 10 c.c. into the abdominal wall, and if necessary the injection is repeated on the following day. In cases treated by Sclavo himself the serum is alone employed, and its action is not aided by the excision of the pustule usually practised. The results obtained have been very good, — Sclavo, out of 164 cases, had only ten deaths or about a fourth of the ordinary mortality in Italy. Sobernheim independently elaborated an almost identical method of com- bining passive with active immunisation for the obtaining of a powerful anti-serum, and he has used this for the protective inoculation of cattle. The technique is to inject the serum into one side of the neck or into one thigh and the culture (Pasteur's second vaccine) into the other side; the doses given are for cattle or horses 5 c.c. of serum and '5 c.c. -culture, and for sheep 4 c.c. of serum and '25 c.c. culture. The method has been widely used in Germany and in Brazil, and its originator claims as its advantages simplification of application, in that one operation instead of two is sufficient, less risk of death following the immunisation procedure, and higher degree and more lasting character of the immunity resulting. Whether this method is really more efficient than that of Pasteur future experience will show, but it might be preferable for developing protection in herds at a time when an epidemic was raging. During the development of active immunity it is likely in every case (see Immunity) that there is a period of increased susceptibility to the disease. Such a period would be more likely to occur with the Pasteur method than with the Sobernheim procedure, where ,the presence in the animal's body of the protective serum might tide it over the stage when the action of the vaccine was lowering its resistance. The effects of the b. anthracis have been much studied with a view to the shedding of light on the processes obtaining in resistance and the development of immunity. Many puzzling METHODS OF EXAMINATION 317 facts have long been known; for example, in the dog, which shows great natural resistance, the serum has little if any bactericidal action, while in the susceptible rabbit there is present a serum capable of killing the organism. Such observa- tions have hitherto been without explanation. Again the properties of the serum of immune animals have been much discussed. Sobernheim and others have been unable to detect in it any trace of special bactericidal action. Sclavo found that the serum when heated to 55° C. did not lose its protective properties ; as the serum might have been complemented (see Immunity) by the serum of the animal into which it was injected, he simultaneously introduced an anti-complementary serum and found that the heated serum was still effectual. From this he deduces that in the action of the serum substances of the nature of immune body and complement are not concerned. Many have thought that the serum had a stimulating effect on the leucocytes, but Cler has brought forward ground for supposing that its effect is a sensitising one on the bacteria, and that thus the effects are to be traced to opsonic action. With regard to the formation of the protective substances, it is stated that the spleen and bone-marrow are richer in these than the blood fluids. In this connection an interesting fact may be mentioned, namely, that Roger and Gamier found evidence of the liver and spleen having special capacities for killing anthrax bacilli ; an otherwise fatal dose could be introduced into the portal vein or the splenic artery without causing death. Methods of Examination. — These include (a) microscopic examination ; (b) the making of cultures ; and (c) test in- oculations. (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 con- clusive 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 bacilli 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 manipulating a pustule before excision, as the diffusion of. the bacilli into the surrounding tissues may be aided and the condition greatly aggravated. The examination 318 ANTHRAX 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 37° 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 injected subcutaneously into a guinea-pig or mouse, 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. ENTEEIC FEVER : GASTRIC FEVER. GERMAN, TYPHUS ABDOMINALIS : ABDOMINALTYPHUS : UNTERLEIBS- TYPHUS. FRENCH, LA FIEVRE TYPHOIDE. Introductory. — The organism now known as the bacillus typhosus was first described in 1880-1 by Eberth, who observed its microscopic appearances in the intestinal ulcers and in the spleen in cases of typhoid fever. It was first isolated (from the spleen) in 1884 by Gaffky, and its cultural characters were then investigated. In 1885 Escherich observed a bacillus, now known as the bacillus coli communis, which occurs in the normal intestine and which both microscopically and culturally closely resembles the typhoid bacillus. Ordinarily the b. coli is no doubt a harmless saprophyte, but under experimental conditions in animals and also naturally in man it may manifest pathogenic properties. Investigation has shown that these two bacilli belong to a widespread group of organisms isolated from various disease conditions, which all bear close resemblances to one another and whose differentiation is often a matter of consider- able difficulty. Other members, of this group are the para- typhoid bacillus, the organism of bacillary dysentery, the b. enteritidis of Gaertner, the psittacosis bacillus, and the bacillus of hog cholera. Bacillus Typhosus. — Microscopic Appearances. — It is some- times difficult to find the typhoid bacilli in the organs of a typhoid patient. Numerous sections of different parts of a spleen, for example, may be examined before a characteristic group is found. The best tissues for examination are a Peyer's patch where ulceration has not yet commenced or where it is 319 320 TYPHOID FEVER 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, e.g. with carbol-thionin-blue, or with Ziehl- Neelsen carbol - fuehsin diluted with five parts of distilled water. As a ^ rule decolorising is not necessary. For the proper V\ observation of the ar- *A rangement of the bacilli in the tissues, paraffin sections should be pre- pared and stained in carbol-thionin-blue for a fewminutes, orinLofner's methylene-blue for one or two hours. The bacilli take up the stain some- what slowly, and as they FIG. 110.-A large clump of typhoid bacilli are also easily decolorised in a spleen. The individual bacilli are the aniline-Oil method ot only seen at the periphery of the mass, dehydration may be used (In this spleen enormous numbers of with advantage (vide p. typhoid bacilli were shown by cultures to g3)> Jn 8uch prepara. be present in a practically pure condition. ) . > r. . Paraffin section ; stained with carbol-thionin- tions the characteristic blue, x 500. appearance to be looked for is the occurrence of groups of bacilli lying between the cells of the tissue (Fig. 110). The individual bacilli are 2 ^ to 4 //, long, with somewhat oval ends, and -5 //. in thickness. Sometimes filaments 8 ^ 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 in a section be viewed endwise, in which case the appearance will be circular. This appearance accounts for some, at least, of the coccus-like forms which have been described. The bacilli are decolorised by Gram'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 ISOLATION AND APPEARANCES OF CULTURES 321 capsule is seared with a cautery to destroy all superficial con- taminating 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 gelatin plates are prepared, or successive strokes made on agar tubes. On the agar media the growths are visible after twenty-four hours' incubation at 37° C. On agar plates the superficial colonies are thin and film-like, circular or slightly irregular at the margins, dull white by reflected light, bluish-grey by transmitted light. Colonies in the substance of the agar are small, and appear as minute round points. When viewed under a low ob- jective, the surface colonies are found to be very transparent (requir- ing 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 than the superficial colonies. On making rovpr ffW nrprarfltinn? Fm< UL— Typhoid bacilli, from a young .over-glass preparations, culture Qn agai. showi some fiiamentous the bacilli are tound to forms. present the same micro- Stained with weak carbol-fuchsin. x 1000. scopic 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. 111). (The same is true of films made from young gelatin colonies) Sometimes the diversity in the length of the bacilli is such as to throw doubt 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 temperature, the bacilli are plump, and the protoplasm stains uniformly. In old cultures, or in cultures which have been exposed to changes of temperature, the protoplasm stains only in parts ; there may be an appearance of irregular vacuola- tion either at the centre or at the ends of the bacilli. There 21 322 TYPHOID FEVER is no evidence that spore -formation occurs in the typhoid bacillus. Mobility. — 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 un- dulating or serpentine motion, and move more slowly. Hanging- drop preparations ought to be made from agar or broth cultures Fm. 1.12. — Typhoid bacilli, from a young culture on agar, showing flagella. Stained by Van Ermengem's method. x 1000. not more than twenty-four hours old. In older cultures the movements are less active. Flagella. — On being stained by the appropriate methods (vide p. 103) the bacilli are seen to possess many long wavy flagella which are attached all along the sides and to the ends (Fig. 112). They are more numerous, longer, and more wavy than those of the b. coli. Characters of Cultures. — Stab cultures in peptone gelatin give a somewhat characteristic appearance. On the surface of the medium growth spreads outwards from the puncture as a thin APPEARANCES OF CULTURES 323 film or pellicle, with irregularly wavy margin (Fig. 113, A). It is semi-transparent and of bluish-white colour. Ultimately this surface growth may reach the wall of the tube. Not infrequently, however, the surface growth is not well marked. Along the stab there is an opaque whitish line of growth, of finely nodose appearance. There is no liquefaction of the medium, and no formation of gas. In stroke cultures there is a thin bluish-white A. Stab culture of the typhoid bacillus in gelatin, five days' growth. B. Stroke culture of the typhoid bacillus on gelatin, six days' growth. C. Stab culture of the bacillus coli in gelatin, nine days' growth ; the gelatin is split in its lower part owing to the formation of gas. film, but it does not spread to such an extent as in the case of the surface growth of a stab culture (Fig. 113, B). In gelatin plates also the superficial and deep colonies present correspond- ing differences. The former are delicate semi-transparent films, with wavy margin, and are much larger than the colonies in the substance, which appear as small round points (Fig. 114). 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 gelatin the surface colonies are 324 TYPHOID FEVER 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 character- istic features. This film is loosely attached to the surface, and can be easily scraped off. The growth on potatoes is important. For several days (at incubation temperature) after inoculation there is apparently no growth. If looked at obliquely, the surface appears wet, and if it is scraped with the platinum loop, a glistening track is left ; a cover-glass pre- paration shows numerous bacilli. Laterfhowever, a slight pellicle with a dull, somewhat velvety surface may appear, and this may even assume a brown appearance) These characteristic ap- pearances are only seen when a fresh potato with an acid reaction has been used. In bouillon incubated at 37° C. for twenty-four hours there is simply a FIG. 114. — Colonies of the typhoid bacillus uniform turbidity (one superficial and three deep) in a gelatin Cover-glass preparations plate. Three days growth at room tern- , ^ perature x 15. made from such some- times show filamentous forms of considerable length without apparent segmentation. Conditions of Growth, etc. — The optimum temperature of the typhoid bacillus is about 37° C., though it also nourishes well at the room temperature. It will not grow below 9° C. or above 42° C. Growth takes place in anaerobic as well as in aerobic conditions. Its powers of resistance correspond with those of most non-sporing bacteria. It is killed by exposure for half an hour at 60° C., or for two or three minutes at 100° C. Typhoid bacilli kept in distilled or in ordinary tap water have usually been found to be dead after three weeks (Frankland). Bacillus coli communis. — This bacillus is the chief organism present in the small intestine in normal conditions, and, with many other bacteria, it also inhabits the large intestine. During typhoid fever, and other pathological conditions affecting the REACTIONS OF B. TYPHOSUS AND B. COLI 325 intestines, it is relatively and absolutely enormously increased in the latter situation, where it may sometimes be almost the only bacillus present. Its relations to various suppurative and inflammatory conditions are described in the chapter on Suppura- tion (p. 185). 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. 115). It is motile, and possesses lateral flagella, which, however, are fewer in number and somewhat 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 twenty- four hours' incubation at 37° C. on agar, there are large superficial colonies and small deep colonies in the plates ; to the naked eye they are denser and more glisten- ing than those of typhoid when viewed by trans- mitted light, and rather of a brownish-white colour. Under a low objective the colonies again appear denser than those of the typhoid bacillus and more granular. On ordinary gelatin 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 impression of having greater vigour. In the case of gelatin stab cultures a few gas bubbles sometimes develop in the medium (Fig. 113, C). On potatoes in forty-eight hours there is a distinct film of growth of brownish tint and moist-looking surface, which rapidly spreads and becomes thicker. This con- trasts very markedly with the colourless film of the b. typhosus. 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 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 cultural characters the growth on FIG. 115. — Bacillus coli com munis. Film preparation from a young culture on agar. Stained with weak carbol-fuchsin. x 1000. 326 TYPHOID FEVER potatoes is the most important. As lias been pointed out by Wathelet, and also by Klein, differences exist in the growth of the two bacilli in melted gelatin. A gelatin tube is inoculated, and instead of being kept at the room temperature, is placed in the incubator at 37° C., at which temperature it is of course fluid. In such cultures, in the case of the b. typhosus, there is a general turbidity of the gelatin, 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 morphological, that importance is to be attached. In detailing the following reactions we must note that all that can be said is that under certain conditions certain effects are obtained. We cannot profess to know the principles which underlie the occurrence of these effects, and it may be that in several apparently diverse reactions the same biological processes are really at work. (1) The Fermentation of Sugars. — Of these one of the most important is the effect on lactose as first pointed out by Chantemesse and Widal. This is usually demonstrated by using a 1 per cent solution of the sugar in peptone-salt solution placed in Durham's tubes (p. 76). If such a medium be coloured with litmus the production of acid and gas by the b. coli can easily be demonstrated. Similar changes caused by this organism can also be observed in litmus milk and in Petruschky's litmus whey. Chantemesse and Widal first showed that the b. typhosus does not act on lactose in bouillon though decolorisation of the litmus may occur. It may be stated that under most conditions of making the test an acid reaction does not result and there is never any formation of gas. This organism is said, however, to break up lactose in litmus milk and in litmus whey with some acid formation. Much would thus seem to depend upon what other constituents are present in the medium, and also, it may be said, on its initial reaction. The lactose fermenting power of the b. coli is of the greatest importance ; and if MacConkey's bile-salt lactose fluid medium be used, this organism and its congeners can be distinguished from the b. typhosus, b. para typhosus, and from the dysentery bacilli (v. infra), none of whose colonies are crimson on this medium. The effects of the b. coli and the b. typhosus on other sugars is also of great importance. As media to which the sugars may be added, either peptone-salt solution or MacConkey's bile-salt media are used (q.v.). To sum up the general results it may be said that b. coli produces acid and gas in bile-salt glucose, REACTIONS OF B. TYPHOSUS AND B. COLI 327 peptone-salt glucose, lactose and mannite, but not in cane sugar,1 while the b. typhosus produces acid without gas in bile-salt glucose, peptone-salt glucose, and mannite, but not in lactose or cane sugar. It can also cause similar changes in arabinose, galactose, and fructose. Gas production by the b. coli can also be demonstrated by means of shake cultures. As ordinary bouillon contains traces of glucose it is best to use peptone-salt solution to which an appropriate sugar has been added and which has been converted into a solid medium with 10 per cent gelatine. If such a medium be inoculated in the fluid condition, shaken and set aside till growth occurs, small bubbles of gas will form all throughout it. In ordinary media inoculated with the b. coli bubbles of gas are often developed along the needle track. In the case of acid production by the b. coli or b. typhosus in ordinary media the acid probably comes, as has been said, from the glucose developed from the muscle sugar, but there may also be a subsidiary acid formation from the breaking up of the proteid elements. In certain members of the coli-typhoid group it has been observed that in such media as litmus milk or litmus whey an acid reaction may be first produced, and this may be followed after a few days by the formation of alkali, and in certain cases this phenomenon may be helpful in differentiating the species. Curdling of Milk by the B. coli. — 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 reaction may be a more complicated one, as milk can be curdled by organisms which do not possess acid-forming properties. In any case the observation of the reaction is important. The typhoid bacillus produces no visible change in milk. (2) Action on Media containing Neutral-Red. — While, as will have been already gathered, neutral-red is used as an indicator, there is some evidence that an actual breaking up of the substance can take place by the action of the coli-typhoid group ; the evidence for this lies in the fact that when the effects of acid formation are observed the tint of the medium cannot be brought back by the addition of alkali. The medium used here is bouillon containing an appropriate sugar and *5 per cent of a 1 per cent watery solution of Grubler's neutral-red. In the case of the typhoid bacillus no change occurs, but in the case of the b. coli there is developed a beautiful canary yellow with a greenish 1 A variety of the organism which does ferment 'cane sugar has been described under the name of the b. coli commuuior. 328 TYPHOID FEVER fluorescence. Fitz- Gerald and Dreyer have shown that an important factor here is the reaction of the medium, and that the effects of the bacteria may be one of degree, — under certain circumstances the effects described as characteristic of the two organisms may be reversed. (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. 77). 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 found that if the typhoid bacillus be grown in peptone solution, after a few generations of three days each it may 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 sugar in a medium retards the production of indol by the b. coli. The indol reaction thus ought to be sought for in a sugar- free medium. (4) The Media of Capaldi and Proskauer. — The first of these ("No. 1 ") is a medium free of albumin, in which b. coli grows well and freely produces acid, while the typhoid bacillus hardly grows at all, and certainly will produce no change in the reaction. Its composition is as follows : asparagin *2 parts, mannite *2, sodium chloride '02, magnesium sulphate '01, calcium chloride '02, potassium monophosphate '2, distilled water to 100 parts. The second medium ("No. 2") contains albumin, and is such that the b. coli produces no acid, while the typhoid bacillus grows well and produces an acid reaction. It consists of Witte's peptone 2 parts, mannite '1, distilled water to 100 parts. After the constituents of each medium are mixed and dissolved, it is steamed for one and a half hours and then made neutral to litmus — the first medium, being usually naturally acid, by sodium hydrate, the second, being usually alkaline, by citric acid. The medium is then filtered, filled into tubes con- taining 5 c.c., and these are sterilised. After inoculation for twenty hours the reaction of the medium is tested by adding litmus. (5) The Application of the Agglutination Test in distinguish- ing B. typhosus from B. coli. — The scope of the application of PATHOLOGICAL CHANGES^ 329 this test will be discussed later (see Immunity). 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. The test is to be taken in conjunction with the other means of differentiating the two organisms (cf. p. 340). It will thus be seen that the diagnosis between the b. typhosus and the b. coli is a matter of no small difficulty. There is no evidence that the one organism ever passes into the other. Great 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 of the characteristic tests, or only gives them very slowly. This is especially true of organisms related to the b. coli. It has consequently become common to speak of the typhoid group and the coli group in order that such varieties may be included. Pathological Changes in Typhoid Fever. — Here we confine our attention solely to the bacteriological aspects of the disease. The inflammation and ulceration in the Peyer's patches and solitary glands of the intestine are the central features. In the early stage there is produced an acute inflammatory condition, attended with extensive leucocytic emigration and sometimes with small haemorrhages. At this period the typhoid bacilli are most numerous in the patches, groups being easily found between the cells. The subsequent necrosis is evidently in chief part the result of the action of the toxic products of the bacilli, which 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 to be remarked that the number of the ulcers arising in the course of a case bears no relation to its severity. Small ulcers may occur in the lymphoid follicles of the large intestine. The mesenteric glands 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, and occasionally necrosis in patches occurs. Sometimes on section the glands are of a pale-yellowish colour, the contents being diffluent and consisting largely of leucocytes. Typhoid 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. 330 TYPHOID FEVER 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 (Fig. 110). 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 demon- strated. The bacillus is found, often in large numbers, in the gall-bladder, where it may persist for years. 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 solid organs which suggest the circulation of soluble poisons in the blood. In the lungs there may be bronchitis, patches of congestion and of acute broncho-pneumonia. In these, typhoid bacilli may sometimes be observed, but evidence of a toxic action depressing the powers of resist- ance of the lung tissue is found in the fact that the pneumococcus frequently occurs in such complications of typhoid fever. The nervous system shows little change, though meningitis associated either with the typhoid bacillus, with the b. coli, or with the streptococcus pyogenes has been observed. The typhoid bacilli probably travel by the blood stream, and they can be isolated from the blood much more readily than was formerly supposed. Considerable quantities of blood (say, 4 c.c.) must of course be taken (v. p. 68). They have been found in the roseolar spots which occur in typhoid fever, but it cannot be yet stated that such spots are always due to the presence of the bacilli. The fact that the typhoid bacilli are usually confined to certain organs and tissues shows that they probably 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 reaction is set up, and which in the intestine is sufficiently 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. The occurrence of bacilli in the blood and organs links typhoid fever with septicaemic processes. Suppurations occurring in connection with Typhoid Fever.— With regard to the relation of the typhoid bacillus to such conditions, 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 PATHOGENIC EFFECTS OF B. TYPHOSUS 331 organism found. This lias been the case in subcutaneous abscesses, in suppurative 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 micrococci, have been obtained, the typhoid bacillus having been searched for in vain. It has, moreover, been experimentally shown, notably by Dmochowski and Janowski, that suppuration 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, the former died out in the pusjnore quickly than the latter. Accordingly, in clinical cases where the typhoid bacillus is present alone, it is improbable that other organisms 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. Attempts to communicate the disease to animals by feeding them on typhoid dejecta have been unsuccessful, and though pathogenic effects have been produced by introducing pure cultures in food, the disease has usually borne no resemblance to human typhoid. The most successful experiments have been those of Remlinger, who, by continuously feeding rabbits on vegetables soaked in water containing typhoid bacilli, produced in certain cases symptoms resembling those of typhoid fever (diarrhoea, remittent pyrexia, etc.). An agglutinating action was observed in the serum, and post mortem there was congestion of the Peyerian patches, and typhoid bacilli were isolated from the spleen. While feeding experiments are thus rather unsatisfactory, the same may be said of the results of subcutaneous or intraperitoneal infection. Here, again, pathogenic effects can easily be produced by the typhoid bacillus, but these effects are of the nature of a short acute illness characterised by pyrexia, rapid loss of weight, inability to take food, and frequently ending fatally in from twenty-four to forty-eight hours. The type of disease is thus very different from what occurs naturally in man. In such injection experiments the results vary considerably, sometimes scarcely any effect being produced by a large dose of a culture. This is no doubt due to the fact that different strains of the bacillus 332 TYPHOID FEVER vary much, in virulence. Ordinary laboratory cultures are often almost non-pathogenic. They can, however, be made virulent in various ways. Sanarelli used the method of injecting sterilised cultures of the b. coli intraperitoneally at the same time as the typhoid bacillus was introduced subcutaneously. After this procedure had been repeated through a series of animals a culture of typhoid was obtained of exalted virulence. Sidney Martin has obtained virulent cultures by passing bacilli, derived directly from the spleen of a person dead of typhoid fever, through the peritoneal cavities of a series of guinea-pigs. Sanarelli, studying the effects of the intraperitoneal injection of a few drops of a culture of highly exalted virulence, found that the Peyer's patches and solitary glands of the intestine were enormously infiltrated, sometimes almost purulent, and that they contained typhoid bacilli, as also did the mesenteric lymphatics and glands, and the spleen. These results are interesting, but have not been confirmed. The Toxic Products of the Typhoid Bacillus. — Here very little light has been thrown on the pathology of the disease, but the general results may be outlined. We may state that there exist in the bodies of typhoid bacilli toxic substances, that in artificial cultures these do not pass to any great degree out into the surrounding medium, and that though they produce effects on the intestine, there is evidence that such effects are not character- istic and not peculiar to the toxins of the b. typhosus. Sidney Martin found that the bodies of bacteria killed by chloro- form vapour were very toxic, — more so than filtered cultures. Diarrhoea was a constant symptom after injection, but no change in the Peyerian patches was observed. Martin found that virulent cultures of the b. coli gave similar results when similarly treated. Allan Macfadyen, by grinding up typhoid bacilli frozen solid by liquid air, produced a fluid whose toxic effect he attributed to the presence of the intracellular poisons. The Immunisation of Animals against the Typhoid Bacillus. — Earlier observers had been successful in accustoming mice to the typhoid bacillus by the successive injections of small and gradually increasing doses of living cultures of the bacillus. Later, Brieger, Kitasato, and Wassermann found that the bacillus when modified by being grown in a bouillon made from an extract of the thymus gland no longer killed mice and guinea- pigs. These animals after injection were moreover immune, 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 PATHOGENICITY OF B. COLI 333 which it would naturally succumb. 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 subcutaneous doses of dead typhoid cultures in bouillon. Experiments performed with serum derived from .typhoid patients and convalescents indicate that similar effects occur in those who have successfully resisted the natural disease. The serum of such patients has antibacterial powers, but there is no evidence that it contains any antitoxic bodies (see chapter on Immunity). Pfeiffer, for example, found on adding serum from typhoid convalescents to the bodies of typhoid bacilli killed by heat, and injecting the mixture into guinea- pigs, that death took place as in control animals which had received these toxic agents alone. Pfeiffer also found that by using the serum of immunised goats, 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. The Pathogenicity of the B. coli and its Relation to that of the Typhoid Bacillus. — We have already seen that the b. coli is probably responsible for the occurrence of some of the abscesses which follow typhoid fever. It is also apparently the cause of some cases of summer diarrhoea (cholera nostras), of infantile diarrhoea, and of some food poisonings. Its numbers in the intestine are greatly increased during typhoid fever, and also during any pathological condition affecting the intestine. Intraperitoneal injection in guinea-pigs is often fatal. Sub- cutaneous injection may result in local abscesses, and sometimes in death from cachexia. Sanarelli found that the b. coli isolated from typhoid stools was much more virulent than when isolated from the stools of healthy persons. He holds that the increase in virulence is due to the effect of typhoid toxins. This increased virulence of the b. coli in the typhoid intestine makes it possible that some of the pathological changes in typhoid may be due, not to the typhoid bacillus, but to the b. coli. Some of the general symptoms may be intensified by the absorption of toxic products formed by it and by other organisms. It is to be noted that lesions produced in guinea-pigs are very similar to those of the b. typhosus. Differences of behaviour of the two bacilli in connection with their pathological effects have been brought forward as confirmatory of the fact of their being distinct species. Thus Sanarelli accustomed the intestinal mucous membrane of guinea-pigs to toxins derived from an old culture of the b. coli, by introducing day by day small quantities of the latter into the stomach. When a relatively large dose could be tolerated, it was found that the introduction in the same way of a small quantity of typhoid toxin was still followed by fatal result. Pfeiffer also found that while the serum of convalescents from typhoid paralysed the typhoid bacilli, it had no more effect on similar numbers of b. coli than the serum of healthy men. 334 TYPHOID FEVER General View of the Relationship of the B. typhosus to Typhoid Fever. — 1. 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 described, and further, it is found by culture and serum reactions to differ from other organisms. Here the important point 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. 2. A difficulty in the way of accepting the etiological relation- ship of the b. typhosus lies in the comparative failure of attempts to cause the disease in animals. We have noted, however, that in nature animals do not suffer from typhoid fever. 3. The observations of Pfeiffer and others on the protective 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 immobilising and causing clumping of the bacilli (vide infra) are also of great importance as indicating an etiological relationship between the bacillus and the disease. Additional important evidence is found in the fact that vaccination by means of the dead bacilli (vide infra) has a marked effect in preventing the disease from arising in a popula- tion exposed to infection, and also in lowering the mortality when the fever attacks those who have been inoculated. These facts may thus be accepted as indirect but practically conclusive evidence of the pathogenic relationships of the typhoid bacillus to the disease. According to our present results we must thus hold that the b. typhosus constitutes a distinct species of bacterium, and that it is the cause of typhoid fever. Evidence of an important nature confirmatory of this view is, we think, found in the fact that cases have occurred where bacteriologists have accidentally infected themselves by the mouth with pure cultures of the typhoid bacillus, and after the usual incubation period has developed typhoid fever. Several cases of this kind have been brought to our notice and are not, we think, vitiated by the fact that other similar instances have occurred without the subsequent development of illness. These latter would be accounted for by PARATYPHOID BACILLUS 335 a low degree of susceptibility on the part of the individual or to a want of pathogenicity in the cultures. The Paratyphoid Bacillus. — This organism, which was when first described often denominated the paracolon bacillus, was primarily isolated from abscesses occurring in apparently non- typhoid cases. Widal noted its resemblances to b. typhosus and b. coli, from the latter of which it differed in not producing indol and in not fermenting lactose. Gwynn first isolated it from the blood of a case presenting typhoid symptoms, and since then it has been recognised as being the probable cause of the disease effects in about 3 per cent of cases which clinically are to be described as typhoid fever. The case-mortality in paratyphoid fever is low, being only from 1 to 2 per cent. The organism has been isolated from the blood, the roseolar spots, and from the stools, — plates of MacCorikey's bile -salt neutral -red agar with 1 per cent lactose added being recommended for use here. Several strains showing slight differences in culture reactions have been obtained. Generally speaking, the cultural reactions resemble those of b. coli, though the growth on potato some- times presents typhoid characteristics. It produces no indol, or at least (with some strains) merely a trace, and its action on sugars also differs. With regard to the latter, different results have been obtained by different observers, but there is general agreement that, like the b. coli, it produces acid and gas in glucose, laevulose, sorbite, mannite, dextrin, maltose, dulcite, galactose, and arabinose; but in lactose, like typhoid, it either originates no change or only slight acid production without any gas formation. It also causes no change in cane sugar, erythrite, salicin, inulin, and raffinose. Probably the most important reactions which will in any case aid in the recognition of the paratyphoid bacillus are the agglutinating reactions. The serum of a paratyphoid patient will agglutinate the bacillus in high dilutions. Observations on the behaviour of such sera towards the b. typhosus have in different cases yielded some discordant results, but to speak generally, it may be said that usually a very much stronger concentration is necessary to give clumping, and often a paratyphoid serum will not clump the typhoid bacillus except in such concentrations as might give similar effects when normal sera are under observation. When any serum clumps both the paratyphoid and the typhoid bacilli the more closely the maximal clumping dilutions correspond, the more likely is the case to be typhoid fever ; on the other hand, if a high dilution will clump the paratyphoid bacillus, while a low dilution is necessary for the typhoid bacillus, then 336 TYPHOID FEVER the case is likely to be paratyphoid fever. With regard to the effects of other sera on the paratyphoid bacillus, it may be said that usually a typhoid serum will require to be used in greater concentrations to clump this bacillus than are necessary to obtain an effect with the typhoid bacillus itself. Similar effects are observed when the sera of animals immunised against Gaertner's bacillus or the bacillus of psittacosis are used. In all serum tests the essential point is that deductions should alone be based on comparative observations of the highest dilutions in which a clumping effect is produced with any series of organisms compared. As has been indicated, a disease resembling typhoid fever is not the only condition originated by the paratyphoid bacillus. The organism has been isolated from cases of bone abscess, from orchitis, and in WidaPs case from a thyroid abscess, and in such cases the history of a previous typhoid-like illness may not be elicited. It has also been found in ordinary faeces. In animal experiments it produces in rabbits and guinea-pigs a fatal ill- ness of a septicaemic type with serous inflammations. Bacillus Enteritidis (Gaertner).— In 1888 Gaertner, in investigating a number of cases of illness resulting from eating the flesh of a diseased cow, isolated, from the meat and from the spleen of a man who died, a bacillus closely resembling the typhoid bacillus. Since then a great number of outbreaks of gastro-enteritis due to eating diseased meat have been inquired into, and very frequently similar bacilli have been found both in the stools and in the organs. These bacilli closely resemble the paratyphoid organism, — indol is not produced, and generally speaking the fermentations of sugars also correspond. With regard to the latter it may, however, be said that, according to some, lactose is fermented, while other observers have found this not to be the case. No doubt different strains differ somewhat from one another. Here again much information may be obtained from the agglutinating properties of the serum and also from the effects on suspicious bacilli of the sera of animals immunised against other strains and other members of the coli group. It has also been found that the serum of persons suffer- ing from meat poisoning sometimes clumps the typhoid bacillus, though a higher concentration is required than in the case of Gaertner's bacillus. The Gaertner group of organisms is very pathogenic for laboratory animals. Often, whatever the channel of infection, there is intense haemorrhagic enteritis, and very usually there is a septicaemia with the occurrence of serous inflam- mations ; the bacilli are recoverable from the solid organs and PSITTACOSIS BACILLUS 337 often from the blood. In man, as the name of the bacillus indicates, the symptoms are centred in the intestine, where there is usually marked inflammation of the mucous membrane, some- times attended with haemorrhage into it ; evidence of a septicaemic condition may also exist. Infection may take place by the bacillus itself where meat has been insufficiently cooked or merely pickled, and here the illness usually appears within twenty-four hours of the food being partaken of, but symptoms may appear almost at once, in which case they are no doubt due to the action of toxins ; here it is important to note that the poisons formed by this group of organisms are relatively heat- resisting, so that boiling for a time does not destroy the toxicity. In cases of Gaertner bacillus poisoning, the animal whose carcase is suspected has usually been found to have been itself suffering from the action of the bacillus, but cases of meat poisoning also occur where the meat of a healthy animal becomes infected subsequently to slaughter with organisms pathogenic to man. In such cases these organisms are often varieties of the b. coli group, and indeed the b. coli itself may be the cause of meat poisoning. The Psittacosis Bacillus. — When parrots are imported from the tropics in large numbers many die of a septicaemia condition in which an enteritis, it may be hsemorrhagic, 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 gelatin, does not ferment lactose, does not curdle milk, and gives no indol reaction. Culturally the organism is practically indistinguishable from the two bacilli last described. The parrot is most susceptible to its action, but it also causes a fatal hsemorrhagic septicaemia in guinea-pigs, rabbits, mice, pigeons, and fowls, the bacilli after death being chieny 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 in- vestigated in Paris. After about ten days' incubation, headache, fever, and anorexia occur, followed by great restlessness, delirium, vomit- ing, 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. The Serum Diagnosis of Typhoid Fever. — This method of 22 338 TYPHOID FEVER diagnosis is based on the fact that living and actively motile typhoid bacilli, if placed in the diluted serum of a patient suffer- ing from typhoid fever, within a very short time lose their motility and become aggregated into clumps. We shall find (see Immunity) 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 in the case of the typhoid bacillus we do not know the true interpretation of some of the facts which have been observed. The methods by which the test can be applied have already been described (p. 109). (1) It will be there seen that the loss of motility and clumping may be observed microscopically. If a preparation be made by the method detailed (typhoid serum in a dilution of, say, 1 : 30 having been employed), and examined at once under the micro- scope, the bacilli will usually be found actively motile, darting about in all directions. In a short time, however, these move- ments 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 applying which has also been described (p. 111). Here at the end of twenty-four hours the bacilli form a mass like a precipitate 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 shows 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. Such is what occurs in the case of a typical reaction. The value of the method as a means of diagnosis largely depends on attention to several details. 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. A race must therefore be selected SERUM DIAGNOSIS 339 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. Continued growth of a race at 37° C. makes it less suitable for use in the test, as the bacilli tend naturally to adhere in clumps, which may be mistaken for those produced by the reaction. Wyatt Johnson recommended that the stock culture should be kept growing on agar at room temperature and maintained by agar sub-cultures made once a month. For use in applying the test, bouillon sub-cultures are made and incubated for twenty-four hours at 37° C. The relation of the dilution of the serum to the occurrence of clumping is most important. It has been found that if the degree of dilution be too small a non-typhoid serum may cause clumping. If possible, observations should always be made with dilutions of 1 : 10, 1 : 30, 1 : 60, 1 : 100. To speak generally, the more dilute the serum the longer time is necessary for a complete reaction. Some typhoid sera have, however, very powerful agglutinating properties, and may in a compara- tively short time produce a reaction when diluted many hundreds of times. With a too dilute serum not only may the reaction be delayed, but it may be incomplete, — the clumps formed being small and 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 incomplete clumping alone. Seeing that low dilutions sometimes give a reaction with non-typhoid sera, it is important to know what is the highest dilution at which complete clumping indicates a positive reaction. The general consensus of opinion, with which our own experience agrees, is (that when a serum in a dilution of 1 : 30 causes complete clumping in half an hour, it may safely be said that it has been derived from a case of typhoid feverj Suspicion should be entertained 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 becomes gradually more marked as the disease advances, and it is still given by the blood of convalescents from 340 TYPHOID FEVER typhoid, but cases occur in which it may permanently disappear before convalescence sets in. How long it lasts after the end of the disease has not yet been fully determined, but in many cases it has been found after several months or longer. As a rule, up to a certain point, the reaction is more marked where the fever is of a pronounced character, whilst in the milder cases it is less pronounced. In certain grave cases, however, the reaction has been found to be feeble or almost absent, and accordingly some hold that (a feeble reaction when the disease is manifestly severe is of bad omenj In some cases which from the clinical symptoms were almost certainly typhoid, the reaction has apparently been found to be absent. Such cases should always be investigated from the point of view of their possibly being paratyphoid fever. 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 by heating at 60° C. for an hour, — if a higher temperature be used, sensitiveness to agglutination is impaired. The capacity is also still retained if a germicide be employed. Here Widal recommends the addition of one drop of formalin to 150 drops of culture. The reaction, however, tends to be less complete. 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 ; sometimes, however, the foetal blood gives 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 tem- peratures, however, cause the property to be lost. The Agglutination of Organisms other than the B. Typhosus by Typhoid Serum. — It was at first thought that' the reaction in typhoid fever would afford a reliable method of distinguishing the typhoid bacillus from the b. coli. Though 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 incomplete.' It has also been found that other organisms belonging to the typhoid group (v. p. 335) react in a similar way. The reaction as a method of distinguishing between these forms is thus not absolutely reliable, but in certain cases it is of great value in giving confirmation to other tests. The im- portant point here is the determination of the highest dilution SEBUM DIAGNOSIS 341 with which clumping is obtained. There 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 circum- stances may have on its subsequent sensitiveness to agglutination by typhoid serum. Again, Christophers has pointed out that a large proportion of sera from normal persons or from those suffering from diseases other than typhoid will clump the b. coli in dilutions of from 1 : 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 latter bacillus. 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 of clumping typhoid bacilli ; there may have been an attack 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 men have not been recognised. The very wide application of the reaction has elicited the fact that it is given in many cases of slight, transient, and ill -denned febriculae, which occur especially when typhoid fever is prevalent. Some of these may be aborted typhoid, some may be paratyphoid. There is no doubt that, if all the facts are taken 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 conclusion here we may say 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. In connection with the phenomenon that a serum either from a normal person or a typhoid patient may clump several varieties of bacteria, some points arise. The theoretical consideration of agglutination is reserved for the chapter on Immunity, but here it may be said that agglutinating properties may be present 342 TYPHOID FEVER normally in a serum or they may be originated by an animal being infected with a particular bacterium. As the result of injecting a bacterium not only may agglutinins capable of acting on that bacterium appear in the serum, but the serum may become capable of agglutinating other, and especially kindred, bacteria ; further, any normal agglutinins for the infecting bacterium present in the serum may be increased in amount. The agglutinin acting on the infecting organism has been called the primary or homologous agglutinin, while the others have been called the secondary or heterologous agglutinins. But besides what we know to be a fact, that infection by a single bacillary species can originate agglutinins acting both on itself and on allied species, we must consider the possibility of infections by more than one species occurring in an animal, e.g. b. typhosus with b. coli or with b. paratyphosus. In such a case each organism may originate its primary agglutinin so that the presence of multiple agglutinins in a serum may really be an indication of a mixed infection. Some attention has been directed to the diagnosis and differentiation of these conditions. Castellani has introduced a method for their investigation. This depends on the capacity manifested by bacteria of absorbing the ag- glutinins from a serum. A small quantity of the agglutinating serum, say '5 c.c., is taken either pure or diluted with bouillon, there are added 4 to 8 loops of an agar culture of the germ which originated it, the mixture is well shaken and set at 37° C. for 12 hours. Clumping of course occurs, and the clumps fall to the bottom of the tube. The supernatant fluid is pipetted off and is available for further tests. Castellani studied the primary and secondary agglutinins produced in infections in rabbits ; he found that when an animal had been infected with b. typhosus this organism would absorb from its serum not only the primary typhoid agglutinins but also such secondary agglutinins as those acting on the b. coli. If, however, an animal had undergone infection with, say, both the b. typhosus and the b. coli, then the b. typhosus could not absorb from its serum the b. coli (primary) agglutinin. Castellani thus put forward the view that by this means primary could be differentiated from secondary agglutinins, and therefore pure could be differentiated from mixed infections. There is little doubt that this view possesses considerable validity, though it is probably not of universal applicability. Safe deductions can only be drawn when any serum is tested with several species of fairly closely related organisms, such as those of the coli group. Especially is it necessary that the highest dilutions in which agglutination occurs should be compared. If VACCINATION AGAINST TYPHOID 343 such precautions be adopted the absorption method can be util- ised for the differentiation of the typhoid and paratyphoid organ- isms and their infections and for similar investigations. Vaccination against Typhoid. — The principles of the im- munisation 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 sloped agar culture when administered hypo- dermically will kill a guinea-pig of from 350 to 400 grammes) Vaccination can be accomplished by such a culture emulsified in bouillon, and killed by heating for five minutes at 60° C. For use, from one-twentieth to one-fourth of the dead culture is injected hypodermically, usually in the flank. The vaccine now used, however, actually consists of a portion of a bouillon culture similarly treated. The effects of the injection are some tenderness locally and in the adjacent lymphatic glands, and it may be local swelling, all of which come on in a few hours, and may be accompanied by a general feeling of restlessness and a rise of temperature, but the illness is over in twenty-four hours. During the next ten days the blood of the individual begins to manifest, when tested, an agglutination reaction, and further, Wright has found that' usually after the injection there is a marked increase in the capacity of the blood serum to kill the typhoid bacillus in vitro. There is little doubt that these observa- tions indicate that the vaccinated person possesses a degree of immunity against the bacillus, and this conclusion is borne out by the results obtained in the use of the vaccine as a prophy- lactic against typhoid fever. Extensive observations have been made in the British army in India, and in the South African War the efficacy of the treatment was put to test. Though in isolated cases not much difference was observed among those treated as compared with those untreated, yet the broad general result may be said to leave little doubt that on the one hand protective inoculation diminishes the tendency for the individual to contract typhoid fever, and on the other, if the disease be contracted, the likelihood of its having a fatal result is diminished. Thus in India of 4502 soldiers inoculated, '98 per cent contracted typhoid, while of 25,851 soldiers in the same stations who were not inoculated, 2 '54 per cent took the disease. In Ladysmith during the siege there were 1705 soldiers inoculated, among whom 2 per cent of cases occurred, and 10,529 uninoculated, among whom 14 per cent suffered from typhoid. Wright has collected statistics dealing in all with 49,600 individuals, of whom 8600 were inoculated, and 344 TYPHOID FEVER showed a case incidence of 2'25 per cent, with a case mortality of 12 per cent; in the remaining 41,000 uninoculated the case incidence was 5 '75 per cent and the case mortality 21 per cent. The best results seemed to be obtained when ten days after the first inoculation a second similar inoculation is practised. Wright has found that in certain cases immediately after inoculation there is a fall in the bactericidal power of the blood (negative phase), and he is of opinion that this indicates a temporary increased susceptibility to the disease. He therefore recommends that when possible the vaccination should be carried out some time previous to the exposure to infection. There can be very little doubt that in this method an important prophy- lactic measure has been discovered. Antityphoid Serum. Chantemesse lias immunised animals with dead cultures of the typhoid bacillus, and having found that their sera had protective and curative effects in other animals, has used such sera in human cases of typhoid with apparent good result. In the hands of others, however, such a line of treatment has not been equally successful. 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, provided the needle be not too large, the procedure is quite safe. Its use, however, is scarcely called for. (b) From the Urine. — Typhoid bacilli are present in the urine in at least 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 suspected urine, see p. 69. (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 MacConkey's lactose bile METHODS OF EXAMINATION 345 salt neutral-red agar, or in the medium of Drigalski and Conradi (v. p. 42). After that period, though the continued infectiveness of the disease indicates that they are still present, their isolation is very difficult. We have seen that after ulceration is fairly estab- lished by the sloughing 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. The microscopic examination of the stools is of course 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 be much more difficult to do so when the latter are enormously diluted by water. The b. typhosus has, how- ever, been isolated from water during epidemics. This 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 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, but the Conradi- Drigalski or MacConkey media may be employed with advantage. Klein 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. From time to time various substances havebeen used with the object of inhibiting the growth of the b. coli without interfering with that of the b. typhosus. Most of these have not stood the test of experience. Lately caffeine has been used for this end. For use in examining waters the following is the method employed. To 900 c.c. of the suspected water there are added 10 grammes nutrose dissolved in 80 c.c. of sterile water, and 5 grammes of caffeine dissolved in sterile distilled water heated to 80° C. and cooled to 55° C. before addition. After mixing the ingredients there is added 10 c.c. of '1 per cent crystal violet. The flask is incubated at 37° C. for 12 hours and then plates of Conradi-Drigalski medium are inoculated from it. For investigation of faeces, a medium made up as above but with ordinary sterile water may be infected and a similar procedure followed. On the whole there is little to be gained from this attempt to isolate the typhoid bacillus from water in any particular case, and it is much more useful for the bacteriologist to bend his energies towards the obtaining of the indirect evidence of contamination of water by sewage, to the nature of which attention has been called in Chap. IV. 346 TYPHOID FEVER BACTERIA IN DYSENTERY. Dysentery has for long been recognised as including a number of different pathological conditions, and within more recent times amoebic and non-amoebic forms have been distinguished. Of the latter bacteria have been believed to be the causal agents, and an organism described by Shiga in 1898 has almost certainly been established as the cause of a large proportion of cases. Shiga's observations were made in Japan, and confirmatory results have been obtained by Kruse in Germany, by Flexner and by Strong and Harvie in the Philippine Islands, and more recently by Vedder and Duval in the United States. It is now further recognised that the epidemics of dysentery which from time to time occur in lunatic asylums are usually due to bacilli of this type, and in America the organism has been demonstrated in the summer diarrhoea of children. The evidence for the relationship of the organism to the disease consists chiefly in the apparently con- stant presence of the organism in the dejecta in this form of dysentery, and the agglutination of the organism by the serum of patients suffering from the disease, but confirmatory evidence has also come from animal experimentation. From different epidemics a great many different strains of the dysentery bacillus have been obtained, but these all possess common characters and are undoubtedly closely related to one another. The various strains resolve themselves into two chief groups, whose differences lie in their behaviour towards certain sugars, in their capacities of originating indol and in their agglutinating reactions. The relation of amcebaB to dysentery will be discussed in the Appendix. Bacillus Dysenteriae (Shiga}. — Morphological Characters. — This bacillus morphologically closely resembles the typhoid bacillus, but is on the whole somewhat plumper, and filamentous forms are comparatively rare. Involution forms sometimes occur, especially in glucose agar. Most observers have found no trace of motility, whilst others say that it is slightly motile. Vedder and Duval have, however, by a modification of Van Ermengen's process, demonstrated in the case of one strain the presence of numerous lateral flagella, which are of great fineness, but of considerable length. No spore formation occurs ; the organism is stained readily by the ordinary dyes, but is decolorised by Gram's method. Cultural Characters. — In these also considerable resemblance is presented to the typhoid bacillus. In gelatin a whitish line of growth occurs along the puncture, but the superficial film-like BACILLUS DYSENTERIC 347 growth is usually absent, or at least poorly marked. In plate cultures also the superficial growths are smaller and have less of the film-like character, than those of the typhoid organism. On agar, growth occurs as a smooth film with regular margins, but after two or three days, especially if the surface be moist, Vedder and Duval describe an outgrowth of lateral offshoots on the surface of the medium. On agar plates the colonies resemble those of the typhoid organism, being of smaller size and less opaque than those of the bacillus coli. 4^ peptone bouillon a uniform haziness is produced. As has been indicated, different strains of the bacillus behave differently towards different sugars, and the results of all observers do not agree, so that only general statements can be made. Without going into the question of the particular strains to be placed in the two groups we may say that, roughly, these may be classified into the Shiga-Kruse group and the Flexner group. All produce acid in peptone-glucose and in taurocholate peptone-glucose, none produce change in lactose or cane-sugar. The Shiga group do not produce acid in maltose or mannite, while the Flexner group do, and, generally speaking, the former do not produce indol while the latter do. Forms inter- mediate between the two groups occur. There is never any evolution of gas observed in sugar media. In litmus milk there is developed at first a slight degree of acidity, which is followed by a phase of increased alkalinity ; no coagulation of the milk ever occurs. On potato the organism forms a transparent or whitish layer, which, however, in the course of a few days assumes a brownish-red or dirty grey colour, with some discoloration of the potato at the margin of the growth. Relation to the Disease. — The organism has been found in large numbers in the dejecta, especially in the acute cases, where it may be present in almost pure culture. In the thirty-six cases examined, Shiga obtained it in thirty-four from the dejecta, and in the two others post mortem from the intestinal mucous membrane. The organism does not appear to spread deeply or to invade the general circulation. In the more chronic cases it is difficult to obtain on account of the large number of the bacillus coli and other bacteria present. Vedder and Duval found agar plates to be the best method of culture, these being incubated at the blood temperature. They also found that if the colonies which appeared at twelve hours were marked with a pencil, there was a greater probability of obtaining the bacillus of dysentery from those which appeared later, most of those appearing early being colonies of the bacillus coli. MacConkey's agar medium with lactose added may be used for isolation from stools. A little of 348 TYPHOID FEVER the faeces is rubbed up in broth and some of the mixture stroked on the medium. The formation of acid by the coli colonies enables them to be excluded, and, therefore, as the b. dysenteriae is not a lactose f ermenter, the colourless colonies which develop after twenty-four hours are picked out for further investigation. As already stated, both acute and chronic cases are marked by the presence of this organism. In the former, where death may occur in from one to six days, the chief changes, according to Flexner, are a marked swelling and corrugation of the mucous membrane, with haemorrhage and pseudo-membrane at places. There is extensive coagulation-necrosis with fibrinous exuda- tion and abundance of polymorpho-nuclear leucocytes, and the structure of the mucous membrane, as well as that of the muscularis mucosae, is often lost in the exudation. There is also great thickening of the sub-mucosa, with great infiltration of leucocytes, these being chiefly of the character of " plasma cells." In the more chronic forms the changes correspond, but are more of a proliferative character. The mucous membrane is granular, and superficial areas are devoid of epithelium, whilst ulceration and pseudo-membrane are present in varying degree. A feature of bacillary dysentery is the fact that abscess of the liver does not occur as a complication. Agglutination. — All the above-mentioned observers agree re- garding the agglutination of this bacillus by the serum — that is, in the cases of dysentery from which the organism can be cul- tivated. The reaction may appear on the second day, and is most marked after from six to seven days in the acute cases ; it is usually given in a dilution of from one in twenty to one in fifty within an hour, though sometimes much higher dilutions give a positive result. In the more chronic cases the reaction is less marked, and here the sedimentation method is to be preferred. It is difficult to make any general statements with regard to the effects of dysenteric sera on the different strains of the bacilli, but it may be said that generally a serum agglutinates the strain which produced it and the other strains of the same group in higher dilutions than it does the strains of the other group. Many observers have found that the serum from a case associated with strains of the Shiga-Kruse group have not agglutinated strains of the Flexner group, and corresponding observations have been made in cases associated with the Flexner group. Often the sera of animals immunised with bacilli have been used for such tests, but apparently great care must be exercised in basing diagnoses on such observations, for some species of animals when treated with a particular strain BACILLUS DYSENTERIC 349 will yield a serum which is active against many more strains than other species will when immunised with that strain. Agglutination of the organism has not been obtained with serum from cases other than those of dysentery, nor has a similar bacillus been cultivated from other such sources. The reaction is also absent in those cases of dysentery which are manifestly of amoebic nature. Pathogenic Properties. — The organism is pathogenic in guinea- pigs and other laboratory animals, but, in these, characteristic changes in the intestine are often awanting. Shiga, however, obtained such effects by introducing the organism into the stomach of young cats and dogs, and confirmatory results were obtained by Flexner. Such attempts have been specially successful when the virulence of the organism has been previously exalted by intraperitoneal passage. In two cases, apparently well authenticated, a dysenteric condition has followed in the human subject from ingestion of pure cultures of the organism. It is probable that in the action of the bacillus a toxin is concerned. It has been found that the nitrate from three weeks' old cultures in alkaline bouillon is very toxic to animals, especially rabbits, and that, however introduced into the body, it causes a haBmorrhagic enteritis with a diphtheritic-like exudate on the surface of the mucous membrane. According to some obser- vers the toxin is more readily obtainable from the Shiga-Kruse strains than from the Flexner strains. The toxin is fairly resist- ant to heat, standing temperatures up to 70° C. without being injured. From the fact, that by the maceration of cultures whose filtrates are relatively non-toxic a stronger poison can be obtained, the dysentery toxin has been thought to be an endotoxin, but on this point no definite opinion can be expressed. Immunisation Experiments. — Both large and small animals have been immunised against the bacillus and also against its toxic filtrates. In the former case the immunisation has been commenced either with non-lethal doses of living cultures or with cultures killed by heat. The nature of the immunisation is probably complex. When cultures have been used, a bactericidal serum, in which immune bodies and complements (vide Immunity) are concerned, is developed. When the toxin is used for immun- isation a serum protecting against the toxin is produced. Ac- cording to some results animals immunised with cultures are immune against the toxin, and vice versa. All races of animal do not lend themselves to immunisation. Large animals (horses, goats) have been immunised with the toxin with a view of ob- taining sera for use in human dysentery, and in certain cases, 350 TYPHOID FEVER notably in the work of Rosenthal, a distinct therapeutic effect has been produced by the subcutaneous administration of the serum, especially in early cases of the disease. It will be seen that the evidence furnished is practically conclusive as to the causal relationship between this bacillus and one form of dysentery, a form, moreover, which is both wide- spread and embraces a large proportion of cases of the disease ; and especially of importance is the fact that observations made independently in different countries have yielded practically identical results on this point. Bacillus Dysenteric (Ogata). — Ogata obtained this bacillus in an extensive epidejnic in Japan in which no amoebae were present. He found in sections of the affected tissues enormous numbers of small bacilli of 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 pathogenic effects. They grew well on gelatin, 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, hsemorrhagic inflammation and ulceration being produced. It still remains to be determined whether this organism has a causal relation- ship to one variety of dysentery. BACILLUS ENTERITIDIS SPOKOGENES. This organism was first isolated by Klein from the evacuations in an outbreak of diarrhoea following the ingestion of milk which contained the microbe, and it was subsequently found by him in certain cases of infantile diarrhoea and of summer diarrhoea, in certain instances in milk, and as a constant inhabitant of sewage (see Chap. IV.). In films made from the stools in diarrhoea cases where it is present it can be micro- scopically recognised as a bacillus 1 "6 /a to 4 '8 ^ in length and '8 n in breadth, staining by ordinary stains and retaining the dye in Gram's method. It often contains a spore near one of the ends, or sometimes nearer the centre. It is slightly motile, and in cultures can be shown to possess a small number of terminal flagella. It grows well under anaerobic conditions in ordinary media, especially on those containing reducing agents. On agar the colonies are circular, grey, and translucent, and under a low power are seen to have a granular appearance. On this medium spore formation does not occur, but is easily obtained if the organism is grown on solidified blood serum, which, further, is liquefied during growth. On gelatin plates liquefaction commences after twenty- four hours at 20° C. It produces acid and gas in bile-salt glucose media, and in peptone salt solution containing glucose or mannite. Spore formation can be seen to take place in 2 per cent dextrose gelatin, but the degree seems to be in inverse ratio to the amount of gas formation. Very typical is the growth on milk, and it is by this medium that isolation can be best effected. A small quantity of the material SUMMER DIARRHCEA 351 suspected to contain the bacillus is placed in 15 to 20 c.c. of sterile milk, which is then heated for ten minutes at 80° C. to destroy all vegetative bacteria ; the tube is cooled, placed under anaerobic con- ditions, and incubated at 37° C. for from twenty-four to thirty-six hours. If the bacillus be present there is abundant gas formation, and almost complete separation of the curd from the whey takes place. The former adheres to the sides of the tube in shreds, and large masses gather with the cream on the top of the fluid, all being torn by the gas evolved. The whey is only slightly turbid and contains numerous bacilli. The growth has an odour of butyric acid. If a small quantity (say 1 c.c.) of the whey be injected into a guinea-pig, the animal becomes ill in a few hours and dies in twenty-four hours. At the point of inoculation the skin and subcutaneous tissues, and sometimes even the subjacent muscles, are green and gangrenous and evil-smelling, there is considerable oedema, and there may also be gas formation. The exudation is crowded with bacilli, which, however, are not generally distributed in any numbers throughout the body. These pathogenic properties of the bacillus enteritidis sporogenes are important in its recognition, for its culture reactions taken alone are very similar to those of the bacillus butyricus of Botkin. SUMMER DIARRHCEA. As has been already stated, both the bacillus of dysentery, b. coli and the b. enteritidis sporogenes have been found associated with epidemics of this disease. This indicates that the condition may be originated by a variety of organisms, and it is further probable that the clinical features in different epidemics vary. The multiple origin of the disease has been illustrated by the work of Morgan, who, in a careful investigation of the disease in Britain, has been unable to find evidence of the dysentery bacillus being present. He has, however, very constantly found in the stools and intestine a bacillus (" Morgan's No. 1 bacillus") which is a motile Gram-negative organism producing acid and slight gas formation in glucose, Isevulose, and galactose, and no change in mannite, dulcite, maltose, dextrin, cane sugar, lactose, inulin, amygdalin, salicin, arabinose, raffinose, sorbite, or ery- thrite ; it further causes indol formation, and in litmus milk slowly originates an alkaline reaction. It produces diarrhoea and death in young rabbits, rats, and monkeys when these animals are fed on cultures. It is thus possible that in this bacillus we have still another cause of the disease. CHAPTER DIPHTHERIA. THERE is no better example of the valuable contributions of bacteriology to scientific medicine than that afforded in the case of diphtheria. Not only has research supplied, as in the case of tubercle, a means of distinguishing true diphtheria from condi- tions which resemble, it, but the study of the toxins of the bacillus has explainer! the manner by which the pathological changes and characteristic symptoms of the disease are brought about, and has led to ttie discovery of the most efficient means of treatment, namely, the an ti- diphtheritic serum. Historical. — The first account of the bacillus now known to be the cause of diphtheria was given by Klebs in 1883, who described its characters in the false membrane, but made no cultivations. It was first cultivated by Loffler from a number of cases of diphtheria, his observations being published in 1884, and to him we owe the first account of its characters in cultures and of some of its pathogenic effects on animals. The organism is for these reasons known as the Klebs- Loffler bacillus, or simply, as Loffler's bacillus. By experimental in- oculation with the cultures obtained, Lb'rfler was able to produce false membrane on damaged mucous surfaces, but he hesitated to conclude definitely that this organism was the cause of the disease, for he did not find it in all the cases of diphtheria examined, he was not able to produce paralytic phenomena in animals by its injection, and, further, he obtained the same organism from the throat of a healthy child. This organism became the subject of much inquiry, but its relationship to the disease may be said to have been definitely established by the brilliant researches of Roux and Yersin which showed that the most important features of the disease could be produced by means of the separated toxins of the organism. Their experiments were published in 1888-90. 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 patho- logical changes in diphtheria, it will be well to mention the out- 352 BACILLUS DIPHTHERIA 353 standing 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 toxins. Other bacteria are, however, concerned in producing various secondary inflammatory complications in the region of the throat, such as ulceration, gangrenous change, and suppuration, which may be accompanied by symptoms of general septic poisoning. The detec- tion of the bacillus of Loffler in the false membrane or secretions of the mouth is to be regarded as supplying the only certain / means of diagnosis of diphtheria. * Bacillus Diphtherias. — Microscopical Characters. — If a film preparation be made from a piece of diphtheria membrane (in the manner described below) and stained with methylene-blue, the bacilli are found to have the following characters. They are slender rods, straight or slightly curved, and usually about 3 //. in length, their thickness being a little greater than that of the tubercle bacillus. The size, however, varies somewhat in different cases, and for this reason varieties have been distin- guished 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 p. Corre- sponding differences in size are found in cultures. 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. 116). 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 com- pared side by side. They usually lie irregularly scattered or in clusters, the individual bacilli being disposed in all directions. Some may be contained within leucocytes. They do not form 23 DIPHTHERIA 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 Bacillus. — The diphtheria bacillus 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. < W, i^^^ff-^m. *H f •* * ik Fro. 116.— Film preparation from diphtheria membrane ; showing 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 pure condition.) Stained with nwthylene-blne. x 1000. It may be mentioned that distinctions formerly drawn between true diphtheria and non-diphtheritic conditions from the appear- ance and site of the membrane, have no scientific value, the only terue criterion being the presence of the diphtheria bacillus. The occurrence of a membranous formation produced by streptococci has already been mentioned (p. 184). In diphtheria the membrane has a somewhat different structure according as it is formed on the surface covered with stratified squamous epithelium as in the pharynx, or on a surface DISTRIBUTION OF THE BACILLUS 355 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 fibriuous exudation. The necrosed epithelium becomes raised up by the fibrin, and its interstices are also filled by it. The FIG. 117. — Sectiou through a diphtheritic membrane in trachea, showing diph- theria 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. fibrinous exudation also occurs around the vessels in the tissue beneath, and in 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 different parts. The membrane lies upon the base- ment membrane, and is less firmly adherent than in the case of the pharynx 356 DIPHTHERIA 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, towards the superficial, that is, usually, the oldest part of the false membrane (Fig. 117). There they may be in a practically pure condition, though streptococci and occasionally 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 accompanied by numerous other organisms. Occasionally a few bacilli have been detected in the lymphatic glands. As Lo'fner first described, they may be found after death in pneumonic patches in the lung, these being due to a secondary extension by the air passages. They have also been occasionally found 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. The diphtheria bacillus may also infect other mucous membranes. It is found in true diphtheria of the conjunctiva, and may also occur in similar affections of the vulva and vagina : some of these cases have been treated successfully with diphtheria antitoxin. The pseudo-diphtheria bacillus, however, may also occur in these situations. Association with other Organisms. — The diphtheria organism is sometimes present alone in the membrane, but more frequently is associated with some of the pyogenic organisms, the strepto- coccus pyogenes being .the commonest. The staphylococci, and occasionally the pneumococcus 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 inflammation 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 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 the action of pyogenic organisms. The extensive swelling of the tissues of the neck, sometimes attended by CULTIVATION OF THE BACILLUS 357" FIG. 118. — Cultures of the diphtheria bacillus on an agar plate ; twenty - six hours' growth. (a) Two successive strokes ; (6) isolated colonies from plate. suppuration in the glands, and also various hsemorrhagic con- ditions, have been found to be as- sociated 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 exten- sive 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 complica- tions produced by other organisms anti-diphtheritic serum produce no favourable effect. Cultivation. — The diphtheria bacillus grows best in cultures at the temperature of the body; growth still takes place at 22° C., but ceases about 20° C. The best media are the following : Loffler's original medium (p. 40), solidified blood serum, alkaline blood serum (Lorrain Smith), blood agar, and ^^iJHlJ^^fe^ the ordinary agar media. If inoculations be made on the surface of blood serum with a piece of diphtheria membranes, colonies of the bacillus *^\ 5^ may appear in twelve \ 5 hours and are well formed «<* .^ *** < I within twenty-four hours IgjNiq^j^ often before any other ^J^jSK growths are visible^ The T§\» Jv?A» 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 mm. in size, but when numerous they FIG. 119.— Diphtheria bacilli from a twenty- four hours' culture on agar. Stained with methylene-blue. x 1000. day they may reach 3 358 DIPHTHERIA remain smaller. •••n Fig. 120. — Diphtheria bacilli of larger size than in previous figure, showing also ir- regular staining of protoplasm. From a three days' agar -culture. Stained with weak carbol-fuchsin. x 1000. On the agar media the colonies have much the same appearance (Fig. 117) but grow less quickly, and sometimes they may be comparatively minute, so as rather to resemble those of the streptococcus pyogenes. In stroke cul- tures the growth forms a continuous layer of the same dull whitish colour, the margins of which often show single colonies partly or completely separated. On gelatin at 22° 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 liquefac- the organism produces a turbidity tion occur. In bouillon which soon settles to the bottom and forms a pow- dery layer on the wall of the vessel. By starting the growth on the surface and keeping the flasks at rest a distinct scum forms, and this is especially suit- able for the development of toxin. 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. 75) the acid reaction Fig. 121.— Involution forms of the diphtheria does not occur. In these media the bacilli show the same characters as in the membrane, but the irregularity in staining is more marked bacillus ; from an agar culture of seven days' growth. Stained with carbol-thionin-blue. x 1000. POWERS OF RESISTANCE OF BACILLUS 359 (Figs. 119, 120). They are at first fairly uniform in size and shape, but later involution forms are present. Many are swollen at their ends into club-shaped masses which stain deeply, and the protoplasm becomes broken up into globules with unstained parts between (Fig. 121). Some become thicker throughout, and segmented so as to appear like large cocci, and others show globules at their ends, the rest of the rod appearing as a faintly- stained line. Occasionally branched forms are met with. The bacilli are non-motile, and do not form spores. Staining. — They take up the basic aniline dyes, e.g. methy- lene-blue in watery solution, with great readiness, and stain deeply, the granules often giving the metachromatic reaction as described. They also retain the colour in Gram's method, though they are more easily decolorised than the pyogenic cocci. By Neisser's stain (p. 108) the granules are stained almost black, the rest of the bacillary substance yellowish brown. Powers of Resistance, etc. — In cultures the bacilli possess long duration of life ; at the room temperature they may survive for two months or longer. In the moist condition, whether in cultures or in membrane, they have a low power of resistance, being killed at 60° 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 98° 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 contagium of diphtheria may be preserved for a long time in the dried membrane. Effects of Inoculation. — In considering the effects produced 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. As Loffler stated in his original paper inoculation of the healthy mucous membranes of various animals with pure cultures causes no lesion, but the formation of false membrane may result when the surface is injured by scarification or otherwise. A similar result may be obtained when the trachea is inoculated after tracheotomy has been performed. In this case the surrounding tissues may become the seat of a blood-stained 360 DIPHTHERIA oedema, and the lymphatic glands become enlarged, the general picture resembling pretty closely that of lar.yngeal diphtheria. The membrane produced by such experiments is usually less firm than in human diphtheria, and the bacilli in the membrane are less numerous. 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. At the site of inoculation there is usually a small patch of greyish membrane, whilst in the tissues around there is extensive inflammatory oedema, often associated with haemorrhages, and there is also some swelling of the corresponding lymphatic glands. The internal organs show general congestion, the suprarenal capsules being especially reddened and often showing haemorrhage, The renal epithelium 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 practically local, cultures made from the blood and internal organs giving usually negative results, though sometimes a few colonies may be obtained. 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 proportionately larger. Roux and Yersin found that -after intravenous injection the bacilli rapidly disappeared from the blood, and when 1 c.c. of a broth culture had been injected no trace of the organisms could be detected by culture after twenty-four hours : nevertheless the animals died with symptoms of general toxaemia, nephritis also being often present (cf. Cholera, p. 408). 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. At the time of death the diphtheria bacilli were still alive and virulent at the site of injection. The most striking result of 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 TOXINS OF DIPHTHERIA 361 the contagion was apparently carried by the milk. Other observers have, however, failed to obtain similar results. Dean and Todd, in investigating an outbreak of diphtheria traceable to milk supplied, found a vesicular eruption on the teats of the udder in which diphtheria bacilli were present. They, however, came to the conclusion that these bacilli were not the cause of the eruption, but were the result of a secondary contamination, probably from the saliva of the milkers. The occurrence of a true infection with the diphtheria bacillus in the horse has been described by Cobbett. The Toxins of Diphtheria. — As in the above experiments the symptoms of poisoning and ultimately a fatal result occur 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 toxins, 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 nitrate when injected into guinea-pigs and other animals produces practically the same effects as the living bacilli ; locally there is fibrinous exudation but a considerable amount of inflammatory cedema, and, if the animal survive long enough, necrosis in varying degree of the superficial tissues may follow. The toxicity may be so great that "01 c.c. or even less may be fatal to a guinea-pig in twenty-four hours. After injection either of the toxin or of the living bacilli, when the animals 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. There is evidence that these paralytic phenomena are produced by toxone (p. 171), as they may occur when there is injected along with the toxin sufficient antitoxin to neutralise the more rapidly acting toxin proper. This toxone is supposed by Ehrlich to have a different toxic action, i.e. a different toxophorous group, from that of the toxin, and to have a weaker affinity for antitoxin ; much of it may thus be left unneutralised. It is to be noted in this connection that paralytic symptoms are of not uncommon occurrence in the human subject after treatment with antitoxin, the explanation of which occurrence is probably the same as that just given. One point of much interest is the high degree of resistance to the toxin possessed by mice and rats. Roux and Yersin, for example, found that 2 c.c. of toxin, which was sufficient to kill a rabbit in sixty hours, had no effect 362 DIPHTHERIA on a mouse, whilst of this toxin even T^ c.c. produced extensive necrosis of the skin of the guinea-pig. Preparation of the Toxin. — The obtaining of a very active toxin in large quantities is an essential in the preparation of anti- diphtheritic serum. Certain 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 toxin 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. The absence of glucose — an all-important point — may be attained by the method described above (p. 75), 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. By this medium he has obtained a toxin of which y^ c.c. is the fatal dose to a guinea-pig of 500 grms. He finds that glucose, glycerin, 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 toxin formation. There is in all cases a period at which the toxicity reaches a maximum, usually in 2-3 weeks, but earlier if the toxin is rapidly formed ; later the toxicity diminishes. Martin found that in his medium the maximum was reached on the 8th- 10th day. It may be added that the power of toxin 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 Toxin. — The toxic substance in filtered cultures is a relatively unstable body. When kept in sealed tubes in the absence of light, it may 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. As will be shown later (p. 472), the toxin probably does not become destroyed, but its toxophorous group suffers a sort of deterioration so that a toxoid is formed which has still the power of combining with antitoxins. Heating at 58° C. for two hours destroys the toxic properties in great part, but not altogether. When, however, the toxin is evaporated to dry ness, it NATURE OF THE TOXIN 363 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 toxin the toxic property disappears, but that it can be in great part restored by again making the fluid alkaline. Guinochet showed that toxin was formed by the bacilli when grown in urine with no proteid bodies present. After growth had taken place he could not detect proteid bodies in the fluid, but on account of the very minute amount of toxin present, their absence could not be excluded. Uschinsky also found that toxic bodies were produced by diphtheria bacilli when grown in a proteid-free medium.1 It follows from this that if the toxin is a proteid, it may be formed by synthesis within the bodies of the bacilli. Brieger and Boer have separated from diphtheria cultures a toxic body which gives no proteid reaction (vide p. 166). 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 has, however, 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 with 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 toxin from cultures, the posterior limbs are first affected ; afterwards the respiratory muscles, and finally the heart, are implicated. 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 ; 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 1 Uschinsky's medium has the following composition : water, 1000 parts ; glycerin, 30-40 ; sodium chloride, 5-7 ; calcium chloride, '1 ; magnesium sulphate, '2- '4 ; di-potassium phosphate, '2- '25; ammonium lactate, 6-7; sodium asparaginate, 3-4. 364 DIPHTHERIA obtained from the spleen, but in higher degree. Martin con- siders 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 are formed by the enzyme which is produced by it locally in the membrane. Cartwright Wood has also found that when diphtheria cultures in an albumin-containing medium are filtered germ-free and exposed to 65° C. for an hour (the supposed ferments being thus destroyed), there still remain albumoses which produce febrile reaction and are active in developing immunity. In the present state of knowledge we are not in a position to give an interpretation of such experiments, and we cannot even say whether the proteids obtained by precipitation from cultures and from the tissues are in themselves toxic, or whether the true 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 high degree of immunity, against both the bacilli and their toxins, can be produced in various animals by gradually increasing doses either of the bacilli or of their filtered toxins (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 glycerin agar. Roux and Yersin found that, when the bacilli were grown at an abnormally high temperature, namely, 39'5° C., and in a current of air, the virulence diminished so much 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 successful for this purpose. If, however, the virulence had fallen very low, even the presence of the streptococci was insufficient to restore it. As a rule, the cultures most virulent to guinea- pigs are obtained from the gravest cases of diphtheria, though to this rule there are frequent exceptions. 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. It has been abundantly established that after the cure of the disease, the bacilli may persist in the mouth for weeks, though they often quickly disappear. Roux and Yersin found, by making cultures BACILLI ALLIED TO DIPHTHERIA BACILLUS 365 at various stages after the termination of the disease, that these bacilli in the mouth gradually become attenuated. L. Martin, moreover, has shown 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, yet their true nature is shown not only by their microscopical characters, etc., but also by the fact that on more prolonged growth they form small quantities of toxin, which is neutralised by diphtheria antitoxin. The persistence of these non-virulent bacilli in the throats of those who have suffered from the disease, and their occasional presence in quite healthy individuals, may manifestly be of importance in relation to the continuance of the infection and the reappearance of epidemics of the disease. Bacilli allied to the Diphtheria Bacillus. Bacteriological examinations carried on within recent times have shown that the diphtheria bacillus is merely a member of a group of organisms with closely allied characters which are of common occurrence and have a wide distribution. The terms "pseudo-diphtheria bacilli "and "diphtheroid bacilli" have been applied in a loose way to organisms which resemble the diphtheria bacillus microscopically, especially as regards the beaded ap- pearance. Such bacilli have been obtained from the mouth, nose, skin, genital organs, and even from the blood in certain diseases. They are to be met with sometimes in conditions of health, and they have been obtained from many diverse morbid conditions — from skin diseases, from coryza, from leprosy, and even from general paralysis of the insane. As has been found with other groups the differentiation is a matter of considerable difficulty. Some are practically identical with the diphtheria bacillus both morphologically and culturally, and a few even give the characteristic reaction with ISTeisser's stain ; others again differ in essential particulars. The fermentative action on sugars T has also been called into requisition as a means of distin- guishing them, but the results obtained cannot be said to be of a definite character, and further work is necessary. It may be stated, however, that most observers have found the diphtheria bacillus of all the members of the group to be the most active acid -producer, though here the difference seems to be one of degree rather than of kind. The absence of the power of fermenting certain sugars, notably glucose, may, however, be accepted in any particular case as sufficient to exclude the 1 Vide a paper by Graham- Smith, Journal of Hygiene, vi. 286. 366 DIPHTHERIA organism from being the diphtheria bacillus. From these facts, and from what has been stated with regard to attenuated diphtheria bacilli, it will be seen that an absolute decision as to the nature of a suspected organism may in some cases be a practical impossibility. It may be that some of the " diphtheroid " organisms cultivated have really been non-virulent diphtheria bacilli. The bearing of this on the practical means of diagnosis will be discussed below. The term " pseudo-diphtheria bacillus " is often restricted by present writers to an organism frequently met with in the throat. This organism, which is also known as Hofmann's bacillus, merits a separate description. Hofmann's Bacillus — Pseudo- Diphtheria Bacillus. — This organism, described by Hofmann in 1888, is probably the same as one observed by Loffler in the previous year, and regarded by him as being a distinct species from the diphtheria bacil- lus. The organism is a shorter bacillus than the , diphtheria bacillus, with usually a single unstained septum running across it, though sometimes there may be more than one (Fig. 122). -The typical beaded appearance is rarely ^^^ seen, and the reaction with Neisser's stain is not given. FIG. 122.— Pseudo-diphtheria bacillus It grows readily on the (Hofmann's). Young agar culture. same media as the diph- Stained with thionin-blue. x 1000. theria baci]luS) but the colonies are whiter and more opaque. It does not form acid from glucose or other sugars, and is non-pathogenic to the guinea-pig. Involution forms may sometimes be produced by it. It is usually a com- paratively easy matter to distinguish this organism from the diphtheria bacillus. Hofmann's bacillus is of comparatively common occurrence in the throat in normal as well as diseased conditions, including diphtheria ; it seems to be specially frequent in poorly nourished children of the lower classes. Cobbett found it 157 times in an examination of 692 persons examined, of whom 650 were not SUMMAEY 367 suffering from diphtheria. Boycott's statistics show that the time of its maximum seasonal prevalence precedes that of the diphtheria bacillus. To what extent, if any, it is responsible for pathological changes in the throat, must be considered a question which is not yet settled. Hewlett and Knight have found evidence that a true diphtheria bacillus may assume the characters of Hofmann's bacillus, but attempts to effect the transformation have met with negative results in the hands of other observers. The general opinion is that the two organisms are distinct species which are comparatively easily distinguished characters. Xerosis Bacillus. — This term has been given to an organism first observed by Kuschbert and Neisser in xerosis of the conjunctiva, and which has been since found in many other affections of the conjunctiva and also in normal conditions. Morphologically it is practically similar to the diphtheria bacillus, and even in cultures presents very minor differences. It is, however, non-virulent to animals, and does not pro- duce an acid reaction in sugar-containing bouillon, or does so to only a slight extent ; in this way it can be distinguished from the diphtheria bacillus. Its morphological characters are shown in Fig. 123. 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 toxins, the following sum- mary may be given of its action in the body. Locally, the bacillus produces in- flammatory change with nbrinous exudation, but at the same time cellular necrosis is also an out- standing feature. Though false membranes have not been produced by the toxins, a necrotic action may result when these are injected subcutaneously. Fm> 123._xerosis bacillus from a young The toxins also act upon agar culture, x 1000. the blood - vessels, and hence cedema and tendency to haemorrhage are produced ; this action on the vessels is also exemplified by the general congestion of organs. The hyaline change in the walls of arterioles and capillaries so often met with in diphtheria is another example of the action of the toxin. The toxins have 368 DIPHTHEKIA 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 dis- integration or by an actual fatty degeneration. 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 in 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 to what extent these nerve lesions are of primary nature or secondary to changes in the nerve cells. Methods of Diagnosis. — The bacteriological diagnosis of diphtheria depends on the discovery of the bacillus. As the bacillus occurs in largest numbers in the membrane, 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), the swab being put into a sterile tube or bottle for transport. A convenient method is to twist a piece of cotton-wool round the roughened end of a piece of very stout iron wire, six inches long, and pass the other end of the latter through a cotton plug inserted in the mouth of a test-tube (compare Fig. 48, the wire taking the place of the pipette), and sterilise. In use the wire and plug are extracted in one piece, and after swabbing are replaced in the tube for transit. A scraping may be made off the swab for microscopic examination, and the swab may be smeared over the surface of a serum tube to obtain a culture. This method of taking and treating swabs is that usually employed in routine public health work. The results obtained ordinarily suffice for the diagnosis of cases suspected to be diphtheritic in nature. The means for identifying the bacillus are (a) By micro- scopical examination. — For microscopical examination it is sufficient to tease out a piece of the membrane with forceps and rub it on a cover-glass ; if it be somewhat dry a small drop of METHODS OF DIAGNOSIS 369 normal saline 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 Loffler's solution. After staining for 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. Neisser's stain (p. 108) may also be used with advantage. 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 sufficiently 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 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 im- purities 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 (p. 357), the same portion of the membrane being always brought into contact with the surface. The tubes are then placed in the incubator at 37° C., and, in the case of the serum media and blood-agar, the circular colonies of the diphtheria bacillus are well formed within 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, Neisser's stain being also applied. When the material has been taken from the throat, an organism with all the morphological and cultural characters of the diphtheria bacillus may for all practical purposes be accepted as the diphtheria bacillus. In cases where a suspicion arises that the organism found is a pseudo-diphtheria bacillus, bouillon containing a trace of glucose should be inoculated and incubated at 37° C. The reaction should be tested after one and after two days' growth. If it remains alkaline, the diphtheria bacillus may be 24 370 DIPHTHERIA excluded. If an acid reaction results, then all the microscopical and cultural characters must be carefully observed, and the virulence of the bacillus may be ascertained by inoculating a guinea-pig, say with 1 c.c. of a broth culture of two days' growth. (See also pp. 359, 365.) A fatal result with characteristic appearances may be taken as positive evidence, but if the animal survive there is still theoretically the possibility that the organism is an attenuated diphtheria bacillus (p. 364). CHAPTER XVI. TETANUS.1 SYNONYMS. LOCKJAW. GERMAN, WUNDSTARRKRAMPF. FRENCH, TETANOS. Introductory. — Tetanus is a disease which in natural conditions affects chiefly: man and theJiorse. Clinically it is characterised by the gradual onset of general stiffness and spasms of the volun- tary 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 denied 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 thejspinal cord, and especially the medulla. Historical. — The general association of the development of tetanus with the presence of wounds, though these might be very small, suggested that some infection took place through the latter, but for long 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 demon- strated the transmissibility of the disease. Nocolaier (1885) infected mice and rabbits with garden earth, and found that many of them developed tetanus. Suppuration occurred in the neighbourhood of the point of 1 This disease is not to be confused with the " tetany " of infants, which in its essential pathology probably differs from tetanus (vide Frankl-Hochwart, " Die Tetanie der Erwachsenen," Vienna, 1907). This remark of course does not exclude the possibility of the occurrence of true tetanus in very young subjects. 371 372 TETANUS inoculation, and in this pus, besides other organisms, there was always present, when tetanus had occurred, a bacillus having certain constant microscopic characters. Inoculation of fresh animals with such pus reproduced the disease. Nicolaier's attempts at its isolation by the ordinary gelatin 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 results were confirmed by Rosenbach, who, though failing to obtain a pure culture, cultivated the other organisms present, and inoculated them, but 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 suppura- tion 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 arising 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. 124). If, however, the tetanus bacilli have not formed spores, they appear as somewhat slender rods, without present- ing any characteristic features. There is 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 be a slender organism, usually about 4 /z to 5 /A in length and '4 //, in thickness, with somewhat rounded ends. Besides occurring as short rods it also develops filamentous forms, the latter being more common in fluid media. It stains readily by any of the usual stains and also by Gram's 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 hanging- drop preparation (p. 64). When stained by the special methods already described, it is found to possess numerous delicate flagella attached both at the sides and at the ends (Fig. 125). These flagella, though they maybe of consider- able 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. 62). As is the case with many other anaerobic flagellated bacteria the flagella, on becoming detached, often become massed together in the form of spirals of striking appearance (Fig. 126). At incubation temperature b. tetani readily forms spores, and then presents a BACILLUS TETANI 373 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 what is usually described as the drumstick form (Figs. 124, 127). In a specimen stained with a watery solution of gentian- violet or methylene-blue, the spores are uncoloured except at the periphery, FIG. 124. — Film preparation of discharge from wound in a case of tetanus, showing several tetanus bacilli of "drumstick" form. (The thicker bacillus with oval and not quite terminal spore, in the upper part 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. 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 become deeply coloured like the bacilli. Further, especially if the preparation be heated, many spores may become free from the bacilli in which they were formed. Isolation. — The isolation of the tetanus bacillus is somewhat difficult. By inoculation experiments in animals, its natural habitat has been proved to be garden soil, and especially the 374 TETANUS contents of dung -heaps, where it probably leads a saprophytic existence, though its 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 FIG. 125. — Tetanus bacilli, showing flagella. Stained by Rd. Muir's method. x 1000. the spores of the bacillus to heat. A sloped tube of inspissated serum or a deep tube of glucose agar is inoculated with the pus and incubated at 37° 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 80° 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 gelatin, and roll-tube cultures are made in the usual way and kept in an atmosphere of hydrogen at 22° C. ; after five days the plates are ready for examination. Kitasato com- ISOLATION OF THE BACILLUS 375 pares the colonies in gelatin plates to those of the b. subtilis. They consist of a thick centre with shoots radi- ating out on all sides. They liquefy the gelatin 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 oc- cur, which grow faster than the tetanus bacillus, and 'thus overgrow it. (2) If in any dis- charge the spore-bearing tetanus bacilli be seen 1 JfP . FIG. 126. — Spiral composed of numerous twisted flagella of the tetanus bacillus. Stained by Kd. Muir's method. x 1000. \ ^ on microscopic examination, then a method of isolation based on the same prin- ciple as the last may be adopted. Inocula- tions with the suspected material are made in half a dozen deep tubes of glucose agar, previ- ously melteTand kept at a temperature of 100° C. After inoculation 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 respect- ively. They are then plunged in cold water till cool, and thereafter placed in the incubator at 37° C., in the hope that in one or other of the tubes all the organisms present will have been FIG. 127. — Tetanus bacilli ; some of which possess spores. From a culture in glucose agar, incubated for three days at 37° C. Stained with carbol-fuchsin. x 1000. 376 TETANUS killed, except the tetanus spores which can develop in pure culture. (3) Some method of anaerobically making plates, such as that of Bulloch, may be employed. The isolation of the tetanus bacillus is in many cases a difficult matter, and various expedients require to be tried. Characters of Cultures. — Pure cultures having been obtained, sub-cultures can be made in deep upright glucose gelatin or agar tubes. On glucose gelatin 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. 128). Slow liquefaction of the gelatin takes place, with slight gas formation. In agar the growth is somewhat similar, consisting of small nodules along the needle track, with irregular short offshoots passing out into the medium (Fig. 131, 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 or- ganism. There is in it at first a slight turbidity, and later a thin layer of a powdery deposit on the walls of the vessel. FIG. 128 -Stab cul- A]1 ^e cultures give out a peculiar burnt ture of the tetanus odour of rather unpleasant character, bacillus in glucose Conditions of Growth, etc. — The b. gelatin, showing tetani grows best at 37° C. The minimum the lateral shoots growth temperature is about 14° C., and (Kitasato). .Natural , -, ^,^0 ^ ^1^1 i i i gize below 22 C. growth takes place very slowly. Growth takes place only in the absence of oxygen, the organism being a strict anaerobe. Sporulation may commence at the end of twenty-four hours in cultures grown at 37° C., — much later at lower temperatures. Like other spores, those of tetanus are extremely resistant. They can usually withstand 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 377 Pathogenic Effects. — The proof that the b. tetani is the cause of tetanus is complete. It can be isolated in pure culture, and when re-injected 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 dis- charge, 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. f[f 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 Ahicker bacilli with oval spores placed at a short distance from their ex- tremities, such forms being common in earth, etc., and also met with in contaminated wounds (Fig. 124). 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 "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, trhere may be haemorrhages in the muscles which have been the subject of the spasms.i 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 fa-regular patches of slight congestion/vhich 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 378 TETANUS 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 toxins of the bacillus. In the case of the cellular degenera- tions the cells have been observed to return to the normal under the curative influence of the antitoxins (vide 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 phenomenon, 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, if ever, are the organisms found anywhere except in the local lesion. (b) The Artificially -produced Disease. — The disease can be com- municated 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 culture. The other animals mentioned require larger doses, and death does not occur so rapidly. (Usually in animals injected subcutaneously the spasms begin in the limb nearest the point of inoculation. In intra- venous inoculation the spasms begin in the extensor muscles of the trunk, as is the case in the natural disease in man.J After death there is found slight hypersemia 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 states that in his 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 toxins 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 80° C. The latter treatment not only killed all the bacilli, but, TOXINS OF THE TETANUS BACILLUS 379 as we shall see, was sufficient to destroy the activity of the toxins. When such splinters are introduced subcutaneously, death results by the development of the spores which they carry, fin this way he completed the proof that the bacilli by themselves can form toxins 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. [Some- times, 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 (vide infra).] By such experiments, supple- mented by the culture experiments mentioned, the natural habitats of the b. tetani, as given above, have become known. The Toxins 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. The earlier results in which certain bases, (tetanin and tetanotoxinj were said to have been isolated, have only a historic interest, as they were obtained by faulty methods. In 1890 Brieger and Fraenkel announced that they had isolated &(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 cultures in bouillon, and was undoubtedly toxic. Within recent years such attempts to isolate tetanus toxins in a pure condition have practically been abandoned, and attention has been turned to the investigation of the physiological effects either of the crude toxin present in filtered bouillon cultures, or of the precipitate produced from the same by ammonium sulphate (cf. p. 167). The toxic properties of bacterium-free nitrates of pure cultures of the b. tetani were investigated in 1891 by Kitasato. This observer 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, file found that guinea-pigs were more susceptible than mice, and rabbits less sol In order that a strongly toxic bouillon be produced, it must originally have been either neutral or slightly alkaline.) fKitasato further found that the toxin was easily injured by heat.) Exposure for a few minutes at 65° C. destroyed it. It was also destroyed by twenty minutes' exposure at 60° C., 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 380 TETANUS sunlight. Behring has more recently pointed out that after the nitration of cultures containing toxin, (the latter may very rapidly lose its power, and in a few days may only possess y^th of its original toxicity. This he attributes to such factors as temperature and light, and especially to the action of oxygen.] The effect of these agents on the crude toxin is undoubtedly to cause a degeneration of the true toxin into a series of toxoids similar to those produced in the case of diphtheria toxin, and it is also true here that the toxoids while losing their toxicity may still retain their power of producing immunity against the potent toxin. Further, altogether apart from the occurrence side by side in the crude toxin of strong and weak poisons, it has been shown that such crude toxin contains toxic substances of probably quite a different nature. (jEhrlich has shown that besides the pre- dominant spasm-producing toxin (called by him tetanospasmin), there exists in crude toxin a poison capable of producing the solution of certain red blood corpuscles. This haemolytic agent he calls tetanolysin) It does not occur in all samples of crude tetanus toxin, nor is it found when a bouillon culture of the bacillus is filtered through porcelain. To obtain it the fresh culture must be treated by ammonium sulphate, as described in the method of obtaining concentrated toxins (p. 167). This substance also has the power of originating an antitoxin so. that certain antitetanic sera can protect red blood corpuscles against its action. Madsen, studying the interactions of this anti- tetanolysin with the tetanolysin, has shown that the phenomena can be demonstrated similar to those noted by Ehrlich as occurring with diphtheria toxin, and which he interpreted as indicating the presence .of degenerated toxins (toxoids) in the crude poison. With tetanus as with diphtheria toxin the action of an acid is to cause an apparent disappearance of toxicity, but if before a certain time has elapsed the acid be neutralised by alkali, then a degree of the toxicity returns. As with other members of the group, nothing is known of the nature of tetanus toxin. Uschinsky has found that the tetanus bacillus can produce its toxin when growing in a fluid containing no proteid matter. The toxin may thus be formed independently of the breaking up of the proteids on which the bacillus may be living, though the latter no doubt has a digestive action on these. The liquefaction (i.e. probable peptonisation) of gelatin cultures advances pari passu with the development of toxins, and filtered bacterium-free cultures will still liquefy gelatin. /It is probable that there is an independent peptic ferment which will, of course, also pass through a filter^ For if equal portions if TOXINS OF THE TETANUS BACILLUS 381 of the filtered culture be left in contact with equal portions of gelatin for various lengths of time, there is no increase of toxicity in those kept longest. There is thus no fresh development of toxin during the advancing liquefaction of the gelatin. Thus peptic digestion and toxin formation are apparently due to different vital processes on the part of the tetanus bacillus. Whatever the nature of the toxin is, it is undoubtedly one of the most powerful poisons known. Even with a 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 have been '23 of a milligramme, or about -^ ^ ^-ths of a grain. Animals differ very much in their susceptibilities to the action of tetanus toxin. According to v. Lingelsheim, if the minimal lethal dose per gramme weight for a horse be taken as unity, that for the guinea-pig would be 6 times the amount, the mouse 12, the goat 24, the dog about 500, the rabbit 1800, the cat 6000, the goose 12,000, the pigeon 48,000, and the hen 360,000. ("A striking feature of the action of tetanus toxin is the occurrence of a definite incubation period between the introduc- tion of the toxin into an animal's body and the appearance of symptoms.J (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. In man the period between the receiving of an injury and the appearance of tetanic symptoms is from two to fourteen daysl With regard to the action of the toxin, 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 toxin is absorbed into 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 some animals the tetanic spasms appear first in the muscles of the part in which the inoculation has taken place. This is not the case with man, in whom usually the first symptoms appear in the neck. In artificial injection of toxin part finds its way into the blood stream, and if infected animals be killed during the incubation period there is often evidence of toxin in the blood and solid organs. In the guinea- 382 TETANUS pig there is little doubt that tetanus toxin has an affinity solely for the nervous system. In other animals, such as the rabbit, an affinity may exist in other organs, and the fixation of the poison in such situations may give rise to no recognisable symptoms. In such an animal as the alligator, it is possible that while some of its organs have an affinity for tetanus toxin its nervous system has none. These facts are of great scientific interest, and a possible explanation of them will be discussed in the chapter on Immunity. If tetanus toxin be introduced into the stomach or intestine, it is not absorbed, but to a large extent passes through the intestine unchanged. Evidence that any destruction takes place is wanting. Within recent years some important light has been shed on the mode of action of tetanus toxin. Marie and Morax studied the path of absorption when the toxin was injected into the muscles of the hind limb. The sciatic nerve in a rabbit was cut near the spinal cord and toxin introduced into the muscles of the same side ; after some hours the nerve was excised and introduced into a mouse — the animal died of tetanus. But if the nerve were cut near the muscles and the same procedure adopted, the mouse did not contract the disease, though no doubt the cut nerve had been surrounded by lymph containing toxin. If the same experiment were performed and an excess of toxin injected into the other limb, still only the nerve which was left in connection with the muscle showed evidence of containing toxin. (From this it was deduced that the toxin was absorbed by the end- plates in the muscle and not from the lymphatics surrounding the nerve.) It was further shown that a nerve in the process of degeneration following section did not absorb toxin after the manner of a normal nerve. By a similar method it was shown that the absorption by the nerve was fairly rapid, as one hour after injection the toxin was present in it, and from other experiments the view was put forth that the toxin was centripetal in its flow and did not pass centrifugally in a nerve to which it artificially gained access. Further observations have been made on this subject by Meyer and Ransom. These observers found evidence that toxin is only absorbed by the motor filaments of a nerve, for while tetanus could .be produced by injection into a mixed nerve like the sciatic the introduction of a lethal dose into such a sensory nerve as the infra-orbital was not followed by disease symptoms. If a small dose of toxin be injected into the sciatic nerve it reaches the corresponding motor cells of the cord, and a local tetanus of the muscles supplied by the nerve results. With a larger dose the poison passes across the commissure to TOXINS OF THE TETANUS BACILLUS 383 the corresponding cells of the other side, and if still further excess is present it passes up the cord to higher centres. The affection of such higher centres can be prevented by section of the cord. Meyer and Ransom £hold that when toxin is injected subcutaneously or intravenously, it only acts by being absorbed by the end-plates in muscles and thence passes to the cord, and they consider that the incubation period is to be explained by the time taken for this extended passage to occur.) In this connection they point out that it is in the larger animals, where the nerve path is longest, that the incubation period is also long. Like Marie and Morax, they believe that absorption of toxin by its bathing the lateral aspects of uninjured nervous structures does not occur, and in support of this they bring forward the observation that when intravenous injection is practised, the occurrence of tetanus in a part of the body can be precipitated by such a slight injury as may be caused by the injection of a drop of normal saline into the corresponding part of the cord. With regard to the action of tetanus toxin, Meyer and Ransom believe that there is a double effect on the nerve cells — ferst, an exaggera- tion of the normal tonus, which accounts for the continuous stiffness of the muscles, and secondly, an increase in reflex irritability, which is a prominent factor in the recurring spasms'^ While no absorption of toxin takes place by sensory filaments, they have found evidence of sensibility of the sensory apparatus in the occurrence of what they call {tetanus dolorosus) This is a great hypersesthesia and a paroxysmal hyperalgesia which can be caused by injecting toxin into the spinal cord or into a sensory root on the spinal side of the posterior root ganglion. These symptoms are unaccompanied by motor spasms, but the animal may die from exhaustion. The same observers have also made interesting observations on the action of antitoxin. They found that the injection of this substance into the course of a mixed nerve could prevent toxin from passing up to the cord, but that if antitoxin were injected even in great excess intravenously, and a short time thereafter toxin were introduced into a nerve, the death of the animal was not prevented. This they attribute to the fact that antitoxin can only neutralise the toxin which is still circulating in the blood. This is a very far-reaching conclusion, as it throws doubt on what has been held to be a possibility, namely, (that toxin can be actually detached from cells in which it is already anchored^ But a still more significant observation was made, for in one case of an animal actively immunised against tetanus, and which contained in its serum a considerable quantity of antitoxin, the injection of toxin 384 TETANUS into the sciatic nerve was followed by tetanus. This would appear to militate against Ehrlich's position that antitoxin is manufactured in the cells which are sensitive to the toxin (see Immunity). Reference may here be made to the effects of injecting tetanus toxin into the brain itself, as investigated by Roux and Borrel. ft was found that the ordinary type of the disease was not produced, but what these observers called "cerebral tetanus. J This consisted of general unrest, symptoms of a psychic character (apparent hallucinations, fear, etc.), and epileptiform con- vulsions. Death occurred in from twelve to twenty hours without any true tetanic spasms. In this manifestation of tetanus the incubation period was much shorter than with subcutaneous injection, and (the fatal dose was one twenty-fifth of the minimal subcutaneous dosel Further, the injection of antitoxin forty-eight to ninety-six hours previously did not prevent an animal from suc- cumbing to the intracerebral inoculation. (En the light of what has been already said these results would seem to indicate a special effect of the toxin when brought into direct contact with the protoplasm of the brain cells) We have seen that unless suitable precautions are adopted, in experimental tetanus in animals death results not from inocula- tion but from an intoxication with toxin previously existent in the fluid in which the bacilli have been growing. According to Vaillard, if spores rendered toxin-free, by being kept for a sufficient time at 80° 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 seat 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 simultaneous 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, fcitasato now holds that in the natural infection in man, along with tetanus spores, the presence of foreign material or of other bacteria is necessaryJ Spores alone or tetanus bacilli without spores die in the tissues, and tetanus does not result. Immunity against Tetanus. — Antitetanic Serum. — The arti- ficial immunisation of animals against tetanus has received much attention. The most complete study of the question is found IMMUNITY AGAINST TETANUS 385 in the work of Behring and Kitasato in 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 toxin. The degree of immunity attained, however, was not high. Subsequent work has shown that the less rich a crude toxin is in modifica- tions of the true toxin the less useful it is for immunisation procedures. In fact it is doubtful if small animals can be immunised at all by fresh filtrates. In some cases it has been found that the injection of non-lethal doses instead of commenc- ing an immunity actually increases the susceptibility of the animal. More successful are the methods of accompanying the early injections of crude toxin with the subcutaneous introduction of small doses of iodine terchloride, or of using toxin which has been acted on with iodine terchloride or with iodine itself. Tizzoni and Cattani have also used the method of administering pro- gressively increasing doses of living cultures attenuated in various ways, e.g. by heat. By any of these methods susceptible animals can be made to acquire great immunity, not only against many times the fatal dose of tetanic toxin, but also against injections of the living bacilli. The degree of immunisa- tion acquired by an animal remains in existence for a very long time. Not only so, but when a high degree of immunity has been produced by prolonged treatment, it is found that the serum of immune animals possesses the capacity, when injected into animals susceptible to the disease, of protecting them against a subsequent infection with a fatal dose of tetanus bacilli or toxin. 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 of success attained depends, however, on the shortness of the time which has elapsed between the infection with the bacilli or toxin and the injection of the serum. In animals where symptoms have fully manifested themselves only a small proportion of cases can be saved. As with other antitoxins there is no evidence that the antitetanic serum has any detrimental effect on the bacilli. It only neutralises the Effects of the toxin. 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 toxin. A mouse weighing twenty grammes would thus require '00002 gramme of the serum to protect it against the minimum lethal dose. In the injection 25 386 TETANUS of such a serum subsequent to infection, if symptoms, have begun to appear, 1000 times this dose would be necessary ; a few hours later 10,000 times, and so on. As the result of his experiments, Behring aimed at obtaining 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, by the injection of toxin 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 toxin. If symptoms had begun to appear, 100 c.c. at once would be necessary, and as the injection of such a quantity might be inconvenient, Behring recommended that for man a more powerful serum should be 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, and the serum is kept in the dark. 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. Intravenous injection of the antitoxin has also been practised, and, in cases which we have seen treated in this way, has seemed to give better results than those obtained by the subcutaneous method. The serum is warmed to the body temperature and slowly introduced into a vein in the arm, the pulse and respiration being carefully watched during the proceeding. Ten to twenty c.c. can be injected every few hours, and in all 100 c.c. should be given in as short a time as possible. Henderson Smith has shown that when antitoxins to toxins of the tetanus group are injected intravenously a high concentration in the body fluid is maintained for some time and the op- portunity for neutralisation of toxin is thus great. He suggests 1 The antitetanic serum sent out by the Pasteur Institute in Paris has a strength of 1 : 1,000,000,000. Of this it is recommended that 50 to 100 c.c. should be injected in one or two doses. IMMUNITY AGAINST TETANUS 387 that vboth intravenous and subcutaneous injections should be simultaneously practised. The former gives the quickly attained concentration which is desirable, and when the antitoxin injected intravenously is beginning to be eliminated that introduced hypodermically comes into the circulation and the concentration is maintained. The antitoxin has also been introduced intra- cerebrally, very slow injection into the brain substance being practised, but no better results have been obtained than by the subcutaneous method. Many cases of human tetanus have been thus treated, but 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 may be 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 treat- ment 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 probable result, the two clinical observations on which, according 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. In addition to these points the duration of the incubation period is of high importance in forming a prognosis. The shorter the time between the infliction of a wound and the appearance of symptoms the graver is the outlook. 388 TETANUS The theory as to the nature of antitoxic action will be discussed later in the chapter on Immunity. 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 corre- sponding to those of the tetanus bacilli, though not absolutely conclusive proof of identification, 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. 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. (b) Cultivation. — The methods to be employed in isolating the tetanus bacilli have already been described (p. 373). It may be added, however, that if the characteristic 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 (EDEMA (Septicemie de Pasteur). The organism now usually known as the bacillus of malignant cedema is the same as that first discovered by Pasteur and named vibrion septique. He described its characters, distin- guishing 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 MALIGNANT (EDEMA 389 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 septicaemia, 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 of the skin FIG. 129. — 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. 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, observed a case in which the bacillus was present in pure condition. Here there occurred intense oadema with swelling and induration of the tissues, and the formation of vesicles on the skin. Those changes were attended with a reddish discoloration, 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 390 MALIGNANT (EDEMA peculiar heavy, but not putrid, odour. The bacillus, which was obtained in pure culture, was present in enormous 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 application 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 pro- duced by inoculating sus- ceptible animals, such as guinea - pigs, wi th garden soil. The bacillus is also of ten present in the intestine of man and animals, and has been described as being present in some gangrenous conditions originating in connection with the in- testine in the human sub- ject. Microscopical Characters. — -The bacillus of malignant oedema is a comparatively large organism, being slightly less than 1 //, in thickness, that is, thinner than the anthrax bacillus. It occurs in the form of single rods 3 //, to 10 /x in length, but both in the tissues and in cultures in fluids it frequently grows out into long filaments, which may be 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 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 FIG. 130. — Bacillus of malignant oedema, showing spores. From a culture iu glucose agar, incubated for three days at 37° C. Stained with weak carbol-fuchsin. x 1000. CHARACTER OF CULTURES 391 bacillus (Figs. 129, 130). 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. — This organism grows readily at ordinary temperature, but only under anaerobic conditions. In a puncture culture in a deep tube of glucose gelatin, the growth A B c FIG. 131.— Stab cultures in agar, five days' growth at 37° C. Natural size. A. Tetanus bacillus. B. Bacillus of malignant oedema. C. Bacillus of quarter-evil (Rauschbrand). 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 nocculent masses of growth at the bottom. At the same time bubbles of gas are given off, which may split up the gelatin. The colonies in gelatin plates under anaerobic conditions appear first as small whitish points which under the microscope show a radiating appearance at the periphery, resembling the colonies of the bacillus subtilis. Soon, however, 392 MALIGNANT (EDEMA 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 extremely rapid. Along the line of puncture, growth appears as a some- what broad white line with short lateral projections here and there (Fig. 131, 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 20° C., and is usually well seen within forty-eight hours at 37° C. The spores 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 example — are susceptible to inoculation with this organism. The ox is said to be quite immune to experimental inoculation, 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 subcutaneous inoculation with garden soil, death usually occurs 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 con- gested, the spleen soft but not much enlarged. In such con- ditions the bacillus of malignant 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 pleurae, 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 directly into 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. BACILLUS BOTULINUS 393 When the bacilli are injected into mice, however, they enter and multiply in the blood stream, and they are found in con- siderable 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 varies con- siderably in different cases, and it always becomes 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 into the tissues, inoculation by scari- fication being followed by no result. A smaller dose produces a fatal result when injected along with various other organisms (bacillus prodigiosus, etc.). Immunity. — Malignant oedema was one of the first diseases against which immunity was produced by injections of toxins. 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. Chamber- land 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 organism 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 any 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 ab- solutely the bacillus of malignant oedema without cultivating it, the presence of spore-bearing bacilli with the characters described above is highly suspicious (Fig. 1 29). In such a case the fluid containing the bacilli should be first exposed to a temperature of 80° 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. BACILLUS BOTULINUS. The term " meat-poisoning " embraces a number of conditions produced by different agents, and the relation of the bacillus of 394 BACILLUS BOTULINUS Gaertner to one class of case has already been discussed. Another group was shown by van Ermengem in 1896 to be caused by an anaerobic bacillus to which he gave the name bacillus lotulinus. He cultivated the organism from a sample of ham, the ingestion of which in the raw condition had produced a number of cases of poisoning, some of them followed by fatal result. The symptoms in these cases closely corresponded with those occur- ring in the so-called " sausage poisoning " met with from time to time in Germany and other countries where sausages and ham are eaten in an imperfectly cooked condition. Such cases form a fairly well-defined group, the symptoms in which are chiefly referable to an action on the medulla, and, as will be detailed below, similar symptoms have been experimentally produced by means of the bacillus mentioned or its toxins. The chief symptoms of this variety of botulismus, as detailed by van Ermengem, are disordered secretion in the mouth and nose, more or less marked ophthalmoplegia, externa and interim (dilated pupil, ptosis, etc.), dysphagia, and sometimes aphagia with aphonia, marked constipation and retention of urine, and in fatal cases interference with the cardiac and respiratory centres. Along with these there is practically no fever and no interference with the intellectual faculties. The symptoms commence at earliest twelve to twenty-four hours after ingestion of the poison. From the ham in question, which was not decomposed in the ordinary sense, van Ermengem obtained numerous colonies of this bacillus, the leading characters of which are given below. It may be added that Homer obtained practically the same results as van Ermengem in a similar condition, and that the bacillus botulinus has been cultivated by Kempner from the intestine of the pig. Microscopical and Cultural Characters. — The organism is a bacillus of considerable size, measuring 4 to 9 //, in length and •9 to 1'2 /* in thickness; it has somewhat rounded ends and sometimes is seen in a spindle form. It is often arranged in pairs, sometimes in short threads. Under certain conditions it forms spores which are oval in shape, usually terminal in position, and a little thicker than the bacilli. It is a motile organism and has 4 to 8 lateral flagella of wavy form. It stains readily with the ordinary dyes, and also retains the colour in Gram's method, though care must be employed in decolorising. The organism can be readily cultivated on the ordinary media, but only under strictly anaerobic conditions. In glucose gelatin a whitish line of growth forms with lateral offshoots, but liquefaction with abundant gas formation soon occurs. In gelatin plates the colonies after four to six days are somewhat MICROSCOPICAL AND CULTURAL CHARACTERS 395 characteristic ; they appear to the naked eye as small semi- transparent spheres, and these on examination under a low power of the microscope have a yellowish-brown colour and are seen to be composed of granules which show a streaming move- ment, especially at the periphery. Cultures in glucose agar resemble those of certain other anaerobes ; there is abundant development of gas, and the medium is split up in various directions. The cultures have a rancid, though not foul, odour, due chiefly to the development of butyric acid. The optimum temperature is below that of the body, viz., between 20° and 30° C. ; at the body temperature growth is slower and less abundant and spore formation does not occur. Pathogenic Effects. — Like the tetanus bacillus the bacillus botulinus has little power of nourishing in the tissues, whereas it produces a very powerful toxin. Van Ermengem found that the characteristic symptoms could be produced in certain animals by administering watery extracts of the infected ham or cultures either by the alimentary canal or by subcutaneous injection. Here also there is a period of incubation of not less than six to twelve hours before the symptoms appear, and when the dose is small a somewhat chronic condition may result in which local paralyses form a striking feature. The characteristic effects can also be produced by means of the filtered toxin by either of the methods mentioned, though in the case of administration by the alimentary canal the dose requires to be larger. Here also, as in the case of the tetanus poison, the potency of the toxin is remarkable, the fatal dose for a guinea-pig of 250 grm. weight being in some instances "0005 c.c. of the filtered toxin. In cases of poisoning in the human subject the effects would accordingly appear to be produced by absorption of the toxin from the alimentary canal ; it is only after or immediately before death that a few bacilli may enter the tissues. Van Ermengem obtained a few colonies from the spleen of a patient who had died from ham-poisoning. The properties of the botulinus toxin have been investigated and have been found to correspond closely, as regards relative instability, conditions of precipitation, etc., with the toxins of diphtheria and tetanus. An antitoxin has also been prepared by Kempner by the .usual methods, and has been shown not only to have the neutralising property but to have considerable therapeutical value when administered some hours after the toxin. The direct combining affinity of the toxin for the central nervous has been demonstrated by Kempner and Schepilewsky by the same methods as Wassermann and Takaki employed in the case of the tetanus toxin. The 396 QUARTER-EVIL condition of the nerve cells in experimental poisoning with the botulinus toxin has been investigated independently by Marinesco and by Kempner and Pollack, and these observers agree as to the occurrence of marked degenerative changes, especially in the motor cells in the spinal cord and medulla. Marinesco also observed hypertrophy and proliferation of the neuroglia cells around them. These observations, therefore, show that in one variety of meat-poisoning the symptoms are produced by the absorption of the toxins of the bacillus botulinus from the alimentary canal, and, as van Ermengem points out, it is of special importance to note that the meat may be extensively contaminated with this bacillus and may contain relatively large quantities of its toxins without the ordinary signs of decomposition being present. The production of an extracellular toxin by this organism, with extremely potent action on the nervous system, is a fact of great scientific interest and has a bearing on the etiology of other obscure nervous affections. QUARTER-EVIL (GERMAN, RAUSCHBRAND ; FRENCH, CHARBON SYMPTOMATIQUE). The characters of the bacillus need be only briefly described, as, so far as is known, it never infects the human subject. The natural disease, which specially occurs in certain localities, affects chiefly sheep, cattle, and goats. Infection takes place by some wound of the surface, and there spreads in the region around, inflammatory swelling attended by bloody oedema and emphysema of the tissues. The part becomes greatly swollen, and of a dark, almost black, colour. Hence the name " black- leg " by which the disease is sometimes known. The bacillus which produces this condition is present in large numbers in the affected tissues, associated with other organisms, and also occurs in small numbers in the blood of internal organs. The bacillus morphologically closely resembles that of malignant oedema. Like the latter, also, it is a strict anaerobe, and its conditions of growth as regards temperature are also similar. It is, however, some- what 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. 132). 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 offshoots, the growth being also more luxuriant (Fig. 131, c). This bacillus is actively motile, and possesses numerous lateral flagella. The disease can be readily produced in various animals, e.g. guinea- pigs, by inoculation with the affected tissues of diseased animals, and also by means of pure cultures, though an intramuscular injection of a considerable amount of the latter is sometimes necessary. The condition produced in this way closely resembles that in malignant oedema, though BACILLUS ^EROGENES CAPSULATUS 397 there is said to be more formation of gas in the tissues. Rabbits are very immune against this disease, whilst they are comparatively suscep- tible to malignant oedema. As in the case of tetanus, inoculation with living spores which have been deprived of adherent toxin by heat does not produce the disease. A toxin can be separated by nitration from bouillon cultures. It is fairly resistant to heat, withstanding two hours at 70-75° C. without being destroyed, and it is also very rapid in its action, being capable in appropriate dose of killing a horse in five minutes. The disease is one against which immunity can be readily produced in various ways, and methods of pre- ventive inoculation have been adopted in the case of animals liable to suffer from it. This subject was specially worked out by Arloing, Cornevin, and Thomas, and later by others. Immunity may be produced by injection with a non-fatal dose of the virus (i.e. the oedematous fluid found in the FIG. 132. — Bacillus of quarter- evil, showing spores. From a culture in glucose agar, incubated for three days at 37° C. Stained with weak carbol-fuchsin. x 1000. tissues of affected animals and which contains the bacilli), or by injection with larger quantities of the virus at- tenuated by heat, drying, etc. It can be produced also by cultures attenuated by heat and by the products of the bacilli obtained by nitration of cultures. An antitoxin has been produced against the toxins of the bacillus, and a method of protection in which the action of this antitoxin is combined with that of the. virus has been used. The antitoxin is said to increase the chemotactic properties of the leucocytes. BACILLUS ^ROGENES CAPSULATUS. This bacillus, though sometimes aiding in the production of patho- logical changes, is chiefly of interest on account of the extensive gaseous development which it gives rise to in the tissues post mortem. It was described by Welch and Nuttall in 1892 ; it is now recognised as being identical with an organism found in gaseous phlegmon by E. Fraenkel, and called by him the bacillus phlegmones emphyscmatosce. The organism is a comparatively large one, measuring 3 to 6 //, in length and having a thickness about the same as that of the anthrax bacillus ; its ends are square or slightly rounded (Fig. 133). It often occurs in pairs, sometimes in chains ; occasionally filamentous forms are met with. It usually shows a well-marked capsule, hence the name ; it is non-motile and does not form spores. It stains readily with the basic aniline dyes and retains the stain in Gram's method. It grows readily on the ordinary media, 398 BACILLUS ^RROGENES CAPSULATUS but only under anaerobic conditions ; the optimum temperature is that of the body, growth at the room temperature being comparatively slow. In a puncture culture in agar there is an abundant whitish line of growth with somewhat indented margin ; the individual colonies are white and of rounded or oval form. There is practically no liquefaction of gelatin, though this medium becomes somewhat softened around the growth. In all cases there is a tendency to abundant evolution of gas in the cultures, and this is especi- ally marked when ferment- able sugars are present. The organism appears to be the most frequent cause of rapid gaseous develop- ment in the blood and organs post mortem, this depending upon an invasion #HH of the blood immediately before death. In such cases, even within twenty - four hours under ordinary con- ditions, large bubbles of gas may be present in the veins, and the organs may be beset with gas-contain- ing spheres of various sizes ; the liver is usually the organ most affected, and its appearance has been com- Fio. 133. — Bacillus aerogeues capsulatu.s ; * £ed to that of Grilyere film preparation from bone- marrow in a cheese. The invasion by case where gas-cavities were present in the this organjsm is met with organs, x 1000. from time to time in puer. peral cases, and also in connection with ulcerative or gangrenous conditions of the intestine ; the bacillus is also found not infrequently in the peritoneum in cases of perforation. . Although the striking changes in the organs are due to a post-mortem development of the bacillus, there is no doubt that its entrance into the blood stream often hastens death, and may in some instances be the cause of it. As already stated, the organism is also met with in some cases of spreading oedema' with emphysema as a leading feature. When tested experimentally the bacillus by itself is found to have little pathogenic action. Injection of pure cultures in rabbits and guinea-pigs may be followed by little result, but sometimes in the latter animals "gaseous phlegmon" is produced, without suppuration unless other organisms are present. If a small quantity of culture be injected intravenously, e.g. in a rabbit, and then the animal be killed, bubbles of gas are rapidly produced in the blood and organs, the picture corre- sponding with that in the human cases. CHAPTER XVII. CHOLERA. Introductory. — It is no exaggeration of the facts to say that previously to 1883 practically nothing of value was known regard- ing 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 also obtained pure cultures of the organism from a large number of cases of cholera, and described their characters. The results of his researches were given at the first Cholera Conference at Berlin in 1884. Since Koch's discovery, 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 invariable 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 con- siderable variations in their characters, and, further, spirilla which closely resemble Koch's cholera spirillum have been cultivated 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 are to be regarded as indicating distinct species or merely 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. These questions will be discussed below. In considering the bacteriology of cholera it is to be borne in mind that in this disease, in addition to the evidence of great intestinal irritation, accompanied by profuse watery discharge, and 399 400 CHOLERA . often by vomiting, there are also symptoms of general systemic disturbance which cannot be accounted for merely by the with- drawal 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-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 .^^••1^^^ with fluid contents. As VJT* xi^ *?i > the characteristic organ- \vJV^ It ~j ''J •*% f^r !S th ~' ' THE CHOLERA SPIRILLUM 401 made from the intestinal contents in typical cases, these organ- isms are present in enor- mous numbers in almost pure culture, most of the spirilla lying with their long axes 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 suit- able methods they are seen to be flagellated. Usually a single terminal flagellum is present at one end only (Fig. 135). It is very delicate, and measures four or five times the length of the organism. In some varieties, however, there may be a flagellum at both ends, or more than one may be present ; cul- tures obtained at different places have shown con- siderable variations in this respect. Cholera spirilla do not form spores. In old cultures the organisms may present great variety in size and shape. Some are irregularly twisted fila- ments, sometimes globose, sometimes clubbed at their extremities, and also show- ing irregular swellings along their course. Others are short and thick, and may FIG. 135. — Cholera spirilla stained to show the terminal flagella. x 1000. have the appearance of large cocci, often staining faintly. All these changes in appear- ance are to be classed to- gether as involution forms. Staining. — -'Cholera spirilla stain readily with the usual basic 26 FIG. 136. — Cholera spirilla from an old agar culture, showing irregularities in size and shape, with numerous faintly - stained coccoid bodies — involution forms. Stained with fuchsin. x 1000. 402 CHOLERA aniline stains, though Lofner's methylene-blue or weak carbol- fuchsin is specially suitable. They lose the stain in Gram's method. Distribution within the Body. — The chief fact in this con- nection 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 Lieberkiihn, and may be seen lying between the basement membrane and the epithelial lining, which becomes loosened by their action. They are, however, rarely found in the connective tissue beneath, and never penetrate deeply. 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 epithelium, the intestinal contents being a com- paratively 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. 413.) 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 16° C., though in some cases it has been obtained at a lower temperature. Peptone Gelatin. — On this medium the organism grows well and produces liquefaction. In puncture cultivations at 22° 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 CULTIVATION 403 appearance : there is at the surface the bubble -shaped de- pression ; below this there is a funnel-shaped area of liquefaction, the fluid being only slightly turbid, but showing at its lower end thick masses of growth of a more or less spiral shape (Fig. 137). The liquefied portion gradually tapers off downwards towards the needle track. (This appearance is, however, in some varieties not produced till much later, especially when the gelatin is very stiff, and, in other varieties which liquefy very slowly, may not' be met with at all.) At a later stage, lique- faction spreads and may reach the side of the, tube. In gelatin plates the colonies are some- what characteristic. They appear as minute whitish points, visible in twenty-four to forty- eight hours, the surface of which, under a low power of the microscope, is irregularly granular or furrowed (Fig. 138, A), and later has an appearance which has been compared to fragments of broken glass. Liquefaction occurs, and the colony sinks into the small cup formed, the plate then showing small sharply - marked rings around the colonies. 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 (Fig. 138, B). The growth of the colonies in gelatin plates constitutes one of the most important means of distinguishing the cholera spirillum from other organisms. On the surface of the agar media a thin almost transparent layer forms, which pre- sents no special characters. 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 organisms. On potato at the ordinary temperature, growth does not take place, but on incubation at a temperature of from 30° to 37° C., a moist layer appears, which assumes a dirty brown colour somewhat like that of the glanders FIG. 137. — Puncture culture of the cholera spirillum in peptone gelatin — six days' growth. Natural size. 404 CHOLERA bacillus ; the appearance, however, varies somewhat 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 37° C. a 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 coagu- lation 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 peptone FIG. 138. — Colonies of the cholera spirillum in a gelatin plate ; three days' growth. A shows the granular surface, liquefaction just commencing ; in B liquefaction is well marked. solution and in bouillon, a circumstance of importance in relation to its separation in cases of cholera (vide p. 413). The cholera organism is one which grows much more rapidly in the presence of oxygen than in anaerobic conditions ; in the complete exclusion of oxygen very little growth occurs. Cholera-red reaction. — 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 test is made by adding a few drops of pure sulphuric acid to a culture in bouillon or in peptone solution (1 per cent) which has been incubated for twenty-four hours at 37° C. ; in the case of the cholera spirillum a reddish-pink colour is produced. This is due to the fact that both 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 POWERS OF RESISTANCE 405 the red colour. Here, as in testing for the production of indol by other bacteria, 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 organism which had not the power of forming nitrites. Hcemolytic Test. — This method introduced by Kraus is performed by means of agar plates, a small quantity of sterile defibrinated blood being added to the agar and thoroughly diffused ; if any organism has hsemolytic properties a clear zone or areola forms around each colony by the diffusion of haemo- globin. In no instance has an undoubted cholera organism been found to produce haemolysis, whereas many species of spirilla closely resembling it possess marked haemolytic action. This test may accordingly be applied along with the others in determining the identity of a supposed cholera organism. Powers of Resistance. — In their resistance against heat, cholera spirilla correspond with most spore-free organisms, and are killed in an hour by a temperature of 55° C., and much more rapidly at higher temperatures. They have comparatively high powers of resistance against great cold, and have been found alive after being exposed for several hours to the temperature of - 10° 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 conditions, 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 exceptional 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, although they may remain alive for a considerable period of time. On moist linen, as Koch showed, they can nourish 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, 406 CHOLERA gradually dimmish 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 conditions for its growth in competition with other organisms. Though we can state generally that the conditions favourable for the growth of the cholera spirillum are a warm temperature, moisture, a good supply of oxygen, and a consider- able proportion of organic material, we do not know the exact circumstances under which it can nourish for an 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 per- sistence 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 conclusion which is well supported by observations on the spread, of the disease. Cholera is practically always transmitted by means of water or food contaminated by the organism, and there is no doubt that con- tamination 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 contaminated 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 agency 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 EXPERIMENTAL INOCULATION 407 experiments on animals by 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 multiplication 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 dogs and rabbits, and they succeeded in producing, 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. Think- ing 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. As this method failed to give positive results he 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 neutral- ising as before with the carbonate of sodium solution. The result was remarkable, as thirty out of thirty -five animals treated died. 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 coiourless fluid with small fiocculi and the cholera organisms in practically pure cultures. These experiments, which have been repeatedly confirmed, therefore demonstrated that the cholera organisms could, under certain conditions, 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 the results obtained with the cholera organism from being used as a demonstration of the specific relation of the latter to the disease. Within later 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 resulting the usual intestinal changes, sometimes with hsemorrhagic peritonitis, the organ- isms, 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 408 CHOLERA transmitted to others by a natural mode of infection. In this affection of young rabbits many of the symptoms of cholera are present. 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 performed 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 evidence obtained from experiments on intestinal infection of animals, though by no means sufficient to establish the specific relation- ship 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. 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. 360). These and other 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 pronounced. Hence arise questions as to the nature and mode of action of toxic bodies produced by the cholera organism. Toxins. — 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 on agar, 1 Pfeiffer obtained his earlier results with a vibrio from Massowah, which is now known (as mentioned above) not to be a true cholera organism. This fact shows that the effects described are not specific to the latter. TOXINS OF KOCH'S SPIRILLUM 409 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 30° 0. before death. Pfeiffer considers that the toxic substances are contained in the bodies of the organisms — that is, they are in- ' tracellular, — 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 filtrate possessed very feeble toxic properties. He showed also that when an animal is inoculated intraperitoneally with the cholera organism, and then some time later anti-cholera serum which produces bacteriolysis is injected, rapid collapse with a fatal result may ensue, apparently due to the liberation of the intra- cellular toxins. 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 60° C., but even after heating at 100° C. a small proportion of toxin remained, which had the same physio- logical action. Recently A. Macfadyen found that the product obtained by grinding up the spirilla frozen by means of liquid air had a very high degree of toxicity when injected intravenously. Like Pfeiffer he found that the " endotoxin " was in great part destroyed at 60° C. On the other hand, other observers (Petri, Ransom, Klein, and others) have obtained toxic bodies from filtered cultures. Metchnikoff, E. Roux, and Taurelli-Salimbeni have demon- strated the formation of such diffusible toxic bodies in fluid media. By means of cultures placed in collodion sacs in the peritoneum of animals, they found that the living organisms produce toxic bodies which diffuse through the wall of the sac and produce toxic symptoms. 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 i c.c. per 100 grm. weight). If the dose of the toxin 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. They found that the toxicity of the filtrate was not altered by boiling; appar- ently the toxic substance is different from Pfeiffer's endotoxin. 410 CHOLERA Attempts to investigate the chemical nature of the toxic bodies have not led to definite results. Experiments on the Human Subject. — Experiments have also been performed in the case of the human subject, both intention- ally 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 enormous numbers. Recovery, how- ever, 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 performed 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 numerous cholera spirilla were present in the stools, though toxic symptoms were proportionately little pronounced. Metchnikoff also, by experiments on himself and others, obtained results which convinced him of the specific relation of the cholera spirillum to the disease. Lastly, we may mention the case of Dr. Orgel in Hamburg, 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 to cholera, and the facts mentioned above are, in our opinion, of the greatest importance 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 (conveniently made into the peritoneum) of non-fatal doses of the spirilla. It is better to IMMUNITY 411 commence the process with non-fatal doses of cultures killed by the vapour of chloroform or by heat, the doses being gradually increased, and afterwards 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 pro- tective 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. Under these circumstances the spirilla undergo a granular transformation and, ultimately, solution ; this phenomenon is generally known as Pfeiffer's reaction, and was applied by him to distinguish the cholera spirillum from organisms resembling it. The following are the details : — Pfeiffers Reaction. — A loopf'ul (2 uigrm.) 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 grm. in weight), and the peritoneal fluid of this animal (conveniently obtained by means of capillary glass tubes inserted into the peritoneum) is examined micro- scopically 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 — negative result. In the former case (positive result) there is, however, still the possibility that the organism is devoid of pathogenic properties and has been destroyed by the normal peritoneal fluid. A control experiment should accordingly be made with '001 c.c. of normal serum in place of the anti-cholera serum. If no alteration of the organism occurs with its use, then it is to be concluded that a true reaction has been given. The serum of an animal immunised by the above method has also marked agglutinative action against the cholera spirillum, and this property closely corresponds with Pfeiffer's reaction as regards specificity. Such a serum has, however, little protective effect against the toxic action of the dead spirilla. On the other hand, Macfadyen by injecting the endotoxin derived from the spirilla by grinding obtained a serum which had antitoxic as well as agglutinative and bacteriolytic properties (vide Immunity). Metchnikoff and others have also obtained antitoxic sera which act on the extra-cellular toxins obtained by filtration. The serum of cholera convalescents has been found to possess properties similar to those of immunised animals ; that is, it affords protection against the cholera spirillum and may also give Pfeiffer's reaction. These properties of the serum may be present eight or ten days after the attack of the disease, but are most marked four weeks after ; they then gradually become 412 CHOLERA weaker and disappear 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 and Bensande). Needless to say, such facts supply strong additional evidence of the relation of Koch's spirillum to cholera. Anti-Cholera Inoculation. — Haffkine's method for inoculation against cholera exemplifies the above principles. It depends upon (a) attenuation of the virus — that is, the cholera organism, and (6) 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 Try the death of the animal, but if the animal be treated first with the attenuated virus, the sub- sequent 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, distinctly encouraging. In the human subject two or sometimes three in- oculations were formerly made with attenuated virus before the virus exalte was used ; now, however, a single injection of the latter is usually practised. Wassermann and Pfeiffer, and also Klein, have found that guinea-pigs immunised by Haffkine's method are not immunised against intestinal infection when the animal is treated by Koch's method (vide p. 407). Notwith- standing this fact Haffkine's method may still have a beneficial effect, though it may not be preventive in all cases. Methods of Diagnosis. — In the first place, the stools ought to be examined microscopically. Dried film preparations should METHODS OF DIAGNOSIS 413 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 con- dition, 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. In the case of the first appear- ance of a cholera-like disease, however, all the other tests should be applied before a definite diagnosis of cholera is made. Dunbar has recently introduced a method for rapid diagnosis which depends on the properties of an anti-cholera serum. Two hang-drop preparations are made, each consisting of a small portion of mucus from the suspected stool broken up in peptone solution. To one a drop of a 50-fold dilution of normal serum is added, to the other a drop of a 500-fold dilution of an active cholera serum. If the spirilla present are cholera organisms they retain their motility in the first preparation, while they lose it and then become agglutinated in the second. By this method a diagnosis may sometimes be given in a few minutes. If the organisms are very numerous, gelatin 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 from 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 spirilla, 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 should 414 CHOLERA 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. The action of an anti-cholera serum, i.e. the serum of animal immunised against the cholera spirillum, should be tested in a similar manner. Up till recent times there had been cultivated from sources other than cholera cases, no organism which gave all the cultural and biological tests (agglutination and Pfeiffer's reaction) of the cholera spirillum. In 1905, however, Gotschlich obtained six different strains of a spirillum which conformed in all these respects. The organisms were obtained at El Tor from the intestines of pilgrims who had died with dysenteric symptoms, and there were no cases of cholera in the vicinity. The organisms in question, however, differ from the cholera organism in having marked hsemolytic action, and also in producing a rapidly acting extra -cellular toxin. There has been diversity of opinion with regard to the nature of these organisms, for while some consider that they are a different species from the cholera organism, others regard them as true cholera spirilla which had been carried by the patients, although no symptoms of cholera resulted from their presence. If they are not to be regarded as cholera organisms, .we have the striking fact that they correspond in the immunity reactions. This instance exemplifies well the great difficulty which may surround the identification of a particular organism obtained from non- cholera cases, and one can hardly doubt that if cholera -like symptoms had been present in the El Tor cases, the spirilla would have been accepted as varieties of the cholera organism, though differing in their haemolytic action. None of the facts ascertained, however, really affect the question as to the causal relationship of Koch's spirillum to cholera, although they indicate the difficulties which may attend the bacteriological diagnosis in isolated cases of the disease. 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 conform closely with Koch's description, though variations undoubtedly occur. Moreover, the facts known with regard to their conditions of growth, etc., are in conformity with the origin and spread of cholera epidemics. SPIRILLA RESEMBLING CHOLERA SPIRILLUM 415 Secondly r, the experiments on animals with Koch's spirillum or its toxins 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 conclusion 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, recognised two main factors in the causation of epidemics, which he designated x and y, and considered 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 admitted to be Koch's spirillum ; the y includes climatic and local con- ditions, e.g. state of ground-water, etc. Other Spirilla resembling the Cholera Organism. — These have been chiefly obtained either from water contaminated 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 berolinensis, cultivated by Neisser from Berlin sewage water, differs from the cholera organism only in the appearance of its colonies in gelatin 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. The organism differed somewhat in the appearance 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 microscopically in the 416 CHOLERA 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 experts, might arise as to whether a certain spirillum were really the cholera organism or a distinct species resem- bling it. A few examples may also be given of organisms cultivated from cases in which cholera-like symptoms were present. The vibrio of Massowah was cultivated by Pasquale from a case during a small epidemic of cholera. The organism so closely resembles Koch's spirillum that it was accepted by several authorities as the true cholera organism, and, as already stated, Metchnikotf produced 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 septicaemia 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. Another organism, the v. Gindha, was cultivated by Pasquale from a well, and was at first accepted by Pfeiffer as the cholera organism, but afterwards rejected, chiefly because it failed to give the specific immunity reaction. It also differs some- what 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 wrhich 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 gelatin. 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. Romanus) 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 Colonel Cunningham in India. It is there- fore quite an open question whether some of the organisms in the former class may not be cholera spirilla which have under- gone variations as a result of the conditions of their growth. That such variations may occur we have a considerable amount of evidence. The great bulk of evidence, however, goes to show METCHNIKOFF'S SPIRILLUM 417 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 organism may be present in the intestinal discharges, though rarely in large numbers. A number of other spirilla have been cultivated, which are of interest on account of their points of resemblance to the cholera organism, though probably they produce no pathological condi- tions in the human subject. Metchnikoff's Spirillum (vibrio Metchnikovi). — This organism was ob- tained by Gamaleia from an epidemic disease of fowls in Odessa, and 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 Vt4T* *"«> their feathers ruffled, and . & usually die within forty- M 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 in the blood in the younger fowls, though generally absent from the blood in the older. Morphologically the or- ganism is practically identi- cal with Koch's spirillum (Fig. 139). It is actively FIG. 139.— Metchnikoflfs spirillum, both in motile, and has the same curved and straight forms ; from an agar staining reactions. Its culture of twenty-four hours' growth, growth in peptone gelatin Stained with weak carbol-fuchsin. x 1000. also closely resembles that of the cholera organism, though it produces liquefaction more rapidly (Fig. 140, A). In gelatin plates the young colonies are, however, smoother and more circular. After liquefaction occurs, some of the colonies are almost identical in appearance with those of the cholera vibrio, whilst others show more uniformly turbid contents. In puncture cultures the growth takes place more rapidly, but in appearance 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, 27 418 CHOLERA of pure culture in pigeons is followed by septicaemia, which produces a fatal result usually within twenty-four hours. Inoculation with the same quantity of cholera culture produces practically no result ; even with large quantities death is rarely produced. The vibrio Metchnikovi produces somewhat similar effects in the guinea-pig to those in the pigeon, subcutaneous inoculation being followed by extensive haemor- rhagic redema 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 toxins of this organism have somewhat the same action as those of the cholera organism. The organism is therefore one which very closely resembles the cholera organism, the results on in- oculating the pigeon offering the most ready means of distinction. It gives a negative reaction to Pfeiffer's test — that is, the properties of an anti- cholera serum are not exerted against it. It may also be mentioned that an organism which is apparently the same as the vibrio Metchnikovi 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 dis- tinguished from it by its micro- scopical characters, although, on the whole, it tends to be rather thicker in the centre and more pointed at the ends (Fig. 141). In cultures, however, it presents marked differences. In puncture cultures on gelatin 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. 140, B). In plate cultures the growth of the colonies is proportionately 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 ; ultimately general liquefaction occurs. On potatoes this organism grows well at the ordinary temperature, and A B FIG. 140. — Puncture cultures in peptone-gelatin. A. Metchnikoffs spirillum. Five days' growth. B. Finkler and Prior's spirillum. Four days' growth. Natural size. DENEKE'S SPIRILLUM 419 X 4 .t 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 fcetid 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 ap - pear. As stated above, Koch succeeded in producing, by this organism, an intes- tinal affection inguinea-pigs after neutralising the stom- ach contents and paralysing the intestine with opium. This occurs in a small pro- portion of the animals ex- perimented on, and the con- ^rv tents of the intestine, unlike what was found in the case of the cholera organism, ^j were turbid in appearance, and had a markedly foetid odour. When tested by in- traperitoneal injection, its effects are somewhat of the same nature as those of the 'V ;^L I \ FIG. 141. — Finkler and Prior's spirillum ; from an agar culture of twenty - four hours' growth. Stained with carbol-fuchsin. x 1000. cholera organism, but its virulence is of a much lower order. An organism cultivated by Miller (" Miller's Spirillum") 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 appearances, though it is rather thinner and smaller. Its growth in gelatin is also somewhat similar, but liquefaction proceeds 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 examination, 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 30° 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 accounts of the organism now known as the influenza bacillus were published simultaneously by Pfeiffer, Kitasato, and Canon, in January 1892. The two first-mentioned observers found it in the bronchial - \r*J ' '*?. sputum, and obtained pure . */ ;J *. i cultures, and Canon ob- j* . % ' Kv f- vv» f^ served it in the blood in a *'A x few cases of the disease. fc * tf, ' fcf&s* " % t It is, however, to Pfeiffer 's >;t ' , , work that we owe most of » i jfv «£**/{' * 1 *..^> our knowledge regarding - « ' s?v'^» | its characters and action. *~" /,*?' S-is results have been ' c* % / amply confirmed by those r of others in various epi- fri? ' f '^\*. .'. V / demies of the disease, and V ^ , « , this organism has been . generally accepted as the cause of the disease, al- FIG. 142. -Influenza bacilli from a culture ^ugh absolute proof is on blood agar. j.-n Stained with carbol-fuchsin. x 1000. stl11 wanting. Microscopical Char- acters.— The influenza bacilli as seen in the sputum are very minute rods not exceeding 1 '5 /A in length and '3 p in thickness. They are straight, with rounded ends, and sometimes stain more deeply at the extremities (Fig. 142). 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 420 CULTIVATION OF BACILLI 421 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. In many cases of the disease, especially in the early stages of the more acute, influenza bacilli are present in large numbers and may be easily found. On the other hand, it is often difficult or impossible to find them, even when the symptoms are severe ; this may be due to the restriction of the organisms to some part not readily accessible, or it may be that they actually die out in great part while the effects of their toxins persist. It has also been observed in recent epidemics, in which the disease has been less widespread and on the whole less severe, that the period during which the bacilli have been readily demonstrable in the secretions has been on the average shorter than in the previous epidemics. Cultivation. — The best medium for the growth of the influenza bacillus is blood agar (see page 38), which was intro- duced by Pfeiffer for this purpose. He obtained growths of the bacilli on agar which had been smeared with influenza sputum, but he failed to get any subcultures 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. In this way he completely succeeded in attaining his object. The blood of the lower animals is suitable, as well as human blood ; and the favouring influences of the blood would appear to be due to the haemoglobin, as a solution of this substance is equally effective. The colonies of the influenza bacilli on blood agar, incubated at 37° C., appear within twenty-four hours, in the form of minute circular dots almost transparent, like drops of dew. 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 or five days. By this method the cultures may be maintained for an indefinite period. Growth on the ordinary agar media is slight and somewhat uncertain ; there is, however, evidence that growth is more marked when other organisms are present, that is, is favoured by symbiosis. Neisser, for example, was able to cultivate the influenza bacfllus on plain agar through several generations by growing the xerosis bacillus 422 INFLUENZA along with it ; dead cultures of the latter had not the sanu- favouring effect. 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 25° to 42° C., the optimum temperature being that of the body. The infliu'ii/a 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 tempera- ture were usually dead in twenty hours, and that if sputum \\viv 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 means of fine particles of disseminated sputum, 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 disappeared* and may still be detected in the sputum. Especially is this the case when there is any chronic pulmonary 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 superficial parts of the mucous membrane. 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 DISTRIBUTION OF MClLLl 423 very grave. Regarding the presence of influenza bacilli in the other pulmonary complications following influenza, much in- formation is still required. Occasionally in the foci of sup- purative softening in the lung the influenza bacilli have been found in a practically pure condition. In cases of empyema the organisms 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. Pfeiffer's 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 conclusions of other observers have, on the whole, confirmed this statement, and it is probable that the chief symptoms in the disease are due to toxins absorbed from the respiratory tract (vide infra). The bacillus may be present in some of the lesions complicating influenza. Pfeiffer found it in inflammation of the middle ear, but in a case of meningitis following influenza FraenkePs diplococcus was present. In a few cases of meningitis, however, the influenza bacillus has been found, sometimes alone, sometimes along with pyogenic cocci ( Pf uhl 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. In a few cases also the bacilli have been found in the brain and its membranes with little tissue change in the parts around. Extensive observations on the bacteriology of the respiratory system show that influenza-like bacilli may be present in a great variety of conditions ; we have, in fact, once more to do with a group of organisms with closely allied characters, of which Pfeiffer's influenza bacillus was the first recognised example. These "pseudo-influenza" bacilli have been obtained from the fauces, bronchi, and lungs in inflammatory conditions, and also in various specific fevers. To this group belongs the bacillus which has been cultivated from cases of whooping-cough by Spengler, Jochmann, Davis, and others, and which is present in considerable numbers in a large proportion of cases of this disease. Wollstein has obtained a marked agglutinative reaction on this organism by the serum of whooping-cough patients, all the sera examined giving a positive reaction in a dilution of at least 1 : 100 on all the strains of the organism isolated ; on the other hand, clumping was never obtained with a normal serum in a greater dilution 424 INFLUENZA than 1:10. Davis, by inoculation of the fauces of a healthy man with this organism, produced inflammatory change with febrile reaction, but not the characteristic symptoms of whooping-cough. There is presumptive evidence in favour of the bacillus being etiologically related to the disease, though the matter cannot yet be considered settled. Miiller's "trachoma bacillus" (p. 192) is a member of the same group. All these organisms are very restricted in their growth, and require the addition of blood or haemoglobin to the ordinary culture media ; hence they are some- times spoken of as hsemophilic bacteria. Some of the examples are a little larger than the influenza-bacillus, and tend to form short filaments, but others are quite indistinguishable. All of them also seem to have very feeble pathogenic properties towards the lower animals. At present it can scarcely be claimed as possible to identify PfeifTer's bacillus by its microscopic and cultural characters. 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. There wras, however, little evidence that the bacilli had under- gone multiplication, the symptoms being apparently produced by their toxins. 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 succeeded in producing infection 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, 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, however, never found in the blood or in other organs. Similar symptoms were also produced by injection of dead cultures, though in this case the dose required to be five or six METHODS OF EXAMINATION 425 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 toxins 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 often in very large numbers. The observed relationships of the organism to lesions in the lungs and elsewhere leave no room for doubt that it is possessed of pathogenic properties, but we cannot yet maintain that its causal relationship to epidemic influenza is absolutely established. A certain amount of confirmatory evidence has been supplied by the results of experiment. Methods of Examination — (a) Microscopic. — A portion of the greenish-yellow purulent material which often occurs in little round masses in the sputum should be selected, and film prepara- tions should be made in the usual way. Films are best stained by Ziehl-Neelsen carbol-f uchsin 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 best brought out, according to Pfeiffer, 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 be allowed to remain till they have a moderately light colour, the time varying according to their appearance. They are then washed in pure alcohol, cleared in xylol, and afterwards mounted in balsam. (b) 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 37° 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 426 PLAGUE Hong Kong in 1894. The results of their investigations, which were published almost at the same time, agree in most of the important points. They cultivated the same organism from a large number of cases of plague, and reproduced the disease in susceptible animals by inoculation of pure cultures. It is to be noted that during an epidemic of plague, sometimes even preceding it, a high mortality has been observed amongst certain FIG. 143. — Film preparation from a plague bubo showing enormous numbers of bacilli, most of which show well-marked bipolar staining. Stained with weak gentian- violet, x 1000. animals, especially rats and mice, and that 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. 143), though considerable variations in size occur. They have rounded ends, and in stained preparations a portion in the middle of the bacillus is BACILLUS OF PLAGUE 427 often left uncoloured, giving the so-called " polar staining." In films from the tissues they are found scattered ~ *•' amongst the cells, for the most part lying singly, though pairs are also seen. On the other hand, in cul- tures in fluids, e.g. bouillon, they grow chiefly in chains, sometimes of considerable length, the form known as a streptobacillus result- ing (Fig. 145). In young agar cultures the bacilli show greater variation in size, and polar staining is less marked than in the tissues: sometimes forms Fm- ^^"^{J^LfaTr6 from a young of considerable length are stained witn wTak'cLbolTchsiu. x 1000. present. After a time in- volution forms appear, especially when the surface of the agar is dry ; but the formation of these is much more rapid and more marked when 2-5 per cent of • \ sodium chloride is added \ to the medium, constitut- ing the so-called " salt- agar " (Hankin and Leu- mann). On this medium, especially with the higher percentage, the in volution forms assume a great size and a striking variety of shapes, large globular, oval, or pyriform bodies resulting (Fig. 146); with FIG. 145.— Bacillus of plague in chains show- about 2 per cent sodium iugboumronaiUiUg' Fr°ma youngculture chloride,aftertwenty-four Stained with thionin-blue. x 1000. hours' incubation, the most striking feature is a general enlargement of all the bacilli. Sometimes in the tissues they are seen to be surrounded by an unstained capsule, though this appearance is by no means common. They do not form 428 PLAGUE 9 * ' spores. Gordon, who has found that they possess flagella which, however, stain with difficulty, states that they are motile. Most observers, however, and with these we agree, have failed to find evidence of true motility. They stain readily with the basic aniline stains, but are decolorised by Gram's method. Cultivation. — From the affected glands, etc., 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 18° C. On agar and on blood serum the colonies are whitish circular discs of somewhat '^ „ „ transparent appearance * y and smooth, shining sur- face. 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 agar cultures are kept at the room temperature, some of the colonies may show a more luxuriant growth with more opaque FIG. 146. — Culture of the bacillus of plague & on 4 per cent salt agar, showing involution appearance than the rest forms of great variety of -size and shape. of the growth, the appear- Stained with carbol-thionin-blue. x 1000. ance in fact being often such as to suggest the presence of impurities in the cultures. In stab cultures in peptone gelatin, growth takes place along the needle track as a white line, composed of small spherical colonies. On the surface of the gelatin a thin, semi-transparent layer may be formed, which is usually restricted to the region of puncture, though sometimes it may spread to the wall of the tube ; some- times, however, there is practically no surface growth. There is no liquefaction of the medium. In gelatin plates the superficial colonies develop first and form slightly raised semi-transparent discs with somewhat crenated margins \ the deeper colonies are smaller and of spherical shape with smooth outline. In bouillon the growth usually forms a slightly granular or powdery deposit at the foot and sides of the flask, somewhat resembling that of DISTRIBUTION OF BACILLI 429 a streptococcus. If oil or melted butter is added to the bouillon so that drops float on the surface, then a striking mode of growth may result, to which the term " stalactite " has been applied. This consists in the growth starting from the under surface of the fat globules and extending downwards in the form of pendulous, string-like masses. These masses are exceedingly delicate, and readily break off on the slightest shaking of the flask ; accordingly during their formation the culture must be kept absolutely at rest. This manner of growth constitutes an important but not absolutely specific character of the organism ; unfortunately it is not supplied by all races of the organism, and varies from time to time with the same race. The organism flourishes best in an abundant supply of oxygen ; in strictly anaerobic conditions almost no growth takes place. The organism in its powers of resistance corresponds with other spore-free bacilli, and is readily killed by heat, an exposure for an hour at 58° C. being fatal. On the other hand it has remarkable powers of resistance against cold ; it has been exposed to a temperature several degrees below freezing-point without being killed. Experiments on the effects of drying have given somewhat diverse results, but as a rule the organism has been found to be dead after being dried for from six to eight days, though sometimes it has survived the process for a longer period ; exposure to direct sunlight for three or four hours kills it. When cultivated outside the body the organism often loses its virulence, but some races remain virulent in cultures for a long period of time. Anatomical Changes and Distribution of Bacilli. — The disease occurs in several forms, the bubonic and the pulmonary being the best recognised ; to these may be added the septiccemic. The most striking feature in the bubonic form is the affection of the lymphatic glands, which undergo intense inflammatory swelling, attended with haemorrhage, 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. The bubo is thus usually formed by a collection of enlarged glands fused by the inflammatory swelling. 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 430 PLAGUE 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 (Fig. 143). In sections of the glands in the earlier stages the bacilli are found to form dense masses in the lymph paths and sinuses FIG. 147. — Section of a human lymphatic gland in plague, showing the injection of the lymph paths and sinuses with masses of plague bacilli — seen as black areas. Stained with carbol-thionin-blue. x 50. (Fig. 147), often forming an injection of them; they may also be seen growing as a fine reticulum between the cells of the lymphoid tissue. At a later period, when disorganisation of the gland has occurred, they become irregularly mixed with the cellular elements. Later still they gradually disappear, and when necrosis is well advanced it may be impossible to find any — a point of importance in connection with diagnosis. In the spleen they may be very numerous or they may be scanty, according to the amount of blood infection which has occurred ; EXPERIMENTAL INOCULATION 431 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 is usually a fairly abundant frothy sputum often tinted with blood, and in it the bacilli may be found in large numbers. Sometimes, however, cough and expectoration may be absent. The disease in this form is said to be invariably fatal. In the septiccemic form proper there is no primary bubo discoverable, though there is almost always general enlargement of lymphatic glands ; here also the disease is of specially grave character. A bubonic case may, however, terminate with septi- caemia ; in fact all intermediate forms occur. 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, however, just before death in very severe and rapidly fatal cases. The examination of the blood by means of cultivation experiments is, however, a much more reliable procedure. For this purpose about 1 c.c. of blood may be with- drawn from a vein and distributed in flasks of bouillon (p. 68). It may be said from the results of different investigators that the bacillus may be obtained by culture in fully 50 per cent of the cases, though the number will necessarily vary in different epidemics. The Advisory Committee, recently appointed, found that in some septica3mic cases the bacilli may be present in the blood in large numbers two, or even three, days before death, though this is exceptional. The above types of the disease are usually classified together under the heading pestis major, but there also occur mild forms to which the term pestis minor is applied. In these latter there may be a moderate degree of swelling of a group of glands, attended with some pyrexia and general malaise, or there may be little more than slight discomfort. Between such and the graver types, cases of all degrees of severity are met with. Experimental Inoculation. — Mice, guinea-pigs, rats, and rabbits are susceptible to inoculation, the two former being on the whole most suitable for experimental purposes. After sub- cutaneous injection there occurs a local inflammatory oedema, which is followed by inflammatory swelling of the corresponding 432 PLAGUE lymphatic glands, and thereafter by a general infection. The lesions in the lymphatic glands correspond in their main characters with those in the human subject, although usually at the time of death they have not reached a stage so advanced. By this method of inoculation mice usually die in 1-3 days, guinea-pigs and rats in 2-5 days, and rabbits in 4-7 days. Post mortem the chief changes, in addition to the glandular enlargement, are congestion of internal organs, sometimes with haemorrhages, and enlargement of the spleen ; the bacilli are numerous in the lymphatic glands and usually in the spleen (Fig. 148), and also, though in somewhat less ^f -f* if* **2t *f\. degree, throughout the mf ^ * fcP^v blood. Infection can also be produced by flf++ * 1 1 *J£I0>\ smearjng the material * '•: 4K, t _ .*•* "* • «• &< ,r • ,- on the conjunctiva or mucous membrane of the nose, and this method of inoculation has been successfully applied in cases where the plague bacilli are present along with other virulent organisms, e.g. in sputum along with pneumococci. FIG. 148. — Film preparation of spleen of rat Rats and mice can also after inoculation with the bacillus of plague, be infected by feeding !^!Yi^^e^1T,bacilli' m°st °f wMch either with pure cultures x 1000. are somewhat plump. Stained with carbol-thionin-blue. or with pieces of organs from cases of the disease, though in this case infection probably takes place through the mucous membrane of the mouth and adjacent parts, and only to a limited extent, if at all, by the alimentary canal. 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. Paths and Mode of Infection. — Plague bacilli may enter PATHS AND MODE OF INFECTION 433 the system by the skin surface through small wounds, cracks, abrasions, etc., and in such cases there is usually no reaction at the site of entrance. This last fact is in accordance with what has been stated above with regard to experiments on monkeys. The path of infection is shown by the primary buboes, which are usually in the glands through which the skin is drained, those in the groin being the commonest site. Absolute proof of the possibility of infection by the skin is supplied by several cases in which the disease has been acquired at post-mortem examinations, the lesions of the skin surface being in the majority of these of trifling nature ; in only two was there local reaction at the site of inoculation. In most of these cases the period of incubation has been from two to three days ; under natural conditions of infection the average period is within five days. While infection may occur by accidental inoculation through small wounds of the skin surface, it appears in the majority of cases to take place by means of the bites of fleas. For some time it had been known that plague bacilli might be found for some time afterwards in the stomach of fleas allowed to feed on animals suffering from plague, and some observers, for example Simond, had succeeded in transmitting the disease to other animals by means of the infected insects. Most observers, however, had obtained negative results, and it was only by the work of the Advisory Committee appointed by the Secretary of State for India in 1 905 l that the importance of this means of infection was established. By carefully planned experiments the Committee showed that the disease could be transmitted from a plague rat to a healthy rat kept in adjacent cages when fleas were present ; whereas this did not occur when means were taken to prevent the access of fleas, though the facilities for aerial infection were the same. The disease can also be produced by fleas removed from plague rats and trans- ferred directly to healthy animals, success having been obtained in fully 50 per cent of experiments of this kind. When plague- infected guinea-pigs are placed amongst healthy guinea-pigs comparatively few of the latter acquire the disease when fleas are absent or scanty ; whereas all of them may die of plague when fleas are numerous. This result demonstrates the com- paratively small part played by direct contact, even when of a close character. Important results were also obtained with regard to the mode of infection in houses where there had been cases of plague. It was found possible to produce the disease in susceptible animals by means of fleas taken from rats in 1 See Journal of Hygiene, vi. 421 ; vii. 323. 28 * 434 PLAGUE plague houses. When animals were placed in plague houses and efficiently protected from fleas they remained healthy; whereas they acquired the disease when the cages were free to the access of fleas in the neighbourhood. The following are some of the experiments which were conducted. A series of six huts were built which only differed in the structure of their roofs. In two the roofs were made of ordinary native tiles in which rats freely lodge ; in two others flat tiles were used in which rats live, but in which they have not such facilities for movement as in the first set, and in the third pair the roof was formed of corrugated iron. Under the roof in each case was placed a wire diaphragm which prevented rats or "their droppings having access to the hut, but which would not prevent fleas falling down on to the floor of the hut. The huts were left a sufficient time to become infected with rats, and then on the floor in each case healthy guinea-pigs mixed with guinea-pigs artificially infected with plague were allowed to run about together. In the first two sets of huts to which fleas had access the healthy guinea-pigs contracted plague, while in the third set they remained unaffected, though they were freely liable to contamination by contact with the Bodies and excreta of the diseased animals. In the third set of huts no infection took place as long as fleas were excluded, but when accidentally these insects obtained admission, then infection of the uninoculated animals commenced. Other experiments were also performed. In one case healthy guinea-pigs were suspended in a cage two inches above a floor on which infected and flea-infested animals were running about. Infection occurred in the cage, but if the latter were suspended at a distance above the floor higher than a flea could jump, then no infection took place. Again, in a hut in which guinea-pigs had died of plague, and which contained infected fleas, two cages were placed, each containing a monkey. One cage was surrounded by a zone of sticky material broader than the jump of a flea. The monkey in this cage remained unaffected, but the other monkey con- tracted plague. Other experiments showed that when plague bacilli were placed on the floors of houses, they died off in a comparatively short period of time. After forty-eight hours it was not found possible to reproduce plague by inoculation with material from floors which had been grossly contaminated with cultures of the bacillus. Afterwards, however, animals placed in such a house might become infected by means of fleas. In all these experi- ments the common rat-flea of India — pulex cheojris (Rothschild) — was used, but it has been shown that this flea, when a rat is not available, will bite a man. These results are manifestly of great practical importance. They show that direct infection by dust and other material through small lesions of the skin plays, probably, a comparatively small part in the spread of the disease, fleas apparently being in the majority of cases the carriers of in- fection. They also point to important preventive measures, which will no doubt be put to a practical test before long. TOXINS, IMMUNITY, ETC. 435 In primary plague pneumonia, from a consideration of the anatomical changes and the clinical facts, the disease may be said to be produced by the direct passage of the bacilli into the respiratory passages. Nevertheless there must be certain factors, still imperfectly understood, which determine the incidence of this form ; as in some epidemics of the highest virulence plague pneumonia has been practically absent, though opportunities for infection by inhalation must have been present. On the other hand, a case of plague pneumonia is of great infectivity in producing other cases of plague pneumonia. If we except infection through the respiratory passages in such cases, it may be said that direct infection from patient to patient is relatively uncommon. This is in accordance with the fact that in bubonic plague the bacilli are not discharged from the unbroken surface of the body, and are only present in the secretions in severe cases. The occurrence of the disease in rats was early recognised, and there is no doubt that it plays a very important part in the spread of epidemics. The disease in these animals has, in fact, been the means of rapidly distributing infection over wide areas of a town or district. This has been abundantly proved in the case of Bombay, where observations have shown that the migration of plague-infected rats to quarters comparatively free from the disease, has been followed by extensive outbreaks in these places. The facts stated above show how the disease is spread among these animals by fleas, and how it is conveyed by them to the human subject. Toxins, Immunity, etc. — As is the case with most organisms which extensively invade the tissues, the toxins in plague cultures are chiefly contained in the bodies of the bacteria. Injection of dead cultures in animals produces distinctly toxic effects ; post mortem haemorrhage in the mucous membrane of the stomach, areas of necrosis in the liver and at the site of inoculation, may be present. The toxic substances are comparatively resistant to heat, being unaffected by an exposure to 65° C. for an hour. By the injection of dead cultures in suitable doses a certain degree of immunity against the living virulent bacilli is obtained, arid, as first shown by Yersin, Calmette, and Borrel, the serum of such immunised animals confers a degree of protection on small animals such as mice. On these facts the principles of preventive inoculation and serum treatment, presently to be described, depend. It may also be mentioned that the filtrate of a plague culture possesses a very slight toxic action, and the Indian Plague Commission found that such a filtrate has practi- cally no effect in the direction of conferring immunity. 436 PLAGUE 1. Preventive Inoculation — Hq/kine's Method. — To prepare the preventive fluid, cultures are made in flasks of bouillon with drops of oil on the surface (in India Haffkine employed a medium prepared by digesting goats' flesh with hydrochloric acid at 140° C. and afterwards neutralising with caustic soda). In such cultures stalactite growths (vide supra) form, and the flasks are shaken every few days so as to break up the stalactites and induce fresh crops. The flasks are kept at a temperature of about 25° C., and growth is allowed to proceed for about six weeks. At the end of this time sterilisation is effected by exposing the contents of the flasks to 65° C. for an hour; thereafter carbolic acid is added in the proportion of '5 per cent. The contents are well shaken to diffuse thoroughly the sediment in the fluid, and are then distributed in small sterilised bottles for use. The preventive fluid thus contains both the dead bodies of the bacilli and any toxins which may be in solution. It is administered by subcutaneous injection, the dose, which varies according to the "strength," being on an average about 7*5 c.c. Usually only one injection is made, sometimes two ; though the latter procedure does not appear to have any advantage. The method has been systematically tested by inoculating a certain proportion of the inhabitants of districts exposed to infection, leaving others uninoculated, and then observing the proportion of cases of disease and the mortality amongst the two classes. The results of inoculation, as attested by the first Indian Commission, have been distinctly satisfactory. For although absolute protection is not afforded by inoculation, both the proportion of cases of plague and the percentage mortality amongst these cases have been considerably smaller in the inoculated, as compared with the uninoculated. Protection is not established till some days after inoculation, and lasts for a considerable number of weeks, possibly for several months (Bannerman). In the Punjab during the season 1902-3 the case incidence among the inoculated was 1*8 per cent, among the uninoculated 7*7 per cent, while the case mortality was 23*9 and 60 '1 per cent respectively in the two classes, the statistics being taken from villages where 10 per cent of the population and upwards had been inoculated. 2. Anti-plague Sera. — Of these, two have been used as therapeutic agents, viz. that of Yersin and that of Lustig. Yersin's serum is prepared by injections of increasing doses of plague bacilli into the horse. In the early stages of immunisation dead bacilli are injected subcutaneously, thereafter into the veins, and, finally, living bacilli are injected intravenously. After a suitable time blood is drawn off and the serum is preserved in the usual way. Of this serum 10-20 c.c. METHODS OF DIAGNOSIS 437 are used, and injections are usually repeated on subsequent days. Lustig's serum is prepared by injecting a horse with repeated and increasing doses of a substance derived from the bodies of plague bacilli, probably in great part nucleo-proteid. Masses of growth are obtained from the surface of agar cultures, and are broken up and dissolved in a 1 per cent solution of caustic potash. The solution is then made slightly acid by hydrochloric acid, when a bulky precipitate forms ; this is collected on a filter and dried. For use a weighed amount is dissolved in a weak solution of carbonate of soda and then injected. The serum is obtained from the animal in the usual way. Extensive observations with both of these sera show that neither of them can be considered a powerful remedy in cases of plague, though in certain instances distinctly favourable results have been recorded. The Indian Com- mission, however, came to the conclusion " that, on the whole, a certain amount of advantage accrued to the patients both in case of those injected with Yersin's serum and of those injected with Lustig's serum." It may also be mentioned that the Commission found, as the result of experiments, that Yersin's serum modified favourably the course of the disease in animals, whereas Lustig's serum had no such effect 3. Serum Diagnosis. — Specific agglutinins may appear in the blood of patients suffering from plague, as also they do in the case of animals immunised against the plague bacillus. It is to be noted, however, that in clinical cases the reaction is not invariably present, the potency of the serum is not of high order, and the carrying out of the test is com- plicated by the natural tendency of the bacilli to cohere in clumps. For the last reason the macroscopic (sedimentation) method is be preferred to the microscopic (p. 111). A suspension of plague bacilli is made by breaking up a young agar culture in 75 per cent sodium chloride solution ; the larger flocculi of growth are allowed to settle, and the fine, supernatant emulsion is employed in the usual way. According to the results of the German Plague Commission and the observations of Cairns, made during the Glasgow epidemic, it may be said that the reaction is best obtained with dilutions of the serum of from 1 : 10 to 1 : 50. Cairns found that the date of its appearance is about a week after the onset of illness, and that it usually increases till about the end of the sixth week, thereafter fading off. It is most marked in severe cases characterised by an early and favourable crisis, less marked in severe cases ultimately proving fatal, whilst in very mild cases it is feeble or may be absent. The method, if carefully applied, may be of service under certain conditions ; but it will be seen that its use as a means of diagnosis is somewhat restricted. Methods of Diagnosis. — Where a bubo is present a little of the juice may be obtained by plunging a sterile hypodermic needle into the swelling. The fluid is then to be examined micro- scopically, and cultures on agar or blood serum should be made by the successive stroke method. The cultural and morpho- logical characters are then to be investigated, the most important being the involution forms on salt agar and the stalactite growth in bouillon, though the latter may not always be obtained with the plague bacillus : the pathogenic properties should also be studied, the guinea-pig being on the whole most suitable for 438 RELAPSING FEVER AND AFRICAN TICK FEVER subcutaneous inoculation. In many cases a diagnosis may be made by microscopic examination alone, as in no other known condition than plague do bacilli with the morphological char- acters of the plague bacillus occur in large numbers in the lym- phatic glands. The organism may be obtained in culture from the blood in a considerable proportion of cases by withdrawing a few cubic centimetres and proceeding in the usual manner. On the occurrence of the first suspected case, every care to exclude possibility of doubt should be used before a positive opinion is given. In a case of suspected plague pneumonia, in addition to microscopic examination of the sputum, the above cultural methods along with animal inoculation with the sputum should be carried out ; subcutaneous injection in the guinea-pig and smearing the nasal mucous membrane of the rat may be recom- mended. Here a positive diagnosis should not be attempted by microscopic examination alone, especially in a plague-free dis- trict, as bacilli morphologically resembling the plague organism may occur in the sputum in other conditions. RELAPSING FEVER AND AFRICAN TICK FEVER. At a comparatively early date, namely in 1873, when practi- cally 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 spirochcete 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 inocula- tions with blood containing the organisms, and a similar con- dition has been produced in apes. Recently it has been shown that the so-called "tick fever" prevalent in Africa is also due to a spirochsete of similar character, and results of the highest importance have been established with regard to the part played by ticks in the trans- mission of the disease. Doubt still obtains as to the relationship of the organisms of the two diseases, but all are agreed that they CHARACTERS OF THE SPIRILLUM 439 are closely similar, if not identical. As a matter of convenience, and in accordance with the history of the investigations, we shall give the chief facts regarding these diseases separately. It has also been shown that spirillar diseases or " spirilloses," as they are called, are widespread in the animal kingdom. Such infections have been described in geese by Sacharoff, in fowls by Marchoux and Salimbeni, in oxen and sheep by Theiler, and in bats by Nlcolle and Comte, and it is interesting to note that in the case of the spirilloses of oxen and fowls the infection is transmissible by means of ticks. The work of Schaudinn(v. p. 548) has led to these spirochsete being regarded by various authorities as members of the protozoal group. As, however, longitudinal division has not been satisfactorily observed and no cycle of development has been /£ determined amongst them, JB we are not justified at fl| present in removing them from the class of bacteria. Characters of the Spirillum. — The organ- £\ isms as seen in the blood J.OI11O CtO OV/V1J- AA-l UAJLV; KS±\S\S\A. during the fever are deli- cate spiral filaments which have a length of from two to six times the diameter of a red blood corpuscle. FlG 149.— Spirilla of relapsing fever in They are, however, exceed- human blood. Film preparation. (After ingly thin, their thickness Koch.) x about 1000. being much less than that of the cholera spirillum. They show several regular sharp curves or windings, of number varying according to the length of the spirilla, and their extremities are finely pointed (Fig. 149). There are often to be seen in the spirals, portions which are thinner and less deeply stained than the rest, and which suggest the occurrence of transverse division. 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. Novy and Knapp have found that there is a single flagellum at one end of the organism. They stain with watery solutions of the basic aniline dyes, 440 RELAPSING FEVER though somewhat faintly, and are best coloured by the llomanowsky method or one of its modifications. 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 considerable degree of vitality, and when kept in sealed tubes they have been found alive and active after many days. They are readily killed at a temperature of 60° C., but may be exposed to 0° 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 temperature 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 other seven days a sharp rise of temperature again takes place, but on this occasion the fever lasts a shorter time, again suddenly disappearing. 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, aud 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, and this experiment has been several times repeated with the same result. Additional proof, that the organism is the cause of the disease has been afforded by experiments on animals. Carter in 1879 was the first to show that the disease could be readily produced in monkeys, and his experi- ments were confirmed by Koch. In such experiments the blood taken from patients and containing the spirilla was injected sub- cutaneously. 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 appear in the blood, and shortly after- wards 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.1 White mice and rats are also susceptible 1 Norris, Pappenheimer, and Flournoy, in their experiments on monkeys in America, found that several relapses occurred. IMMUNITY 441 to infection. In the former animals the disease is characterised by several relapses, in the latter there is, however, no relapse. 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 multiplication took FIG. 150. — Spirillum Obermeieri in blood of infected mouse. x 1000. place. Recently Norris, Pappenheimer, and Flournoy have found that a considerable amount of multiplication may take place in the citrated blood of man and the rat. Immunity. — Metchnikoff found that during the fever the spirilla were practically never taken up by the leucocytes in the circulating blood, but that at the time of the crisis, on dis- appearing from the blood, they accumulated in the spleen and were ingested in large numbers by the microphages or poly- morphonuclear leucocytes. Within these they rapidly under- went degeneration and disappeared. It is to be noted in this connection that swelling of the spleen is a very marked feature in relapsing fever. These observations were entirely confirmed 442 RELAPSING FEVER by Soudake witch, who also produced the disease in two monkeys (cercocebus fuliginosus) from which the spleen had been previously removed, the animals having been allowed to recover completely from the operation, and found that 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. Recent observations, however, in- dicate that, as in the case of so many other diseases, the all- important factor in the destruction of the organisms is the development of antagonistic substances in the blood. Lamb found in the case of the monkey (macacm radiatus) that the removal of the spleen of an animal rendered immune by an attack of the disease did not render it susceptible to fresh inoculation, and attributed the immunity to the presence of bactericidal bodies in the serum. He found, for example, that in vitro the serum of an immune animal brought the movements of the spirilla to an end, clumped them, and caused their dis- integration ; and further, that when the spirilla and the immune serum were injected in one case into a fresh monkey no disease developed. In opposition to Soudakewitch, Lamb found that with a monkey from which the spleen had been removed death did not occur after it was inoculated with the spirilla. Sawt- schenko and Milkich found that there are developed during the disease an immune body and an agglutinin, while Novy and Knapp in their recent important work distinguish germicidal, immunising, and agglutinating substances. They found that the blood of the rat has no germicidal properties during the onset of the disease, but that these appear and become well marked during the decline. They produced a " hyper-immunity " in rats by repeated injections of blood containing the spirilla, and found that the serum of such animals had a markedly cura- tive effect, and could cut short the disease in rats, mice, and monkeys. In the case of the human subject it has been found that a second attack of the disease can follow the first after a com- paratively 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 or in tissues where they escape the bactericidal action of the serum. With the disappearance of the immunity the organ- AFRICAN TICK FEVER 443 isms 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 to allow all the organisms to be killed. The production of anti-substances during the febrile attack is an established fact, and the experi- mental results above detailed show that the disease as met with in the human subject will probably be eminently amenable to serum therapeutics. The fact that other like spirillar diseases may be conveyed by the bites of insects makes it extremely probable that relaps- ing fever may also be transmitted in this way, and a number of facts point to the bed-bug as the means of transmission. The presence of the spirilla within the bodies of bugs has been demonstrated, and it has also been shown that they may be present for a considerable time after the insects have sucked the blood, — according to Karlinski for forty days. Tictin, by inject- ing the blood removed from a number of bugs which had been allowed to bite infected monkeys, produced the disease in other healthy monkeys, but so far as we know the crucial experiment of infecting man by means of the bites of these insects has not yet been successfully carried out. African Tick Fever. The disease long known by this name as prevalent in Africa has also been shown to be caused by a spirillum or spirochaete. Organisms of this nature had been seen in the blood of patients in Uganda by Greig and Nabarro in 1903, and Milne and Ross in the end of 1904 recorded a series of observations which led them to the conclusion that tick fever was due to a spirochsete. It is, however, chiefly owing to the work of Button and Todd in the Congo Free State, on the one hand, and of Koch in German East Africa, on the other; that our knowledge of this disease has been thoroughly established. The former gave a full account of the organism, and by means of experiments showed that the disease could be transferred by means of ticks to healthy animals. The latter published interesting observations on the infection of the ticks and the transmission of the organisms to the young, and also important facts with regard to the extent to which ticks were infected in certain districts. The following are the chief facts regarding this fever. Clinically the fever closely resembles relapsing fever, but the periods of fever are somewhat shorter, rarely lasting for more than two or three days. It is rarely attended with a fatal result 444 AFRICAN TICK FEVER unless in patients debilitated by other causes. The spirilla are considerably fewer in the blood than in the European relapsing fever, and sometimes a careful search may be necessary before they are found. Morphologically they are said to be practically identical, although Koch thought that the organisms in tick fever tended on the whole to be slightly longer ; the average length may be said to be 15 to 35 /x. Dutton and Todd showed that it was possible to transmit the disease to certain monkeys .• /* FIG. 151. — Film of human blood containing spirillum of tick fever, x 1000. * (cercopitheci) by means of ticks which had been allowed to bite patients suffering from the disease, the symptoms in these animals appearing about five days after inoculation. The disease thus produced is characterised by several relapses, and often leads to a fatal result. In one case they produced the disease by means of young ticks hatched from the eggs of ticks which had been allowed to suck the blood of fever patients, and they came to the conclusion that the spirilla were not simply carried mechanically by the ticks, but probably underwent some cycle of 1 We are indebted to Colonel Leishman, R.A.M.C., for the preparations from which Figs. 150-152 were taken. AFRICAN TICK FEVER 445 development in the tissues of the latter. The species of tick concerned is the ornithodorus moubata. These results were con- firmed and extended by Koch. He found that after the ticks had been allowed to suck the blood containing the organisms, these could be found for a day or two in the stomachs of the insects. After this time they gradually disappeared from the stomach, but were detected in large numbers in the ovaries of the female ticks, where they sometimes formed felted masses. FIG. 152. — Spirillum of human tick fever (Spirillum Duttoni) in blood of infected mouse. x 1000. He also traced the presence of the spirilla in the eggs laid by the infected ticks, and in the young embryos hatched from them. He was thus able to demonstrate how the infection might be continued within the tissues of ticks from generation to generation ; in the process of transmission, however, the spirillar form was always observed, and there was no evidence that the organism went through a cycle of change. Koch also made extensive observations on the ticks in German East Africa, and found that of over six hundred examined 11 ,per cent of these insects along the main caravan routes contained spirilla, and in some localities almost half of the ticks were infected. In 446 MALTA FEVER places removed from the main lines of commerce he still found them, though in smaller number. It has also been demonstrated that in some places the ticks are found to be infected with the spirilla although the inhabitants do not suffer from tick fever, a circumstance which is probably due to an acquired immunity against the disease. Although our knowledge regarding the relationships of these to other spirilla is far from complete, certain differences between the organisms of European relapsing fever and of African tick fever have been established. Zettnow, for example, has found that the organism of tick fever possesses numerous lateral flagella, whereas, as already stated, the sp. Obermeieri has a single terminal flagellum. This observation, however, has not yet been confirmed. Differences are also brought out by animal inoculation. In addition to the more severe illness produced by the spirillum of tick fever in monkeys, it has been found by Breinl and Kinghorn that a considerable number of animals are susceptible to the African spirillum, including rabbits and guinea-pigs, which appear to be refractory to the organism of relapsing fever. Breinl also compared the immunity conferred by the sp. Obermeieri and by the tick fever spirillum, and found that each conferred a relative active immunity against itself, but not against the other. It is thus highly probable that they represent two distinct species. Spirillar fever has also been found in India, but its relations to the European and African fevers have not yet been fully worked out. 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. Since its bacteriology has been worked out, it has been found to occur also in India, China, and in some parts of North and South America, its distribution being much wider than was formerly supposed. Although from its symptomatology and pathological anatomy it had been re- cognised as a distinct affection, and was known under various names, its precise etiology was unknown till the publication of the researches of Colonel 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 MICROCOCCUS MELITENSIS 447 means of inoculation experiments established its causal relation- ship to the disease. Wright and Semple applied the agglutina- tion test to the diagnosis of the disease, while within recent years the mode of spread of the disease has been fully studied by a Commission, and it has been demonstrated that goat's milk is the chief means of infection. 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 con- tinued type with irregular remissions. In addition 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 '4 /A 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. 153). (Durham has shown that in old cultures kept at the room temperature bacillary forms appear, and we have noticed indications of such in comparatively young cultures ; the usual form is, however, that of a coccus.) It stains 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 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. It may be cultivated from the blood during life in a considerable proportion of cases; for this purpose 5-10 c.c. of blood should be withdrawn from a vein and distributed in small flasks of bouillon. The micrococcus was found by the members of the Commission in the urine of Malta fever patients in 10 per cent of the cases examined ; it was sometimes scanty, but some- times present in large numbers. 448 MALTA FEVER Cultivation. — This can usually readily be effected by making stroke cultures on agar tubes from the spleen pulp and incub- ating at 37° C. The colonies, which are usually not visible before the third or fourth day, appear as small round discs, slightly raised and of somewhat transparent appearance. The maximum size — 2-3 mm. in diameter — is reached about the ninth day ; 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 Hfefe^ cultures assume a buff tint. The optimum temperature is 37° C., but growth still occurs down to about 20° C. On gelatin at summer temperature growth is ex- tremely slow — after two or three weeks, in a puncture culture, there is a delicate line of growth along the needle track and a small flat expansion of growth „ * on the surface. There is no liquefaction of the rfrn medium. In bouillon there FIG. 153. — Micrococcus rnehtensis, from a . . ,., two days' culture on agar at 37° C. occurs a general turbidity Stained with fuchsin. x 1000. with flocculent deposit at the bottom ; on the surface there is no formation of a pellicle. The reaction of the media ought to be very faintly alkaline, as marked alkalinity interferes with the growth. On potatoes no visible growth takes place even at the body temperature, though the organism multiplies to a certain extent. Outside the body the organism has considerable powers of vitality, as it has been found to sur- vive in a dry condition in dust and clothing for a period of two months. 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 first showed that by inoculation with even comparatively small doses of pure cultures the disease could be produced in monkeys, sometimes with a fatal result. And it has now been fully established that inocula- MODE OF SPREAD OF THE DISEASE 449 tion with the minutest amount of culture, even by scarification, leads to infection both in monkeys and in the human subject. Rabbits, guinea-pigs, and mice are insusceptible to inocula- tion by the ordinary method. Durham, by using the intra- cerebral method of inoculation, has, however, succeeded in raising the virulence so that the organism is capable of produc- ing in guinea-pigs on intra-peritoneal injection illness with some- times 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. Mode of Spread of the Disease. — The work of the recent Commission has resulted in establishing facts of the highest importance with regard to the spread of the disease. In the course of investigations Zammitt found that the blood of many of the goats agglutinated the micrococcus melitensis, and Horrocks obtained cultures of the organism from the milk. Further observations showed that agglutination was given in the case of 50 per cent of the goats in Malta, whilst the organism was present in the milk in 10 per cent. Sometimes the organism was present in enormous numbers, and in these cases the animal usually appeared poorly nourished, whilst the milk had a some- what serous character. In other cases, however, the organism was found when the animals appeared healthy and there was no physical or chemical change in the milk. It was also determined that the organism might be excreted for a period of two to three months before any notable change occurred in the milk. Agglutination is usually given by the milk of infected animals, and this property was always present when the micro- coccus was found in the milk. It was, moreover, found that mon- keys and goats - could be readily infected by feeding them with milk containing the micrococcus, the disease being contracted by fully 80 per cent of the monkeys used. It was therefore rendered practically certain that the human subject was infected by means of such milk, and the result of preventive measures by which milk was excluded as an article of dietary amongst the troops in Malta has fully borne out this view. After such measures were instituted, the number of cases in the second half of 1906 fell to 11 per thousand, as contrasted with 47 per thousand in the corresponding part of the preceding year. The various facts with regard to the epidemiology of the disease have thus been cleared up. For example, it is more prevalent in the summer months, when more milk is consumed, and there is a larger proportion of cases amongst those in goad social 29 450 MALTA FEVER position, the officers, for example, suffering more in proportion than the privates. Another interesting fact, pointed out by Horrocks, is that the disease has practically disappeared from Gibraltar since the practice of importing goats from Malta has stopped. The work of the Commission, so far as it has gone, has been to exclude other modes of infection as being of practical importance, by dust, by the bites of mosquitoes, etc., and if it is conveyed by contact at all, this is only when the contact is of an intimate character, and even then it is probably of rare occurrence. Although numerous patients suffering from the disease come to England, there is no known case of fresh infection arising under natural conditions. Agglutinative Action of Serum. — The blood serum of patients suffering from Malta fever possesses the power of agglutinating the micrococcus melitensis in a manner analogous to what has been described in the case of typhoid fever. The reaction appears comparatively early, often about the fifth day, and may be present for a considerable time after recovery — sometimes for more than a year. Distinct agglutination with a 1 : 20 dilution of the serum in half an hour may be taken as a positive reaction, sufficient for diagnosis. The reaction is, however, usually given by much higher dilutions, e.g. 1 : 500, and even higher. It is to be noted that normal serum diluted 1 : 2 may produce some agglutination. As regards relation to prognosis, the observations of Birt and Lamb and of Bassett-Smith have given results analogous to those obtained in typhoid (p. 340). The Commission has recently found that vaccination with dead cultures of the micrococcus confers a certain degree of protection amongst those exposed to the disease. As a rule two injections were made, 200-300 million cocci being the dose of the first injection, and about 400 million of the second. The use of vaccines has also been carried out in the treatment of the disease, but the observations are not sufficiently numerous to allow a definite statement to be made as to its value. Methods of Diagnosis. — During life the readiest means of diagnosis is supplied by the agglutinative test just described (for technique, vide p. 109). Cultures are most easily obtained from the spleen either during life or j>o*t mortem. Inoculate a number of agar tubes by successive strokes and incubate at 37° C. Film preparations should also be made from the spleen pulp and stained with carbol-thionin-blue or diluted carbol-fuchsin (1 : 10). YELLOW FEVER 451 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 infected. 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, and 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 may be grouped with the " bilious fevers " prevalent in yellow-fever regions. This would explain the degree of immunity which is shown during a serious epidemic by the older immigrants. Great variations are observed in the clinical types under which the disease presents itself. Usually after from two to six days' incubation a sudden onset in the form of a rigor occurs. The temperature rises to 104°- 105° F. The person is livid, with outstanding bloodshot eyes. There are present 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 improvement 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, hemorrhages occur from all the mucous surfaces, causing, in 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. Post mortem the stomach is found in a state of acute gastritis, and contains much altered blood derived from hemorrhages 452 YELLOW FEVER 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. Etiology of Yellow Fever. — Although a large amount of bacteriological work has been done on yellow fever, this has now chiefly a historical interest, as it is now known that the causal agent is not one of the ordinary bacteria, but belongs to the group of ultra-microscopic organisms.1 A mosquito acts as the intermediate host, and the facts detailed below point to the organism passing through some cycle of development in the body of the insect. The analogy of malaria makes it extremely probable that the organism is a protozoon, but as this has not yet been completely proved we have not felt justified in altering the position of the disease and placing it amongst the protozoal infections. As bacteriological work led up to the establishment of our knowledge regarding the nature of the disease, some reference must be made to it. A very full research into the bacteriology of yellow fever was that of Sternberg,- the result of which was that of the varied organisms isolated, one which he called the bacillus x appeared possibly to have some relationship to the disease. Sanarelli in 1897 obtained cultures of an organism which he called bacillus icteroides, and which he considered to be the cause of yellow fever ; it is probably identical with the bacillus x of Sternberg. Subsequent observations made by others gave conflicting results, some finding this bacillus, others failing to do so. The bacillus icteroides, as described by Sanarelli, belongs to the paratyphoid group, possessing lateral flagella, growing on gelatin without liquefaction, and fermenting glucose but not lactose. Reed and Carroll found that it was practically identical with the bacillus of swine cholera. It 1 In several diseases the existence of such causal factors is suspected. Other examples are foot and mouth disease, South African horse-sickness, and the contagious pleuro-pneumonia of cattle. ETIOLOGY OF YELLOW FEVER 453 must now be considered merely as an organism which may occur in the organs and tissues in yellow fever as a secondary infection, but without any etiological significance. The facts of importance which have been established regarding the etiology of the disease are due to the labours of the United States Army Commission, which began its work in 1900. The members of the Commission first directed their inquiries towards determining whether the bacillus icteroides was present in the blood during life, and a series of cases were investigated bacteriologically, with entirely negative results in each instance. They then resolved to test the hypothesis of Dr. Carlos Finlay of Havana, promulgated several years pre- viously, that the disease was carried by mosquitoes. Selecting mosquitoes which they reared from eggs, they allowed them to bite yellow fever patients and then to bite healthy men. Of several experiments of this nature two were successful in the first instance, the first individual to be infected in this way being Dr. James Carroll, a member of the Commission, who passed through a severe attack of typical yellow fever. Experi- ments were then performed on a larger scale, with completely confirmatory results, as to the conveyance of the disease by mosquitoes. Of twelve non-immunes living under circumstances which excluded natural means of infection, ten contracted yellow fever after having been bitten by mosquitoes which had previously bitten yellow fever patients; happily all of these recovered. Two of the men who were thus infected had been previously exposed to contact with fomites from yellow fever patients without results. These results were confirmed by Guiteras, whose investigations were carried out along similar lines; of seventeen individuals bitten by infected mosquitoes, eight took yellow fever, and three of these died. The species of mosquito used by the American Commission was the Stegomyia fasciata, and up to the present time no other species has been found capable of carrying the infection. It has also been determined that a certain period must elapse after the insect has bitten a yellow fever patient before it becomes infec- tive to another subject. In summer weather this period is about twelve days ; at a lower temperature somewhat longer. This probably means that, as in the case of malaria, the parasite must pass through certain stages of development before it reaches the salivary gland and is thus in a position to be transferred to a fresh subject. Infected mosquitoes, however, retain the power of infection for a considerable time afterwards, probably as long as sixty days. It has also been shown that mosquitoes may become 454 YELLOW FEVER infective after biting a patient on the first, second, or third day of the disease, but at a later period the results are usually negative, apparently because the virus is no longer present in the blood. Interesting results were also obtained with regard to the communication of the disease directly from patient to patient, the conclusion arrived at, after careful experiments, being that the disease cannot be transferred in this way, even when the contact is of a close character. In a specially constructed house seven men were exposed to the most intimate contact with the fomites of yellow fever patients for a period of twenty days each, the soiled garments worn by the patients being in some cases actually slept in by these men ; the result was that not one of them thus exposed contracted the disease. The conclusions on this point have been subsequently confirmed by other workers. The American Commission also found it possible to transmit yellow fever to a healthy man by injecting small quantities of blood or of serum taken from a yellow fever patient at any period up till the third day of the disease. The period of incubation in this case is somewhat shorter than when the disease is conveyed by the bite of mosquitoes, the average duration in the former case being about three days, and in the latter about four days, though these times may be considerably exceeded. It is also interesting to know that in these experimental injec- tions the blood or serum used was found to be free from bacteria. Up till the present time, we know of only these two methods of infection, namely, indirectly by the bite of a mosquito infected with the yellow fever germ, or directly by the injection of some of the blood from a yellow fever patient. In these respects there is a striking similarity to what has been established in the case of malarial fever. Experiments with regard to the nature of the yellow fever organism were carried out by Reed and Carroll, and interest- ing results were obtained. They found that the organism of the disease was very easily killed by heat, as blood from a yellow fever patient lost its infective power on being heated to 55° C. for ten minutes. On the other hand, blood or serum was found to be still infective after having been passed through a Berkefeld filter. This has been confirmed by the French Commission, with the additional result that the virus passes through a Chamberland F filter, but not through a Chamber- land B. These facts would show that the parasite is of extremely minute size, and apparently belongs to the group of ultra - microscopic organisms. Up till the present time all ETIOLOGY OF YELLOW FEVER 455 attempts to find by microscopic examination the yellow fever parasite, either in the blood of patients suffering from the disease, or in the tissues of infective mosquitoes, have been attended with negative results. Though nothing has been determined regarding the actual nature of the virus, yet the results already obtained have supplied the basis for preventive measures against the disease, these being directed towards the destruction of mosquitoes and the protection of those suffering from yellow fever, and also the healthy, against the bites of these insects. Already a striking degree of success has been obtained in Havana. Such measures came into force in February 1901, and in ninety days the town was free of yellow fever and f9r fifty-four days later no new cases occurred; and although subsequently the disease was reintroduced into the town, no difficulty was experienced in stamping it out by the same measures. In recent years the results have also been highly gratifying, and the disease may be said to be practically eradicated from Havana. In other places also successful results have been obtained, and epidemics would appear to be now under control if the proper measures are taken. 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 toxins of an organism. 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 is to be noted that man and the lower animals may be exempt from certain diseases under natural conditions, and yet the causal organisms of these diseases may produce pathogenic effects when injected in sufficient quantity. Immunity is, in fact, of very varying degrees, and accordingly the use of the term has a correspondingly relative significance. This is not only true of infection by bacteria, but of toxins also : — when the resistance of an animal to these is of high degree, the resistance may in certain cases 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 toxin, 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, 456 ARTIFICIAL IMMUNITY 457 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 con- sidered highly probable that the passing through an attack of an acute disease produced by an organism, confers immunity for a longer or shorter period. The immunity is not, however, to be regarded as the result of the disease per se, but of the bacterial products introduced into the system ; as will be shown below, by suitable gradation of the doses of such products, or by the use of weakened toxins, a high degree of immunity may be attained without the occurrence of any symptoms whatever. 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 inoculated 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 con- dition than that produced by vaccination, and it is found that the degree of protection or immunity resulting occupies an inter- mediate position. Immunity and Recovery from Disease. — Recovery from an acute infective disease shows that in natural conditions the vims 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 Antikb'rper) appear in the blood, which are antagonistic either to the toxin or to the vital activity of the organism. In such cases a process of immunisation would appear to be going on during the pro- gress of the disease, and when this immunisation has reached a certain height, the disease naturally comes to an end. It can- not, however, be said as yet that such antagonistic substances are developed in all cases ; though the results already obtained make this probable. ARTIFICIAL IMMUNITY. Varieties. — According to the means by which it is produced, immunity may be said to be of two kinds, to which the terms 458 IMMUNITY active and passive are generally applied, or we may speak of immunity directly, or indirectly, produced. We shall first give an account of the established facts, and afterwards discuss some of the theories which have been brought forward in explanation of these facts. 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 " toxins," 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 ; or, what practically amounts to the same, an organism of greater virulence or a toxin of greater strength may be used. A proportionate degree of resistance or immunity can thus be developed, which degree in course of time may reach a very high level. Such methods constitute the means of preventive inoculation or vaccination. Immunity of this kind is comparatively slowly produced and lasts a consider- able 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. Such a serum, generally known as an anti-serum, may exert its effects if introduced into an animal at the same time as infection occurs or even a short time afterwards ; it 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 toxin it is called antitoxic ; when, with little or no antitoxic power, it protects 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 merely for purposes of convenience and to aid subsequent description ; the principles underlying all the methods are the same. ARTIFICIAL IMMUNITY. A. Active Immunity — i.e. produced in an animal by an in- jection, or by a series of injections, of non-lethal doses of an organism or its toxins. ARTIFICIAL IMMUNITY 459 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 antiseptics, 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. toxins ; or of chemical substances derived from such filtrates. These methods may also be combined in various ways. B. Passive Immunity, i.e. produced in one animal by injection 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 toxin. 2. By antibacterial serum, i.e. the serum of an animal highly immunised against a particular bacterium 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 investigators had to deal with organisms of high virulence, which had accordingly to be reduced before the organisms could be injected in the living state. It is now found most convenient as a rule to start the process of active immunisa- tion with the injection of dead cultures. The principle is the same as that of vaccination, and both attenuated cultures and also the dead cultures used for injection 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 culti- vated for some time outside the body, loses its virulence, and in the case of some this is very marked indeed, e.g. the pneumo- 460 IMMUNITY coccus. Pasteur found in the case of chicken cholera, that when cultures were kept for a 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 protective 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 (p. 412). (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 its being passed through guinea-pigs, the disease produced in the ox by inoculation from the guinea-pig being a non-fatal one. This dis- covery was confirmed by Greenfield, who showed 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. The method of vaccina- tion against smallpox depends upon the same principle. There is also evidence to show that the virulence of the tubercle bacillus becomes modified according to its host, being often diminished for other animals. (3) Many organisms become diminished in virulence when grown at an abnormally high temperature. The method of Pasteur, already described (p. 314), 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, as was found by Toussaint in the case of anthrax. (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 Koux, for example, succeeded in attenuating the anthrax bacillus by growing it in a medium containing carbolic acid in the proportion of 1 : 600. These examples will serve to show the principles underlying attenuation of the virulence of an organism. There are, how- ever, still other methods, most of which consist in growing the organism in conditions somewhat unfavourable to its growth, e.g. under compressed air, etc. BY LIVING CULTURES 461 (b) Immunity 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, and then to continue with living cultures. 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 conveniently 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 staphylococci, 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 injecting 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 accompanying in- jection 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 com- bined in various ways. By repeated injections of cultures at first in the dead condition, then living and attenuated and 462 IMMUNITY afterwards more virulent, and by increasing the doses, a high degree of immunity may be obtained. 2. Immunity by Dead Cultures of Bacteria.— In some 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. In this method the so-called endotoxins will be injected along with the other substances in the bacterial protoplasm, but the resulting immunity is chiefly directed against the vital activity of the organisms — is antibacterial rather than antitoxic (vide infra). The cultures when dead produce, of course, less effect than when living, and this method may be conveniently used in the initial stages of active immunisation, to be afterwards followed by injections of the living cultures. The method is extensively used for experimental purposes, and is that adopted in anti- plague and anti-typhoid inoculations. 3. Immunity by the Separated Bacterial Products or Toxins. — 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 toxins, 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 toxin is started by small non-lethal doses of the strong toxin, or by larger doses of toxin the power of which has been weakened by various methods (vide infra). Afterwards the doses are gradually increased. This method was carried out with a great degree of success in the case, of diphtheria, tetanus, malignant oedema, etc. It appears capable of general application in the case of organisms where it is possible to get an active toxin from the filtered cultures. It has also been applied in the case of snake poisons by Calmette and by Fraser, and a high degree of im- munity has been produced. Immunity may also be obtained by means of certain chemical substances separated from filtered bacterial cultures, 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. The following may be mentioned as some of the most important examples of the practical application of the principles of active immunity, i.e. of protective inoculation : — (1) Inocula- tion of sheep and oxen against anthrax (Pasteur) (p. 314); (2) Jennerian vaccination against smallpox (p. 503) ; (3) Anti- BY BACTERIAL PRODUCTS OR TOXINS 463 cholera inoculation (Haffkine) (p. 412); (4) Anti-plague inoculation (Haffkine) (p. 434) ; (5) Anti-typhoid inoculation (Wright and Seinple) (p. 343) ; (6) Pasteur's method of inocu- lation against hydrophobia, which involves essentially the same principles (p. 516). Vaccines as a Method of Treatment. — Up till recently the principles of active immunity had not been directly applied in the treatment of an existing disease except in the case of tuberculosis. The work of Wright, however, shows that active immunisation in such circumstances is not only possible, but is also probably capable of wide application. From his study of the part played by phagocytosis in the successful combat of bacteria by the body, he was led to advocate the treatment of bacterial infections by carrying on an active immunisation against the causal agents by the injection of dead cultures of the latter. The justification for such a procedure lies in his contention that in many cases infections are to be looked on as practically entirely localised, e.g. the cases of an acne pustule, or a boil. The reason for the local growth of bacteria in the part of the body affected is that there is for unknown causes a deficiency of the opsonic power (vide p. 483) of the body fluids, which is essential for the phagocytosis of the invading bacteria. Still more marked in such cases is the deficiency in the opsonic qualities of the fluids in the actual site of infection. Any procedure which will raise the opsonic power of the body fluids as a whole, and therefore of the fluids in the focus of infection, will aid the destruction of the bacteria by sensitising them to phagocytic action. Such a procedure is found in the active immunisation which results from the injection of a vaccine consisting of a dead culture of the causal bacteria. The appli- cation of a vaccine of this kind must, however, be controlled by constant observation of the opsonic index of the patient's serum during the treatment. When a local infection occurs the general opsonic index is usually found to be below unity. If dead bacteria be injected into the individual there may occur during the following few days a further fall in the opsonic index, — what Wright calls the occurrence of a negative phase. In a case where the treatment is successful, this negative phase is succeeded by a rise in the opsonic index above its original level, — ocurrence of positive phase, — and with this reaction there is an improve- ment in the local condition. Usually in such cases repeated injections are required to effect a cure, and the important point, according to Wright, is to avoid giving an injection when a negative phase is in progress. If this point is not attended to 464 IMMUNITY only an aggravation of symptoms is to be looked for (vide pp. 194, 261). With regard to the details of the preparation of the vaccines, an agar culture is taken, the growth removed into normal saline and killed by steaming for a sufficient time — say 1J hours. The efficiency of the sterilisation is tested by inoculating tubes of appropriate media. The strength of the emulsion is then estimated by the method of counting dead bacteria described on p. 67. The number of bacteria employed for a vaccination is usually from 250,000,000 to 500,000,000, and in the details of the measurement of this quantity and in its injection, every aseptic precaution must, of course, be adopted. Such vaccines have been used extensively in the treatment of acne, boils, sycosis, infections of the genito-urinary tract by the b. coli, infections of joints by the gonococcus, and in many cases con- siderable success has followed the treatment. 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. 169). In the course of some weeks' treatment in this way the resulting immunity was of so high a degree that the animals could tolerate on subcutaneous inoculation 400 times the dose originally fatal. Fraser also found in the case of snake venom that rabbits could be immunised, by feeding with the poison, against several times the lethal dose of venom injected into the tissues. By feeding animals with dead cultures of bacteria or with their separated toxins, a degree of immunity may in some 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 may be regarded as specific, that is, is exerted only towards the organism or toxin by means of which it has been produced. A certain degree of immunity, or rather of increased general resistance of parts of the body (for example the peri- toneum), 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. B. Passive Immunity. Action of the Serum of Highly-Immunised Animals. — 1. The serum of an animal A, treated by repeated and gradually PASSIVE IMMUNITY 465 increased doses of a toxin of a particular microbe, may protect an animal B against a certain amount of the same toxin 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 curative or palliative power. Seeing that the serum of animal A appears to neutralise the toxin, the term antitoxic has been applied to it. 2. The serum of an animal A, highly immunised against a bacterium by repeated and gradually increasing doses of the organism, may protect an animal B against an infection by the living organism when injected under conditions similar to the above. This serum is therefore antimicrobic, or antibacterial, i.e. preventive against invasion by a particular organism. (In addition to the preventive or protective action in vivo, such a serum may exert certain recognisable effects on the corresponding organism in vitro. Thus (a) it may lead to the death or solution of the organism — bactericidal or lysogenic action', (6) it may produce an increased susceptibility to ingestion by phagocytes — opsonic action ; (c) it may lead to the clumping of the organism — agglutinative action.) These two kinds of anti-sera — antitoxic and antibacterial — exert their effect when injected along with the toxin or organism respectively or some time previously ; as would be expected, they have less effect when injected some time afterwards, though even then they may have a certain degree of curative or palliative power. The two properties, antitoxic and antibacterial, are essen- tially different in kind, the former leading to a neutralisation of the toxin, the latter to some alteration in the vital activity of the bacterium ; in other words, the point of attack in the case of the two sera is different. A serum may, however, possess both properties in varying degree. The fundamental fact in passive immunity, viz. that immunity can be transferred to another animal, shows that the serum in question differs from the serum of a normal animal in containing antagonistic sub- stances to the toxin or bacterium as the case may be, — these being generally spoken of as anti- substances. It will accordingly . be convenient to speak of anti-substances in general. The development of anti-substances, first observed in the case of the injection of toxins, is found to occur when a great many different substances are introduced into the tissues of the living- body. We can, in fact, divide organic molecules into two classes — those which give rise to the production of anti-substances, and those which have not this property. Amongst the former are 30 466 IMMUNITY various toxins, ferments, molecules of tissue cells, bacteria, red corpuscles, etc. They are all probably of proteid nature, though their true constitution is not known, and none of them have been obtained in a pure condition. Amongst the latter may be placed the various poisons of known constitution, glucosides, alkaloids, etc. We may .also state at present that the anti- substance forms a chemical or physical union with the particular substance which has led to its development, and shall discuss the evidence for this later. Furthermore, the anti-substance has apparently a specific combining group which fits, as it were, a group in the corresponding substance, the two groups having been compared to a lock and key. It is, however, to be noted that this specificity is a chemical one rather than a biological one. An anti-serum, for example, developed by the injection of bacterium A may also have some effect on bacterium B, and thus appear not to be specific. We have, however, evidence to show that some of the preponderating molecules in bacterium A are also present in bacterium B, and thus the theory of chemical specificity is not invalidated. The number of different anti- substances, as judged by their combining properties, would appear to be almost unlimited, a fact which throws new light on the complexity of the structure of living matter. When anti- substances are studied as regards their action on the substances with which they combine, they may be conveniently arranged in three classes corresponding to Ehrlich's three classes of receptors (vide p. 491). In the first place, the anti-substance may simply combine with the substance without, so far as we know, producing any change in it, and to this group the anti- toxins and anti-ferments belong. In the second place, the anti-substance, in addition to combining, may produce some recognisable physical alteration. In other words, it possesses an active or zymotoxic group as well as a combining group. The agglutiniris may be mentioned as examples of this group. In the third place, the anti-substance after combination leads to the combination of another body normally present in serum called complement or alexine, and this latter, which has a con- stitution very similar to that of a toxin, may lead to physical change, for example, death or solution of a cell. Anti-substances of this class are known as immune -bodies or amboceptors (Ehrlich) or as sensitising substances — substances sensibilisatrices of French writers. Anti-substances of the second and third groups are met with especially, though not exclusively, when .f formed elements such as bacteria, red corpuscles, or tissue cells, etc., are injected, the anti-serum developed possessing agglu- ANTITOXIC SERUM 467 tinating, solvent, or other properties towards the particular substance. After this preliminary statement in explanation we shall consider the actual properties of the two classes of serum, and later we shall resume the theoretical consideration. Antitoxic Serum. — The best examples are the antitoxic sera of diphtheria,. ^nd^tatanus, though similar principles and methods are involved in the case of the anti-sera to ricin and abrin, and to snake poison. We shall here speak of diphtheria and tetanus. The steps in the process of preparation may be said to be the following : First, the preparation of a powerful toxin. Second, the estimation of the power of the toxin. Third, the development of antitoxin in the blood of a suitable animal by gradually increasing doses of the toxin. Fourth, the estima- tion from time to time of the antitoxic power of the serum of the animal thus treated. 1. Preparation of the Toxin. — The mode of preparation and the conditions affecting the development of diphtheria toxin have already been described (p. 362). In the case of tetanus the growth takes place in glucose bouillon under an atmosphere of hydrogen (vide p. 60). In either case the culture is filtered through a Chamberland filter when the maximum degree of toxicity has been reached. The term " toxin " is usually applied for convenience to the filtered (i.e. bacterium-free) culture. 2. Estimation of the Toxin. — The power of the toxin 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, of course, varies in proportion to the weight of the animal, and is expressed accord- ingly. In the case of diphtheria, in Ehrlich's standard, the minimum lethal dose — known as M.L.D. — is the smallest amount which will certainly cause death in a guinea-pig of 250 grms. within four days. Behring uses the term " normal diphtheria toxin of simple strength" (DTN1), as indicating a toxin of which '01 c.c. is the minimum lethal dose under these conditions. A toxin of which the minimum lethal dose is '02 will be of half normal strength (DTN>5); and so on. The testing of a toxin directly is a tedious process, and in actual practice, where many toxins 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" (p. 468). 3. Development of Antitoxin. — The earlier experiments on tetanus and diphtheria were performed on small animals, such 468 IMMUNITY 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 toxin or a powerful toxin modified by certain methods. Such methods are the addition to the toxin 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 toxins weakened to varying degrees by being exposed to different temperatures, viz. 60°, and 55°, and 50° C. In the case of large animals immunisation is sometimes started with small doses of unaltered toxin ; and the doses are gradu- ally increased. The toxin is at first injected into the sub- cutaneous tissues, later into a vein. Ultimately 300 c.c., or more, of active diphtheria toxin 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 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 (v. p. 494). (In immunisation of small animals an indication of their general condition may be obtained by weighing them from time to time.) 4. Bttwn&ting 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 toxin. Various standards have been used, of which the two chief are that of Ehrlich and that of Roux. Ehrlich has adopted as the immunity unit the amount of antitoxic serum which will neutralise 100 times the minimum lethal dose of toxin, serum and toxin being mixed together, diluted up to 4 c.c. and injected subcutaneously into a guinea-pig of 250 grms. weight, the prevention of the death of the animal within four days being taken as the indication of neutralisation, 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. With such a standard test-serum any newly prepared serum can readily be compared. A " normal " antitoxic serum is one of which 1 c.c. contains an immunity unit. 1 c.c. of a serum, of which '02 c.c. will protect from a hundred times USE OF ANTITOXIC SERA 469 the lethal dose, will possess 50 immunity units, and 20 c.c. of this serum 1000 immunity units. Sera have been prepared of which 1 c.c. has the value of 800 units or even more. Roux adopts a standard which represents the animal Aveight 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 grins, 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 antitoxin 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 antitoxic sera are prepared in a corresponding manner. Some further facts about antitetanic serum are given on p. 384. 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 anti- toxic 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. A strong serum prepared by Behring contains 3000 units in 5-6 c.c., but even stronger sera may be obtained. 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 ad- ministered, 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 vegetable toxins, 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 470 IMMUNITY 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 corresponding antagonistic body, to which Fraser has given the name "antivenin," appears in the blood of animals in the process of immunisation against snake poison. Theso investigations are specially instructive, as such vegetable and animal poisons, both as regards their local action and the general toxic phenomena produced by them, present, as we have seen, an analogy to various toxins of bacteria. Nature of Antitoxic Action.— This subject is only part of the general question with regard to the relation of anti-substances to their corresponding substances, but it is with regard to anti- toxic action that most of the work has been done. We have to consider here two points, viz. (a) the relation of antitoxin to toxin, and (b) the source of the antitoxin. With regard to the former subject there has been much diversity of opinion, but the evidence now available goes to show that the antagonism between toxin and antitoxin is not a physiological one but that the two bodies unite in vitro to form a compound inert towards the living tissues, there being in the toxin molecule an atom group which has a specific affinity for the antitoxin molecule or part of it. 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 toxins, of snake poisons, etc. When toxin and antitoxin are brought together in vitro it can be proved that their behaviour towards each other resembles what is observed in chemical union. Thus it has been found that a definite period of time elapses before the neutralisation of the toxin is complete, that neutralisation takes place more rapidly in strong solutions than in weak, and that it is hastened by warmth and delayed by cold. C. J. Martin and Cherry, and also Brodie, have shown, that in the case of diphtheria toxin and in that of 'an Australian snake poison the toxin molecules will pass through a colloid membrane (p. 166), whilst those of the corresponding antitoxin will not. Now if a mixture of equivalent parts of toxin and antitoxin is freshly prepared and at once filtered, a certain amount of toxin will pass through, but the longer such a mixture is allowed to stand before nitration the less toxin passes, till a time is reached when no toxin 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 NATURE OF ANTITOXIC ACTION 471 animal no symptoms take place ; this shows that after a time neutralisation is complete. Again, in cases when the toxin has some definite physical effect demonstrable in vitro, e.g. lysis, agglutination, coagulation, or the prevention of coagulation, its action can be annulled by the antitoxin ; in such circumstances manifestly no physiological action of antitoxin through the medium of the cells of the body can come into play. These facts are practically conclusive in favour of antitoxin action depending upon a direct union of the two substances concerned. The evidence usually brought forward against the direct union of toxin and antitoxin rests chiefly on certain observations of Calmette, who found that the antitoxin to a snake venom was more easily destroyed by heat than the toxin, and stated that when a neutral mixture of the two was heated at a temperature sufficient to destroy free antivenin, the toxic properties in part returned. Hence he concluded that the two bodies existed in an uncombined condition in the mixture. Martin and Cherry, however, on repeating these experiments, found that the above result was not obtained if sufficient time for complete combination was allowed ; but if this precaution was not taken, then the presence of the free toxin was revealed when the antitoxin was destroyed by heat. Even, however, if Calmette's results were quite correct, they cannot be considered to constitute a proof that chemical union does not occur : they would only prove that the toxin has not been destroyed. If two complicated chemical compounds of unequal stability are in loose chemical union, it is quite conceivable that the less stable may be destroyed (e.g. by heat), whilst the more stable escapes. Although practically all authorities are now agreed as to the direct combination of toxin and antitoxin there is still much uncer- tainty as to the exact nature of this union. Controversy on this subject may be said to date from the important work of Ehrlich on the neutralisation of diphtheria toxin. Using an immunity unit of antitoxin (the equivalent of 100 doses of toxin) he deter- mined with any example of crude toxin the largest amount of toxin which could be neutralised completely, so that no symptoms resulted from an injection of the mixture. This amount he called the limes null dose, expressed as L0. He then investigated the effects of adding larger amounts of toxin to the immunity unit and observed the quantity which was first sufficient to produce a fatal result, that is, which contained one M.L.D. of free toxin ; this amount he called the limes todtlich, fatal limit, expressed as Lt. Now if, as he supposed, the union of toxin and antitoxin resembled that of a strong acid and base, Lt - L0 ought to be the equivalent of a minimum lethal dose of the toxin alone. This, however, was never found to be the case, the difference being always considerably more than one M.L.D. For example, in the case of one toxin, M.L.D. = '0165 gr., Lt = 1*26 gr., L0 = 472 IMMUNITY •9 gr. ; difference = -36 gr., i.e. 21 '9 M.L.D. This, in brief, is what is known as the "Ehrlich phenomenon," and it has been explained by him as the result of the presence of toxoids (vide p. 171), i.e. toxin molecules in which the toxophorous group has become degenerated. He distinguishes three possible varieties of such bodies according to the affinity of the haptophorous group, namely prototoxoid with more powerful affinity than the toxin molecule, epitoxoid with less powerful affinity, and syntoxoid with equal affinity. The presence of epitoxoids would manifestly explain the above phenomenon. The L0 dose would represent toxin + epitoxoid molecules all united to antitoxin molecules and the addition of another M.L.D. of toxin would not result in there being a free fatal dose, but in the added toxin taking the place of epitoxoid. Several lethal doses would need to be added before the mixture was sufficient to produce a fatal result ; that is, Lt - L0 would equal several M.L.D.s. Ehrlich observed another fact strongly in favour of the existence of toxoids, namely that in the course of time the toxin might become much weakened, so that in one case observed the M.L.D. was three times the original fatal dose, and still the amount of antitoxin necessary to neutralise it completely was the same as before. Ehrlich also investigated the effects of partial neutralisation of the L0 amount of toxin, that is, he added to this amount different fractions of an immunity unit and estimated the toxicity of the mixture. He found by this method that the neutralisation of the toxin did not take place gradually, but as if there were distinct bodies present with different combining affinities — the graphic repre- sentation of the mixture not being a curve but a step-stair line. Thus he distinguished proto-, deutero-, and trito-toxins (with corresponding toxoids). It will thus be seen that Ehrlich regards the combination toxin-antitoxin to be a firm one, and that the neutralisation phenomena are to be explained by the complicated constitution of the crude toxin. The chief criticism of Ehrlich's views has come from the important work of Madsen and Arrhenius. Their main con- tention is that the toxin-antitoxin combination is not a firm one but a reversible one, and is governed by the laws of physical chemistry. For example, in the case of a mixture of ammonia and boracic acid in solution, there is a constant relation between the amounts of each of the substances in the free condition and the amounts in combination, — the combination is reversible, so that if some of the free ammonia were removed a certain amount of the combined ammonia would become dissociated to take its place ; further, if to the mixture, in a state of equilibrium, MODE OF PRODUCTION OF ANTITOXINS 473 more ammonia or more boracic acid were added, part would remain free while part would combine. Accordingly, if toxin and antitoxin behaved in a similar manner an explanation of the Ehrlich phenomenon would be afforded. Madsen and Arrhenius have worked out the question in the case of a great many toxins and find that the graphic representation of neutralisation is in every case a curve which can be represented by a formula. It should be noted in connection with this controversy that there are two questions which may be independent of each other, viz. (1) does the "toxin" in any particular case represent a single substance or several ? (2) What is the nature of the combination of any one constituent substance and its anti-substance — is it reversible or is it not ? It may be said that it is practically impossible to explain the facts with regard to diphtheria toxin on the hypothesis of a single substance even if this should have its combining and toxic actions equally weakened ; " toxoids " in Ehrlich's sense must in our opinion be supposed. Then there is an important fact established by Danysz and by v. Dungern, namely that the amount of toxin neutralisable by a given amount of antitoxin is different according as the toxin is added in several moieties or all at once — in the latter case the amount of toxin neutralisable is greater. There seems no explanation of this according to the view of Madsen and Arrhenius as the same state of equilibrium ought to be reached in the two cases, that is, the amounts of toxin neutralised should be the same. On the other hand we have instances of the combination of a substance and its anti-substance being reversible — the example of a haemolytic immune-body may be cited (p. 481) — and there is no doubt that there are varying degrees of firmness of the union. It is quite evident that if there should be several toxic bodies in a "toxin," and that if the union of some of these with antitoxin should be reversible, the problem, becomes one of extreme complexity. There has recently been a tendency on the part of some authorities to consider that the union of toxin-antitoxin does not correspond to what takes place in ordinary chemical union, but is a physical interaction of bodies in a colloidal state, the action being one of the so-called absorption phenomena. The smaller toxin molecule becomes entangled, as it were, in the larger antitoxin one, very much as a dye becomes attached to the structure of a thread. Bordet has long maintained a theory of this nature and gives reasons for believing that there is no definite quantitative relationship in the combination of the molecules of the two substances, different amounts of antitoxin affecting in varying degree all the molecules of a given amount 474 IMMUNITY of toxin. A statement on the general question is at present impossible ; we can only say that combination of the two bodies does occur ; that sometimes, probably often, the " toxin " con- tains different toxic bodies with varying affinity ; and that in a few instances the combination has been proved to be reversible, but to what extent this is the case remains still to be determined. The next question to be considered is the source of antitoxin. The following three possibilities present themselves : (a) antitoxin may be formed from the toxin, i.e. may be a " modified toxin " ; (6) antitoxin may be the result of an increased formation of molecules normally present in the tissues ; (c) antitoxin may be an entirely new product of the cells of the body. It can now be stated that antitoxin is not a modified toxin. It has been shown, for example, that the amount of antitoxin produced by an animal may be many times greater than the equivalent of toxin injected ; and further, that when an animal is bled the total amount of antitoxin in the blood may some time afterwards be greater than it was immediately after the bleeding, even although no additional toxin is introduced. This latter circumstance shows that antitoxin is formed by the cells of the body. If antitoxin is a product of the cells of the body, we are almost com- pelled, on theoretical grounds, to conclude that it is not a newly- manufactured substance, but a normal constituent of the living cells which is produced in increased quantity. We have, however, direct evidence of the presence of antitoxin under normal con- ditions,— the presence of such being sho\vn by its uniting with toxin and rendering it inert. Normal horse serum, to mention an example, may have a varying amount of antitoxic action to the diphtheria poison, ox-bile has a similar action to snake poison, whilst in the case of other anti-substances — such as agglutinins, bacteriolysins, hasmolysins, etc. — whose production is governed by the same laws, numerous examples might be given. It is, however, rather to the protoplasm of living cells than to the serum that we must look for the source of antitoxins. In the first place, we have evidence that in the living body bacterial toxins enter into combination with, or, as it is often expressed, are fixed by the tissues — presumably by means of certain combining affinities. This has been shown by the experiments of Donitz and of Heymans with tetanus toxin. We have, in such cases, however, no evidence as to where the toxin is fixed beyond that supplied by the occurrence of symptoms. Another line of research which has been followed is to bring emulsions of various organs into contact with a given toxin and observe whether any of the toxicity is removed. This was first carried out by Wassermann CHEMICAL NATURE OF ANTITOXINS 475 and Takaki, who investigated the action of emulsions of the central nervous system of the susceptible guinea-pig on tetanus toxin. They found in this way that the nervous system con- tained bodies which had a neutralising effect on the toxin. For example, it was shown that 1 c.c. of emulsion of brain and spinal cord was capable of protecting a mouse against ten times the fatal dose of toxin. These observations have been confirmed, though their significance has been variously interpreted. It would, however, be out of place to discuss at length the opposing views, and we accordingly simply state the facts ascertained. We may note, however, that it is not a serious objection that in certain animals other tissues than that of the central nervous system can combine with tetanus toxin — this might take place with or without resulting symptoms ; the important fact is that in the nervous system certain molecules have an affinity for the toxin. It will be seen from what has been stated with regard to the relation of toxin and antitoxin, that the fixation of toxin by the tissues leads up theoretically to the possible production of anti- toxin. In other words, the substance which, when forming part of the cells, fixes the toxin and thus serves as the means of poisoning, may act as an antitoxin when free in the blood. This will be discussed below in connection with Ehrlich's theory of passive immunity. We may conclude by saying that anti- toxin is probably represented by molecules normally present in the cells or (more rarely) in the fluids of the body. Of the chemical nature of antitoxins we know little. From their experiments C. J. Martin and Cherry deduce that while toxins are probably of the nature of albumoses, the antitoxins probably have a molecule of greater size, and may be allied to the globulins. Hiss and Atkinson have also come to the con- clusion that antitoxin belongs to the globulins. They found that the precipitate with magnesium sulphate from anti- diphtheria serum contained practically all the antitoxin, and that any substance obtained which had an antitoxic value gave all the reactions of a globulin. They also found that the per- centage amount of globulin precipitated from the serum of the horse increased after it was treated in the usual way for the production of antitoxin. Such a supposed difference in the sizes of the molecules might explain the fact, observed by Fraser and also by C. J. Martin, that antitoxin is much more slowly absorbed when introduced subcutaneously than is the case with toxin. Antitoxin, when present in the serum, leaves the body by 476 IMMUNITY the various secretions, and in these it has been found, 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 antitoxin in the yolk of eggs of hens whose serum contained antitoxin. Bulloch also found in the case of hremolytic sera (vide infra) that the anti-substance (" immune -body ") is trans- mitted from the mother to the offspring. Antibacterial Serum. — The stages in the preparation of antibacterial sera correspond to those in the case of antitoxic sera, but living, or, in the early stages, dead cultures are used instead of toxin separated by nitration, and in order to obtain a serum of high antibacterial 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 accompanied by the appearance in the blood of protective substances, which can be transferred to another animal. The law enunciated by Behring regarding immunity against toxins 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 in- jected along with them. Here again is presented the remark- able potency of the antagonising substances in the serum, which in this case lead to the destruction of the corresponding microbe. The anti-streptococcic serum of Marraorek may be briefly described, as PROPERTIES OF ANTIBACTERIAL SERUM 477 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. 41). 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. Streptococci 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 con- siderable 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 toxin 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 Antibacterial Serum. — We have here to consider the three main actions mentioned above, viz. (a) bactericidal and lysogenic action, (6) opsonic action, and (c) agglutinative action. (a) Bactericidal and 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 immedi- ately, gradually became granular, swollen, and then disappeared in the fluid — these changes constitute " Pfeiffer's phenomenon." Further, he found that the same phenomenon was witnessed if a minute quantity of the anti-serum was added to a certain quantity of the corresponding organisms, and the mixture injected into the peritoneal cavity of a non-treated animal. Pfeiffer found that the serum of convalescent cholera patients gave the same reaction as that of immunised animals. He obtained the same reaction also in the case of the typhoid bacillus and other organisms. From his observations he con- cluded that the reaction was specific, and could be used as a means of distinguishing organisms wilich resemble one another. He accordingly considered that a specific substance was developed in the process of immunisation and that this was rendered actively bactericidal by the aid of the living cells of the body. It was subsequently shown, however, by Metchnikoff and by Bordet that lysogenesis might occur outside the body by the 1 A true antitoxic cholera serum has been prepared by Metchnikoff, E. Roux, and Taurelli-Salimbeni. 478 IMMUNITY addition of fresh peritoneal fluid or normal serum to the heated immune-serum. PfeifFer also found that an anti-serum heated to 70° C. for an hour produced the reaction when injected with the corresponding organisms into the peritoneum of a fresh animal. The outcome of these and subsequent researches is to show that when an animal is immunised against a bacterium a substance appears in its serum with combining affinity for that particular organism. This substance which is generally known as the immune-body, amboceptor (Ehrlich), or substance sensibilisatrice (Bordet) is comparatively stable, resisting usually a temperature of 70° C for an hour. It cannot produce the destructive effect alone but requires the addition of a substance normally present in the serum, which is spoken of under various names — com- plement (Ehrlich), alexine or cytase (French writers). The com- plement is relatively unstable, being rapidly destroyed by a temperature of 60° C., and it is not increased in amount during the process of immunisation. Though ferment-like in its in- stability, it differs from a ferment in being fixed or used up in definite quantities. The phenomenon of lysogenesis is, however, only seen in the case of certain organisms when an animal is highly immunised against them ; the typhoid and cholera group are outstanding examples. It is also to be noted that it sometimes is seen in the case of a normal serum (vide Natural Immunity). In other oases the bactericidal effect of a serum may occur without the rapicLdis- solution characteristic of lysogenesis though other structural changes may be produced. In still other cases a bactericidal effect may be wanting ; nevertheless it may be shown that an immune-body is developed by the process of immunisation. This may be done by observing the increased amount of complement which is fixed through the medium of the anti-serum (immune- body), sensitised red corpuscles being used as the test for the presence of free complement. The following scheme will show the mode of experiment, which is carried out in a series of small test-tubes : — (1) Bacteria + immune-body (anti-serum heated at 55° C.) + complement. (The same amount of bacteria and immune-body in each tube, varying amounts of complement in different tubes.) (2) Incubate at 37° C. for one and a half hours. (3) Add to each tube red corpuscles treated with the corresponding immune-body, and incubate for another hour. The " immune-body " is in each case the anti-serum deprived of complement (by heating at 55° C.), obtained from animals injected with the bacteria and red corpuscles respectively. The ILEMOLYTIC AND OTHER SERA 479 control is got by substituting in another experiment the same amount of heated normal serum for the anti-serum. If there is free complement left there will be corresponding lysis of the red corpuscles ; if the complement has all been fixed there will be no lysis. To take an example from Muir's experiments,— it was found that an emulsion of the bacteria alone took up "03 c.c. of guinea- pig's complement, whilst the same amount of bacteria treated with immune-body took up '13 c.c. The all-important action of the immune-body is thus to bring an increased amount of com- plement into union with bacteria ; whether death of the bacteria will result or not will depend ultimately on their sensitiveness to the action of the particular complement. It is to be noted that with a bactericidal serum there is an optimum amount of immune-body which gives the greatest bactericidal effect. If this amount be exceeded the bactericidal action becomes diminished and may be practically annulled. This result, which is generally known as the Neisser-Wechsberg phenomenon, has been the subject of much controversy, and cannot yet be said to be satisfactorily explained. It would accordingly be out of place to discuss here the different views with regard to it. (Regarding some theoretical considerations as to the therapeutic applications of antibacterial sera, vide p. 489.) The laws of lysogenesis are, however, not peculiar to the case of solution of bacteria by the fluids of the body, but, as has been shown within the last few years, hold also in the case of other organised substances, red corpuscles, leucocytes, etc., when these are introduced into the tissues of an animal as in a process of immunisation. Of such sera the hsemolytic have been most fully studied, and owing to the delicacy of the reaction and the ease with which it can be observed, have been the means of throwing much light on the process of lysogenesis, and thus on one part of the subject of immunity. A short account of their properties may now be given. Hcemolytic and other Sera. — It has been known for some time that in some instances the blood serum of one animal has, in certain degree, the power of dissolving the red corpuscles of another animal of different species ; in other instances, how- ever, this property cannot be detected. Bordet showed that if one animal were treated with repeated injections of the corpuscles of another of different species, the serum of the former acquired a marked haemolytic property towards the corpuscles of the latter, the property being demonstrated when the serum is added to the corpuscles. Bordet also found that the hsemolytic 480 IMMUNITY property disappeared when the haeraolytic serum was heated at 55° C., but, as in the case of a bacteriolytic serum, was regained on the subsequent addition of some serum from a fresh (i.e. non- treated) animal. These observations have been fully confirmed and greatly extended. Ehrlich and Morgenroth analysed the phenomena in question, and showed that the specially developed and heat-resisting substance, "immune-body," entered into com- bination with the red corpuscles at a comparatively low temperature, namely at 0° C. ; whereas complement does not combine at this temperature. In this way a method is supplied by which the immune-body can be removed from a hsemolytic serum while the complement is left. They came to the con- clusion that immune - body combined with the complement though the combination was less firm and only occurred at a higher temperature — best about 37° C. They therefore consider that the immune-body acts as a sort of connecting link between the red corpuscle and the complement, hence the term " amboceptor " which Ehrlich afterwards applied. It may be stated, however, that the direct union of complement and immune-body has not been conclusively demonstrated. Bordet, on the other hand, holds that the immune-body acts merely as a sensitising agent — hence the term substance sensibilisatrice — and allows the ferment-like complement to act. It is quite evident from his writings, however, that he does not mean, as is often assumed, that the immune-body causes some lesion in the corpuscle which allows the complement to act, but simply that it produces in the molecules (receptors) of the red corpuscles an avidity for complement. All that we can say definitely at present is that the combination of receptor + immune-body takes up complement in firm union while neither does so alone ; whether the immune-body acts as a link between the two or not must be left an open question. Even after the corpuscles are laked with water the receptors are not destroyed: Muir and Ferguson have shown that they can still take up immune- body and, through its medium, complement, just as the intact corpuscles do. Ehrlich and Morgenroth showed that in some cases the red corpuscles can take up much more immune-body than is necessary for their lysis, and Muir found in one case studied, that each further dose of immune-body led to the fixation of more complement, so that as many as ten times the haemolytic dose of complement might thus be used up. It is a matter of considerable importance that the union of immune-body and red corpuscles can be shown to be a reversible action. If, as was found by Morgenroth and Muir independently, corpuscles treated ELEMOLYTIC AND OTHER SERA 481 with several doses of immune-body and then repeatedly washed in salt solution be mixed with untreated corpuscles and allowed to remain for an hour, then sufficient immune-body will pass from the former to the latter, so that all become lysed on the addition of sufficient complement. The combination of complement, on the other hand, is usually of very firm nature. It has been a disputed point whether there are several distinct complements in a normal serum with different relations to different immune-bodies, for which Ehrlich and his co-workers have brought forward a large amount of evidence, or whether, as Bordet holds, there is a single complement which may, how- ever, show slight variations in behaviour towards different immune-bodies. There is at least no doubt that all the com- plement molecules in a serum are not the same. Workers of the French school also hold that complement does not exist in the free condition in the blood, but is liberated from the leucocytes when the blood is shed ; though this cannot be held as proved, there is evidence that the amount of free complement increases after the blood is shed and some time later gradually diminishes. The hsemolytic action of a normal serum can be shown in many cases to be of the same nature as that of an immune- serum, that is, complement and the homologue of an immune- body can be distinguished. For example, the guinea-pig's serum is hsemolytic to the ox's corpuscles ; if a portion of serum be heated at 55° C. the complement will be destroyed ; if another portion be treated with ox's corpuscles at 0° C., the natural immune-body will be removed and only complement will be left. Neither portion is in itself hsemolytic, but this property becomes manifest again when the two portions are mixed. Haemolytic sera are of great service in the study of the question of specificity. Each is specific in the sense already explained (p. 466), but the serum developed against the corpuscles of an animal may have some action on those of an allied species, that is, some receptors are common to the two species. This fact can be readily shown by the usual absorption tests, for example, in the case of an anti-ox serum tested on sheep's corpuscles. A close analogy holds to what has been established in the case of agglutinins. It is further of great interest to note that by the injection of red corpuscles into an animal its serum not only becomes haemolytic, but in many cases when heated at 55° C. possesses also agglu- tinating and opsonic properties towards the red corpuscles used. And further, it would appear that in some cases at least the immune-body, haBmagglutinin, and hsemopsonin are distinct 31 482 IMMUNITY substances. These facts abundantly show how close an analogy obtains between anti-bacterial and hsemolytic sera, and how important a bearing haemolytic studies have on the questions of immunity in general. In addition to hsemolytic sera, anti-sera have been obtained by the injection of leucocytes, spermatozoa, ciliated epithelium, liver cells, nervous tissue, etc. The laws governing the pro- duction and properties of these are identical, that is, each serum exhibits a specifk-4)roperty towards the body used in its produc- tion— i.e. dissolves leucocytes, immobilises spermatozoa, etc. The specificity is, however, not so marked as in the case of sera produced against red blood corpuscles ; thus a serum pro- duced against tissue cells is often hsemolytic ; this is probably due to various cells of the body having the same receptors. Here again when the anti-serum produces -no destructive effect on the corresponding cells, the presence of an immune-body may be demonstrated by the increased amount of complement which is taken up through its medium. It may also be mentioned that each anti-serum usually exhibits toxic properties towards the animal whose cells have been used in the injections, e.g. a hsemolytic serum may produce a fatal result, with signs of extensive blood destruction, haemoglobinuria, etc., i.e. it is haemotoxic for the particular animal ; a serum prepared by injection of liver cells has been found to produce on injection necrotic changes in the liver in the species of animal whose liver cells were used. These are mentioned as examples of a very large group of specific activities. With regard to the sites of origin of immune-bodies our information is still very deficient. Pfeiffer and Marx brought forward evidence in the case of typhoid, and Wassermann in the case of cholera, that the immune-bodies are chiefly formed in the spleen, lymphatic glands, and bone-marrow. According to certain workers of the French school, the chief source of anti- substances acting on cells such as red blood corpuscles is the large mononucleated leucocytes, whilst those acting on bacteria are chiefly derived from the polymorpho-nuclear leucocytes (vide p. 495). Another view is that immune-bodies are chiefly formed by the large mononucleated leucocytes, whilst complements are products of the polymorphs. That these cells are concerned in the production of antagonistic and protective substances is almost certain, though another possible source of wide extent, viz. the endothelium of the vascular system, has been largely overlooked. As yet, definite statements cannot be made on this point. OPSONIC ACTION 483 Methods of Hsemolytic Tests. — A hsemolytic serum is usually pre- pared by injecting the red corpuscles of an animal into the peritoneum of an animal of different species — the corpuscles of the ox are most frequently used, and the rabbit is the most suitable animal for injection. The corpuscles ought to be completely freed of serum by repeatedly washing them in sterile salt solution and centrifugalising. An injection of the corpuscles of 5 c.c. of ox's blood followed by two injections, each of 10 c.c., at intervals of ten days, will usually give an active serum. The animal should be killed by bleeding it, aseptically as far as possible, seven to ten days after the last injection ; the serum which separates may be collected in suitable lengths of quill glass-tubing drawn out at the ends, which are afterwards sealed in the flame. To ensure sterility when the serum is to be kept some time, it is advisable to heat it for an hour at 55° C. on three successive days ; we have always found that serum treated in this way remains sterile. It is of course devoid of complement. The test amount of corpuscles is usually 1 c.c. of a 5 per cent suspension of corpuscles in '8 per cent sodium chloride solution : that is, the corpuscles of 5 c.c. blood are completely freed of serum by repeatedly washing in salt solution, and then salt solution is added to make up 100 c.c. In any investigation it is necessary to obtain the minimum haemolytic dose (M.H.D.) of the immune-body and of the complement to be used. (It is to be noted that as complement does not increase during immunisation, the hsemolytic dose of the fresh serum will come far short of representing the amount of immune-body present.) In testing the dose of immune-body, the fresh serum to be used as com- plement must be devoid of hsemolytic action (in the present instance rabbit's serum will be found suitable) and more than sufficient to produce lysis with immune-body is added to each of a series of tubes. Varying amounts of immune-body are added to the tubes, the contents are shaken, made up to 1*5 c.c. and incubated for two hours. The amount of lysis is then noted and the tubes are placed in a cool chamber till next morning, when a final reading is takeri. The smallest amount of immune-body which gives complete lysis is of course the M.H.D. : sometimes this may be as low as '001 c.c. for the test amount of corpuscles. When further observations are to be continued on the same day, the reading after incubation must be taken as the working standard. To estimate the M.H.D. of complement, proceed in a corresponding manner ; to each of a series of tubes add several doses of immune-body, and then to the several tubes different amounts of com- plement. The activity of a serum as complement varies considerably, and each sample must be separately tested. The above will serve as an indication of the fundamental methods ; for further details special papers on the subject must be consulted. (b) Opsonic Action. — The presence of a substance in an immune-serum which makes the corresponding organism sensitive to phagocytosis was first demonstrated by Denys and Leclef in 1895, in the case of an anti-streptococcal serum. They also showed that the serum produced this effect by acting on the organism, not on the leucocytes. It is, however, chiefly to the researches of Wright and his co-workers that this subject has come into special prominence. Wright and Douglas in their first paper showed that the phagocytosis of staphylococci by 484 IMMUNITY leucocytes depended on a body in the normal serum which became fixed to the cocci and made them a prey to the phagocytes. To this they gave the name of " opsonin " (vide pp. 194, 261). There is no phagocytosis of cocci by leucocytes washed in salt solution ; normal serum heated to 55° C. is also without effect in inducing this phenomenon. They could not demonstrate any effect of the opsonin on the leucocytes. On the other hand, if bacteria be exposed to the fresh serum, and they be freed from the excess of serum and then exposed to phago- cytes also washed free from serum, they will be readily taken up by the cells. It has been abundantly shown that the opsonic action of the serum is increased by the process of immumsa/EToh against an organism, and the opsonic index represents the degree of immunity in one of its aspects as already explained (p. 111). The matter has, however, become complicated by the circum- stance that in an immune-serum an opsonin may still be present after the serum is heated at 55° C., as has been shown by Dean and others. Some observers consider that this opsonin is simply an immune-body, but the results brought forward by others would point to their being different substances, at least in certain cases, notably in haemolytic sera. We are, however, probably safe in saying that the thermostable opsonin of an immune-serum is a true anti-substance\ possessing the specific characters of anti- substances in general and comparable in this respect and in its mode of production with an agglutinin. Muir and Martin have, however, found that the thermolabile opsonin of a normal serum has different characters. For example, when a normal serum is tested on a particular bacterium, the opsonic effect on that bacterium may be removed by treating the serum with other bacteria ; in other words, the thermolabile opsonin of normal serum does not possess the specific character of the opsonin developed in the process of immunisation./ They have also found that various substances or combinations of substances which act as "complement -absorbers" also remove the opsonic property from a normal serum, while they have no effect on an immune-opsonin. According to this view the opsonic effect of the unheated serum of an actively immunised animal or person would represent the sum of the effects of the two kinds of opsonin. Further study will be necessary before the exact relationships of these substances are fully understood, and other questions with regard to them have as yet scarcely been touched upon. Increased phagocytic action had long been known by the work of Metchnikoff to be associated with the development of active AGGLUTINATION 485 immunity and the theory of stimulation of leucocytes was supported by many. The work on opsonins has caused a swing of the pendulum in the other direction, and points to the development of anti-substances in the serum as the all-important factor. It remains to be determined to what extent the opsonic and directly bactericidal properties taken together will explain the phenomena of natural and acquired immunity. (c) Agglutination. — Charrin and Koger in 1889 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, found that when a small quantity of an anti-serum is added to an emulsion of the corresponding bacterium, the organisms become agglutinated into clumps, this phenomenon depending upon the presence of definite bodies in the serum called agglutinins. 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, obtained independ- ently by Griinbaum and Widal, but first published by the latter, was to show that the serum possessed this specific action shortly after infection had taken place; in other words, the develop- ment of this variety of anti- substance can be demonstrated at an early stage of the disease. Agglutination is also observed in the case of cholera, Malta fever, bacterial dysentery, glanders, plague, infection by Gartner's bacillus, b. coli, etc. Furthermore the phenomenon is not peculiar to bacteria ; it is seen, for example, when an animal is injected with the red corpuscles of another species, haemagglutinins appearing in the serum, which have a corresponding specificity. The physical changes on which agglutination depends cannot as yet be said to be fully understood. Gruber and Durham considered that the agglutinin produced a change in the envelope of the bacterium, causing it to swell up and become viscous, and the facts first established appeared to be in favour of this view. On the other hand, this is not the full explanation, as it has been shown by Nicolle and by Kruse that if an old bacterial culture be filtered through porcelain, the addition of some of the corresponding anti-serum produces a sort of granular precipitate in it, and that when, as in the agglutination of bacteria, 486 IMMUNITY minute inorganic particles are added to the mixture they become aggregated into clumps. The phenomenon would thus appear to be the result of the interaction of the agglutinin and some substance in the bacterial cell which is known as the agglutin- able substance or as the agglutinogen, seeing that it is probably the element in the bacterial structure which in the tissues of the animal or person leads to the development of the agglutinin. Joos has found in the case of the typhoid bacillus that there are two agglutinable substances which differ in their resistance to heat — a- and ^-agglutinogen, and that they give rise to corresponding agglutinins. Further, as the result of a com- parative study of the agglutinins of a motile and a non-motile variety of the hog cholera bacillus Theobald Smith has come to the conclusion that there is an agglutinin which is produced by and acts on the flagella and another which is similarly related to the bacterial bodies. The former acts in very much higher dilutions than the latter, and this is regarded as an explanation of the fact that in the case of non-motile organisms the agglutinating serum acts only in proportionately high concentra- tion as compared with the case of most motile forms. Another factor necessary for the phenomenon of agglutination is a proper salt content. Bordet showed that if the clumps of agglutinated bacteria are freed from salt by washing in distilled water they become resolved, and that on the addition of some sodium chloride they are formed again, and Joos has also brought forward striking confirmatory evidence as to the necessity for the presence of salts. It is thus probable that in the pheno- menon of agglutination as ordinarily understood more than one factor is concerned, and it is possible that in part it may depend on some altered molecular relationship of the bacteria to the surrounding fluid analogous to altered surface tension. t? } As stated above, the agglutinins are usually placed in the second order of anti-substances, and are regarded as possessing a combining group and an active or agglutinating group. The constitution would thus be analogous to that of a toxin, and in conformity with this view Eisenberg and Volk consider that the agglutinating group may be destroyed while the combining group remains, the result being an agglutinoid. The evidence for this lies in the fact that when an agglutinating serum is heated to a certain temperature, not only does it lose its agglutinating action but when the bacteria are treated with such a serum their agglutination by active serum is interfered with, a sort of plugging up of the combining molecules having apparently taken place. Other facts have, however, been AGGLUTINATION 487 brought forward in opposition to this view, and the existence of agglutinoids cannot be said to be completely proved. Like immune-bodies agglutinins are not destroyed at 55° C. (a temperature sufficient to annul bactericidal action), and the question arises as to the relation of the two bodies ; discussion has also taken place as to how far agglutination is an indication of immunity. It may be said that in the case of certain sera investigated it has been shown that the immune-body and the agglutinin are separate substances, and that the agglutinative power does not vary pari passu with the degree of immunity — a serum may be strongly agglutinative and feebly bactericidal and vice versa. But while probably as a rule the two substances are distinct, it would not be justifiable to say this is always the case — that is, that an immune-body never has an agglutinating action. And while the agglutinative power cannot in itself be taken as the measure of the degree of immunity, agglutinins and immune-bodies are the products of corresponding reactive pro- cesses, and their formation is governed by corresponding laws. Agglutinins become fixed in definite proportion by the receptors of the bacteria ; that is, the agglutinin becomes used up in the process of agglutination, and it has been shown that bacteria may take up many times the amount necessary to their agglutination — a corresponding fact to what has been established with regard to immune -bodies of hasmolytic sera. The agglutinins are specific in the sense which has been explained above (p. 466). It can be shown by the method of absorption that in an agglutinating serum there may be several agglutinins with different combining groups, some of which may be taken up by organisms allied to that which has given rise to the anti- serum. Whether or not the combination of an agglutinin with the bacterial receptors is a reversible action must be left an open question. Besides those stated above, other phenomena have been observed in the interaction of anti-sera and the corresponding 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 proportion of the homo- logous serum, their morphological characters may be altered, growth taking 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. Precipitins. — This subject does not strictly belong to bacteriology, but the general phenomena are so closely allied to those just described, 488 IMMUNITY especially to agglutination, that some reference may be made to it. When the serum of an animal is injected in repeated doses into another animal of different species, after the type of an immunisation, there appears in the serum of the animal treatea a substance called precipitin, which causes a cloudiness or precipitate when added to the serum used. This precipitate results from the union of the precipitin in the anti-serum with a body in the homologous serum, the latter being known as the precipitinogen. (In the case of rabbits doses of 3 to 4 c.c. of the serum may be injected intraperitoneally at intervals of four to five days, a precipitin usually appearing at the end of about three weeks.) The reaction, which is a very delicate one, is conveniently observed by adding a given amount of the anti-serum, say '05 c.c., to varying amounts of the homologous serum '1, *01, etc. c.c., in a series of small test tubes, the volume being then made up with salt solution to 1 c.c. In this way a definite reaction may be observed with '001 c.c. of the serum or even less. The reaction is specific in the sense explained above. It is always most marked towards the serum of the species used in the immunisation ; but while this is so, there may also be a slight reaction .towards animals of allied species. An anti-human serum, for example, gives the maximum reaction with human serum, but also a slight reaction with the serum of monkeys, especially of anthropoid apes ; it, however, gives no reaction with the serum of other animals. The precipitin test has thus come to be employed as a means of differentiating human from other bloods. Another interesting phenomenon is what is known as the "deviation of complement," which is produced by the combination of the two sub- stances in the serum and anti-serum respectively. If mixtures be made according to the above method, and then a small quantity of complement, say fresh guinea-pig serum, be added, it will be found that the comple- ment becomes absorbed, as may be shown by subsequently adding a test amount of sensitised red blood corpuscles. This deviation phenomenon is even a more delicate reaction than the precipitin test, it being often possible to demonstrate by its use from a tenth to a hundredth of the smallest amount of serum which will give a perceptible precipitate ; it also is specific within the same limits.1 Therapeutic Effects of Anti-Sera. — As will have been gathered, the chief human diseases treated by anti-sera are diphtheria, tetanus, streptococcus infection, pneumonia, plague, 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 antitoxin came into general use about October 1894, and the statistics published by Behring towards the end of 1895 indicated 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, 14*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 1 For an account of precipitins, vide Nuttall, "Blood Immunity and Relationships," Cambridge 1904 ; and of complement deviation, Muir and Martin, Journ. of Hyg. vi., 1906, p. 265. THERAPEUTIC EFFECTS OF ANTI-SERA 489 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 instances 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. Another effect of the antitoxic treatment has been that when tracheotomy is necessary the percentage of recoveries is now much higher, being 73 per cent instead of 27 per cent in a group of cases collected by the American Pediatric Society. In the London fever hospitals since 1894 the recoveries after tracheotomy have been 5 6 '4 as compared with 32'1 per cent previous to the intro- duction of antitoxin. One of the most striking 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 here 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 treat- ment should be commenced whenever the disease is recognised. Behring 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, whilst after the fifth day it was 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 tho 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 antitoxin the improvement in results has no? been marked, but some chronic cases have been benefited, and as already stated (p. 386) better results are obtained in acute cases if intravenous in- jection be practised. 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 anti- streptococcic and anti-pneumonic sera, though in the case of the first mentioned numerous cases of apparently successful result have been recorded. With regard to anti-venin, Lamb has shown that, if a cobra with full glands bites a man, many times the minimal lethal dose are probably injected. In cases of slight bite, however, benefit may accrue from the use of the anti- serum. 490 IMMUNITY As has been shown above, antibacterial sera require for their complete action a sufficiency of complements, and as these diminish in amount when a serum is kept, the unsatisfactory results with this class of sera may be due to a deficiency of complement. Or it may be as Ehrlich has suggested, that the complement naturally existing in human serum does not suit the immune-body in the anti-serum — that is, is not taken up through the medium of the latter and brought into combination with the bacterium. And there is still the further possibility that even though the complement should be taken up, the zymotoxic group of the latter is not sufficiently active towards the bacterium to effect its death. In both cases it will appear that an extracellular bactericidal action cannot be produced by the particular immune-body in association with the complement of the animal in question. There is no doubt that this question of complements is one of importance, and that both combining affinity and toxic action of complements must be considered in each case. 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. Only a short reference need be made to these. The theory of exhaustion, with which Pasteur's name is associated, supposed that in the body of the living animal there are substances necessary for the existence of a particular organism, which become used up during the sojourn of that organism in the tissues ; this pabulum being exhausted the organisms die out. Such a supposition is, of course, quite disproved by the facts of passive immunity. According to the theory of retention the bacteria within the body were considered to 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. Such a theory only survives now in the view that antitoxins are modified toxins, the evidence against which has already been discussed (p. 470). There then came the humoral theory and the theory of phagocytosis, but neither of these is tenable in its pure form, and the distinction between them need not be maintained. For, on the one hand, any substance with specific property in the serum must be the product of cellular activity, and on the other hand, the facts with regard to passive immunity go far beyond the ingestive and digestive properties of phagocytes, though these cells may EHRLICH'S SIDE-CHAIN THEORY 491 be in part the source of important bodies in the serum. At the present time interest centres around two theories, viz. Ehrlich's side-chain theory and Metchnikoff 's phagocytic theory as further developed. These will now be discussed, and it may be noted that the ground covered by each is not coextensive. For the former deals chiefly with the production of anti-substances and its biological significance, the latter deals with the defensive properties of cells, either directly by their phagocytic activity or indirectly by substances produced by them after the manner of digestive ferments. It will be seen, however, that each has a normal process as its basis, viz. that of nutrition. 1. Ehrlich's Side-Chain Theory. — This may be said to be an application of his views regarding the nourishment of proto- plasm. A molecule of protoplasm (in the general sense) may be regarded as composed of a central atom group or executive centre (Leistungskern) with a large number of side- chains (Seitenketten), i.e. atom groups with combining affinity for food-stuffs. It is by means of these latter that the living molecule is increased in the process of nutrition, and hence the name receptors given by Ehrlich is on the whole preferable. These receptors are of three chief kinds corresponding to the classes of anti-substances described (p. 466) ; the first has a single unsatisfied combining group and fixes molecules of simpler constitution — receptor of the first order : the second has a combining group for the food molecule, and another active or zymotoxic group, which leads to some physical change in it — receptor of the second order ; the third has two com- bining groups, one for the food molecule and another which fixes a ferment in the fluid medium around — receptor of the third order or amboceptor. These latter receptors come into action in the case of larger food molecules which require to be broken up by ferment -action for the purposes of the cell economy. In considering the application of this idea to the facts of passive immunity, it must be kept in view that all the substances to which anti-substances have been obtained are, like proteids, of unknown but undoubtedly of very complex chemical constitution, and that in apparently every case the anti-substance enters into combination with its corresponding substance. The dual constitution of toxins and kindred substances, as already described (p. 170), is also of importance in this connection. Now, to take the case of toxins, when these are introduced into the system they are fixed, like food-stuffs, by their haptophorous groups to the receptors of the cell protoplasm, but are unsuitable for assimilation. If they are in sufficiently large amount the 492 IMMUNITY toxophorous part of the toxin molecule produces that disturb- ance of the protoplasm which is shown by symptoms of poisoning. If, however, they are in smaller dose, as in the early stages of immunisation, fixation to the protoplasm occurs in the same way ; and as the combination of receptors with toxin is supposed to be of firm nature, the receptors are lost for the purposes of the cell, and the combination R.-T. (receptor + toxin) is shed off into the blood. The receptors thus lost become replaced by new ones, and when additional toxin molecules are introduced, these new receptors are used up in the same manner as before. As a result of this repeated loss the regeneration of the receptors becomes an over-regeneration, and the receptors formed in excess appear in the free condition in the blood stream and then constitute antiiojdn_jiiojejcules. There are thus three factors in the process, namely, (1) fixation of toxin, (2) over-production of receptors, (3) setting free of receptors produced in excess. Accordingly these receptors which, when forming part of the cell protoplasm, anchor the toxin to the cell, and thus are essential to the occurrence of toxic phenomena, in the free condition unite with the toxin, and thus prevent the toxin from combining with the cells and exert- ing a pathogenic action. The three orders of receptors, when separated from the cells, thus give the three kinds of anti- substances. Ehrlich does not state what cells are specially concerned in the production of anti-substances, but from what has been stated it is manifest that any cell which fixes a toxin molecule, for example, is potentially a source of antitoxin. Cells, to whose disturbance, resulting from the fixation of toxin, characteristic symptoms of poisoning are due, will thus be sources of antitoxin, e.g. cells of the nervous system in the case of tetanus, though the cells not so seriously affected by toxin fixation may act in the same way. The experimental investiga- tion of the source of antitoxins has, however, yielded little result, and no definite statement can be made on the subject. When we come to consider how far Ehrlich's theory is in harmony with known facts, we find that there is much in its favour. In the first place, it explains the difference between active and passive immunity, e.g. difference in duration, etc. ; in the former the cells have acquired the habit of discharging anti- substances, in the latter the anti-substances are simply present as the result of direct transference. It is also in harmony with the action of antitoxins, etc., as detailed above, and especially it affords an explanation of the multiplicity of anti-substances. For, if we take the case of antitoxins, we see that this depends EHELICH'S SIDE-CHAIN THEORY 493 uppn the combining affinity of the toxin for certain of the cells of the body, and this again is referred back to the complicated constitution of living protoplasm. Furthermore, the biological principle involved is no new one, being simply that of over- regeneration after loss. It would appear likely that the integrity of the executive centres of the protoplasm molecules would be essential to the satisfactory production of side- chains, and this would appear in accordance with the fact that antitoxin formation occurs most satisfactorily when there is no marked disturbance of the health of the animal. It is to be noted, however, that it does not explain active immunity apart from the presence of anti-substances in the serum. For example, an animal may be able to withstand a much larger amount of toxin than could be neutralised by the total amount of antitoxin in its serum. This might theoretically be explained by supposing a special looseness of the cell re- ceptors so that the toxin-receptor combination became readily cast off. The question, however, arises whether there may not be really an increased resistance of the cells to the toxophorous affinities. An observation recently made by Meyer and Ransom (v. p. 383) is also difficult of explanation according to the view that antitoxin is formed by the cells with which the toxin combines and on which it acts. They found that in an animal actively immun-J ised against tetanus and with antitoxin beginning to appear in its blood, the injection of a single M.L.D. of tetanus toxin into a peripheral nerve brought about tetanus with a fatal result. On the other hand, the injection of antitoxin into the sciatic nerve above the point of injection of toxin prevented the latter from reaching the cells of the cord. One can scarcely imagine an explanation of these facts if antitoxin molecules were in process of being shed off by the cells of the nervous system. Further, when the serum of an animal contains a large amount of anti- toxin, how does the additional toxin injected reach the cells in order to influence them as we know it does? This also is difficult to understand unless the toxin has a greater affinity for the receptors in the cells than for the free receptors (antitoxin) in the serum. A supersensitiveness of the nerve-cells of an animal to tetanus toxin, sometimes observed even when there is a large amount of antitoxin in the serum, has been often brought forward as an objection. But this also may perhaps be explained by there having occurred a partial damage of the cell protoplasm by the toxophorous action in the process of immunisation — an explanation which, of course, demands that in some way the freshly introduced toxin may reach the cells in spite of the anti- 494 IMMUNITY toxin in the blood. Further investigation alone will settle these and various other disputed points, and may remove many of the apparent objections. At present we may say, however, that Ehrlich's theory is the only one which even attempts to explain the cardinal facts of this aspect of immunity. In connection with the condition of supersensitiveness referred to above, an interesting phenomenon has recently been -described by Theobald Smith, and is now generally known as "serum anaphylaxis," or "Theobald Smith's phenomenon." It is briefly the following : — If a guinea-pig be injected with a quantity, say 5 c.c., of horse serum, no disturbance follows ; if, however, the animal be previously treated, say fourteen davs before, with a very small quantity of horse serum, '001 c.c. (even less is sufficient), and then the 5 c.c. of serum be injected, the animal usually dies within an hour with characteristic symptoms. The general lesions are of hsemorrhagic nature, as pointed out by Gay and Southard,1 and occur especially in the stomach. The condition of super- sensitiveness to the horse serum lasts fora long period of time. Accord- ing to Gay and Southard the phenomenon depends upon a substance in the horse serum which they call anaphylactin, and which persists for a long period of time in the blood of the guinea-pig. This body they con- sider to act as a slight irritant to the cells of the guinea-pig, and to produce an increased affinity for the molecules in the horse serum. Ac- cordingly when the second injection is made the rapid combination of these substances with the cells result in the disturbances referred to. What- ever may be the explanation, the phenomenon is of extreme importance as showing the profound alterations in metabolism which may be induced by a minute quantity of serum of a normal animal. The facts relating to hypersensitiveness raise the question of whether in any immunisation procedure an injury may not be constantly done to the cells forming the anti-substances. We have already drawn attention to the occurrence of what Wright has called the negative phase in the course of the increase of the opsonic power of the serum aimed at in a bacterial vaccination. There is evidence that such negative phases are common in all immunisations. They have been also noted in the formation of antitoxins, of immune-bodies, and of agglutinins. Thus in the case of the first, Salamonsen and Madsen showed that the fall in the content of an animal's serum in antitoxin after a fresh toxin injec- tion was greater than could be accounted for by the neutralisa- tion of the free antitoxin in the blood by the toxin introduced, Iand they attributed the occurrence to an injury to the producing 1 cells temporarily diminishing the productive activity. The4 normal course of every immunisation may be said to consist in a succession of positive and negative phases, and an effective immunisation is one where each succeeding positive phase brings 1 Vide Gay and Southard, Journ. Med. Research, xvi., 1907, 143. THE THEORY OF PHAGOCYTOSIS 495 the serum to a higher content in anti-body. Again, in no case is the capacity of producing anti-bodies unlimited. In certain reactions the limit of possible increase is less than in others. Thus it is not possible to raise the opsonic power of a serum higher than a not very great multiple of its original opsonic content. On the other hand, when we are dealing with the reaction against bacterial toxins we find that the mechanism producing antitoxin can react in an extraordinary degree, and a serum many thousand times stronger than that produced during the early days of immunisation may ultimately be attained. The animal body also exhibits great power of forming agglutinins, and the capacity of forming immune bodies seems to occupy an intermediate position between the opsonic reaction and the antitoxin reaction. But even in the antitoxin reaction a time comes in a high immunisation when evidence of exhaus- tion of the producing mechanism is manifest, so that the injection of fresh toxin is no longer efficient, and the negative phase is not followed by a positive phase. From the practical stand- point it is the aim of the immuniser to select the time just preceding such an event for the bleeding of an animal. If the cells of the latter be given a few months rest then the capacity for producing antitoxin usually reappears. But such facts emphasise what we have said as to the possibility of every immunisation entailing the infliction of an injury on some bodily mechanism. 2. The Theory of Phagocytosis.— This theory, brought forward by Metchnikoff to explain the facts of natural and acquired immunity, has been of enormous influence in stimu- lating research on the subject. Looking at the subject from the standpoint of the comparative 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 digest and destroy them. On extending his observations to what occurred in disease, he came to the conclusion 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 " polyinorpho-nuclear " finely granular leucocytes of the blood, and (b) the macrophages, which include the larger hyaline leucocytes, 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 rapid activity on the part of the phagocytes, different varieties 496 IMMUNITY being concerned in different cases, — an activity which may rapidly destroy the bacteria and prevent even local damage. If the organisms are introduced into the tissues of a moderately susceptible animal, 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 showed that when a naturally susceptible animal was immunised, the process was accompanied by the appearance of an active phagocytosis. The ingestion of bacteria by phagocytes is undoubtedly a phenomenon of the greatest importance in the defence of the organism. It is known that amoebae and allied organisms have digestive properties which are specially active towards bacteria, and from what can be directly observed, as well as indirectly inferred, there can be no doubt that such a faculty is also possessed by the phagocytes of the body. Thus bacteria within these cells are in a position favourable to their destruction and do in many instances become destroyed. In fact, observations on phagocytosis in vitro show that such destruction may in the case of some organisms occur so rapidly that the actual number observable in the leucocytes is no indication of the activity of the process. In other instances, e.g. in gonorrhoea, the ingested organisms would appear to survive a considerable time without undergoing change. Undoubtedly phagocytosis is of the highest importance in active immunity, as. by its means organisms which would not undergo an extra-cellular death may be killed off. In the process of immunisation of a susceptible animal we see a negative or neutral chemiotaxis becoming replaced by positive chemiotaxis. This has been explained by Metchnikoff as due to an education or stimulation of the phagocytes. The recent work on opsonins shows, however, that this is not the case, as leucocytes from an immunised animal are not more active in this direction than those of a normal animal, the all-important factor being the development of an opsonin in the immune animal. Thus this phase of immunity comes to be merely a part of the subject of anti-substances in general. The digestive ferments of phagocytes or cytases are, according to Metchnikoff, retained within the cells under normal conditions, but are set free when these cells are injured, for example, when the blood is shed. They then become free in the serum by THE THEORY OF PHAGOCYTOSIS 497 the breaking up of the cells— the process known as phagolysis — and they then constitute the alexines, or complements of Ehrlich. Of these, as has already been said, Metchnikoff thinks there are probably two kinds — one called macrocytase, contained in the macrophages, which is specially active toward the formed elements of the animal body, protozoa, etc. ; and the other, microcytase, contained within the poly morpho-nucl ear leucocytes, which has a special digestive action on bacteria. It is the microcytase which gives blood serum its bactericidal properties. It appears to us, however, that Metchnikoff has gone too far in distinguishing the activities of the two classes of cells so much as he has done. When the properties of antibacterial sera, as above described, are considered in relation to phagocytosis, Metchnikoff gives the following explanation. He admits that the immune -body is fixed by the bacteria (or red corpuscles, as the case may be), though he does not state that a chemical combination takes place ; hence he calls it a fixative (fixateur). The immune-bodies are to be regarded as auxiliary ferments (ferments adjuvants) which aid the action of the alexine. Unlike the latter, however, they are formed in excess during immunisation and set free in the serum. He compares their action to that of enterokinase, a ferment which is produced in the intestine and which aids the action of trypsin. Thus, when the bacteria have fixed the immune- body their digestion is facilitated either within the phagocytes, or outside of them when the alexine has been set free by phagolysis. He, however, maintains that extracellular digestion or lysogenesis does not take place without the occurrence of phagolysis. The source of immune-bodies is, in all probability, also the leucocytes, as these substances are specially abundant in organs rich in such cells — spleen, lymphatic glands, etc. ; here again the mono- nuclear leucocytes are probably the source of the immune-bodies concerned in haemolysis, the polymorpho-nuclear leucocytes the source of those concerned in bacteriolysis. Although the im- mune-bodies are usually set free in the serum, this is not always the case ; sometimes they are contained in the cells, and this probably occurs when there is a high degree of active immunity against bacteria without a serum having an antibacterial action, the powers of intracellular digestion being in such cases increased. In this way the facts of immunity can be explained so far as these concern the destruction of bacteria. Metchnikoff 's work has less direct bearing on the production of antitoxins. He admits the fixation of the toxin by the anti- toxin to form a neutral compound, and he apparently considers 32 498 IMMUNITY that leucocytes may also be concerned in the production of antitoxins. Apart, however, from antitoxin formation, he con- siders the acquired resistance of the cells themselves of high importance in toxin immunity. When we consider MetchnikofFs theory as thus extended to cover recently established facts, it must be admitted that it affords a rational explanation of a considerable part of the subject, though the elucidation of the chemiotactic phenomena during immunisation as explained above detracts from the importance which he attached to the leucocyte. It, however, does not afford explanation of the multiplicity and specificity of antitoxins as Ehrlich's does ; on the other hand, it is more concerned with the cells of the body as destroyers or digesters of bacteria. As regards the subject of antibacterial sera, the results of these two workers may be said to be in harmony in some of the funda- mental conceptions. And it is of interest to note that Metchni- koff, starting with the phenomena of intracellular digestion, has arrived at the giving off of specific ferments by phagocytes ; whilst Ehrlich, from his first investigations on the constitution of toxins, has arrived at an explanation of antitoxins and immune-bodies also with a theory of cell-nutrition as its basis. Within the last few years marked progress has thus been made towards the establishment of the fundamental laws of 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 destroy- ing 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 possessed by an animal of destroying the organisms when introduced into its tissues. It might also possibly be due to an insusceptibility to, or power of neutralising, the toxins of NATURAL BACTERICIDAL POWERS 499 the organism. For the study of the various diseases shows that the toxins (in the widest sense) are the weapons by which morbid changes are produced, and that toxin-formation is a property common to all pathogenic bacteria. There is, moreover, no such thing known as a bacterium multiplying in the living tissues without producing local or general changes, though, theoretically, there might be. As a matter of fact, however, natural immunity is in most cases one against infection, i.e. consists in a power possessed by the animal body of destroying the living bacteria when introduced into its tissues : such a power may exist though the animal is still susceptible to the separated toxins. We shall now look at these two factors separately. 1. Variations in Natural Bactericidal Poivers. — The funda- mental fact here is that a given bacterium may be rapidly destroyed in one animal, whereas in another it may rapidly multiply and produce morbid effects. The special 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 and destroyed 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. Variations in phagocytic activity are found to correspond more* or less closely with the degree of immunity present, but are probably in themselves capable of explanation. The fundamental observations of Wright and Douglas show that in many cases at least, leucocytes do not ingest organisms in a neutral saline solution, and that this is not due to the medium in which they are, is readily shown by subjecting the organisms to the action of fresh serum and then washing them ; thereafter, they are rapidly taken up by the leucocytes in salt solution. In most cases this result is due to the labile opsonin of normal serum which has combining affinities for a great many organisms as already stated. In other cases more specific substances may be concerned. But the all-important fact is that whether phagocytosis occurs or not, appears to depend upon certain bodies in the serum. As yet we cannot say whether the phagocytosis in a given serum, observed according to the opsonic technique, always runs parallel with phagocytosis in the tissues of the animal from which the serum has been taken. This is a subject on which extended observations are necessary. But whether or 500 IMMUNITY not phagocytosis in vivo corresponds with that in vitro it is probably to be explained in the same way ; that is, it probably depends upon the content of the serum. The composition of the latter, no doubt, is the result of cellular activity, and in this the leucocytes themselves are in all probability concerned, but the movements and phagocytic activity of these cells seem to be chiefly if not entirely controlled by their environments. Ingestion is, however, only the first stage in the process ; intra- cellular destruction is the second, and is of equal importance. What may be called intracellular bactericidal action probably varies in the case of leucocytes of different animals, but regarding this our knowledge is deficient, and, further, bacteria may some- times survive the cells which have ingested them. (b) When it had been shown that normal serum possessed bactericidal powers against different organisms, the question naturally arose as to whether this bactericidal power varied in different animals in proportion to the natural immunity enjoyed by them. The earlier experiments 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 remark- able immunity to anthrax, had greater bactericidal powers than that of other animals investigated. Further investigation, how- ever, has shown that this is not an example of a general law, and that the bactericidal action of the serum does not vary pari passu with the degree of immunity. In many cases, however, non-pathogenic and also attenuated pathogenic bacteria can be seen to undergo rapid solution and disappear when placed in a drop of normal serum. The bactericidal action of the serum was specially studied by Nuttall, and later by Buchner and Hankin, who believe that the serum owes its power to certain substances in it derived from the spleen, lymphatic glands, thymus, and other tissues rich in leucocytes. To these substances Buchner gave the name of alexines] as already explained, they correspond with MetchnikofFs cytases and Ehrlich's complements described above. They can be pre- cipitated by alcohol and by ammonium sulphate, and in this respect and in their relative lability correspond with enzymes or unorganised ferments. Variations in bactericidal power of the serum as tested in vitro, however, do not explain the presence or absence of natural immunity against a living bacterium. In some cases, for example, it has been found to be considerable, while the organisms flourish in the body, and the animal has no immunity. In such a case Metchnikoff says that there occurs in the living body no liberation of alexines by the phagocytes, and NATURAL SUSCEPTIBILITY TO TOXINS 501 hence no bactericidal action such as occurs when the blood is shed. In the case of the haemolytic action of a normal serum, it has been shown in many instances that in addition to complement a natural immune-body is also concerned (p. 481), and this would appear to be the rule ; the process being analogous to what is seen in the case of an artificially developed hsemolytic serum. In certain instances an analogous condition appears to obtain in a normal bactericidal serum. For example the dog's serum heated at 58° C. contains a natural immune-body to anthrax which can be activated by the addition of normal guinea-pig's serum so as to produce a bactericidal action, though the latter is by itself with- out any such effect. At present, however, the possibility of bactericidal action by complement alone cannot be excluded, as it appears to combine with many bacteria without any inter- mediary. Further work is necessary to determine whether all the facts regarding natural immunity are explainable by the opsonic and bactericidal properties of the serum. 2. Variations in Natural Susceptibility to Toxins. — We must here 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-suscepti- bility 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 toxin of the tetanus bacillus may be here mentioned (v. p. 381), and large amounts of this poison can be injected into the scorpion without producing any effects whatever; the high resistance of the pigeon to morphia is a striking example in the case of vegetable poisons. This variation in resistance to toxins 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 toxins such as that of diphtheria. We must take this natural resistance for granted, though it is possible that ere long it will be explained. According to Ehrlich's view of the constitution of toxins, it might be due to the want of combining affinity between the tissue cells and the haptophorous group of the toxin ; or, on the other hand, supposing this affinity to exist, it might be due to an innate non-susceptibility to the action of the toxophorous group. Certain investigations have been made in order to determine the combining affinity of the nervous system of the 502 IMMUNITY fowl with tetanus toxin, as compared with that obtaining in a susceptible animal, but the results have been somewhat contra- dictory. Accordingly, a general statement on this point cannot at present be made, though in all probability variations in the susceptibility to the toxophorous group will be found to play a very important part. It has been shown by Muir and Brown- ing by means of hsemolytic tests that the toxic activity of com- plement, after it has been fixed to the corpuscles, varies very much; in some instances an amount of complement, which would rapidly produce complete lysis of one kind of corpuscle, may have practically no effect on another, even though it enters into combination. These results are of importance in demonstrating how the corresponding molecules of different animals may vary in sensitiveness to toxic action. 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 introduced by Jenner. Though there is little doubt that a contagium vivum is concerned in its occurrence, the etiological relationship of any particular organism to smallpox has still to be proved ; and with regard to Jennerian vaccination, 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 satis- factorily settled. We cannot 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 inoculation (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 also had this effect. This inoculation method had long been practised in various parts of the world, and had considerable 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 503 504 SMALLPOX AND VACCINATION were insusceptible to subsequent infection from smallpox. In the horse there occurs a disease known as horsepox, especially tend- ing 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, or more or less deep ulcers are formed at their sites. From such a lesion the hands of the milkers may become infected through abrasions, and a similar local eruption occurs, with general symptoms in the form of slight fever, malaise, and loss 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 smallpox, 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 .4 ft Inquiry into the Causes and Effects of the Variola Vaccince. Though from the first Jennerian vaccination had many opponents, it gradually gained the con- fidence of the unprejudiced, and became extensively 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 usual 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 most of the strains at present in use have probably 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 hefe, in numerous instances, it is only the unvaccinated individuals who have contracted the disease. RELATIONS OF SMALLPOX TO COWPOX 505 While vaccination is undoubtedly efficacious in protecting against smallpox, Jenner was wrong in supposing that a vaccina- tion in infancy afforded protection for more than a certain number of years thereafter. It has been noted in smallpox epidemics which have occurred since the introduction of vaccina- tion, that whereas young unprotected subjects readily contract the disease, those vaccinated as infants escape more or less till after the thirteenth to the fifteenth years. It has become, therefore, more and more evident that revaccination 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 was investigated in this country in 1896 by a Royal Commission, whose general conclusions were as follows. Vaccination diminishes the liability to attack by smallpox, and when the latter does 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 an attack outlasts its power wholly to ward it off. Revaccination restores protection, but this operation must be from time to time repeated. Vaccination 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 introduced 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 506 SMALLPOX AND VACCINATION 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 of conferring immunity against another. The most difficult 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 experiments 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 produced 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 variola- tion of calves. The criticism of these experiments which has been offered, namely, that since many of them were performed in vaccine establishments, the calves were probably 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 extraordinary 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 crucis for establishing the identity of the two diseases would of 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 BACTERIA IN SMALLPOX 507 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 susceptibilities 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 small- pox 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 vaccina- tion similar to those observed in cattle -plague have been reported. Smallpox, cowpox, cattle-plague, horsepox, 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 Sander- son 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 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. Streptococci have also been described as agglutinable by the sera of smallpox patients and of vaccinated persons ; such sera it may be said had no effect on other strains of streptococci. Calmette and Guerin have described very minute granules in the lymph which could not be cultivated but which persisted after all the bacteria had been removed. (The method by which the latter was accom- plished was by exciting a leucocytosis in a rabbit's peritoneum and 508 SMALLPOX AND VACCINATION then introducing the vaccinal lymph ; the leucocytes phagocyted the bacteria so that the lymph no longer gave cultures on ordinary media. It was, however, still potent to produce vaccinia.) Klein and also, independently, Copeman, have observed an 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 Lbffler'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 inoculating 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 been fully investigated, and thus the identity of this bacillus with that seen in the tissues is not proved. Various 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 of 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 are described as multiplying by simple division, and in the living condition exhibiting amoeboid movement. Similar bodies have been described by Reed in the blood of smallpox patients and of vaccinated children and calves. These are probably the bodies described by Guarnieri and to which considerable attention has been paid. They are from 1-8 /A in diameter, are round, oval, or sickle -shaped, and stain by ordinary nuclear dyes. They lie in the cells in spaces often near the nucleus, and are readily demonstrable in vaccine pustules and also in the experimental lesions which can be produced in the rabbit's cornea, the larger bodies being defined in the cells towards the centre of the lesion. These bodies NATURE OF VACCINATION 509 have been looked on by many as protozoa, and Guarnieri himself stated that multiplication could be seen occurring in them in fresh lymph, but Ewing and also Prowazek have brought forward strong evidence for the appearances being due to nuclear changes, Still the question of the specificity of these changes to variolous lesions remains, and here it may be said Wasielewski has shown that they persist through 46 transfers on the cornea of the rabbit and further no similar appearances have been found in other skin lesions. Prowazek examined material fixed in a hot mixture of two- thirds saturated perchloride of mercury and one -third 90 per cent, alcohol, washed in 40 per cent, iodine alcohol and stained in Grenacher's haematoxylin, and found bodies in the epithelial cells 1-4 fj. in size, sharply contoured and having ragged edges as if made up of massed chromosomes. These were often broader at one end than at the other, and appearances have been seen which suggest longitudinal division. Prowazek has also seen these " lymph-bodies " as he has called them in the lymph, and he inclines to the idea that they may be protozoa. Bonhoff and also Carini have described spirochaetes as occurring in variolous lesions, but this has not been confirmed. Future investigations must show what significance is to be attached to these various observations. The causal organism of smallpox is probably very small as, though there has been some difference in opinion on this point, there is little doubt that it will pass through the coarser porcelain filters. The Nature of Vaccination. — As we are ignorant of the cause of smallpox, we can only conjecture what the nature of vaccina- tion 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 immunity 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. 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, especi- ally 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 individual to individual by the same paths of infection. Thus it occurs epidemically from time to time in cattle, sheep, horses, and deer, and can be communicated to man. It is questionable whether infection can take place from man to man as the saliva of a person suffering from hydrophobia appears not to contain the virus. It is to be noted that the virus is apparently extremely potent, as cases of infection taking place through an unabraded mucous membrane by the licking of a rabic animal are on record and the experimental applications of the virus to such surfaces as the mucous membrane of the nose or the conjunctiva is often followed by infection. 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 manifestations 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, (1) 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 stille Wuth). The disease, however, is essentially the same in both cases. In the dog the furious form is the 510 PATHOLOGY OF HYDROPHOBIA 511 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 special fear of water is, however, a myth, — it fears to swallow at all. 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 reflexes 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 widespread and progressive paralysis is the outstanding feature. In man then also occur cases where the cerebellum and also the sympathetic system seem to be specially affected. The Pathology of Hydrophobia.— In hydrophobia as in tetanus, to which it bears more than a superficial resemblance, the appearances presented in the nervous system, to which all 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 peri- 512 HYDROPHOBIA vascular 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. Round the nerve cells in the grey matter of the cord and medulla Babes described accumulations of newly-formed cells, and Van Gehuchten observed a phagocytosis of the cells in the posterior root ganglia and also in the sympathetic ganglia. Both of these conditions were at one time thought to be specific of rabies, but this has been found not to be the case. 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 there is no evidence that they are really of this nature. 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 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 THE VIRUS OF HYDROPHOBIA 513 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 jsubcutaneously in one of its limbs, with virulent material, ^tf now the animal be killed before symptoms have manifested themselves, rabies can be produced by subdural inoculation from the r^erves 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, paralysis, 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 injection of the virus, on the other hand, differs from the other modes of infection in that it more frequently gives rise to paralytic rabies. This fact Pasteur explained by supposing that the whole of the nervous system in such a^ case becomes simultaneously affected. In certain animals 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 connection various organisms have been described as being associated with the disease. 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 con- firmation 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 hydrophobia virus by means of pure cultures of the alleged causal organism. With regard to other possible 33 514 HYDROPHOBIA causal agents, Grigorjew thinks such may be found in a protozoon which he has constantly observed after inoculation in the cornea. In 1903 Negri described certain bodies as occurring in the nervous system in animals dying of rabies to which considerable attention has since been devoted, and regarding the significance of which opinion is still divided. It may be said that Negri's observations have been generally confirmed, and as it is probable, whatever the final opinion as to the nature of the bodies may be, that their occurrence is specific to the disease and hence may be used for diagnosis, we shall describe the methods for their demonstration. In doing so we shall chiefly follow the work of the American observers, Williams and Lowden, who, more than any others who have confirmed Negri, have used methods widely employed in the investigation of similar appearances. Their chief method is to take a piece of the brain tissue, to squeeze it between a slide and cover-glass, and, sliding off the latter, to make a smear which is then fixed in methyl alcohol for five minutes and stained by Giemsa's stain (p. 107) for half an hour to three hours ; the prepara- tion is then washed in tap water for 2-3 min. and dried. For rapid work, after fixation, equal parts of distilled water and stain are used instead of the more dilute mixture. For sections the tissues are left in Zenker's fluid l for 3-4 hours, then placed in tap water for five minutes, 80 per cent alcohol with enough iodine added to give it a port wine colour for 24 hours ; 95 per cent alcohol and iodine, 24 hours ; absolute alcohol, 4-6 hours ; cleared with cedar oil and embedded in paraffin of melting point 52° C. ; sections should be 3-6 /u, thick. For staining, Mallory's methylene - blue eosin is recommended ; the steps are as follows : xylol ; absolute alcohol ; 95 per cent alcohol and iodine, £ hour ; 95 per cent alcohol, ^ hour ; absolute alcohol, £ hour ; eosin solution (5-10 per cent aqueous solution), 20 minutes ; rinse in tap water ; Unna's polychrome raethylene-blue solution diluted 1-4 with distilled water, 15 minutes ; differentiation in 95 per cent alcohol for 1-5 minutes (the preparation being kept in motion and its progress watched with a low power) ; rapid and careful dehydration and clearing. The bodies vary much in size, measuring from -5 //- to 25 p. They are round, oval, or angular in outline. They are found in the protoplasm of the nerve cells and of their processes. They have a hyaline appearance with a sharply -defined outline, and in their substance they contain granular material. Taking for granted their cellular structure we may say that with the Giemsa mixture 1 Zenker's fluid is of the following composition : potassium bichromate 2'5 gr., sodium sulphate 1 gr., perchloride of mercury 5 gr., glacial acetic acid 5 c.c., water to 100 c.c. Dissolve the perchloride of mercury and the bichromate of potassium in the water with the aid of heat and add the acetic acid. THE VIRUS OF HYDROPHOBIA 515 their cytoplasm stains blue and the granules a blue-red, — by Mallory's stain the cytoplasm is magenta and the granules a deep blue. The cytoplasm is homogeneous, and in it is a nucleus -like body whose chromatin particles in the larger individuals are arranged round the periphery, there being a clear centre containing a nucleolus ; in the smaller forms the nucleus is a mere chromatin spot. Round the central definite nuclear body are some chromatoid particles which are irregular in outline and size, are sometimes elongated, and do not take on such a pure chromatin stain as the nucleus. There is evidence of division of the nucleus, and sometimes there may apparently be three or four nuclei in one body without division of the protoplasm having occurred. Sometimes the chromatin appears to fragment and break up into a large number of small particles, and in such bodies active budding of the protoplasm may be seen. Sometimes the bodies seem to go on dividing again and again, with the result that some very small forms may be produced, these sometimes appearing in mulberry masses. The Negri bodies have been found in nearly all cases of street-rabies examined by many observers, and have never been found in other conditions of brain disease. They occur in all parts of the central nervous system, but are said to be most abundant in the cells of the cornu Ammonis. They are apparently not so readily found, at least in their larger forms, in animals dying from the inoculation of virus fixe. What the significance of these bodies is, it is at present, impossible to say ; but whatever may be their nature, there is now considerable evidence that their presence is specific of rabies, and that thus in their recognition a much quicker means of diagnosis is possible than by the longer method of awaiting symptoms in an in- oculated rabbit. Many have looked on these bodies as protozoa, and their appearance is not inconsistent with such a view. . The objection which has been raised, that if they were protozoa they could not pass through a porcelain filter (vide infra) as the virus does, is met by the fact of the occurrence of minute forms, and by the fact that similar small forms probably exist in certain trypanosomes (see Appendix E). The occurrence of minute forms would also account for the non-recognition of the parasite in the more acute forms of the disease where there had been an active vegetative condition, and thus no time for the larger forms to develop. 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 516 HYDROPHOBIA constitute the great work of Pasteur ; and everything points to a micro-organism being the cause. The organism, whatever it is, is, in its infective form, probably very small, as it can pass through the coarser Berkefeld niters, and also occasionally through the coarser Chamberland candles. Evidence that it is the organism itself which passes through, is found in the fact that when an animal dies from infection with the nitrate, a small portion of its central nervous system will originate the disease in a fresh animal. Judging from our knowledge of similar diseases we would strongly suspect that it is actually present in a living condition in the central nervous system, the saliva, etc., which yield what we have called the hydrophobic virus, for by no mere toxin could the -disease be transmitted through a series of animals, as we shall presently see can be done. A toxin may, however, be concerned in the production of the pathogenic effects. Remlinger found that death with paralytic symptoms sometimes followed the injection of filtered virus, but that the nervous system of the dead animals did not reproduce rabies. He explains this occurrence by supposing that the filtrate contained a toxin but not the actual infective agent. The resistance of the virus to external agents varies. Thus a nervous system containing it is virulent till destroyed by putre- faction ; it can resist the prolonged application of a temperature of from - 10° to - 20° 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 undoubtedly 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 PROPHYLACTIC TREATMENT OF HYDROPHOBIA 517 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 dogs is constant in the dog, the virus has been from time immemorial of constant strength. Such a virus, of what might be called natural 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 inoculation 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 vims — 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 writh 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 con- tained 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 comparatively long period of incubation in man could not be taken advantage of to "vaccinate" him against the disease before its gravest manifesta- tion 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 the cord used, and inject it under the 518 HYDROPHOBIA 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 gradually increased, as shown in the table : — July 7, 1885, 9 A.M., cord of June 23, i.e. 14 days old. , 7 6 P.M. „ 25 12 8 9 A.M. ,, 27 11 8 6 P.M. ,, 29 9 9 11 A.M., cord of July 1 8 10 3 7 11 5 6 12 7 5 13 9 4 14 11 3 15 13 2 16 15 1 day' old. The patient never manifested the slightest symptom of hydro- phobia. Other similarly favourable results followed ; and this prophylactic treatment of the disease quickly gained the con- fidence of the scientific world, which it still maintains. (The principal 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 lias 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 treat- ment are condensed. Thus on the first day, say at 11 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, cords of 1 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 ten days. In each case the average dose is about 2 c.c. of the emulsion. The success of the treatment has been very marked. The statistics of the cases treated in Paris are published quarterly in the Annales de I'Institut 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, 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 the central nervous system of the diseased animal. Class B includes those bitten by dogs that a competent 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 ; 940 belonging to class B, with two deaths ; and 449 belonging to Class C, METHODS 519 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. Sometimes during or after treatment there appear slight paralytic symptoms with obstinate constipation and it may be retention of urine, but these pass off within a few weeks and leave behind no ill etfects. Antirabic Serum. — In the early part of the nineteenth century an Italian physician, Valli, showed that immunity against rabies could be conferred by administering through the stomach pro- gressively increasing doses of hydrophobic virus. Following up this observation, Tizzoni and Centanni have attenuated rabic virus by submitting it to peptic digestion, and have immunised animals by injecting gradually 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 immunity in other animals ; and further, that if injected into animals from seven to fourteen 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. 454), and they recommended the use, in severe cases, of the serum of such animals in addition to the treatment of the patient by the Pasteur method. A like serum has been obtained from animals treated by the ordinary Pasteur method. Methods, (a) Diagnosis. — When a person is bitten by an animal suspected to be rabid, the latter must under no circum- stances 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 develop- ment of the characteristic symptoms can be watched, and death will occur in not more than five 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 520 HYDROPHOBIA trephine which is made for the purpose. Symptoms usually occur in from ten to twenty-three days and death in fifteen to twenty-five days. 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 glycerin in well-stoppered bottles. When the brain of the suspected dog is available either through its death or its being killed, the Negri bodies should be sought for especially in the cornu Ammonis by the methods described above. (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 treat- ment. 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. IT has now been conclusively proved that the cause of malarial fever is a protozoon of which there are several species. They belong to the hsemosporidia (a sub-class of the sporozoa) which are blood parasites, infecting the red corpuscles of mammals, reptiles, and birds. The parasite was formerly known as the hcematozoon or plasmodium malarias, although the use of the latter term is incorrect ; the term hcemamoeba is, however, now generally employed. The parasite was first observed by Laveran in 1880, and his discovery received confirmation from the in- dependent researches of Marchiafava and Celli, and later from the researches of many others in various parts of the world. Golgi supplied valuable additional information, especially in relation to the sporulation of the organism and the varieties in different types of malarial fever. In this country valuable work on the subject was done by Manson, and to him specially belongs the credit of regarding the exflagellation of the organism as a preparation for an extra-corporeal phase of existence. By in- duction he arrived at the belief that the cycle of existence outside the human body probably took place in the mosquito. It was specially in order to discover, if possible, the parasite in the mosquito, that Ross commenced his long series of observations, which were ultimately crowned with success. After patient and persistent search, he found rounded pigmented bodies in. the wall of the stomach of a dapple-winged mosquito (a species of Anopheles) which had been fed on the blood of a malarial patient. The pigment in these bodies was exactly similar to that in the malarial parasite, and he excluded the possibility of their representing anything else than a stage in the life cycle of the organism. He confirmed this discovery and obtained cor- responding results in the case of the proteosoma infection of birds, where the parasite is closely related to that of malaria. 521 522 MALARIAL FEVER In birds affected with this organism, he was able to trace all stages of its development, from the time it entered the stomach along with the blood, till the time when it settled in a special form in the salivary glands of the insect. Ross's results were published in 1898. Exactly corresponding stages were after- wards found in the case of the different species of the human parasite, by Grassi, Bignami, and Bastianelli ; and these with other Italian observers also supplied important information regarding the transmission of the disease by infected mosquitoes. Abundant additional observations, with confirmatory results, were supplied by Koch, Daniels, Christophers, Stephens, and others. Wherever malaria has been studied the result has been the same. Lastly, we may mention the striking experiment carried out by Manson by means of mosquitoes fed on the blood of patients in Italy suffering from mild tertian fever. The insects, after being thus fed, were taken to London and allowed to bite the human subject, Hanson's son, Dr. P. Thurburn Manson, offering himself for the purpose. The result was that infection occurred ; the parasites appeared in the blood, and were associated with an attack of tertian fever. Ross's discovery has not only been a means of elucidating the mode of infection, but, as will be shown below, has also supplied the means of successfully combating the disease. From the zoological point of view the mosquito is regarded as the definitive host of the parasite, the human subject as the intermediate host. But in describing the life history, it will be convenient to consider, first, the cycle in the human body, and, secondly, that in the mosquito. Various terms have been applied to the various stages, but we shall give those now generally used. The Cycle in the Human Subject. — With regard to this cycle, it may be stated that the parasite is conveyed by the bite of the mosquito in the form of a small filamentous cell — sporozoite or exotospore, which penetrates a red corpuscle and becomes a small amoeboid organism or amoebula. There is then a regularly repeated asexual cycle of the parasite in the blood, the length of which cycle determines the type of the fever. During this cycle there is a growth of the amcebulae or trophozoites within the red corpuscles up to their complete development ; sporulation or schizogony then occurs. The onset of the febrile attack corresponds with the stage of sporulation and the setting free of the spores (enhsemospores or merozoites), i.e. with the production of a fresh brood of parasites. These spores soon become attached to, and penetrate into the interior FORMS OF THE MALARIAL PARASITE 523 of the red corpuscles, becoming intra-corpuscular amoebulse ; the cycle is thus completed. The parasites are most numerous in the blood during the development of the pyrexia, and, further, they are also much more abundant in the internal organs than in the peripheral blood ; in the malignant type, for example, the process of sporulation is practically confined to the former. In addition to these forms which are part of the ordinary asexual cycle, there are derived from the amoebulse other forms, which are called gametocytes, or sexual cells. These remain unaltered during successive attacks of pyrexia, and undergo no further change until the blood is removed from the human body. In the simple tertian and quartan fevers (vide infra] the gameto- cytes resemble somewhat in appearance the fully developed amcebulas before sporulation, whereas in the malignant type they have a characteristic crescent-like or sausage-shaped form ; hence they are often spoken of as " crescentic bodies." The various forms of the parasite seen in the human blood may now be described more in detail. 1. The Enhoemospores (Lankester) or Merozoites are the youngest and smallest forms resulting from the segmentation of the adult amoebula — sporocyte or schizont. They are of round or oval shape and of small size, usually not exceeding 2 //. in diameter; the size, however, varies somewhat in the different types of fever. A nucleus and peripheral protoplasm can be distinguished (Fig. 159). The former appears as a small rounded body which usually remains unstained, but contains a minute mass of chromatin which stains a deep red with the Romano wsky method ; the peripheral protoplasm is coloured fairly deeply with methylene-blue. The spores show little or no amoeboid movement ; at first free on the plasma, they soon attack the red corpuscles, where they become the intra-corpuscular amcebulre. If the blood, say in a mild tertian case, be examined in the early stages of pyrexia, one often finds at the same time sporulating forms, free spores, and the young amcebulas within the red corpuscles. 2. Intra-corpuscular Amoebulce or Trophozoites. — These include the parasites which have attacked the red corpuscles ; they are at first situated on the surface of the latter but afterwards 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, of about the same size as the spores. As seen in fresh blood, they exhibit more or less active amoeboid movement, showing marked variations in shape. The amount and character of the 524 MALARIAL FEVEK amoeboid 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. 155, 156). 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 elaborated from the haemoglobin of the red corpuscles, the parasite growing at the expense of the latter. The red corpuscles thus invaded may remain unaltered in appearance (quartan fever), may become swollen and pale (tertian fever), or somewhat shrivelled and of darker tint (malignant fever). In stained specimens a nucleus may be seen in the parasite as a pale spot containing chromatin which may be arranged as a single concentrated mass or as several separated granules, the chromatin being coloured a deep red by the Romanowsky method. The protoplasm of the parasite, which is coloured of varying depth of tint with methylene-blue, shows great variation in configuration (Fig. 156). The young parasites not unfrequently present a "ring-form," a portion of the red corpuscle being thus enclosed by the parasite. These ring-forms are met with in all the varieties of the parasite, but they are especially common in the case of the malignant parasite, where they are of smaller size and of more symmetrical form than in the others (Fig. 160); the pigment is usually collected in a small clump at one side. Within the red corpuscles the parasites gradually increase in size till the full adult form is reached (Fig. 157). In this stage the parasite loses its amoeboid movement more or less completely, has a somewhat rounded form, and contains a considerable amount of pigment. In the malignant form it only occupies a fraction of the red corpuscle. The adult parasites may then undergo sporulation, but not all of them do so ; some become degenerated and ultimately break down. 3. Sporocytes or Schizonts. — In the process of schizogony the chromatin becomes divided into a number of daughter nuclei which are scattered through the protoplasm ; the latter then undergoes corresponding segmentation and the small merozoites or enhsemospores result. The pigment during the process becomes aggregated in the centre and is surrounded by a small quantity of residuary protoplasm. Schaudinn has found in the case of the tertian parasite that schizogony begins by a sort of primitive mitosis, which is then followed by simple multiple fission. The spores or merozoites are of rounded or oval shape, as above described, and are set free by the rupture of the FIG. 154. FIG. 155. FIG. 156. FIG. 157. , t* FIG. 158. FIG. 159. FIGS. 154-159. — Various phases of the benign tertian parasite. Fig. 154. Several young ring-shaped amcebulae within the red corpuscles, one of the latter enlarged and showing a dotted appearance. Fig. 155. A larger amcebula con- taining pigment granules. Fig. 156. Two large amoebulse, exemplifying the great variation in form. Fig. 157. Large amcebula assuming the spherical form and showing isolated fragments of chromatin — preparatory to sporulation. Fig. 153. Sporocyte or schi/ont, which has produced eighteen spores, each of which contains a small collection of chromatin. Fig. 159. A number of spores which have just been set free in the plasma, x 1000. FIG. 160. FIG. 161. FIG. 162. FIG. 163. • FIG. 164. FIG. 165. FIGS. 160-165. — Exemplifying phases of the malignant parasite. Fig. 160. Two small ring-shaped amcebulse within the red corpuscles. Fig. 161. A "crescent" or gamete showing the envelope of the red corpuscles; also an amoebula. Figs. 162-165 illustrate the changes in form undergone by the crescents outside the body. In the interior of the spherical form in Fig. 164 evidence of the flagella can be seen. Fig. 165. A male gametocyte which has undergone exflagellation, showing the thread-like microgametes or spermatozoa attached at the periphery. xlOOO. (The figures in this plate are from preparations kindly lent by Sir Patrick Hanson.) FORMS OF THE MALARIAL PARASITE 527 envelope of the red corpuscle. The pigment also becomes free and may be taken up by leucocytes. The number and arrange- ment of the spores within the sporocyte vary in the different types. In the quartan there are 6-12, and the segmentation is in a radiate manner, giving rise to the characteristic daisy-head appearance; in the tertian they number 15-20 or more, and have a somewhat rosette-like arrangement (Fig. 158) ; in the malignant there are usually 6-12 spores of small size and somewhat irregularly arranged. Gametocytes. — As stated above, these are sexual cells which are formed from certain of the amcebulae, and which undergo no further development in the human subject. In the mild tertian and quartan fevers they are rounded and resemble some- what the largest amcebulae. The female cells, macrogametocytes, are of large size, measuring up to 1 6 ft in diameter ; they con- tain coarse grains of pigment, and the protoplasm stains somewhat deeply with methylene-blue. The male cells, microgametocytes, are smaller, and the protoplasm stains faintly ; the nucleus, generally in the centre, is rich in chromatin. In the malignant fevers the gametocytes have the special crescentic form mentioned above. They measure 8-9 ^ in length, and occasionally a fine curved line is seen joining the extremities on the concave aspect, which represents the envelope of the red corpuscle (Fig. 161). They are colourless and transparent, and are enclosed by a distinct membrane ; in the central part there is a collection of pigment and granules of chromatin. It is stated that the male crescents can be distinguished from the female by their appear- ance. In the former the pigment is less dark and more scattered through the cell, and there are several granules of chromatin ; in the latter the pigment is dark and concentrated, often in a small ring, and there are one or two masses of chromatin in the centre of the crescent. According to the Italian observers the early forms of the crescents are somewhat fusiform in shape and are produced in the bone-marrow. The fully developed crescents do not appear in the blood till several days after the onset of the fever, and they may be found a considerable time after the disappearance of the pyrexial attacks. They are also little, if at all, influenced by the administration of quinine. It is well known that after a patient has apparently recovered from malarial fever a relapse may take place without fresh infection occurring, and Schaudinn has published interesting observations bearing on this point. He has found that the macrogametocyte of tertian fever may by a process of partheno- genesis give rise to merozoites, which in their turn infect the red 528 MALARIAL FEVER corpuscles and start the cycle again. As described and figured by him, the chromatin of the macrogametocyte divides first into two portions, one of which is smaller and stains more deeply than the other. This more deeply-staining portion then divides, and the protoplasm becomes segmented as in ordinary schizogony, and a young brood of parasites results. The more faintly-staining chromatin along with part of the protoplasm breaks up and disappears. The Cycle in the Mosquito. — As already explained, this starts from the gametocytes. After the blood is shed, or after it is swallowed by the mosquito, two important phenomena occur, viz. (a) the full development of the sexual cells or gametocytes, and (b) the impregnation of the female. If the blood from a case of malignant infection be examined in a moist chamber, preferably on a warm stage, under the microscope, both male and female gametocytes may be seen to become oval and afterwards rounded in shape (Figs. 162-164). Thereafter, in the case of the male cell, a vibratile or dancing movement of the pigment granules can be seen in the interior, and soon several flagella-like structures shoot out from the periphery (Fig. 165). They are of considerable length but of great fineness, and often show a somewhat bulbous extremity. By the Romanowsky method they have been found to contain a delicate core of chromatin, which is covered by protoplasm. They represent the male cells proper, that is, they are sperm-cells or spermatozoa ; they are also known as microgametes. They become detached from the sphere and move away in the surrounding fluid. The female cell also assumes the rounded form, and maturation- takes place by the giving off of part of the nuclear chromatin. Impregnation occurs by the entrance of a microgamete, the chromatin of the two cells afterwards becoming fused. Impregnation was first observed by MacCallum in the case of halteridium, and he found that the female cell afterwards acquired the power of independent movement or became a "travelling vermicule." He also observed the impregnation of the malignant parasite. The fertilised female cell is now generally spoken of as a zygote or ookinete. It has been established that the phenomena just described occur within the stomach of the mosquito, and that the fertilised cell or zygote penetrates the stomach wall and settles between the muscle fibres ; on the second day after the mosquito has ingested the infected blood small rounded cells about 6-8 // in diameter and containing clumps of pigment may be found in this position. (It was in fact the character of the pigment VARIETIES OF THE MALARIAL PARASITE 529 which led Ross to believe that he had before him a stage in the development of the malarial parasite.) A distinct membrane called a sporocyst forms around the zygote, and on subsequent days a great increase in size takes place, and the cysts come to project from the surface of the stomach into the body cavity. The zygote divides into a number of cells called blastophores or sporoblasts, and these again divide and form a large number of filiform cells which have a radiate arrangement; these were called by Ross "germinal rods," but are now usually known as sporozoites or exotospores (in contradistinction to the enhaemospores of the human cycle). The full development within the sporocyst occupies, in the case of proteosoma, about seven days, in the case of the malarial parasites a little longer. When fully developed the cyst measures about 60 /* in diameter, and appears packed with sporozoites. It then bursts, and the latter are set free in the body cavity. A large number settle within the large veneno-salivary gland of the insect, and are thus in a position to be injected along with its secretion into the human subject. The sporozoites enter red corpuscles and become amcebulse as above described. Daniels found that in the case of the malignant parasite an interval of twelve days at least intervened between the time of feeding the mosquito and the appearance of the sporozoites in the gland. It will thus be seen that in the human subject the parasite passes through an indefinite number of regularly recurring asexual cycles, with the giving off of collateral sexual cells, and that in the mosquito there is one cycle which may be said to start with the impregnation of the female gamete. Varieties of the Malarial Parasite. — The view propounded by Laveran was 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. It may, however, now be accepted as proved that there are at least three distinct species which infect the human subject. Practically all are agreed as to a division into two groups, one of which embraces the parasites of the milder fevers — " winter-spring " fevers of Italian writers — there being in this group two distinct species, for the quartan and tertian types respectively ; whilst the other includes the parasites of the severer forms — " sestivo- autumnal " fevers, malignant or pernicious fevers of the tropics, or irregularly remittent fevers. There is still doubt as to whether there are more than one species in this latter group. Formerly Italian writers distinguished (1) a quotidian ; (2) a non-pi gmented quotidian ; and (3) a malignant tertian parasite, 34 530 MALARIAL FEVER though the morphological differences described were slight. Further observations have, however, thrown doubt on this dis- tinction, and the evidence rather goes to show that there is a single species. Opinion also varies as to the cjcle of this parasite ; according to some observers it is twenty-four hours, according to others forty-eight hours, though it is generally admitted that variations occur. The fever is often of an irregular type and multiple infection is probably common. Although the question cannot be considered as finally settled, we shall speak of three species of human parasites. The zoological position may be shown by the following scheme, generally followed by English writers, the terminology being chiefly that of Grassi and Feletti : — Family : H^MAMCEBID^ (Wasielewski). Genus I. Hsemamoeba. The mature gametes resemble in form the schizonts before segmentation lias occurred. Species 1. Ncemamceba Danilewski or halteridium. Parasite of pigeons, crows, etc. Species 2. Hcemamceba relida or proteosoma. Parasite of sparrows, larks, etc. Species 3. fTcemamceba malarice. Parasite of quartan fever of man. Species 4. ffcemamceba vivax. Parasite of tertian fever of man. Genus II. Haemomenas. The ganietocytes have a special crescentic form. Species : Hcemomenas prcecox. Parasite of malignant orsestivo-autumnal fever of man. In addition there are other species belonging to the same family of blood parasites, which infect frogs, lizards, bats, etc., especially in malarial regions. We shall now give the chief distinctive characters of the three human parasites. 1. Parasite of Quartan Fever, — The cycle of development in man is seventy-two hours, and produces pyrexia every third clay ; double or triple infection may, however, occur. In fresh speci- mens of blood the outline is more distinct than that of the tertian parasite, and amceboid movement is less marked. Only the smaller forms show movement, and this is not of active character. The infected red corpuscles do not become altered in size or appearance, and the pigment within the parasite is in the form of coarse granules, of dark brown or almost black colour. The VARIETIES OF THE MALARIAL PARASITE 531 fully developed schizont has a "daisy-head" appearance, dividing by regular radial segmentation into six to twelve merozoites which, on becoming free, are rounded in form. 2. The Parasite of Mild Tertian Fever. — The cycle of develop- ment is completed in forty-eight hours, though a quotidian type of fever may be produced by double infection. The amoebulae have a less refractile margin than in the quartan type, and are thus less easily distinguished in the fresh blood ; the amoeboid movements are, however, much more active, while longer and more slender processes are given off. The infected corpuscles become swollen and pale, and may show deeply stained points by the Romahowsky method — " Schiiffner's dots." The pig- ment within the parasite is fine and of yellowish-brown tint. The mature schizont is rather larger than in the quartan, has a rosette appearance, and gives rise to fifteen to twenty merozoites, though sometimes even more occur ; these have a somewhat oval shape. In both the quartan and tertian fevers all the stages of development can be readily observed in the peripheral blood. 3. The Parasite of Malignant or ^Estivo-autumnal Fever, or Tropical Malaria. — The cycle in the human subject probably occupies forty-eight hours, though this cannot be definitely stated to be always the case (vide supra). The amoebulae in the red corpuscles are of small size, and their amoeboid movements are very active ; they often, however, pass into the quiescent ring form (Fig. 160). The pigment granules, even in the larger forms, are few in number and very fine ; the infected red corpuscles have a tendency to shrivel and assume a deeper or coppery tint. The fully developed schizont occupies less than half the red corpuscle, and gives rise to usually from six to twelve merozoites, somewhat irregularly arranged and of minute size. Schizogony takes place almost exclusively in the internal organs, spleen, etc., so that, as a rule, no sporocytes can be found in the blood taken in the usual way. The proportion of red corpuscles infected by the amcebula3 is also much larger in the internal organs. The gametes have the crescentic form, as already described. Cases of infection with the malignant parasite sometimes assume a pernicious character, and then the number of organisms in the interior of the body may be enormous. In certain fatal cases with coma the cerebral capillaries appear to be almost filled with them, many parasites being in process of sporulation ; and in so-called algid cases, characterised by great collapse, a similar condition has been found in the capillaries of the 532 MALARIAL FEVER omentum and intestines. The process of blood destruction, present in all malarial fevers, reaches its maximum in the malignant class, and the brown or black pigment elaborated by the parasites— in part after being taken up by leucocytes, chiefly of the mononuclear class — becomes deposited in various organs, spleen, liver, brain, etc., especially in the endothelium of vessels and the peri vascular lymphatics. In the severer forms also brownish-yellow pigment is apparently derived from liberated haemoglobin, and accumulates in various parts, especially in the liver cells ; most of this latter gives the reaction of haemosiderin. General Considerations. — The development of the malarial parasites in the mosquito and infection of the human subject through the bites of this insect, have, by the work of Ross and others, as detailed above, become established scientific facts. These facts, moreover, point to certain definite methods of pre- vention of infection, which have to a certain extent already been practically tested. The extensive observations recently carried out go to show that all the mosquitoes which act as hosts of the parasite belong to the genus anopheles] of these there are a large number of species, and in at least eight or nine the parasite has been found. Some of these anopheles occur in England, especially in regions where malaria formerly prevailed. The opportunity for infection from cases of malaria returning from the tropics to this country thus exists, and such infection has occurred. The breeding-places of the insects are chiefly in stagnant pools and other collections of standing water, and accordingly the removal, where practicable, by drainage of such collections in the vicinity of centres of population, and the killing of the larvae by petroleum sprinkled on the water, have constituted one of the most important measures. This procedure has been carried out in various places with marked success. Another measure is the protection against mosquito bites by netting, it being fortunately the habit of the anopheles to rarely become active before sundown. The experiments of Sambon and Low in the Campagna proved that individuals using these means of protection may live in a highly malarial district without becoming infected. The administration of quinine to persons living in highly malarial regions, in order to prevent infection, has also been recommended and carried out. In the tropics the natives in large proportion suffer from malarial infection, and one would accordingly expect that infection of the mosquitoes in the neighbourhood of native settlements will be common. This has been found to be actually the case, and it has accordingly been suggested that the dwellings of whites THE PATHOLOGY OF MALARIA 533 should as far as possible be at some distance from the native centres of population. So far as is known none of the lower animals have been found to take the place of man as intermediate host to the parasite of malaria, but the possibility of such being the case cannot be as yet definitely excluded. On the death of infected mosquitoes the exotospores or sporozoites will become set free, and therefore theoretically there is a possibility that they may enter the human subject by inhalation or by some other means. We have no facts, however, to show that this really occurs, and the evidence already obtained establishes the bites of mosquitoes as the most important if not the only mode of infection. It may also be mentioned as a scientific fact of some interest, though not bearing on the natural modes of infection, that the disease can also be communicated from one person to another by injecting the blood containing the pa.rasites. Several experi- ments of this kind have been performed (usually about J to 1 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 from seven to fourteen days, after which the fever occurs; the same type of fever is re- produced as was present in the patient from whom the blood was taken. The Pathology of Malaria. — While much work has been done on the malarial parasite, relatively less attention has been directed to the processes by which it produces its pathogenic effects. It may be said that the organisms are not always equally prevalent in the circulating blood, and probably at certain stages tend to be confined in the solid organs ; thus they may be scanty at the height of the paroxysm. Some of the pathogenic effects are probably associated with particular stages in the life cycle. Thus the pyrexia occurs when the stage of sporulation is actively in progress. No opinion can be stated, however, as to the cause of the fever, — whether it is due to a toxic process or to general disturbance of metabolism. We can better explain the anaemia which is so pronounced in cases where the disease is of long standing, and which is due to the actual destruction of red blood corpuscles. The parasite in its sojourn in these cells absorbs their pigment and thus destroys their function ; this is further evidenced by the activity displayed by the red marrow in its attempts to make good the loss sustained by the blood. One of the most interesting events in malaria, and one that links it with bacterial infections, is the reaction of the colourless cells of the blood. It has been shown that during 534 MALARIAL FEVER the apyrexial stages the total number of leucocytes may be diminished but that there is always an increase of the mono- nuclear cells, these frequently numbering 20 per cent of the whole, and sometimes even outnumbering the polymorphs. This is such an important feature that in cases where the parasites themselves cannot be demonstrated in the blood, the mononuclear reaction, along with the presence of pigment in the mononuclear cells (due to phagocytosis of pigmented parasites), has been taken as evidence that the case is really one of malaria. The mononuclear reaction is specially interesting from the fact that in other protozoal diseases an activity of the same elements has been observed. The question of the possibility of immunity to malaria being developed naturally arises, and this is specially interesting in the light of the leucocytic reaction which we have seen must be looked on as an element in immunity against bacterial infection. With regard to Europeans developing immunity it is difficult to speak. In such a malaria-stricken region as the West Coast of Africa the death-rate in residents of more than four years' standing is less than in the previous years, but this may be due to the survival of the more resistant immigrants. But there can be little doubt that malaria in the negro is a much less serious condition than in the European. Koch from his observations in New Guinea attributes this to the infection of the native children leading to the development of immunity in the adult community. He found, what has been independently noted by Stephens and Christophers in West Africa, that the greater number of the children harboured malarial parasites in their blood. The wide- spread presence of parasites in children might appear to preclude the immunity of the adult being due to survival of the most resistant, but the infant mortality in these regions may be very high, and such a survival may be the real explanation. On the other hand, Koch states that while an immunity appears to exist in native adults in malarial districts, this is only true of those born in the locality ; natives coming from neighbouring non- malarial districts into the malarial region being liable to contract the disease. At present it must be held that the facts available do not enable us to determine the relative parts played by the development of artificial immunity on the one hand, and the existence of a natural immunity on the other, in apparent un- susceptibility to malaria. Our knowledge on the relationship of blackwater fever to malaria is also in an unsatisfactory condition. Blackwater fever is a condition often occurring, especially in Europeans, in tropical METHODS OF EXAMINATION 535 countries. It is characterised by pyrexia, darkly-coloured urine, — the colour being due to altered haemoglobin pigment, — delirium and collapse, frequently ending in coma and death. By some the condition has been looked on as a separate disease, by others as the terminal stage of a severe malaria. With regard to the former view no special parasite has yet been demonstrated. Stephens sums up the evidence for the second view by saying that malaria, apart from the occurrence of blackwater fever, is a relatively non-fatal disease, that in the great majority of cases there is direct or indirect evidence of the subject of the condition having suffered from repeated attacks of malaria, that while in all cases there must be an agent at work causing hadinolysis, there is evidence that in many cases there is the possibility of that agent being quinine. This last point is of great interest. It has been shown that in certain individuals the taking of this drug is sometimes followed by hsemoglobinuria. The conditions under which this occurs are unknown, and in the case of black- water patients, neither is the serum haemolytic for normal corpuscles, nor do the red corpuscles seem to be specially sensitive to haemolysis by quinine, in fact, the latter do not appreciably differ from ordinary red cells. The whole subject of the pathology of the condition is thus very obscure. 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 preferred. The amoaboid 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 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 (a piece of cigarette-paper also does well), 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. 87), or by placing in 536 MALARIAL FEVER absolute alcohol for five minutes (Manson). The films thus prepared and fixed may be stained for two or three minutes in a saturated watery solution of methylene blue or in carbol-thionin- blue (p. 98) ; the solutions must be carefully filtered (especially the latter), and the films must be washed well after staining. They are then dried and mounted in balsam. In the case of thionin-blue, sharper results are obtained by dehydrating in alcohol and clearing in xylol before mounting. The best results are, however, obtained by one of the Romanowsky methods as described on p. 106. The fact that in many cases the parasites may be few in number led Ross to devise a method for making their recognition more easy by using blood films of unusual thickness. Here about as much blood as is used in a haemoglobin determination (20 c.mm.) is taken on a slide, and, being spread out only so much as to occupy the area of an ordinary cover-glass, is allowed to dry. There is then dropped on it by means of a glass rod a little of the watery eosin used in making up the Romanowsky dye (vide p. 106). This is allowed to act for about a quarter of an hour, and then very gently washed off with distilled water. The Romanowsky methylene-blue solution is then applied for a few seconds and also carefully washed off, and the preparation dried and mounted. The haemoglobin of the red corpuscles is washed out by the eosin solution, and the smaller forms of the malarial parasite stand out as round circles containing the char- acteristic chromatin dots ; and in consequence of the greater number present in an area of unit size as compared with an ordinary preparation their recognition is very easy. For the large forms of the parasite Ross has found it useful to make such a film and, haemolysing the red cells with distilled water, to examine it unstained. The presence of pigment in the para- sites enables these to be readily seen. APPENDIX D. AMCEBIC DYSENTERY. IN a previous chapter it has been pointed out that the term "dysentery" has been applied to a number of conditions of different etiology, and the relations of bacteria as causal agents have been there discussed (vide p. 346). We shall here consider that variety of tropical dysentery which is believed to be due to an amoeba, and hence often known as amoebic dysentery. Amongst the early researches on the relation of organisms to dysentery probably the most important are those of Losch, who noted the presence and described the characters of amoebae in the stools of a person suffering from the disease, and considered that they were probably the causal agents. Further observations on a more extended scale were made by Kartulis with confirma- tory results, this observer finding the same organisms also in liver abscesses associated with dysentery. Councilman and Lafleur, working in Baltimore, showed that this variety of dysentery can be distinguished from other forms, not only by the presence of amoebae but also by its pathological anatomy. The intestinal lesions, to which reference is made below, are of a grave character, mortality is relatively high, and recovery, when it occurs, is protracted on account of the extensive tissue changes. The subject was, however, complicated by the fact that a similar organism — the amoeba coli — had been previously found in the intestine in normal conditions and in other diseases than dysentery (by Cunningham and Lewis and others), and additional research confirmed these results. It may now be regarded as established that the amoeba of dysentery and the common amoeba of the colon are two distinct species. This has especially been shown by the researches of Schaudinn, who has given the terms entamoeba histolytica and entamoeba coli to the two organisms. Entamoeba histolytica as seen in the dysenteric stools occurs 537 538 AMCEBIC DYSENTERY in the form of rounded, oval, or pear-shaped cells, measuring 12-30 /x. in diameter. When at rest a somewhat clear, highly refractile ectoplasm and a granular endoplasm can be distin- guished, a feature which differentiates the organism from the eritamoeba coli. The nucleus is rounded, or oval, and is seen with difficulty ; its position is usually eccentric arid is sometimes quite at the margin of the ectoplasm. In the fresh condition, and especially when examined on a warm stage, the organism shows very active amoeboid movements. The pseudopodia, which are quickly protruded and retracted, are blunt and appear to be of tough consistence (Fig. 166), a property which Schaudinn considers of importance as enabling the organism to penetrate the mucous membrane, etc. The amoebic movements are often m Flo. 166. — Anioebse of dysentery. a and b, amoeb;e 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. of an active kind and locomotion may be fairly rapid ; not infrequently red corpuscles, bacteria, cells, etc. may be seen in the interior. The organism usually dies and undergoes disinte- gration in a comparatively short time after being removed from the body ; the stools ought therefore to be examined in as fresh a state as possible. Multiplication takes place by simple amitotic division and also by budding. The entamceba coli is an organism of about the same size. When at rest it shows no differentiation into ectoplasm and endoplasm, and the nucleus, usually situated in the centre, shows a highly refractile membrane with chromatiri masses scattered in the interior. During amoeboid movement some delicate processes of ectoplasm come into view. DISTRIBUTION OF THE AMCEB^E 539 Both organisms have now been shown to pass into a resting stage with formation of cysts, the character and mode of forma- tion of which are markedly different in the two cases. The cyst formation of the entamoeba histolytica is specially seen when the disease is in process of cure and the stools are beginning to have a less fluid character. In the earliest stage of the change the nuclear membrane becomes broader and fades into the proto- plasm, whilst the chromatin becomes dispersed through the entoplasm in the form of small chromidia. Buds then form on the surface and into these some of the chromatin passes. Around these buds concentric striation can be seen, and then a hyaline cyst wall is formed, which is highly refractile in character. The cyst then becomes separated from the rest of the cell. Several cysts which measure 2-7 /x in diameter may be formed from the same amoeba, and the remnant of the cell under- goes disintegration. These cysts, as will be shown below, repre- sent a resting stage with high powers of resistance to external agencies, and are concerned in producing infection of another subject. The cellular changes in the encysting of the entamceba coli have also been worked out by Schaudinn. They are of a somewhat complicated character, involving the formation of reduction bodies and copulation of nuclei, but the ultimate result is the formation of a fairly large cyst, which contains eight small cells. The process of cyst formation accordingly in the two organisms is of a widely different character. Cultivation. — Various attempts have been made to cultivate the amoeba of dysentery, and Kartulis considered that he obtained growth in straw infusions. Recently Lesage has announced that he has obtained cultures on plates of agar which had been washed in wTater for eight days. Both the vegetative and the cystic forms were used for inoculation. In some cases a growth of a colon bacillus was made on the agar and afterwards removed, this procedure interfering with the development of such bacilli present in the material used for inoculation. The plates were kept in the sloped position, and the inoculations were made in the lower part ; the amoeba? moved to the upper part, where they were got in pure .condition. He succeeded in obtaining cultures in seven out of thirty cases, and in some instances cultivated the organisms for more than sixty generations. The amoebae multi- plied by simple amitotic division, and in certain cases produced small cysts. These cysts, as described and figured by him, correspond in all important respects with the changes observed by Schaudinn in dysenteric stools. Distribution of the Amoebae. — As already stated, they are 540 AMCEBIC DYSENTERY usually found in large numbers in the contents of the large intestine, in tropical amoebic dysentery. They also, however, penetrate into the tissues, where they appear to exert a well- marked action. In this disease the lesions are chiefly in the large intestine, especially in the rectum and at the flexures, though they may also be present in the lower part of the ileum. At first there are seen local swellings on the mucous surface, chiefly clue to a sort of inflammatory gelatinous oedema with little leucocytic infiltration; soon, however, the mucous membrane becomes partially ulcerated, more or less extensive necrosis of the subjacent tissues oc- curs, and gangrenous sloughs result. The ulcers thus come to have irregular and overhanging margins, and the excavation below is often of wider extent than the aperture in the mucous membrane. The amoebae are found in the mucous membrane when ulcers are being formed, but their most character- istic site is beyond the ulcerated area, where they may be seen penetrating FIG. 167. — Section of wall of liver abscess, deeply into the submucous, showing an amoeba of spherical form with and even into the muscular vacuolated protoplasm. From a case T .-, ... published by Surgeon-Major D. G. C0ats' In these Positions Marshall, x 1000. they may be unattended by any other organisms, and the tissues around them show oedematous swelling and more or less necrotic change without much accompanying cellular reaction, beyond a certain amount of swelling and proliferation of the connective-tissue cells. This action of the amoebae on the tissues explains the character of the ulcers as just de- scribed. These lesions are considered to be characteristic of amoebic dysentery. As a complication of this form of dysentery, liver abscesses are of comparatively common occurrence. They are usually single and of large size, sometimes there are more than one, and occasionally numerous small ones may be present. The contents are usually a thick pinkish fluid of somewhat slimy consistence and are largely constituted by necrosed and liquefied tissue with EXPERIMENTAL INOCULATION 541 admixture of blood in varying amount. Microscopic examination shows chiefly necrosed and granular cells and debris resulting from their disintegration, whereas ordinary pus corpuscles are scanty or may be practically absent. In such abscesses associated with dysentery the amoebae are usually to be found, and not infrequently are the only organisms present, no cultures of bacteria being obtainable by the ordinary methods (Fig. 167). They are most numerous at the spreading margin, and this probably explains a fact pointed out by Manson, that examination of the contents first removed may give a negative result, while they may be detected in the discharge a day 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. Kartulis records two cases of brain abscess occurring secondarily to dysentery in which numerous amoebae were present. Experimental Inoculation. — The anatomical changes in dysentery, as above described, gives strong presumptive evidence as to the causal relationship of the amoebae, and practically con- clusive evidence is afforded by animal experiments. Dysentery occurs occasionally in animals, but it is of rare occurrence. The disease sometimes results in the dog by experimental inoculation with dysenteric material. Kartulis, for example^ records two cases, in one of which liver abscess was present. Cats are, however, found to be more susceptible, especially young animals. Dysenteric changes have been produced in this animal by Kartulis, Kruse and Pasquale, and others. The method generally adopted is the introduction of a small quantity of mucus from a dysenteric case into the rectum. The resulting disease is of an acute character, and sometimes leads to a fatal result. The changes in the large intestine resemble those found in the human disease, and microscopic examination shows the amoebae penetrating the wall of the bowel in the characteristic manner. Kruse and Pasquale obtained corresponding results when the material from a liver abscess, containing amoebae without any other organisms, was injected. Quincke and Koos obtained no effects when the amoebae were administered by the mouth, but they obtained a fatal result in two out of four cases when the cyst-like forms were given. They also found that the cysts, unlike the amoebae, were still present even after the material had been kept for two or three weeks. Extremely important confirmatory evidence with regard to infection by the cysts has 542 AMCEBIC DYSENTERY been supplied by experiments of Schaudinn. Dysenteric material was obtained from China, and portions of it which were found to contain the cysts were thoroughly dried. Some of this material was given with food to cats by the mouth, and typical dysentery resulted, the amoebae being found in the stools. No results follow when the material ingested merely contains the vegetative form of the organism, as it is readily destroyed in the contents of the stomach. Investigations with regard to entamoeba coli show that it is a harmless organism and that it is frequently present in the intestines of healthy individuals. Schaudinn found that in East Prussia as many as 50 per cent of the population were infected with it. The administration of the amoebae, or of the cysts by the methods mentioned above, produces no result in animals. It has, however, been shown that when the eight-celled cysts are swallowed by persons who are free from the parasite, the entamceba coli appears in the large intestine in a comparatively short period of time. It accordingly appears that in the case of both organisms it is the cysts alone which give rise to infection. From the above facts, all of which have received ample confirmation, there can be no doubt that the amoeba described is the cause of the form of dysentery with which it is associated. We are still ignorant whether the organism has any life history outside the body, but it has been shown that the cysts have high powers of endurance and almost certainly form the means of infection when they are swallowed in drinking-water or in food. It is also of importance to note that the serum of patients suffering from amoebic dysentery gives no agglutinating reaction with Shiga's bacillus of dysentery (vide p. 348). Methods of Examination. — The faeces in a case of suspected dysentery ought to be examined microscopically as soon as possible after being passed, as the amoebae disappear rapidly, especially when the reaction becomes acid. A drop is placed on a slide without the addition of any reagent, a cover-glass is placed over it but not pressed down, and the preparation is examined in the ordinary way or on a hot stage, preferably by the latter method, as the movements of the amoebae become more active, and it is difficult to recognise them when they are at rest. Hanging-drop preparations may also be made by the methods described. Dried films are not suitable, as in the preparation of these the amoebae become broken down ; but wet films may be fixed with corrosive sublimate or other fixative (vide p. 88). In sections of tissue the amoebae may be stained by methylene-blue, by safranin, by haematoxylin and eosin, etc. Benda's method of METHODS OF EXAMINATION 543 staining with safranin and light-green is also a very suitable one. Sections are stained for several hours in a saturated solution of safranin in aniline oil water (p. 98), they are then washed in water and decolorised in a \ per cent solution of light-green in alcohol till most of the safranin is discharged, the nuclei, however, remaining deeply stained. In this method the nuclei of the amoebae are coloured red (like these of the tissue cells), the protoplasm being of a purplish tint. APPENDIX E. TRYPANOSOMIASIS— KALA-AZAR—PIROPLASMOSIS. THE PATHOGENIC TRYPANOSOMES. THE trypanosomata are protozoal organisms belonging to the sub-class Flagellata, and during the last decade several members of the genus have come to be recognised as living in the blood and tissues in various animals and as causing important disease conditions. As long ago as 1878 the Trypanosoma Leivisi was observed infesting the blood of rats, and it has been found to be sometimes capable of causing death. Other diseases in which similar organisms have been found are Surra, which occurs in cattle, horses, and camels in India, and which is associated with the Tr. Evansi ; Dourine, a condition affecting horses in especially the Mediterranean littoral (Tr. equiperdum or Rougeti) ; Mai de Caderas, a disease of South American horses (Tr. equinum or Elmassiani) ; Tse-tse Fly Disease or Nagana, affecting horses and herbivora in South Africa (Tr. Brucei) ; trypanosomiasis of African cattle (Tr. Theileri) ; and — most important from the human standpoint — the trypanosomiasis and sleeping sickness of West and Central Africa associated with the Tr. gambiense and Tr. ugandense, which are now believed to be the same organism. These diseases present many general resemblances to one another. They tend to be characterised by wasting, cachexia, anaemia, fever often of an intermittent type and irregular oedemas, and often lead to a fatal result. In many cases the infective agent is conveyed from a diseased to a healthy animal by the agency of blood-sucking insects. General Morphology of the Trypanosomata. — If a drop of blood containing trypanosomes be examined, the organism will be seen to be a fusiform mass of protoplasm which at one end passes into a pointed flagellum. In the living condition the trypanosome is usually actively motile by an undulatory move- 544 THE PATHOGENIC TRYPANOSOMES 545 ment of its protoplasm and a lashing of the flagellum. The size varies, but those mentioned above are about 30 //. long and about 1 '5 to 3 //, broad. From the fact that in progression the flagellum is in front, the flagellated end is denominated the anterior end of the organism. It is stated that the method of examining the fresh blood merely allowed to spread itself out in a fairly large drop beneath a cover-glass is more likely to reveal the presence of trypanosomes, if these are present in small numbers, than is the examination of stained specimens; but the minuter structure of the organisms can best be studied in dried preparations stained by Romanowsky dyes such as those of Leishman or Giemsa. For staining trypanosomata (or the Leishman-Donovan bodies) in sections so as to bring out the chromatin structures, Leishman recom- mends the following method: — Sections of 5 //. thickness are made and carefully fixed on slides. The paraffin is very thoroughly removed by melting it before applying the first xylol, and then washing with alternate baths of alcohol and xylol three or four times. The last alcohol is thoroughly washed off by distilled water, and the excess of water is removed with cigarette paper. A drop of fresh blood serum is then placed on the preparation and allowed to soak in for five minutes. The excess is removed by blotting, and the remainder is allowed to dry on the section, which is now treated with a mixture of two parts of Leishman's stain and three of distilled water, and placed in a Petri dish for 1 to 1^ hours. The preparation is very deeply stained, the nuclei being almost black and decolorisation and differentiation are effected by alternately applying the acetic acid and caustic soda solutions (commencing with the acid) used in the application of the stain to ordinary histological sections (v. p. 106), the effects being carefully watched with a low power. The essential part of the method is the application of the blood serum, though what effect this has is not known ; Leishman suggests that it restores the normal alkalinity of the tissue. In preparations stained by the above methods the protoplasm of trypanosomata stains blue, and in some species some parts are more intensely coloured than others. Sometimes it contains violet-coloured granules (chromatin granules), and occasionally there appears in it slight longitudinal striation. Two bodies are always present in the protoplasm. Usually near the middle there is an oval granular body staining purple, — the nucleus or macro- nucleus, — and towards the posterior end is a minute intensely stained purple granule known as the micronucleus or centrosome (that this body represents the centrosome is strongly held by Laveran from the analogy of appearances in certain spermatozoa which closely resemble trypanosomes in structure). This micro- nucleus is often surrounded by an unstained halo, and in its neighbourhood, in certain species, a vacuole has been described 35 546 TRYPANOSOMIASIS as existing ; this has been considered by some to be analogous to the contractile vacuole present in many protozoa, and its shape and position have been made the basis of specific dis- tinctions ; Laveran, however, thinks it is an artefact. From the micronucleus or from its neighbourhood there arises an important structure in the trypanosome, — the undulatory membrane. This is of varying breadth, has a sharp undulating free margin, and surmounts the protoplasm of the organism like a cock's comb; it narrows towards the anterior end where it passes into the flagellum. Motion is chiefly effected by the undulations of this membrane and of the flagellum. The latter is continuous with the protoplasm of the body of the organism ; it stains uniformly like it, except the free edge which has the reddish hue of the chromatin. In different species of trypanosomes variations occur in shape, in length, in breadth, in the position of the micronucleus (and therefore in the length of the undulating membrane), in the breadth ,of the membrane, in the length of the free part of the flagellum, in the shape of the posterior end, which is sometimes blunt, sometimes sharp, and in the presence or absence of free chromatin granules in the protoplasm. Multiplication in the body fluids ordinarily occurs by longitudinal, amitotic division (see Fig. 168). First of all the micronucleus divides, sometimes transversely, sometimes longitud- inally, then the nucleus and undulating membrane, and lastly the protoplasm. In some species the root of the flagellum only divides so that in the young trypanosomes the flagellum is short and subsequently increases in length (Tr. Lewisi) ; usually the whole flagellum takes part in the general splitting of the organism. In the cases of several of the trypanosomata it has been found possible to cultivate them outside the body, the chief work here having been done by Novy and MacNeal, who have succeeded with the Tr. Lewisi, Tr. Evansi, and Tr. Brucei. The most suitable medium is made as follows : — 125 grammes rabbit or ox flesh is treated with 1000 c.c. distilled water, as in making ordinary bouillon, and there are added to the meat extract 20 gr. Witte's peptone, 5 gr. sodium chloride, 20 gr. agar, and 10 c.c. normal sodium carbonate. The medium is placed in tubes and sterilised in the autoclave at 110° C. for thirty minutes. It is cooled to 50° C. , and there is added to the medium in each tube twice its volume of defibrinated rabbit blood, which has been prepared with all aseptic precautions ; the tubes are allowed to set in the inclined position. In inoculating such tubes they are placed in an upright position for a few minutes and then the infective material is introduced. THE PATHOGENIC TRYPANOSOMES 547 Multiplication goes on readily on such, a medium, the organisms dividing longitudinally as in the tissues. Sometimes very small forms result, and often these are found in rosettes which are formed by a number of individuals arranging them- selves in a circle with the flagella directed towards the centre of the agglomeration. By repeated sub-cultures several of the trypanosomata named have been kept alive for more than a year, and when re-introduced into appropriate hosts have been found not to have lost their infective properties. Within recent years considerable attention has been directed to the question of whether in the trypanosomes a sexual cycle occurs. It cannot be said that the existence of such has been definitely established, and we shall merely give a short account of certain views which have been advanced. The starting point lies in the slight differences in form which have been observed in the organisms in the body fluids of the vertebrate hosts. Such differences have been described in Tr. Lewisi and Tr. Brucei by Prowazek, and in Tr. ugandense by Minchin, and have been made the basis of a classification into three types which are looked on as representing male, female, and indifferent individuals. The male type is rather slender both in body and in nucleus, the free part of the flagellum is longer than the body and the protoplasm is free from granules ; the female is broader, its nucleus is larger and rounder, the undulating membrane narrower, the free part of the flagellum is shorter than the body, and the protoplasm contains many chromatin granules, which are looked upon as reserve food material. The indifferent individuals present intermediate characters. All multiply by fission as described, and the indifferent individuals can on occasion become differentiated into male or female forms. The females are the most hardy, and next come the indifferent individuals. If all but the females die out, these can undergo parthenogenesis, and representatives of all three types can be again reproduced. The sexual cycle is represented as occurring in the invertebrate host. In Tr. Lewisi, according to Prowazek, this is found in the rat louse, hcematopinus spinulosus. When this insect sucks the blood of an infected rat, copulation occurs by the male trypanosome entering the female near the micronucleus and the various parts of the two individuals becoming fused. A non-flagellated ookinete results, which, passing through a spindle-shaped gregarine-like stage, can develop into a trypanosome in the stomach of the louse. A resting-stage in an immature trypanosome -like form is described as occurring between or attached to the intestinal 548 TRYPANOSOMIASIS epithelium, and the parasite is supposed to reach the body cavity, and ultimately the pharynx of the insect, and thus to find the opportunity for passing into the body of a fresh host. A still further development of the views held as to the life-history of the trypanosomes is found in the work of Schaudinn, who investigated . the trypanusoma noctuce found in the owl (athenc noctua), and which is carried from bird to bird by the common mosquito (culex pipiens). In the blood of the owl is a halteridium haemamoeba showing pigmented male and female forms, closely corresponding to those observed by Macallum in the crow. These, according to Schaudinn, on reaching the mosquito's stomach undergo ordinary changes — the microgametocyte develops microgametes, one of which fertilises the macrogamete. An oval motile ookinete results, and in the formation of its nucleus from the male and female,' elements a reduction of chromosomes takes place, while the superfluous nuclear structures along with the pigment are cast out of the cell. In these ookinetes a differentiation into male, female, and indifferent forms can be recognised, but the important new departure is that each can go on to develop into a trypanosome. In the indifferent ookinete a portion of the new nucleus breaks off by a sort of hcteropolar division, and the broken-otf part becomes the micronucleus or blepharo- plast of the trypanosome and gives origin to the undulatory membrane. Longitudinal division of these forms in the mosquito's intestine may occur, and here it may be said that Schaudinn differs from other observers in holding that on division the membrane does not split, but that one of the individuals gets its membrane by this being laid down along the root of that already in existence. Further, a resting-stage of the trypanosome may occur, in which it becomes attached to the intestinal epithelium, and losing more or less its flagellate form, may resemble a gregarine. The female ookinete is plumper and contains more chromatin granules in its protoplasm than the male and indifferent forms, gives rise to a trypanosome after a more complicated division of its nucleus, is less motile, does not reproduce itself by longitudinal fission, soon attaches itself to the intestinal epithelium, is in virtue of the reserve material in its protoplasm much more resistant than the male or indifferent forms ; when these die out, as they do when, for instance, the insect is starved, it reproduces all three forms by a process of parthenogenesis. In the female ookinete the smaller nucleus which goes to form the blepharoplast of the indifferent trypanosome divides into eight small nuclei, all of which perish, and the blepharoplast and membrane are formed by a fresh division in the large nucleus remaining. In the male ookinete, which differs from the female in the clearness of its protoplasm, a similar heteropolar mitosis takes place, and again eight small nuclei are produced. These evidently represent the essentially male element, for they persist, and each, appropriating to itself a portion of the cellular protoplasm, detaches itself so that eight small trypanosomes are budded off from the ookinete. This male ookinete Schaudinn holds to be homologous with the microgametocyte occurring in the blood of the .owl, and the small trypanosomes are similarly homologous with the microgametes formed when the blood of the host reaches the stomach of the mosquito. These small trypanosomes and the male trypano- somes readily die, probably because, by a reduction process in their genesis, the assimilative powers of the larger nucleus have been diminished. In degenerating they often are found in the intestinal THE PATHOGENIC TRYPANOSOMES 549 tract of the mosquito arranged in rosettes, with sometimes the anterior ends, sometimes the posterior ends, directed towards the centre of the rosette. The next stage of development takes place when the parasites by the mosquito's bite reach the blood of the vertebrate host, and it is in connection with this stage that Schaudinn's observations are very far-reaching. The indifferent forms by this time, by repeated longi- tudinal division, have become small. In the owl they attach them- selves to red blood corpuscles, which they penetrate, and, assuming a halteridium form, grow in size for twenty-four hours ; they then leave the cells, elongate, again assume the flagellate form, move freely in the blood till the next night, when they again enter fresh cells. This cycle is repeated for six nigiits, the organism gaining in size with each sojourn in a corpuscle. When the full size is thus attained, repeated longi- tudinal division occurs, and when the smallest forms are again reached the intra- cellular/ cycle recommences. The largest female forms prob- ably cannot pass through the mosquito's proboscis, but when small forms reach the owl they enter the red corpuscles. They assimilate food material, and appear not to migrate so frequently as the indifferent forms. They lose their capacity of assuming the free trypanosoma form, and ultimately their capacity of migration, so that they are found lying surrounded by the remains of the last cell they entered. The male forms when they reach the blood of the host rapidly die, and the microgarnetocytes are always recruited from the indifferent forms, or from parthenogenesis of the female forms. It may be said that according to Schaudinn the trypanosomes gain access' to the tissues of the mosquito by perforating the intestinal wall, and, passing through the body till they reach the wall of the pharynx, they burst through this, and are in a position to be ejected when next the insect bites. Such are the views put forward by this observer on the cycle of life-history of the Tr. noctuse. It will be recognised that the essential point is the occurrence, both in the vertebrate and invertebrate host, of halteridium stages alternating with those in which the trypanosome form is assumed. It is evident that if this were substantiated important effects would follow in our views as to the morphology not only of the trypanosome group, but also of that to which the malarial parasites belong. Certain criticisms of these results have been made, especially by Novy and MacNeal, who sought confirmatory evidence by means of their culture method. These observers are of opinion that the appearances described by Schaudinn were due to his dealing with a mixed infection of the owls by trypanosomes on the one hand and haemamoebae of the halteridium type on the other. They examined a very large number of different species of birds, and established the fact that infection with halteridium parasites on the one hand and with trypanosomes on the other is extremely common, and further, that in the blood of the same bird both halteridium and trypanosome infection could be observed. The bird trypanosomes could be readily cultivated, and it was observed that no cultures were obtainable from birds in which halteridia were alone found, and further, that when a trypanosome isolated from a case where both forms of parasite had been seen was injected into a fresh 550 TRYPANOSOMIASIS bird, only trypanosome forms were found to develop in its body. These results are, of course, not quite conclusive, as the halteridium stage might only follow on the sexual portion of the cycle. In this connection, however, the fact may be mentioned that both with Tr. Brucei and Tr. ugandense attempts to produce disease by means of the contents of the stomachs of infected insects have failed. Bruce was of opinion that infection took place in nagana by means of the insect carrying the trypanosomes in the tube of its proboscis, where he observed them to be freely motile for forty-eight hour_s after the insect had fed, and with regard to the sleeping sickness organism Minchin held a similar opinion, and showed that if a glossina bit an infected animal, and then in succes- sion two healthy animals, only the first of the latter would contract the disease — the proboscis being apparently cleaned by the biting process. Reference may here be made to the views put forward by Schaudinn regarding the relationship of certain spirilla to the trypanosomes. In the athene noctua, besides the Tr. noctuae already referred to, there is a protozoal parasite infesting the leucocytes known as spirillum Ziemanni or leucocytozoon Ziemanni, whose invertebrate host is also the culex pipiens. Ziemann had described the male and female forms in the owl, and microgametes had been observed forming from the micro- gametocytes. Schaudinn observed the formation of an ookinete in the mosquito ; in certain cases this ookinete elongates, and the vermicule rolls itself up into a ball with great proliferation of the nucleus. Each little nucleus attaches to itself a portion of the protoplasm, and becoming a miniature trypanosome, swarms off and becomes free. These minute trypanosomes elongate and develop into typical spirilla by rolling their ribbon- shaped bodies spirally along their longitudinal axes, the individuals possessing male, female, or indifferent characters, just as in Tr. noctuae. - These 'spirilla multiply by longitudinal division, and often after fission the two individuals remain attached to each other by their posterior ends, and in this way there is made possible what is often seen in spirilla, namely, a capacity to move in either direction. The spirilla often divide so frequently that ultimately the individuals become invisible by means of the microscope and can only be seen when lying in clumps. In this stage Schaudinn thinks the organism would be able to pass through a Chamberland filter, and this may be a very important observation, as throwing light on the etiology of certain diseases, such as yellow fever, in which no visible parasites have been found. Schaudinn's views on the trypanosomal characteristics of Sp. Ziemanni have raised important questions regarding the morphology of other similar forms which have been long familiar, such as Sp. Obermeieri, and also of the Spirochsete pallida which Schaudinn himself .discovered. TRYPANOSOMA LEWISI 551 It is as yet too soon to express any opinion on the ultimate effect of these views. According to Schaudinn a trypanosomal spirillum consists of a central thread which represents the posterior nucleus of the trypanosome ; round this thread the undulating membrane is spirally wound, and the principal nucleus is represented by minute chromatin dots sometimes seen in the course of the spiral. Whether all spirilla have this structure must be left for future investigation to determine. Difficulties arise with regard to the significance of an undulatory membrane as a spirillary characteristic and also with regard to the terminal flagellum which Schaudinn himself found in Spirochaete pallida, and which he had previously thought did not occur in protozoal spirilla. It is possible that two groups of organisms have hitherto been classed together under the name spirillum, and that one of these may still have to be placed with the bacteria. Trypanosoma Lewisi. — In 1878 Lewis described in rats in India the occurrence of the parasite which now goes by his name, and since that time this trypanosome has been found to be very common in the blood of rats all over the world, though the percentage of animals affected varies in different localities. The condition is of great interest, as, though the infection runs a very definite course, it is very rarely fatal ; in fact, many observers have been unable to' produce death by infecting even large series of animals. There is, however, little doubt that a fatal issue does occur sometimes in young individuals, especially when these are infected with strains of the organism imported from other localities. The trypanosome, which is actively motile, is of the usual length but is somewhat narrow, and its protoplasm does not contain any granules. It multiplies by fission, of which Laveran describes two varieties. In one, the organism splits longitudinally and gives rise to smaller individuals than the parent. In the other, the trypanosome loses its ordinary shape and becomes more oval; nuclear division, which is often multiple, then takes place, and on subsequent division of the protoplasm a number of small flagellate organisms result ; these last may attain the full form and size before dividing again, or they may divide when still small. When a rat is infected by injection into the peritoneum active multi- plication goes on in the cavity for a few days and then comes to an end. Very soon after infection the organisms begin to appear in the blood and there rapid multiplication occurs, the extent of which is sometimes so great that the trypanosomes may seem to equal the red blood corpuscles in number. The animal usually shows no symptoms of illness. The infection goes on for about two months, and then the organisms gradually dis- appear from the blood. In the great majority of cases the rat is now immune against fresh infection. If trypanosomes be 552 TRYPANOSOMIASIS introduced into its peritoneum they are, according to Laveran, taken up by mononucleate phagocytes and destroyed. The serum of a rat which has been infected shows agglutinating capacities towards the trypanosomes, causing them to agglomer- ate in rosettes in which the flagella are directed outwards, and the serum of immune rats has a certain degree of protective action if injected along with the organism into a susceptible animal. As has already been noted, this trypanosome has been cultivated on artificial media, on which it multiplies freely, large numbers of small forms being often produced. These when injected into rats give rise to the usual infection, but not so rapidly as when blood from an infected animal is used. The organism multiplies at the body temperature, but a lower temperature is preferable, and at 20° C. Novy and MacNeal succeeded in carrying a growth through many sub-cultures. This trypanosome is very resistant to cooling, and has been exposed for fifteen minutes to the temperature of liquid air ( - 191° C.) without being killed. The means by which the rat becomes infected naturally is not known, but probably this comes about by the bite of a flea or louse. Nagana or Tse-tse Fly Disease. — This is a disease affecting under natural conditions chiefly horses, cattle, and dogs ; it is prevalent especially in certain regions of South Africa, though it probably may occur elsewhere. In the horse the chief symptoms are the following : — The animal is observed to be out of condition, its coat stares, it has a watery discharge from the eyes and nose, and the temperature is elevated ; swellings appear on the under surface of the abdomen and in the legs, it gradually becomes extremely emaciated and anaemic, and dies after an illness of from two or three weeks to two or three months. In other animals the symptoms are of the same order, though the duration of the disease varies much ; thus in the dog the illness does not last more than one or two weeks, while in cattle it may continue for six months. It is doubtful whether a domestic animal attacked by the disease ever recovers. The popular idea regarding the etiology of the disease was that it was contracted by animals passing through certain rather restricted and sharply defined areas or belts characterised by heat and damp, usually lying beside rivers, and always infested by the tse-tse fly (glossina morsitans), to the bite of which the disease was attributed ; in this connection it is important to note that though man is frequently bitten by the tse-tse fly he does not contract nagana. Modern know- ledge on the subject dates from the discovery made by Bruce NAGANA OR TSE-TSE FLY DISEASE 553 in 1894 that the blood of animals suffering from nagana swarmed with a trypanosome now known as the Tr. Brucei, and in 1895 he was instructed by the Governor of Natal to undertake the investigation which led him to work out the true etiology of the disease. It may be said that this research forms the starting-point of the important work done during the last decade with regard to infections by trypanosomes. In his earlier work Bruce found that the parasite was present in the blood of every animal suffering from nagana and absent from the blood of healthy animals in the affected districts; further, that the fever which marks the onset of the disease was accompanied by the appearance of the trypanosome in the blood; and finally, that the transference of the smallest quantity of blood from an affected to a healthy animal origin- ated the disease. He then proceeded to investigate the part played by the tse-tse fly in the condition. He found that if flies taken from the fly belt were transported to a place where nagana did not occur, kept for a few days, and then allowed to bite susceptible animals, the latter did not contract the disease — this result showing that it was not, as had been supposed by some, a poison natural to the insect which was the pathogenic agent. But if such a fly was allowed to bite a dog suffering from the disease and then to bite a healthy dog, the latter contracted the malady and abundant trypanosomes were found in its blood. Again, threads dipped in the blood of an infected animal and allowed to dry caused the disease in healthy animals up to but rarely beyond 24 hours after being dried ; if, however, the blood were kept moist, then it retained its infectiveness up to between 4 and 7 days; up to 46 hours living trypano- somes could be seen in the tube of the fly's proboscis. This corresponds roughly with what was found regarding the limits of the infectiveness of a fly, in that 24 hours after it has been fed on an infected animal its bite is usually innocuous. Further, Bruce showed that infection did not occur by any food or water partaken of by an animal while going through a fly belt, for he took horses through such a region without allowing them to eat or drink, and found that they still contracted the infection, if during their few hours' journey through the belt they had been bitten by the tse-tse fly. Finally, he showed that if flies were taken from an infected area to a healthy one a few miles off and allowed at once to bite infected animals, the latter contracted nagana. By those experiments it was thus determined that nagana could be transmitted by the blood of the infected animal, that 554 TRYPANOSOMIASIS is, without the agency of the fly ; that the latter had no inherent power to produce the disease ; that it could, however, by successively biting infected and healthy animals transmit the disease to the latter ; and that specimens of the insect caught in infected areas harboured the parasite and were thus infective. The question remained as to how the flies might become infected in nature. It had been observed that in districts where the FIG. 168. — Trypanosoraa Brucei from blood of infected rat. Note hi two of the organisms commencing division of micronucleus and undulating membrane, x 1000. tse-tse fly lived the prevalence of the disease in imported animals was related to the presence in the locality of wild herbivora. Bruce now found that, if considerable amounts of the blood of the latter were taken to another locality and' injected into dogs, these in a proportion of cases contracted nagana, and from this he deduced that the wild animals harboured the parasites in small numbers in their blood and thus kept up the possibility of infection. A further and as yet unexplained fact was that other blood -sucking flies besides the tse-tse NAGANA OR TSE-TSE FLY DISEASE 555 appeared incapable of acting as carriers of infection. Brace's work as a whole pointed to the trypanosome as the cause of nagana, and this has since been finally established by the origination of the disease by artificial cultures of the organism. The Tr. Brucei (Fig. 168), according to Laveran, measures in the horse from 28-33 /x long and from 1*5 to 2*5 /x broad ; in the rat and dog it is somewhat shorter. It is motile, but its activity is less than that of Tr. Lewisi. When stained it presents the usual appearances ; its posterior end is usually blunt, and the body often contains granules in the anterior portion of its protoplasm. It divides longitudinally, and according to Bradford and Plimmer a form of longitudinal conjugation occurs in the blood. According to the same observers, it can be kept alive for 5-6 days in blood outside the body. It is less resistant to the action of cold than Tr. Lewisi, perishing in a few days at 5-7° C., but like the other organism it can withstand short exposures to temperatures down to -191° C. ; it is quickly killed at 44-45° C. Novy and MacNeal succeeded in cultivating this trypanosome also, though here it was very difficult to obtain a first growth from the blood on their blood-agar medium ; once started, however, it was kept alive through many sub-cultures, the optimum temperature of growth being 25° C., and it was from these sub-cultures that the infection was obtained which definitely proved the organism to be the cause of the disease. In cultures, as with Tr. Lewisi, short forms occur, and there is sometimes a rosette formation with the flagella directed outwards ; agglutina- tion phenomena are also observable in defibrinated blood. Under unfavourable conditions involution forms occur, the organism dividing frequently to form round flagellated individuals. Nearly all laboratory animals are susceptible to infection, and the duration of the illness corresponds to what has been observed in the natural infection of these animals. The rat has been largely used for experiment and usually succumbs in about ten days, there being very few symptoms up till a few hours before death. A very important fact has been observed with regard to this animal, namely, that individuals which have gone through infection with Tr. Lewisi and which are immune are still susceptible to the Tr. Brucei; from this it has been deduced that the two organisms are to be looked on as -distinct species. Trypanosoma of Sleeping Sickness. — Since the year 1800 the disease called sleeping sickness, sleeping dropsy, or negro 556 TRYPANOSOMTASIS lethargy has been recognised as prevailing on the West Coast of Africa from the Senegal to Lagos and in the parts lying behind the coast between these regions. It has also been found to be rife from Cameroon to Angola and in the Congo valley, and to a less extent up the Niger and its tributaries. In 1901 it began to appear in the Uganda Protectorate, and it is in that region that the investigations have been carried on which have led to a knowledge of its cause ; here it has wrought very serious havoc amongst the native population. It is characterised in the early stages by a change in disposition leading to moroseness, apathy, disinclination for work or exertion, and slowness of speech and gait. There may be headache, indefinite pains about the body, the evening temperature may be elevated several degrees, the pulse tends to be soft and rapid, and in a very large number of cases the superficial glands of the body are enlarged. In a rapid case the lethargy becomes more pronounced ; fine tremors, especially of the tongue and arms, develop; pro- gressive emaciation occurs ; blood changes appear, consisting of a progressive diminution of the red cells and of the haemoglobin, and of a lymphocytosis in which the percentage of both the large and small mononuclear cells is increased, so that the former may constitute from 20 to 30 and the latter from 30 to 40 per cent of all the white cells present. As the disease progresses the drowsiness increases till it deepens into a coma from which the individual cannot be roused. Often during the disease there occur irregular cedematous patches on the skin and sometimes erythematous eruptions, and effusions into the serous cavities. Not every case runs a progressively advancing course. Some- times along with enlargement of glands the chief early feature is the occurrence from time to time of attacks of fever which may be mistaken for malaria, and from these apparently com- plete recovery may take place ; recurrence, however, follows as a rule, and ultimately the typical terminal phenomena may commence. Such cases may go on for years, and it is probable that many patients die of pneumonia without exhibiting typical manifestations of the malady from which they really suffer. The disease is an extremely fatal condition, and probably no case where the actual lethargy is developed ever recovers. On considering the disease from the standpoint of patho- logical anatomy there is little to be said. As Mott described, the most striking feature is the presence of a chronic meningo- encephalitis and meningo-myelitis. The pia-arachnoid is some- times opaque and slightly thickened and may be adherent to the brain, and its vessels usually show some congestion. TKYPANOSOMA OF SLEEPING SICKNESS 557 The sub-arachnoid fluid is sometimes in excess and occasionally may even be purulent. The membranes of the spinal cord show similar changes. The chief other feature is the presence of enlarged lymphatic glands in the body, but otherwise there is nothing special to note. With regard to the microscopic changes the chief feature, according to Mott, is a proliferation and overgrowth of the neuroglia cells, especially of those which FIG. 169. — Trypanosoma gambiense from blood of guinea-pig. x 1000. are related to the sub-arachnoid space and the perivascular lymph spaces, with accumulation and probably proliferation of lympho- cytes in the meshwork. He further points out that the changes in the lymph glands are of similar nature and resemble the in- filtration of the perivascular lymphatics of the central nervous system. These changes are specially significant in view of the lymph ocytosis present in the blood, which has already been noted, and which so often occurs in protozoal infections. In the nervous structures there is comparatively little change, there being merely, according to Mott, some atrophy of the dendrons 558 TRYPANOSOMIASIS of the nerve cells, a diminution of Nissl's granules, and an excentricity of the nucleus. Trypanosoma gambiense. — Before going further we must refer to the observation of a trypanosome in the blood of persons not evidently suffering from sleeping sickness. The first case of this was recorded by Button in 1901, the patient being a European then living at Bathurst on the Gambia. The progress of the disease was here very slow, and was characterised by general wasting and weakness, irregular rises of temperature, local oedemas, congested areas of the skin, enlargement of spleen, and increased frequency of pulse and respiration ; death occurred a year after the case came under observation after an access of fever, and a striking fact was the absence of any gross causal lesion. During the time the patient was under observation trypanosomes were repeatedly demonstrated in the peripheral blood, and they also developed in the bodies of monkeys and white rats inoculated with the blood. Pursuing further in- quiries, Dutton and Todd demonstrated similar parasites in other Europeans and in several natives in the Gambia region, whilst about the same time Manson reported a case of the same kind in the wife of a missionary on the Congo. It thus came to be recognised that in man there occurred a disease having characters somewhat resembling nagana and in which trypanosomes could be demonstrated in the blood, and this was usually referred to as human trypanosomiasis, or trypanosoma fever, — the trypanosome being named the Tr. gambiense. Relation of Trypanosomes to Sleeping Sichiess. — Several views as to the etiology of this disease had been advanced. A Portuguese Commission in 1902 described a diplococcus tending to grow in chains which they isolated from the cerebro-spinal fluid taken from cases during life, and to which they were inclined from the constancy of its occurrence to attribute a causal role. The seriousness of the epidemic in Uganda had led the Royal Society of London in 1902, at the instigation of the Foreign Office, to despatch a Commission to investigate the condition on the spot. Soon after commencing work, Dr. Castellani found in some cases in the cerebro-spinal fluid, especially when this was centrifugalised, living trypanosomes resembling the Tr. gambiense ; he also found in 80 per cent of the cases post mortem a coccus resembling if not identical with that observed by the Portuguese Commissioners. At first Castellani was inclined to look on the presence of the protozoan as accidental, but Colonel Bruce on going out with Nabarro and Greig in 1903 to pursue the work of the Commission TRYPANOSOMA OF SLEEPING SICKNESS 559 realised the significance of the observation, urged Castellani to further inquiries, which he himself continued after the departure of the latter, with the result that in a series of examinations carried out in several infected localities, the trypanosome was demonstrated in every case of the disease. This work formed the starting-point for inquiries, the results of which make it practically certain that the parasite is the causal agent of the condition. The organisms were not demonstrable in the cerebro- spinal fluid of patients dying of other diseases in the sleeping sick- ness area. On the other hand, it was found that if cerebro-spinal fluid withdrawn from cases of the disease was injected into monkeys (especially macacus rhesus) trypanosomes appeared in the blood, and in many cases in three or four months the animals died of an illness indistinguishable from sleeping sickness and with the para- sites in the central nervous system. It was further found that in the parts round the north end of Victoria Nyanza where sleeping sickness was rife, the distribution of the disease exactly corre- sponded with the distribution of a blood-sucking insect, the glossina palpalis, a species closely allied to the glossina morsitans of nagana. It was found that, when one of these flies was fed on a sleeping sickness patient and then allowed to bite a monkey, frequently trypanosomes appeared in the animal's blood, and that when fresh flies caught in the sleeping sickness area were placed on a monkey a similar occurrence took place. The trypanosome of sleeping sickness is 17-28 /A long and 1*4-2 //. broad (Fig. 169) (when about to divide it is both longer and broader). According to Laveran the free part of the flagellum often equals a fourth of the whole length, but occasion- ally the body protoplasm extends quite to the end of the organism. The undulating membrane is narrow, and the posterior end may be either sharp or blunt. The trypanosome contains the macro- and micronucleus characteristic of the group, and the protoplasm often shows chromatin granules. Castellani attached great importance to a vacuole often seen in the neigh- bourhood of the micronucleus, but, as stated above, Laveran holds this to be an artefact. The organism divides longitudin- ally in the usual manner, and often two can be seen to approach each other and lie more or less side by side, but whether this indicates conjugation or not is not known. The organism does not usually long survive removal from the body, but it has been found to be motile for nineteen days when kept on rabbit-blood agar at 22° C. As we have said, when Tr. ugandense is in- oculated into monkeys they often contract an illness which ultimately presents the features of typical sleeping sickness. 560 TRYPANOSOMIASIS Inoculation of other species of animals is not usually so successful, though in nearly every case, e.g. in the guinea-pig, a proliferation of the parasite, as indicated by its appearing in the blood, takes place ; but often either no disease occurs or this runs a very chronic course. The relative insusceptibility of animals, especially of the dog, to the Tr. ugandense is taken as evidence that this organism is essentially different from Tr. Brucei. By means of microscopic examination the organisms may be found in the cerebro-spinal fluid, the blood, or the juice of glands. In the case of the first about 10 c.c. of the fluid is to be centrifuged for fifteen minutes and the deposit placed under a cover-glass for examination ; it is better to make a little cell on a slide by painting a ring of ordinary embedding paraffin, to place the droplet of fluid in its centre, and to support the cover- glass on the paraffin ; in this way injury to the delicate structure of the organism is avoided. In fresh cerebro-spinal fluid the trypanosomes can be seen to be actively motile ; the number in which they occur varies very much, and the same is true to a greater degree of the blood, in which they are, however, usually very scanty. With regard to the examination of the blood Bruce and Nabarro state that it is difficult by ordinary centri- fugalisation to concentrate the organisms, as they are not readily precipitated. They accordingly recommend that the blood be mixed with citrate of sodium solution (equal parts of blood and of a one per cent citrate solution) and centrif ugalised for ten minutes, that the plasma be removed and centrifugalised afresh for the same time, and that this be repeated three times, the deposit from each centrifugalisation after the first being carefully examined. Greig and Gray have insisted that the examination of the glands in a suspected case forms the most ready means of arriving at a diagnosis, and this opinion has found strong support from the work of Button and Todd. The method is to push a hypodermic needle into the gland, suck up a little of the juice, and blow it out on to a slide. In all cases where films of any kind are to be prepared the staining methods of Leishman or Giemsa are to be recommended. Often in cerebro-spinal fluid and gland juice the staining of the chromatin is difficult, but good preparations are obtained by the procedure recommended by Leishman for studying the parasite in sections (p. 545). Greig and Gray have studied the trypanosome in the body of the glossina. They found evidence of its multiplying in the stomach of the insect, and it also was seen to undergo changes not elsewhere observed. These consisted in alterations in the position of the micronucleus, which often became anterior to the TRYPANOSOMA OF SLEEPING SICKNESS 561 macronucleus ; there also occurred rosettes, consisting of from four to twenty individuals attached by their posterior extremities. Oval forms were also observed. It was found that monkeys could not be inoculated with the trypanosomes from the stomach of the fly, and this observation corresponds with what Bruce found to be true of the trypanosome of nagana in glossina morsitans. Early in the Uganda investigations the question arose as to whether the trypanosoma of sleeping sickness was different from Tr. gambiense. This was forced on the inquirers by the fact that a very large proportion of the natives in the sleeping sickness area were found to harbour trypanosomes in their blood, although not apparently suffering from the disease. Several cases were carefully examined in which trypanosomes were constantly present in the blood, but in which the patients from time to time suffered from fever, and during these pyrexial periods trypanosomes were found in the cerebro- spinal fluid. It was suggested that these cases were on the way to develop sleeping sickness. A very important observation was that while in sleeping sickness areas a large proportion of the native population harboured trypanosomes, this was not the case where sleeping sickness did not occur. Further, it was found that trypanosomes from the cerebro -spinal fluid of sleeping sickness cases and from the blood of persons harbouring try- panosomes, but not suffering from disease symptoms, gave rise in monkeys to the same group of chronic effects which resembled the last stages of the disease in man. These facts led the Commissioners to incline to the idea that trypanosoma fever and sleeping sickness are due to the same cause, and represent different stages of the same disease. It has already been pointed out that a fatal termination can occur in trypanosoma fever by an acute febrile attack or from intercurrent disease, and thus the terminal lethargic stage may only develop in a certain proportion of cases. The view of the identity of the two conditions has continued to gain ground. The best authorities are agreed that morphologically no difference between the two organisms can be recognised, and the con- tinued observation of prolonged cases of trypanosoma fever, both in Uganda by Greig and Gray and in this country by Manson, has shown that sometimes the termination of a case is by the onset of typical sleeping sickness. The prevalence of trypanosomes in the blood of apparently healthy natives has raised the question of the possibility of tolerance existing and of immunity being established. It is 36 562 TRYPANOSOMIASIS possible that both phenomena occur, that not every infection results in multiplication of the parasite in the body of the victim, and that in certain cases where multiplication does occur a resistance is developed which enables the body to kill the parasites. The occurrence of the mononuclear reaction is here significant ; it has been suggested that, when this resist- ance is weak, the organism gains entrance to the spinal canal, and that then sleeping sickness results. The whole of the recent work on the disease is of the highest interest and importance. Short of the prolonged subculture of the parasite and the reproduction of the disease by such cultures, the strongest evidence may be said to exist that the Tr. ugandense is the cause of sleeping sickness. Other Pathogenic Trypanosomata. — It is beyond the scope of this work to deal at length with the other diseases of animals caused by trypanosomes. The chief of these have been mentioned in the opening paragraph, but it may be said that many others have been described in various species of mammals, birds, and fishes, and that these are spread either by flies or by leeches. The most interesting of those mentioned is Dourine, a condition resembling in many ways nagana. It, however, presents this peculiarity, that infection does not take place by an intermediate host, but apparently directly through coitus, as it occurs only in stallions and in mares covered by these. In several of the trypanosomal infections of animals it appears as if, as in the case of Tr. Lewisi, the animal suffers little inconvenience from the presence of the parasite in its blood, and the view has even been put forward that with all pathogenic trypanosomes there exists a host which carries the organism without being affected by its presence more, for example, than is the rat by Tr. Lewisi. Though no opinion can be expressed on this point, it is necessary to bear the fact in mind that either natural or acquired immunity can exist against such protozoa. Not only is this important from the point of view of the investigation of the conditions under which such tolerance arises, but also from the bearing which the existence of this tolerance may have on the spread in nature of the parasites to a susceptible species from immune animals which still harbour trypanosomes in their blood. We are, however, as yet quite ignorant of many of the processes at work in the body during a trypanosomal infection, and of the causes of the symptoms and other morbid effects. KALA-AZAR 563 Ki.LA-AZAK. (Synonyms : Cachectic Fever, Dum-Dum Fever, Non-malarial Remittent Fever.) Leishman noticed in several soldiers invalided from India for remittent fever and cachexia that the most careful examination of the blood failed to reveal the presence of the malarial parasite. From the fact that such patients had almost invari- ably been quartered during their service at Dum-Dum, an unhealthy cantonment near Calcutta, he suspected he had to deal with an undescribed disease. In 1900 he noticed in the spleen of such a case peculiar bodies which, from comparison with certain appearances found in degenerating forms of Tr. Brucei, he suggested might be trypanosomes, and on publishing his observations in 1903 he put forward the view that trypanosomiasis might prevail in India and account for the aberrant cases of cachexial fever met with there. Soon after Leishman's paper appeared, his observations were confirmed in India by Donovan, and the bodies associated with the disease are usually called the " Leishman " or the " Leishman-Donovan " bodies. They were found by Bentley, and later by Rogers, in the disease known in Assam as Kala-azar, the pathology of which had long puzzled those who had worked at it, from the fact that, while it resembled malaria in many ways, no parasite could be demonstrated to occur in those suffering from it. Kala-azar (or "black disease," — so called from the hue assumed by chocolate-coloured patients suffering from it) has been known since 1869 as a serious epidemic disease in Assam, where it has spread from village to village up the Brahmaputra valley. The disease is now known to occur in various sub- tropical centres south of the forty-ninth parallel — cases where the Leishman bodies have been found having been met with in many parts of India, China, North Africa, and Arabia. The disease is characterised by fever of a very irregular type, by progressive cachexia, and by anaemia associated with enlargement of the spleen and liver, and often with ulcers of the skin and dropsical swellings. Rogers has pointed out that there occurs a leucopenia which differs from that of malaria in that it is almost always more marked, — the leucocytes usually numbering less than 2000, and further in that they are always reduced in greater ratio than the red corpuscles, which condition, again, does not occur in malaria. The disease is chronic, 564 KALA-AZAR often going on for several years, and is extremely fatal, — in fact, it is difficult to say if recovery can ever take place. Post mortem, there is little to note beyond the enlargement of the liver and spleen, but in the intestine, especially in the colon, there are often large or small ulcers, and there is evidence of proliferation in the bone marrow, the red marrow encroaching on the yellow. In a film made from the spleen and stained by Leishman's Fia. 170. — Leishman- Donovan bodies from spleen smear. x 1000. stain, the characteristic bodies can be readily demonstrated (Fig. 170). They are round, oval, or, as Christophers has pointed out, cockle-shell shaped, and usually 2*5 to 3 -5 //, in diameter, though smaller forms occur. The protoplasm stains pink, or sometimes slightly bluish, and contains two bodies taking on the bright red colour of nuclear matter when stained by the Romanowsky combination. The larger stains less intensely than the smaller, is round, oval, heart-shaped, or bilobed, and lies rather towards the periphery of the body — in the region of the " hinge " in the cockle-shaped individuals. KALA-AZAR 565 The other chromatin body is usually rod-shaped, and is set perpendicularly or at a tangent to the larger mass, with which only exceptionally it appears to be connected. Usually the protoplasm contains one or two vacuoles. Though in spleen smears many free bodies are seen, the study of sections shows that ordinarily their position is intra-cellular, — the cells con- taining them being of a large mononuclear type (Fig. 171). The view held is that on their entering the circulation they are taken up by the mononuclear leucocytes and by such cells as the endothelial lining of the splenic sinuses or by those lining capillaries or lymphatics, that in these cells multiplication takes place — it may be to such an extent as to rupture ^ the cell, — and that if thus the bodies become free, they are taken up by other cells and the process is repeated. The clusters of bodies some- times seen in smears are probably held together by the remains of rup- tured phagocytes. In capillaries the endothelial cells after phagocyting the bodies probably be- come detached from the capillary wall, as they f/ , T £ FlG. 171. — Leishmau-Donovan bodies within are often observed free endothelial cell in spleen, x 1000. in the lumen of the vessel, — this being well seen in the hepatic capillaries. In the body generally the parasites are found in greatest abundance in the spleen, liver, and bone marrow, and also in mesenteric glands, especially in those draining one of the intestinal ulcers • less frequently they occur in the skin ulcers, and in other parts of the body. Whether they can be demon- strated microscopically in the blood is disputed. Donovan described them as occurring in the blood, and also as being present within red blood corpuscles, but though Laveran agreed with Donovan's description, the observation has not been confirmed by other observers. In the body the parasite multiplies by simple fission, both nuclei dividing amitotically, and two new individuals being formed ; but sometimes a multiple division takes place, each 566 KlLA-AZAR nucleus dividing several times within the protoplasm and a corresponding number of new parasites resulting. In view of Leishman's original opinion an extremely important discovery was made by Rogers and later confirmed by Leishman himself, to the effect that in cultures a flagellate organism developed from the Leishman-Donovan body. Cultivation was effected by taking spleen juice containing the parasite, placing it in 10 per cent sodium citrate solution and keeping at 17-24° C. Under such conditions there occurs an enlargement of the organism, but especially of the larger nucleus. This is followed by the appearance of a pink-staining vacuole in the neighbourhood of the smaller nucleus. Along with these changes, in from 24 to 48 hours the parasite becomes elongated and the smaller nucleus and its vacuole move to one end ; from the vacuole there then appears to develop a red-staining flagellum, which when fully formed seems to take its origin from the neighbourhood of the small nucleus. The body of the parasite is now from 20-22 //, long and 3-4 p broad, with the flagellum about 22 /x long. The whole development occupies about 96 hours. The formation of an undulating membrane was not observed, and, although the flagellated organism moved flagellum first, like a trypanosome, it is evident that here the relationship of the micronucleus is different as this structure lies anterior to the macronucleus. In his cultures, which kept alive for four weeks, Leishman made a still further important observation. In certain of the flagellate forms he saw chromatin granules develop in the protoplasm often in couples, a larger and a smaller. There then occurred a very unequal longitudinal division of the protoplasm, and a hair-like undulating individual containing one of the pairs of chromatin granules would be split off. At first these would be non- flagellate, but later a red-staining flagellum would appear at one end. The analogies between these observations and those of Schaudinn (v. p. 550) on the relations of spirochaetes to trypanosomes will be at orice apparent; the further develop- ment of these spirillary forms in Leishman's organism could not, however, be traced. The facts just detailed have caused considerable discussion as to the classification of the organism, which now usually goes by the name Leishmania donovani, originally given to it by Ross. According to one view, it is to be looked on as a trypanosome, and although, as we have noted, its flagellated form differs from the typical trypanosoma form, it bears considerable resemblance to the members of this group, and as Leishman has pointed out, his cultures may not represent the full development of the KALA-AZAR 567 organism in the trypanosoma direction. Others have looked on it as a piroplasma, but Minchin suggests that in the present incomplete state of knowledge it may be well to place it in a provisional genus, Leishmania, of the flagellata. In this genus there would be at present two species, the Leishmania donovani, and the organism seen in Dehli sore, Leishmania tropica, presently to be alluded to. The question arises, given that the Leishmania donovani is the cause of kala-azar, how is infection spread1? On this we have as yet no certain information. The fact that in some centres of the disease natives who are supplied with good water are less liable than those who rely on the ordinary polluted native cisterns, has led to the opinion that water may be the carrier of infection. On the other hand, the possible relation- ship of the organism to the trypanosomata naturally suggests the idea of an insect as an intermediary, and Rogers has brought forward some evidence that the bed-bug is the extra-human host, but the organism has not as yet been demonstrated in the body of this insect. It has been objected to the theory of an insect carrier that the organism probably does not occur in the blood, but it has been pointed out that invisible spirillary forms may be the instruments of infection, and that such may exist in the blood. It may be said here that all attempts to communicate the disease to animals have been hitherto unsuccessful. With regard to kala-azar as a whole, we may say that we are dealing with a disease fairly widespread in various sub-tropical regions, which is, there is every reason to believe, a separate entity. All attempts to include it among the malarial cachexias, which clinically it so much resembles, have failed. In this atypical cachexial fever there is always present a parasite of very special characters belonging or closely allied to a group which contains many varieties capable of giving rise to similar diseases. Beyond this we cannot go, but at present we must admit that there is strong presumptive evidence of the parasite described being the cause of the disease. Methods of Examination. — The Leishmania donovani can be readily seen in films or sections of the organs in which we have mentioned its occurrence. These should be stained by the Romanowsky stains. Fluid taken from the enlarged spleen during life may be examined, but it is probable that in this disease puncture of the spleen may not be a very safe operation, as death from haemorrhage from this organ is a not uncommon natural terminal event. During life the main points on which a pathological diagnosis may be based are the absence of 568 KALA-AZAR malarial parasites from the blood, and the features of the leucopenia which have been alluded to. Delhi Sore. — In various tropical and sub-tropical regions there is widely prevalent a variety of very intractable chronic ulceration which goes by various names in different parts of the world, Delhi sore, tropical ulcer, Aleppo boil, etc. Various views have been held as to the pathology of the condition, but the work of J. H. Wright, which has been confirmed by other observers, makes it extremely probable that a protozoal parasite is concerned in its etiology. In the discharge from the ulcer and in sections of a portion of tissue excised from a case coming from Armenia, Wright observed great numbers of round or oval, sharply defined bodies, 2-4 ya in diameter. When stained by a Romanowsky combination there was found to be a peripheral portion coloured a pale blue and a central portion tending to be unstained; there were also two chromatin bodies, one larger, occupying a fourth or a third of the whole and situated in the periphery, another smaller, round or rod-shaped, and of a deeper colour than the larger mass. It was found that the bodies were usually intra-cellular in position in the lesion, as many as twenty being in one cell, and that the type of cell containing them was, as in kala-azar, that derivable from endothelial tissues. There can be little doubt that these bodies are of the same type as those occurring in kala-azar, and the question of the identity of the two parasites has been raised. At present the tendency is to regard them as distinct. As we have seen, although skin ulcers are common in kala-azar, it is difficult to find the parasite in this lesion of the disease, while, on the other hand, in Wright's case at least the number of organisms present in the ulcer was enormous. Provisionally Minchin calls this parasite the Leishmania tropica and includes it as the second species of the genus Leishmania. PlROPLASMOSIS. Up to the present no human disease has been proved to be associated with the presence of piroplasmata. The observations of Donovan, which seemed to indicate that the parasite of kala-azar might be found within the red blood corpuscles, and which led Laveran to denominate the Leishmania donovani the piroplasma donovani, have, as already indicated, not been confirmed ; the same is true of the association of piroplasms with the occurrence of the Kocky Mountain spotted fever sometimes prevalent in Montana. But several important diseases of the lower animals are almost certainly caused by protozoan parasites of this group, and a short account of the organisms may be given. The piroplasmata are pear-shaped unicellular organisms about 1-1 '5 /* PIROPLASMOSIS 569 longhand varying in breadth. The peripheral part is denser than the central, which often appears as if vacuolated, and at the broad end there is a well-staining chromatin mass. Sometimes irregular and ring-, rod-, or oval-shaped individuals occur. The organisms are found within the red blood corpuscles of the infected animal and also free in the blood. In the former situation there is sometimes only one within a cell, but the numbers vary under different circumstances and in different species. Multiplication takes place by fission, and the new individuals, remaining for longer or shorter times in apposition, account for some of the appearances seen in cells. Especially in the forms free in the blood pseudopodial prolongations of the protoplasm, usually from the pointed end, are developed, and it may be by means of such pseudopodia that entrance to the red cells is obtained. Infection is usually carried from infected animals by means of ticks. In one case Koch has described the development in the organism, in the stomach of the tick, of spiked proto- plasmic processes sprouting out from the broad end of the piroplasm, and the occurrence of conjugation of two such individuals by their narrow ends to form a zygote. Further observations, however, here are necessary, and nothing is known of the further history of the parasite within the insect except that the eggs in the ovary may become infected, so that insects developed from these can carry infection to animals. Frequently when an animal has passed through an attack of a piro- plasmosis it is immune to the disease, and with regard to this immunity in certain cases very interesting facts have been observed. For instance, the condition may not be associated with the disappearance of the parasite from the blood of the immune animal, and the latter may thus be a source of danger to other non-immune animals with which ticks harboured by it may come in contact. The following are the chief piroplasmata causing disease in animals : — (1) Piroplasma bigeminum. This was first described by Theobald Smith and is the cause of Texas or red-water fever, a febrile condition associated with hsemoglobinuria, which occurs in the Southern States of America, the Argentine, South and Central Africa, Algeria, various parts of Northern Europe, and in Australia. The organism gets its name of bigeminum from the fact that it is often present in the red cells in pairs, which may be attached to one another by a fine thread of protoplasm ; this probably results from the complete separation of two individuals being delayed after division has occurred. Infection is here spread by the tick boophilus boms, and some of the characteristics of the disease epidemiologically are explained by the fact that this insect goes through all its moultings on the same individual host. (2) Piroplasma parvum. This organism was discovered by Theiler in the blood of cattle suffering from African East Coast fever, a disease closely resembling Texas fever, which prevails endemically in a narrow strip along a long extent of the east coast, and which occurs epidemically inland. As its designation implies, the organism is small, and it is also attenuated. Its insect host is the tick rhipicephalus appetidiculatus, and it may be noted that this tick drops off the animal on wThich it may be feeding when it is about to go through one of its several moultings. It can thus carry an infection much more quickly and widely through a herd than can the carrier of ordinary red- water fever. It may be said that in England there occurs a red-water fever also associated with the presence of a piroplasm in the blood, but the, relationship of this organism to the other varieties has not yet been fully worked out. (3) Piroplasma equi. This organism gives rise to biliary fever in horses, another South African disease, and it is carried 570 PIROPLASMOSIS by the tick rhipicephalus evertsii. In this disease Theiler made the interesting observation that when the blood of a donkey which had recovered from the disease was injected into a horse the latter suffered a slight illness only, although the organisms were present in the blood injected. Such a fact is of importance, as attenuation of virulence in pathogenic protozoa seems, so far as our present knowledge goes, a not very common event. (4) Piroplasma canis. This causes a piroplasmosis occurring in dogs. With regard to the pathology of infection by piroplasmata we know nothing. The diseases are often extremely fatal, carrying off nearly every individual attacked, but we do not know the nature of the changes originated. BIBLIOGRAPHY. GENERAL TEXT- BOOKS.— In English the student may consult the follow- ing : "Micro-organisms and Disease," E. Klein, 3rd ed., London, 1896. " Bacteriology and Infective Diseases," Edgar M. Crookshank, London, 1898. "A Manual of Bacteriology," George M. Sternberg, New York, 1st ed. 1893, 2nd ed. 1896 (this book contains a full bibliography). "Text- Book upon the Pathogenic Bacteria," Joseph M'Farland, London, 5th ed., 1906. "Practical Bacteriology," A. A. Kanthack and J. H. Drysdale, London, 1895. " Bacteria and their Products," G. S. Woodhead, London, 1891. "Bacteriological Technique," Eyre, London, 1902. 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. For non-pathogenic bacteria occurring in connection with pathological work consult Heim, op. cit. infra. 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," ii., transl. by Garnsey and Balfour, Oxford, 1887. In German: "Die Mikroorganismen," by Dr. C. Fliigge, 3rd ed., Leipzig, 1896. " Lehrbuch der pathologischen Mykologie," by Baum- garten, Braunschweig, 1890. " Handbuch der pathogenen Mikro- organismen," Kolle and Wassermann, Fischer, Jena, 1904. In French : Roger, " Les maladies infectieuses," Paris, 1902. PERIODICALS.— For references to current work see (1) Centralbl. f. Bakteriol. u. Parasitenk., Jena. This publication commenced in 1887. Two volumes are issued yearly. In 1895 it was divided into two parts. Abtheilung I. deals with Medizinisch-hygienische Bakteriologie und thierische Parasitenkunde. The volumes of this part are numbered con- secutively with those of the former series, the first issued thus being vol. xvii. Commencing in 1902 with volume xxxi., each volume of Ab- theilung I. was further divided into two parts, one consisting of Originate the other of Referate. Abtheilung II. deals with Allgeineine landwirt- schaftlich-technologische Bakteriologie, Garungs-physiologie und Pflanzen- pathologie. The first volume is entitled Zweite Abtheilung, Bd. I. It contains original articles, JRcferate, etc. (2) Bull, de I'lnsi. Pasteur, Paris, Masson. Besides bacteriological abstracts this journal contains many valuable reviews and analyses relating to protozoology. (3) "Ergeb- nisse der allgemeinen Pathologic," Lubarsch and Ostertag, Wiesbaden, Bergmann. This from time to time contains valuable critical reviews. The most complete account of the work of the year is found in the Jahresb. u. d. Fortschr. . . . d. path. Mikroorganismen, conducted by 571 572 BIBLIOGRAPHY Baumgarten, and published in Braunschweig. This publication com- menced 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 and Internat. Cat. Sc. Lit. (Section, Bacteriology). The chief bacteriological periodicals are the Journ. Path, and Bacterial., Cambridge, edited by G. Sims Woodhead ; the Ztschr. f. Hyg. u. Infec- tionskrankh., Leipzig, edited by Koch and Fliigge, and the Ann. de I'Inst. Pasteur, Paris, edited by Duclaux ; Journ. Exper. Med. , New York, edited by Flexner ; Journ. Hyg., Cambridge, edited by Nuttall ; Journ. Med. Research, Boston, edited by Ernst ; Journ. Infect. Diseases, Chicago, edited by Hektoen ; Journ. of Royal Army Medical Corps, edited by Bruce. Valuable papers also from time to time appear in the Lancet, Brit. Med. Journ., Deutsche med. Wchnschr., Berl. klin. Wchnschr., Semaine med., Arch. f. Hyg., Arch. f. exper. Path. u. Pharmakol. Besides these periodicals the student may have to consult the Reports of the Med. Off. of the Local Government Board, which contain the reports of the medical officers, also the Proc. Roy. Soc. London, the Compt. rend. Acad. d. sc. Paris, the Compt. rend. Soc. de biol. Paris, and the Arb. a. d. k. Gsndht- samte. (the first two volumes of the last were denominated Mitthcilungen}. 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 Morphologic der Spaltpflanzen," Leipzig, 1882; "Beitr. z. Physiologic uud Morphologic niederer Organismen," 5th ed., Leipzig, 1895. Cohn, Beitr. z. Biol. d. Pftanz., Bresl. (1876), ii. v. Nageli, "Die niecleren Pilze," Munich, 1877; " Untersuchungen iiber niedere Pilze," Munich, 1882. Migula, "System der Bakterien," Jena, 1897. Duclaux, "Traite de microbiologie," Paris, 1898-99. For general morphological relations see Marshall Ward, art. "Schizomycetes," Ency. Brit., 9th ed. xxi.' 398 ; xxvi. 51. Engler and Prantl, "Die natiirlichen Pflanzenfamilien," Lieferung, 129. " Schizophy ta " (by W. Migula). STRUCTURE or BACTERIAL CELL. — Biitschli, "Uber den Bau der Bakterien," Leipzig, 1890; "Weitere Ausfuhrungen iiber den Bau der Cyanophyceen und Bakterien," Leipzig, 1896. Fischer, op. cit. in text. Buchner, Longard and Riedlin, Centralbl. f. Bakteriol. u. Para- sitenk. ii. 1. Ernst, Ztschr. f. Hyg. v. 428 ; Babes, ibid. v. 173. Neisser, ibid. iv. 165. MOTILITY. — Klein, Biitschli, Fischer, Cohn, loc. cit. Loftier, Centralbl. f. Bakteriol. u. Parasitenk. vi. 209 ; vii. 625. PIGMENTS. — Zopf, loc. cit. ; Galeotti, Ref. in Centralbl. f. Bakteriol. u. Parasitenk. xiv. 696. Babes, Ztschr. f. Hyg. xx. 3. SPORULATION. — Prazmowski, Biol. Centralbl. viii. 301. A. Koch, Botan. Ztg. (1888), Nos. 18-22. Buchner, Sitzungsb. d. math.-phys. Cl. d. k. layer. Akad. d. Wissensch. zu Munchen, 7th Feb. 1880. R. Koch, Mitth. a. d. k. Gsndhtsamte. i. 65. CHEMICAL STRUCTURE OF BACTERIA. — Nencki, Ber. d. deutsch. chem. Gesellsch. (1884), xvii. 2605. Cramer, Arch. f. Hyg. xvi. 154. Buchner, Berl. klin. Wchnschr. (1890), 673, 1084 ; vide Fliigge, op. cit. CLASSIFICATION OF BACTERIA. —For general review see Marshall Ward, Ann. of Botany, vi. 103 ; Migula, loc. cit. supra. BIBLIOGRAPHY 573 FOOD OF BACTERIA. — Nageli, Cohn, op. cit. Pasteur, "Etudes sur la biere," 1876. Hueppe, Mitth. a. d. k. Gsndhtsamte. ii. 309. RELATIONS TO OXYGEN. — Pasteur, Compt. rend. Acad. d. sc. Hi. 344, 1142 ; Kitasato and Weyl, Ztschr. f. Hyg. viii. 41, 404 ; ix. 97. TEMPERATURE. — Vide Fliigge, op. cit. ; for thermophilic bacteria, Rabinowitsch, Ztschr. f. Hyg. xx. 154 ; Macfadyen and Blaxall, Journ. Path, and Bacterial, iii. 87. ACTION OF BACTERIAL FERMENTS. — Salkowski, Ztschr. f. Biol., N.F., vii. 92 ; Pasteur and Joubert, Compt. rend. Acad. d. sc. Ixxxiii. 5 ; Sheridan Lea, Journ. Physiol. vi. 136 ; Beijerinck, Centralbl. f. Bak- teriol. u. Parasitenk., Abth. II. i. 221 ; E. Fischer, Ber. d. deutsch. chem. Gcsellsch. xxviii. 1430 ; Liborius, Ztschr. f. Hyg. i. 115 ; see also Pasteur, "Royal Society Catalogue of Scientific Papers." VARIABILITY. — 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, iv. 213, 257, 760 ; v. 92, 577. Maze, ibid. xi. 44 ; xii. 1, 263. 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. METHODS OF STERILISATION. — R. Koch, Gatt'ky, and Lbffler, Mitth. a. d. k. Gsndht- samte. i. 322 ; Koch and Wolffhiigel, ibid. i. 301. CULTURE MEDIA. — See text -books, especially Kanthack and Drysdale, Eyre ; Pasteur, "Etudes sur la Mere," Paris, 1876; R. Koch, Mitth. a." d. k. Gsndht- samte. i. 1 ; Roux et Nocard, Ann. de Vlnst. 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. Pub. Health Ass. xx. 381. Theobald Smith, Centralbl. f. Bakteriol. u. Parasitenk. vii. 502 ; xiv. 864. Durham, Brit. Med. Journ. (1898), i. 1387. "Report of American Committee on Bacteriological Methods," Concord, 1898. MacConkey, Thompson- Yates and Johnston Lab. Rep. vol. iii. pt. iii. 151 ; vol. iv. pt. i. p. 151 ; Journ. Hyg. v. 333. Drigalski and Conradi, Ztschr. f. Hyg. xxxix. 283. CHAPTER III. — MICROSCOPIC METHODS, ETC. Consult text-books, especially Klein, Kanthack and Drysdale, Hueppe, Gunther, Heim ; also Bolles Lee, "The Microtomist's Vademecum, " London, 1905 (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 Vlnst. Pasteur, ix. 666 ; Kiihne, " Praktische Anleituug zum mikroscopischen Nachweis der Bakterien im tierischen Gewebe," Leipzig, 1888 ; van Ermengem, ref. Centralbl. f. Bakteriol. u. Parasitenk. xv. 969 ; Richard Muir, Journ. Path, and Bacterial, v. 374 ; Mann, " Physiological Histology," Oxford, 1902. For Romanowsky methods see Jenner, Lancet (1899), 574 BIBLIOGRAPHY i. 370 ; Leishman, Brit. Med. Journ. (1901), i. 635 ; (1902), ii. 757 ; Journ. of Roy. Army Med. Corps (1904), ii. 669 ; Geirasa, Deutsche Med. Wchnschr. (1905), 1026 ; Ann. de I'Inst. Pasteur, xix. 346 ; MacNeal, Journ. Inf. Dis. iii. 412; Wright, J. H., Journ. Med. Res. vii. 138. AGGLUTINATION.— Delepine, Brit. Med. Journ. (1897), ii. 529, 967. Widal and Sicard, Ann. de I'Inst. Pasteur, xi. 353. Wright, Brit. Med. Journ. (1897), i. 139 ; (1898), i. 355. CHAPTER IV. — BACTERIA OF AIR, SOIL, WATER. ANTISEPTICS. AIR, SOIL, AND WATER. — Petri, Ztschr. f. Hyg. iii. 1; vi. 233. Fliigge, ibid. xxv. 179. Sticher, ibid. xxx. 163. Weyl, " Handbuch der Hygiene," Jena, 1896, et seq. Houston, Rep. Med. Off. Local Gov. Bd. xxvii. (1897-98) 251 ; xxviii. (1898-99) 413, 439, 467 ; xxix. (1899- 1900) 458, 489. Sidney Martin, ibid. xxvi. (1896-97) 231 ; xxvii. (1897-98) 308; xxviii. (1898-99) 382. Horrocks, "Bacteriological Examination of Water," London, 1901. Percy and G. C. Frankland, "Micro-organisms in Water," London, 1894. Dibdin, " Purification of Sewage and Water," London, 1897. Ann. Rep. Bd. Health, Mass., Boston, 1890, ^ seq. Savage, " The Bacteriol. Exam, of Water Supplies," London, 1906. Lewis, Rideal, and Walker, Journ. Roy. San. Inst. (1903), xxiv. 424. ANTISEPTICS. — R. Koch, Mitth. a. d. k. Gsndhtsamte. i. 234. Behring, Ztschr. f. Hyg. ix. 395. Ritchie, Trans. Path. Soc. London, 1. 256. Rideal, "Disinfection and Disinfectants," London, 1898. 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. CHAPTER VI. — INFLAMMATORY AND SUPPTJRATIVE CONDITIONS. Ogston, Brit. Med. Journ. (1881), i. 369. Rosenbach, " Mikro- organismen bei den Wundinfectionskrankheiten des Menschen," Wies- baden, 1884. Passet, Fortschr. d. Med. (1885), Nos. 2 and 3. W. Watson Cheyne, "Suppuration and Septic Diseases," Edinburgh, 1889. Grawitz, Virchow's Archiv, cxvi. 116 ; Deutsche med. Wchnschr. (1889), No. 23. Steinhaus, Ztschr. f. Hyg. v. 518 (micrococcus tetragenus) ; '•'Die Aetiologie der acuten Eiterung," Leipzig, 1889. Christmas- Dirckinck-Holmfeld, " Recherches experimentales sur la suppuration," Paris, 1888. Muir, Journ. Path, and Bacteriol. vii. 161 ; Trans. Path. Soc. London, 1902. Garre, Fortschr. d. Med. (1885), No. 6. Marmorek, Ann. de I'Inst. Pasteur, ix. 593. Petruschky, Ztschr. f. Hyg. xvii. 59 ; xviii. 413; xxiii. 142; (with Koch, xxiii. 477). Liibbert. " Biologische Spaltpilzuntersuchung," Wiirzburg, 1886. Krause, Fortschr. d. Med. (1884), Nos. 7 and 8. Ribbert, Fortschr. d. Med. (1886), No. 1. Widal and Besaii9on, Ann. de I'Inst. Pasteur, ix. 104. .v. Lingelsheim, Ztschr. BIBLIOGRAPHY 575 /. Hyg. x. 331 ; xii. 308. Behring, Centralbl. f. BaTcteriol. u. Parasitenk. xii. 192. Thoinot et Masselin, Rev. de med. (1894), 449. Orth and Wyssokowitsch, Centralbl. f. d. med. Wissensch. (1885), 577. Netter, Arch, de physiol. norm, et path. (1886), 106. Weichselbauni, Wien. med. Wchnschr. (1885), No. 41 ; (1888), Nos. 28-32 ; Centralbl. f. Bakteriol. u. Parasitenk. ii. 209 ; Beitr. z. path. Anat. u. z. allg. Path. iv. 127. Becker, Deutsche med. Wchnschr. (1883), No. 46. Lannelongue et Achard, Ann. de I'lnst. Pasteur, v. 209. Fehleisen, "Die Aetiologie des Erysipels," Berlin, 1883. Welch, Am. Med. Journ. Sc. (1891), 439. Lemoine, Ann. de I'lnst. Pasteur, ix. 877. Kurth, Arb. a. d. Tc. Gsndhtsamte. vii. 389. Knorr, Ztschr. f. Hyg. xiii. 427. Bulloch, Lancet (1896), i. 982, 1216. Bordet, Ann. de I'lnst. Pasteur, xi. 177. Booker (streptococcus enteritis), Johns Hopkins Hosp. Rep. vi. 159. Hirsch, Centralbl. f. Bakteriol. u. Parasitenk. xxii. 369. Libman, ibid. xxii. 376. Wright and Douglas, Proc. Roy. Soc. Lond. Ixxiv. 147. Wright, Clinical Journal (1906), May 16. Streptococci. — Hiss, Journ. Exper. Med. vi. 317. Schottmiiller, Munchen. med. Wchnschr. (1903), 849. Gordon, Reports Med. Officer Local Gov. Board (1905), 388 ; Lancet (1905), ii. 1400. Andrewes and Horder, Lancet (1906), ii. Ruediger, Journ. Infect. Dis. iii. 755. Besredka, Bull, de I'lnst. Pasteur, iii. Conjunctivitis.— Morax, Ann. de I'lnst. Pasteur (1896), x. 337. Eyre, Journ. Path. and. Bacteriol. vi. 1. Miiller, Wien. med. Wchnschr. 1897 ; Inglis Pollock, Trans. Ophthalm. Soc. 1905 ; Axenfeld, in Lubarsch and Ostertag, "Ergebnisse der allgem. Pathol. u. Path. Anat.," 1901 ; "Die Bakteriologie in der Augeuheilkunde," 1907 (full references). Acute Rheumatism. — Triboulet and Cay on, Bull. Soc. med. d. h6p. de Paris (1898), 93. Westphal, Wassermann, and Malkoff, Berl. klin. Wchnschr. (1899), 638. Poynton and Paine, Lancet (1900), ii. 861, 932 (full references). Trans. Path. Soc. Lond. (1902), liii. 221 ; Lancet, December 1905. Beaton and Walker, Brit. Med. Journ. (1903), i. 237. Shaw, Journ. Path, and Bacteriol. (1903), ix. 158. Beattie, ibid. ix. 272 ; Journ. Med. Research, xiv. 399 ; Journ. Exper. Med. ix. 186. Cole, Journ. Infect. Dis. i. 714. CHAPTER VII. — INFLAMMATORY AND SUPPURATIVE CONDITIONS, CONTINUED : ACUTE PNEUMONIAS, EPIDEMIC CEREBRO - SPINAL MENINGITIS. Friedlander, Fortschr. d. Med. i. No. 22 ; ii. 287 ; Tirchow's Archiv, Ixxxvii. 319. A. Fraenkel, Ztschr. f. klin. Med. (1886), 401. Salvioli and Zaslein, Centralbl. f. d. med. Wissensch. (1883), 721. Ziehl, ibid. (1883), 433 ; (1884), 97. Klein, ibid. (1884), 529. Jiirgensen, Berl. klin. Wchnschr. (1884), 270. Seibert, ibid. (1884), 272, 292. Senger, Arch, f. exper. Path. u. Pharmakol. (1886), 389. Weiohselbaum, Wien. med. Wchnschr. xxxvi. 1301, 1339, 1367 ; Monatschr. f. Ohrenh. (1888), Nos. 8 and 9 ; Centralbl. f. Bakteriol. u. Parasitenk. v. 33. Gamaleia, Ann. de I'lnst. 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, Centralbl. f. Bakteriol. u. Parasitenk. x. 817. Lannelongue, Gaz. d. Mp. (1891), 379. Netter, Bull, et mim. Soc. med. d. hdp. de Paris (1889) ; Cpmpt. rend. Acad. d. sc. (1890) ; Compt. rend. Soc. de. biol. Ixxxvii. 34. 576 BIBLIOGRAPHY G. and F. Klemperer, Berl. klin. Wchnschr. (1891), 893, 869. Fok and Bordoni-Uffreduzzi, Deutsche med. Wchnschr. (1886), No. 33. Emmerich, Miinchen. med. Wchnschr. (1891), No. 32. Issaeff, Ann. de I'lnst. Pasteur, vii. 260. Grimbert, Ann. de I'lnst. Pasteur, xi. 840. Wash- bourn, Brit. Med. Journ. (1897), i. 510 ; (1897), ii. 1849. Eyre and Washbourn, Journ. Path, and Bacterial, iv. 394 ; v. 13. See also Brit. Med. Journ. (1901), ii. 760; Neufeld and Rimpau, Ztschr. f. Hyg. Ii. 283. Meningitis. — Weichselbaum, Fortschr. d. Med. (1887), v. 573, 620. Jaeger, Ztschr. f. Hyg. xix. 351. Councilman, Mallory, and Wright, "Epidemic Cerebro-spinal Meningitis," Rep. Bd. Health Mass., Boston, 1898 (full references). Gwyn, Johns Hopkins Hosp. Bull. (1899), 109. v. Lingelsheim, Klin. Jahrb. xv. 373. Kolle and Wassermann, ibid. p. 507. Kutscher, Deutsche med. Wchnschr. (1906), 1071. Bettencourt and Franca, Ztschr. f. Hyg. xlvi. 463. Durham, Journ. Infect. Dis. Suppl. No. 2, p. 10. Goodwin and von Sholly, ibid. p. 21. Arkwriglit, Journ. of Hyg. vii. 145. Flexner, Journ. Exper. Med. ix. 105. Van- steenberghe et Grysez, Ann. de I'lnst. Pasteur, xx. 69. CHAPTER VIII.— GONORRHOEA, SOFT SORE, SYPHILIS. GONORRHOEA. — Neisser, Centralbl. f. d. med. Wissensch. (1879), 497 ; Deutsche med. Wchnschr. (1882), 279 ; (1894), 335. Bumm, " Der Mikro- organismus der gonorrhoischen Schleimhauterkrankungen," Wiesbaden, 1885, 2nd ed. 1887 ; Miinchen. med. Wchnschr. (1886), No. 27 ; (1891), Nos. 50 and 51 ; Centralbl. f. Gynak. (1891), No. 22 ; Wien. med. Presse (1891), No. 24. Bockhart, Monatsh. f. praTct. Dermat. (1886), v. No. 4 ; (1887), vi. No. 19. Steinschneider, Berl. Tclin. Wchnschr. (1890), No. 24 ; (1893), 'No. 29 ; Verhandl. d. deutsche dermat. Gesellsch. I. Congress, Wien (1889), 159. Wertheim, Wien. Tclin. Wchnschr. (1890), 25 ; Deutsche med. Wchnschr. (1891), No. 50 ; Arch. f. Gynak. xli. Heft 1 ; Centralbl. f. Gynak. (1891), No. 24 ; (1892), No. 20 ; Wien. klin. Wchnschr. (1894), 441. Gerhardt, Charity-Ann. (1889), 241. Leyden, Ztschr. f. klin. Med. xxi. 607 ; Deutsche med. Wchnschr. (1893), 909. Bordoni-Uffreduzzi, ibid: (1894), 484. Councilman, Am. Journ. Med. Sc. cvi. 277. Finger, Ghon, and Schlagenhaufer, Arch. f. Dermat. it. Syph. xxviii. 3, 276. Lang, ibid. (1892), 1007 ; Wien. med. Wchnschr. (1891), No. 7; "Der Venerische Katarrh, dessen Pathologic und Therapie," Wiesbaden, 1893. Klein, Monatschr. f. Geburtsh. u. Gynaek. (1895), 33. Michaelis, Ztschr. f. klin. Med. xxix. 556. Heiman, New York Med. Eec. (1895), 769 ; (1896), Dec. 19. Foulerton, Trans. Brit. Inst. Preven. Med. i. 40. De Christmas, Ann. de I'lnst. Pasteur, xi. 609. Nicolaysen, Centralbl. f. Bakteriol. u. Parasitenk. xxii. 305. Rendu, Berl. klin. Wchnschr. (1898), 431. Wassermann, Ztschr. f. Hyg. xxvii. 298 ; Miinchen. med. Wchnschr. (1901), No. 8. Lenhartz, Berl. klin. Wchnschr. (1897), 1138. Thayer and Lazear, Journ. Exper. Med. iv. 81. Kb'nig, Berl. klin. Wchnschr. (1900), No. 47. De Christmas, Ann. de I'lnst. Pasteur\(19QQ), xiv. 331. Raskai, Deutsche med. Wchnschr. (1901), No. 1. Jundell, Centralbl. f. Bakteriol. u. Parasitenk. xxix. 224. Colombini, ibid. xxiv. 955. Bressel, Miinchen. med. Wchnschr. (1903), No. 13. Holler, Arch. f. Dermat. u. Syph. (1904), Ixxi. 269. Wynn, Lancet (1905), i. 352. Prochaska, Arch. f. klin. Med. Ixxxiii. Heft 1-2. Strong, Journ. Am. Med. As., May 1904. BIBLIOGRAPHY 577 SOFT SORE. — Ducrey, Monatsh. f. prakt. Dermat. 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), 61. Quinquand, Semaine mtd. (1892), 278. Petersen, Centralbl. f. Bakteriol. u. Parasitenk. xiii. 743 ; Arch. f. Dermat. u. Syph. (1894), 419. Audrey, Monatsh. f. prakt. Dermat. (1895), 267. Colombini, Centralbl. f. Bakteriol. u. Parasitenk. xxv. 254. Nicolle, Presse medicale (1900), 304. Bezan9on, Griffon, and Le Sourd, Ann. de dermat. et de syphilolog. (1901); tome ii. 1. Lenglet, ibid. (1901), tome ii. 209. Simon, Compt. rend. Soe. Biol. (1902), 547. Tomasczewski, Ztschr. f. Hyg. (1903), Bd. 43, p. 327. Davis, Journ. of Med. Research (1903), ix. 401. 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. di sc. 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. Van Niessen, Centralbl. f. Bakteriol. u. Parasitenk. xxiii. 49. Metchnikoff and Roux, Ann. de I Inst. Pasteur, xvii.-xix. Lassar, Berl. klin. Wchnschr. (1903), 1189. Neisser, Deutsche med. Wchnschr. (1904), 1369, 1431. Schaudinn and Hoffmann, Arb. a. d. kaiserl. Gesundheitsamte (1905), Bd. 22 ; Deutsche med. Wchnschr. (1905), No. 18 ; Berl. klin. Wchnschr. (1905); Nos. 22, 23. Schaudinn, Deutsche med. Wchnschr. (1905), No. 22. Hoffmann, Berl. klin. Wchnschr. (1905), No. 46; "Selected Essays on Syphilis and Smallpox," New Sydenham Society, 1906. Metchnikoff, La Semaine med. (1905), 234. Levaditi, ibid. (1905), 247. Siegel, Miinchen. med. Wchnschr. (1905). 1321, 1384. Herxheimer, ibid. (1905), 1857. Shennan, Lancet (1906), i. 663, 746. Maclennan, Brit. Med. Journ. (1906), i. 1090. Levaditi, Ann. de I'Inst. Pasteur (1906), 41. CHAPTER IX.— TUBERCULOSIS. 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Parasitenk. ix. 189, 685 ; x. 33, 576 (Ref.) ; xi. 325 ; Berl. klin. Wchnschr. (1894), 732. Madsen, Ztschr. f. Hyg. xxxii. 214. Ritchie, Journ. of Hyg. i. 125. Danysz, Ann. de I'lnst. Pasteur, xiii. 155. Marie and Morax, Ann. de I'lnst. Pasteur, Paris, xvi. 818. Meyer and Ransom, Proc. Roy. Soc. London, Ixxii. 26 ; Arch. f. exper. Path. u. Pharmakl., Leipzig, xlix. 269. Roux and Borrel, Ann. de I'lnst. Pasteur, Paris, xii. 225 ; Henderson Smith, Journ. Hyg. vii. 205. Kitt, see ref. in Centralbl. f. Bakteriol. u. Parasitenk. Jena, Referate, xxxii. 359. MALIGNANT OEDEMA. — Pasteur, Bull. Acad. de med., 1881, 1887. Koch, Mitth. a. d. k. Gsndhtsamte. i. 54. Kitt, Jahresb. d. k. Centr.- Thierarznei-Schule in Munchen, 1883-84. W. R. Hesse, Deutsche med. Wchnschr. (1885), No. 14. Chauveau and Arloing, Arch. vtt. (1884), 366, 817. Liborius, Ztschr. f. Hyg. i. 115. Roux and Chamberland, Ann. de I'lnst. Pasteur, i. 562. Charrin and Roger, Compt. rend. Soc. de biol. (1877), ser. viii. vol. iv. p. 408. Kerry and S. Fraenkel, Ztschr. f. Hyg. xii. 204. Sanfelice, ibid. xiv. 339. Leclainche and Velle, Ann. de I'lnst. Pasteur, xiv. 202, 590. BACILLUS BOTULINUS. — v. Ermengem, Centralbl. f. Bakteriol. u. Parasitenk. xix. 443 ; Ztschr. f. Hyg. xxvi. 1. Kempner, ibid,, xxvi. 481. Kempner and Schepilewsky, ibid, xxvii. 213. Kempner and Pollack, Deutsche med. Wchnschr. (1897), No. 32. Brieger and Kempner, ibid. (1897), No. 33. Marinesco, Compt. rend. Soc. de biol. (1896), No. 31. Schneidemiihl, Centralbl. f. Bakteriol. u. Parasitenk. xxiv. 577, 619. Romer, ibid, xxvii. 857. QUARTER-EVIL. — See Nocard and Leclainche, "Les maladies micro- biennes des animaux," Paris, 1896. Arloing, Cornevin, et Thomas, " Le charbon symptomatique du bceuf," Paris, 1887. Nocard and Roux, Ann. de I'lnst. Pasteur, i. 256. Roux, ibid. ii. 49. See also Journ. Comp. Path, and Therap. iii. 253, 346 ; viii. 166, 233. BACILLUS ^ERG-GENES CAPSULATUS. — Welch and Nuttall, Bull. Johns Hopkins Hosp. (1892), 81. Welch and Flexner, Journ. Exper. Med. i. 5. 584 BIBLIOGRAPHY E. Fraenkel, Centralbl. f. Bakteriol. u. Parasitenk. xiii. 13. Durham, Bull. Johns Hopkins Hosp. (1897), 68. Norris, Am. Journ. Med. Sc. cxvii. 172. CHAPTER XVII.— CHOLERA. Koch, Rep. of 1st Cholera Conference, 1884 (v. " Microparasites in Disease," New Sydenham Soc., 1886). Nikati and Rietsch, Compt. rqnd. Acad. d. sc. xcix. 928, 1145. Bosk, Ann. de I'Inst Pasteur, ix. 507. Pettenkofer, Munchen. med. Wchnschr. (1892), No. 46 ; (1894), No. 10. Sawtschenko, Centralbl. f. Bakteriol. u. Parasitenk. xii. .893. Pfeiffer, Ztschr. f. Hyg. xi. 393. Kolle, ibid. xvi. 329. Issaeff and Kolle, ibid. xviii. 17. Wassermann, ibid. xiv. 35. Sobernheim, ibid. xiv. 485. Metchnikoff, Ann. de I'Inst. Pasteur, vii. 403, 562 ; viii. 257, 529. Fraenkel and Sobernheim, Hyg. Rundschau, iv. 97. Duribar, Arb. a. d. k. Gsndhtsamte. ix. 379. Pfeiffer and Wassermann, Ztschr. f. Hyg. xiv. 46. Wesbrook, Ann. de V Inst. Pasteur, viii. 318. Scholl, Berl. klin. Wchnschr. (1890), No. 41. Griiber and Wiener, Arch.f. Hyg. xv. 241. Cunningham, Sclent. Mem. Med. Off. India, 1890 and 1894. Hueppe, Deutsche med. Wchnschr. (1889), No. 33. Klemperer, ibid. (1894), 435; Berl. klin. Wchnschr. (1892), 969. Lazarus, ibid. (1892), 1071. Reincke, Deutsche med. Wchnschr. (1894), 795. Koch, Ztschr. f. Hyg. xiv. 319. Voges, Centralbl. f. Bakteriol. u. Parasitenk. xv. 453. Pastana and Bettencourt, Centralbl. f. Bakteriol. u. Parasitenk. xvi. 401. Dieudonne, ibid. xiv. 323. Celli and Santori, ibid. xv. 289. Neisser, ibid. xiv. 666. Sanarelli, Ann. de I'Inst. Pasteur, vii. 693. Ivanoff, Ztschr. f. Hyg. xv. 485. Issaeff, ibid. xvi. 286. Pfuhl, ibid. x. 510. Rumpel, Deutsche med. Wchnschr. (1893), 160. Klein, Rep. Med. Off. Loc. Govt. Board, 1893 ; "Micro-organisms and Disease," London, 1896. Haffkine, Brit. Med. Journ. (1895), ii. 1541 ; Indian Med. Gaz. (1895), No. 1 ; "Anti-cholera Inoculation," Rep. San. Com. India, Calcutta, 1895. Pfeiffer in Fliigge, "Die Mikroorganismen," 3rd ed. 1896; Gamaleia, Ann. de I'Inst. Pasteur, ii. 482, 552. Achard and Bensande, Semaine med. (1897), 151. Rumpf, "Die Cholera Asiatica und Nostras," Jena, 1898. Kraus and Pribram, Centralbl. f. Bakteriol. xli. (Orig.), 15, 155. Kraus and Prantschoff, ibid. 377, 480. A. Macfadyen, ibid. xlii. (Orig.), 365. Gotschlich, Scientific Reps. Sanitary, Maritime, and Quarantine Council of Egypt, Alexandria, 1905, 1906. For discussion, vide Supple- ment to Centralbl. f. 'Bakteriol. Referate, xxxviii. 84. Dunbar, Berlin, klin. Wchnschr. (1902), No. 39. CHAPTER XVIII.— INFLUENZA, ETC. INFLUENZA. — Pfeiffer, Kitasato, and Canon, Deutsche med. 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, Rep. Med. Off. Loc. Govt. Board (1893), 85. Pfeiffer, Ztschr. f. Hyg. xiii. 357. Huber, Ztschr. f. Hyg. xv. 454. Kruse, Deutsche med. Wchnschr. (1894), 513. Pelicke, Berl. klin. Wchnschr. (1894), 524. Pfuhl and Walter, Deutsche med. Wchnschr. (1896), 82, 105. Cantani, Ztschr. f. Hyg. xxiii. 265. Pfuhl, Ztschr. f. Hyg. xxvi. 112. Wassermaim, Deutsche med. Wchnschr. (1900), No. 28. Clemens, Munchen. med. Wchnschr. (1900), No. 27. BIBLIOGRAPHY 585 Wynecoop, Journ. Med. Ass., February 1903. Neisser, Deutsche med. Wchnschr. (1903), No. 26. Auerbach, Ztschr. f. Hyg. (1904), xlviii. 259. WHOOPING-COUGH. — Jochmann, Arch. f. klin. Med. Ixxxiv. 470. Spengler, Deutsche med. Wchnschr. (1897), 830. Davis, Journ. Infect. Dis. iii. 1. Bordet and Gengou, Ann. de I'lnst. Pasteur, xx. 731. PLAGUE. — Kitasato, Lancet (1894), ii. 428. Yersin, Ann. de I'lnst. Pasteur, viii. 662. Lowson, Lancet (1895), ii. 199. Yersin, Calmette, and Borrel, Ann. de I'lnst. Pasteur, ix. 589. Aoyama, Centralbl. f. Bakteriol. u. Parasitenk. xix. 481. Zettnow, Ztschr. f. Hyg. xxi. 164. 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(1906), vi. 422 ; (1907), vii. 323. RELAPSING FEVER.— Obermeier, Centralbl. f. d. med. Wissensch. (1873), 145 ; and Berl. klin. Wchnschr. (1873), No. 35. Miinch, 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, Virchow's Archiv, xcviii. 160. Metchnikoff, ibid. cix. 176. Soudake- witch, Ann. de I'lnst. Pasteur, v. 545. Lamb, Scient. Mem. Med. Off. India (1901), pt. xii. 77. Sawtschenko and Melkich, Ann. de I'lnst. Pasteur, xv. 497. Tictin, Centralbl. f. Bakteriol. xxi. 179. Karlinski, Centralbl. f. Bakteriol. (1902), Orig. xxxi. 566. Gabritschewsky, Ztschr. f. klin. Med. (1905), Bd. 56. Norris, Pappenheimer, Flournoy, Journ. Infect. Dis. iii. 266. Novy and Knapp, ibid. 291. Zettnow, Ztschr. f. Hyg. (1906), Iii. 485; Deutsche med. Wchnschr., 1906. AFRICAN TICK FEVER.— Ross and Milne, Brit. Med. Journ. (1904), ii. 1453. Button and Todd, Thompson- Yates Laboratory Rep. (1905), vi. pt. ii. Koch, Deutsche med. Wchnschr., 1905 ; Berl. klin. Wchnschr., 1906. Hodges and Ross, Brit. Med. Journ. (1905), i. 713. Breuil and Kinghorn, ibid. i. 668. Breuil, Lancet (1906), i. 1806. Levaditi, Compt. rend. Soc. biol., 1906. MALTA FEVER.— Bruce, Practitioner, xxxix. 160 ; xl. 241 ; Ann. de I'lnst. Pasteur, vii. 291. Bruce, Hughes, and Westcott, Brit. Med. Journ. (1887), ii. 58. Hughes, Ann. de I'lnst. 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. 1512. Gordon, ibid. (1899), i. 688. Durham, Journ. Path, and Bacterial, v. 377. Bruce in Davidson's "Hygiene and Diseases of Warm Climates," Edinburgh and London, 1893. Birt and Lamb, Lancet (1899), ii. 701. Brunner, Wien. klin. Wchnschr. (1900), No. 7. Bruce, Journ. Roy. Army Med. Corps (1904), ii. 487, 731 ; (1907), viii. 225. Horrocks, Proc. Roy. Soc. London, Series B (1905), Ixxvi. 586 BIBLIOGRAPHY 510. "Reports of the Commission on Mediterranean Fever," 1904-1907 (reprinted in Journ. Roy. Army Med. Corps). Eyre in Kolle and Wassermann's Handbuch d. Pathoq. Mikro-organismen, Erganzungsband, 1906. YELLOW FEVER. — Sternberg, Rep. Am. Pub. Health Ass. xv. 170. Sanarelli, Ann. de I'Inst. Pasteur, xi. 433, 673, 753 ; xii. 348. David- son, art. in Clifford Allbutt's "System of Medicine," vol. ii., London, 1897. Sternberg, Centralbl. f. Bakteriol. u. Parasitenk. xxii. 145 ; xxiii. 769. Sanarelli, ibid. xxii. 668. Reed and Carroll, Medical News, April 1899. Reed, Journ. of Hyg. ii. 101 (with full references). Durham, Thompson- Yates Laboratory Rep. (1902), iv. pt. ii. 485. Gorgas, Lancet, 1902, Sept. 9 ; 1903, March 28. Marchoux, Salimbeni, and Simond, Ann. de I'Inst. Pasteur, xvii. 665 ; xx. 16, 104, 161. Bandi, Ztschr. f. Hyg. (1904), xlvi. 81. Otto and Neumann, Ztschr. f. Hyg. (1905), Ii. Heft 3. 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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'Inst. Pasteur, viii. 275 ; xi. 95. Fraser, Proc. Roy. Soc. Edin. xx. 448. Marmorek, Ann. de I'Inst. Pasteur, ix. 593. Metchnikoff, Roux, and Taurelli-Salimbeni, ibid. x. 257. Charrin and Roger, Compt. rend. Soc. de biol. (1887), 667. Griiber and Durham, Miinchen. med. Wchnschr. (1896), March. Durham, Journ. Path, and Bacteriol. iv. 13. Cart- wright Wood, Lancet (1896), i. 980 ; ii. 1145. 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, 1135. Discus- sion on Immunity, Path. Soc. London, Brit. Med. Journ. (1892), i. 373. Fodor, Deutsche med. Wchnschr. (1887), No. 34. Hueppe, Berl. klin. Wchnschr. (1892), No. 17. Nicholle, Ann. de I'Inst. Pasteur, 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 I'Inst. Pasteur, vi. 160, 604 ; viii. 275 ; ix. 225 ; x. 675 ; xi. 214 ; xii. 343. C. J. Martin, Journ. Physiol. xx. 364 ; Proc. Roy. Soc. BIBLIOGRAPHY 587 London, Ixiv. 88. C. J. Martin and Cherry, ibid. Ixiii. 420. Gautier, "Les Toxines microbiennes et animales," Paris, 1896. Wassermann, Berl. klin. Wchnschr. (1898), 1209. Pfeiffer and Marx, Ztschr. f. Hyg. xxvii. 272. Bordet, Ann. de I'Inst. Pasteur, xii. 688. Ehrlich, Deutsche med. Wchnschr. (1898), xxiv. 597. "Die Wertbemessung des Diph- therieheilserums," Jena, 1897 ; Croonian Lecture, Proc. Roy. Soc. London, Ixvi. 424 ; Deutsche med. Wchnschr. xxvii. (1901), 866, 888, 913; Nothnagel's " Specielle Pathologie und Therapie," Bd. viii. Schlussbetrachtungen. Ehrlich and Morgenroth, Berl. klin. Wchnschr. (1899), xxxvi. 6, 481 ; (1900), xxxvii. 453, 681 ; (1901), xxxviii. 251, 569, 598. Weigert, in Lubarsch and Ostertag, " Ergebnisse der Allgemeinen Pathologie " (1897), iv. Jahrg. (Wiesbaden, 1899). Morgen- roth, Centralbl. f. Bakteriol. u. Parasitenk. xxvi. 349. Bulloch, Trans. Jenner Inst. 2nd ser. p. 46. Dbnitz, Deutsche med. Wchnschr. (1897), xxiii. 428. Bordet, Ann. de rinst. Pasteur, xii. 688 ; xiii. 225, 273 ; xiv. 257 ; xv. 303 ; xvii. 161 ; xviii. 593 ; Metchnikoff, ibid. xiii. 737 ; xiv. 369 ; xv. 865. Gengou, ibid. xv. 232. Sawtschenko, ibid. xvi. 106. Ritchie, Journ. of Hyg. ii. 215, 251, 452 (with full references) ; "General Pathology of Infection," in Clifford Allbutt's "System of Medicine," 2nd ed. 1906, vol. ii. pt. i. p. 1. 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Muir and Martin, Brit. Med. Journ. 1906, ii. ; Proc. Eoy. Soc. London, Ixxix. 187. APPENDIX A. — SMALLPOX. Jenner, "An Inquiry into the Causes and Effects of the Variola Vaccinte," London, 1798. Creighton, art. "Vaccination" i\\Ency. Brit., 9th ed. Crookshank, " Bacteriology and Infective Diseases." M'Vail, "Vaccination Vindicated." Chauveau, Viennois et Mairet, "Vaccine et variole, nouvelle etude experimentale sur la question de 1'identite de ces deux affections," Paris, 1865. Klein, Hep. Med. Off. Loc. Oovt. Board (1892-93), 391 ; (1893-94), 493. Copeman, Brit. Med. Journ. (1894), ii. 631 ; Journ. Path, and Baderiol. ii. 407 ; art. in Clifford Allbutt's "System of Medicine," vol. ii. L. Pfeiffer, "Die Protozoen als Krank- heitserreger," Jena, 1891. Ruffer, Brit. Med. Journ. (1894), June 30. Beclere, Chambon, and Menard, Ann. de I'Inst. Pasteur, x. 1 ; xii. 837. Copeman, "Vaccination," London, 1899. Calmette and Guerin, Ann. de I'Inst. Pasteur, xv. 161. Guarnieri, Centralbl. f. Bakteriol. u. Para- sitenk. xvi. 299. Ewing, Journ. Med. Research, xiii. 233. Prowazek, 588 BIBLIOGRAPHY Arb. a. d. kaiserl. Gesundheitsamte, xxii. 535 ; xxiii. 525. Wasielewski, Ztschrft. f. Hyg. xxxviii. 212. Bonhoff', Berl. klin. Wchnschrft. 1905, p. 1142. Carini, Centralbl. f. BakterioL u. Parasitenk. (Orig.) xxxix. 685. 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 I'lnst. Pasteur, iii. 644. Fleming, Trans. 7th Internal. Cong. Hyg. and Demog. iii. 16. Helman, Ann. de I'lnst. 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. Remlinger, Ann. de I'lnst. Pasteur, xvii. 834 ; xviii. 150 ; xix. 625. Negri, Ztschrft. f. Hyg. u. Infectionskrankh. xliii. 507 ; xliv. 519. Williams and Lowden, Journ. Inf. Dis. iii. 452. APPENDIX C. — MALARIAL FEVER. ^ Laveran, Bull. Acad. de med. (1880) ser. ii. vol. ix. 1346 ; "Traitedes fievres palustres," Paris, 1884 ; "Du paludisme et de son hernatozoaire," Paris, 1891. Marchiafava and Celli, Fortschr. d. Med., 1883 and 1885 ; also in Vircliows Festschrift. Golgi, Arch, per le sc. med., 1886 and 1889 ; Fortschr. d. Med. (1889), No. 3 ; Ztschr. f. Hyg. x. 136 ; Deutsche med. 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. Koc. Philadelphia, xii. xiii. Grass! and Feletti, Kiforma med. (1890), ii. No. 50. Canalis, Fortschr. d. Med. (1890), Nos. 8, 9. Danilewsky, Ann. de I'lnst. Pasteur, xi. 758. "Parasites of Malarial Fevers," New. Syd. 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. Wchnschr. (1899), 69. Ross, Indian Med. Gaz. xxxiii. 14, 133, 401, 448. Nuttall, Centralbl. f. BakterioL u. Parasitenk. xxv. 877, 903 ; xxvi. 140 ; xxvii. 193, 218, 260, 328 (with full literature). Manson, Lancet (1900), i. 1417; (1900) ii. 151. Gray, Brit. Med. Journ. (1902), i. 1121. Leishman, ibid. (1901), i. 635 ; ii. 757. Daniel, ibid. (1901), i. 193. Celli, ibid. (1901), i. 1030. Nuttall and Shipley, Journ. of Hyg. i. 45, 269, 451 (with literature). Ross, Nature, Ixi. 522 ; "Mosquito Brigades and how to organise them," London, 1902. Celli, "Malaria," trans, by Eyre, London, 1900. Lankester, Brit. Med. Journ. (1902), i. 652. Ewing, Journ. Exper. Med. v. 429; vi. 119. Schaudinn, Arbeit, ans d. kaiserl. Gesundheitsamte, xix. ; Argutinsky Archiv mikroskop. Anat. lix. 315 ; Ixi. 331. Ruge in Kolle and Wassermanns Handbuch d. pathogen. Mikroorganismen, Erganzungsband, 1907 (full literature). Ross, Lancet (1903), i. 86. Minchin, "The Sporozoa," London, 1903. Stephens, art. " Blackwater Fever," in Allbutt's "System of Medicine," vol. ii. pt. ii., London, 1907. BIBLIOGRAPHY 589 APPENDIX D. — DYSENTERY. Losch, Virchow's Archiv, Ixv. 196. Cunningham, Quart. Journ. Micr. Sc., N.S. xxi. 234. Kartulis, Virchow's Archiv, cv. 118 ; Centralbl. f. BakterioL u. Parasitenk. ii. 745 ; ix. 365. Koch, Arb. a. d. k. Gsndhtsamte. iii. 65. Councilman and Lafleur, Johns Hopkins Hosp. Rep. (1891), ii. 395. Maggiora, Centralbl. f. BakterioL u. Parasitenk. xi. 173. Ogata, ibid. xi. 264. Schuberg, ibid. xiii. 598, 701. Quincke and Roos, Berl. klin. Wchnschr. (1893), 1089. Kruse and Pasquale, Ztschr. f. Hyg. xvi. i. Ciechanowski and Nowak, Centralbl. f. BakterioL u. Parasitenk. xxiii. 445. Howard and Hoover, Am. Journ. Med. Sc. (1897), cxiv. 150, 263. Harris, Vir chow's Archiv, clxvi. 67. Schaudinn, Arbeit, ans d. kaiserl. Gsndhtsamte. (1903), xix. 547. Lesage, Ann. de VInst. Pasteur (1905), xix. 9. Kartulis in Kolle and Wassermann's Handbuch d. pathoy. Mikroorganismen, Erganzungsband, 1906 ; Cen- tralbl. f. BakterioL (Originate) 1904, xxxvii. 527. APPENDIX E. — TRYPANOSOMIASIS, ETC. GENERAL. — Laveran and Mesnil, Trypanosomes et trypanosomiasis, Paris, Masson, 1904. Minchin, in Clifford Allbutt's "System of Medicine," 2nd ed. vol. ii. pt. ii. p. 9, London, Macmillan, 1907. Schaudinn, Arbeit, a. d. kaiserl. Gesundheitsamte, xx. 387. Mense, Handbuch der Tropenkrankheiten, Leipzig, 1906, Barth. Novy and MacNeal, Journ. Inf. Dis. ii. 256. Leishman, Journ. Hyg. iv. 434. SLEEPING SICKNESS. — Mott, Reports of the Sleeping Sickness Com- mission of the Royal Society, pt. vii. No. 15, London, Bale, Sons and Danielsson, 1906. Dutton and Todd, Brit. Med. Journ. (1903), i. 304. Button and Todd, Thompson- Yates Lab. Rep. v. pt. ii. i. ; v. pt. ii. 97. Dutton, Todd, and Christy, ibid. vi. pt. i. p. 1. Manson and Daniels, ibid. (1903), i. 1249. Idem, ibid. (1903), ii. 1461. Low and Mott, ibid. (1904), i. 1000. Bettencourt, Kopke, Resende, and Mendes, ibid. (1903), i. 908. Castellani, Reports of the Sleeping Sickness Commission of the Royal Society, No. 1, i. 1, London, Harrison and Sons, 1903. Bruce and Nabarro, ibid. (1903), No. 1, ii. 11. Bruce, Nabarro, and Greig ibid. (1903), No. 4, viii. 3. Greig and Gray, ibid. (1905), No. 6, ii. 3. Leishman, Journ. Hyg. iv. 434. Minchin, Gray, and Tulloch, Reports of the Sleeping Sickness Commission of the Royal Society, No. 8, xxi. 122, London, H.M. Stationery Office, 1907. Manson, Brit. Med. Journ. (1903), ii. 1249, 1461. See Discussions at British Medical Association, Brit. Med. Journ. (1903), ii. 637 ; (1904), ii. 365. Thomas, Thompson- Yates Lab. Rep. vi. pt. ii. 1. KALA-AZAR. — Leishman, Brit. Med. Journ. (1903), i. 1252. Idem, Clifford Allbutt, "System of Medicine," 2nd ed. vol. ii. pt. ii. 226, London, Macmillan, 1907. Idem, Mense, Handbuch der Tropenkrankheiten, iii. 591, Leipzig, Barth., 1906. Leishman and Statham, Journ. of Roy. Army Med. Corps, iv. 321. Donovan, Brit. Med. Journ. (1903), ii. 79. Rogers, Quart. Journ. Micr. Soc. xlviii. 367. Idem, Brit. Med. Journ. (1904), i. 1249 ; ii. 645. Idem, Proc. Roy. Soc. Ixxvii. 284. Bentley 590 BIBLIOGRAPHY Brit. Med. Journ. (1904), ii. 653 ; ibid. (1905), i. 705. Christophers, Scicntif. Mem. by Off. of the Med. and San. Dept. of the Govt. of India, Nos. 8, 11, 15. Ross, Brit. Med. Journ. (1903), ii. 1401. See discussion at Brit. Med. Assoc., Brit. Med. Journ. (1904), ii. 642. DELHI SORE. — Wright, J. H., Journ. Med. Research, x. 472. PIROPLASMOSIS. — See Minchin, loc. cit. supra. Koch, Deutsche, med. Wchnschrft. (1905), No. 47 ; Ztschrft. f. Hyg. u. InfektionsTcrankh. liv. i. Nuttall, Journ. Hyg. iv. 219. Nuttall and Graham -Smith, ibid. v. 237 ; vi. 586. INDEX INDEX Abrin, 169 immunity against, 464, 469 Abscesses (v. also Suppuration) bacteria in, 174 in dysentery, 540 Absolute alcohol, fixing by, 89 Acid-fast bacilli, 239, 252 stain for, 100 Acid formation, observation of, 44, 77 Acquired immunity in man, 456 theories of, 490 Actinomyces, 15 characters of, 287 cultivation of, 292 inoculation with, 296 varieties of, 294 Actinomycosis, 286 anaerobic streptothrices in, 293 diagnosis of, 296 lesions in, 290 origin of, 292 Active immunity, 458, 459 Aerobes, 17 culture of, 57 separation of, 51 ^Estivo-autumnal fevers, 529 Agar media (v. also Culture media), 35 separation by, 55 Agglutinable substance, 486 Agglutination by sera, 485 in relapsing fever, 442 methods, 109 of b. mallei, 282 of b. typhosus, etc., 337 of cholera vibrio, 412 of m. melitensis, 450 of plague bacillus, 437 of red blood corpuscles, 481, 485 theories regarding, 485 38 593 Agglutinins, primary (homologous), 342 secondary (heterologous), 342 Agglutinogen, 486 Agglutinoids, 486 Aggressins, 164 Air, bacteria in, 126 examination of, for bacteria, 126 Albumose of anthrax, immunity by, 312 Albumoses, 165 in diphtheria, 363 Alcohols, higher, fermentation of, 75 Aleppo boil, 568 Alexines, 478, 500 Amboceptors, 480, 491 Amoebic dysentery, 537 Amcebulae of malaria, 523 Anaerobes, 17 cultures of, 60 separation of, 57 toxins of, 60 Anaerobic Esmarch's tubes, 60 Anaerobic fermentation tubes, 60 Anaerobic plate cultures, Bulloch'.s apparatus for, 58 Anaesthetic leprosy, 269 Aniline oil, dehydrating by, 93 water, 98 Aniline stains, list of, 94 Animals, autopsies on, 123 inoculation of, 120 Anthrax, 300 anti-serum, 315 bacillus, 301 biology of, 304 cultivation of, 302 inoculation with, 308 toxins of, 312 594 INDEX Anthrax, diagnosis of, 317 in animals, 306 in man, 309 protective inoculation, 314 spread of, 313 Anti-abrin, 470 Anti-anthrax serum, 315 Anti-bacterial sera, 476 properties of, 477 Anti-cholera vaccination, 412 Anti-diphtheritic serum, 467 Antikorper, 457 Anti-plague inoculation, 436 Anti-plague sera, 436 Antipneumococcic serum, 210 Antirabic serum, 519 Anti-ricin, 470 Antiseptics, 141 actions of, 144 standardisation of, 143 testing of, 142 Antisera, therapeutic action of, 488 Antistreptococcic serum, 489 Antitetanic serum, 384 preparation of, 467 et seq. Antitoxic action, nature of, 470 bodies in normal tissues, 474 sera, use of, 469 serum, 467 cholera, 411 standardisation of, 468 Antitoxins, chemical nature of, 470 origin of, 474 Antitubercular serum, 264 Antityphoid serum, 344 Appendicitus, 185 Arthrospores, question of occurrence of, 7 Artificial immunity, varieties of, 457 et seq. Attenuation of virulence, 457 Autoclave, 29 Autolysis of bacteria, 162 Autopsies on animals, 123 Avian tuberculosis, 250 Bacilli, acid-fast, 239, 252 stain for, 100 characters of, 12 Bacillus acidi lactici, 20 aerogenes capsulatus, 188, 397 anthracis, 301 botulinus, 393 Bacillus coli communis, lesions caused by, 183 et seq. characters of, 324 comparison with b. typhosus, 325 coli in soil, 133, 134 pathogenicity of, 333 diphtherias, 353 dysenteric Shiga-Flexner, 346 enteritidis (Gaertner), 336 enteritidis sporogenes, 350 in soil, 133, 134 of glanders, 277 icteroides, 452 of influenza, 420 Koch- Weeks, 191 lactis aerogenes, 180 lacunatus, 193 of leprosy, 269 of malignant oedema, 390 Miiller's, 192 mycoides in soil, 132 ozoenae, 285 paratyphosus, 335 of plague, 426 pneumonice, 199 pseudo-diphthericus, 366 of psittacosis, 337 pyocyaneus, 180 agglutination of, 485 occurrence of, 185 pyogenes fcetidus, 174 of quarter-evil, 396 of rhinoscleroma, 284 of smegma, 254- of soft sore, 227 subtilis, 57 of syphilis, 229 tetani, 372 of Timothy grass, 253 of tubercle, 237 of typhoid, 319 differentiation from b. coli, 325 of xerosis, 367 Bacteria, action of dead, 154 aerobic (v. Aerobes), 17 anaerobic (v. Anaerobes), 17 biology of, 16 capsulated, 3 chemical action of, 21 composition of, 9 classification of, 11 cultivation of, 25 death of, 141 effects of light on, 19 INDEX 595 Bacteria, food supply of, 16 higher, 14 lower, 11 microscopic examination of, 85 morphological relations of, 2 motility of, 7 movements of, 19 multiplication of, 4 nitrifying, 23 parasitic, 21 pathogenic, action of, 149 effects of, 155 saprophytic, 21 separation of, 51 species of, 23 spore formation in (v. also Spores), 5, 56 structure of, 3 sulphur- containing, 9 temperature of growth of, 18 toxins of, 161 variability among, 23 virulence of, 150, 461 Bacterial ferments, 22, 168 pigments, 10 protoplasm, structure of, 8 treatment of sewage, 139 Bactericidal powers of serum, 477 substances, 477 Bacteriological diagnosis, 118 examination of discharges, 116 Beer wort agar, 44 Beggiatoa, 15 Behring ondmmunity, 385, 468 Bile-salt media, 43 Bismarck-brown, 95 Blackleg, 396 Blackwater fever, 534 Blastophores (malaria), 529 Blood-agar (v. also Culture media), 00 DO Blood, examination of, 68, 88 in malarial fever, 521 in relapsing fever, 438 serum, coagulated, as medium, 40 Bone-marrow in leucocytosis, 156 Bordet's phenomenon, 477 Botulism, bacillus of, 393 Bouillon (v. also Culture media), 32 Bovine tuberculosis, 248 Bread paste, 47 Brieger and Boer, 166 Fraenkel, 162 Buboes, 227 Bubonic pest, 429 Buchner on alexines, 500 Bulloch's apparatus for anaerobic culture, 58 Biitschli on bacterial structure, 9 Butter bacilli, acid fast, 253 Calmette, 435, 462, 469, 471 Canon on influenza, 420 Cantani on influenza, 424 Capaldi and Proskauer, media of, 328 Capsules, staining of, 102 Carbol-fuchsin, 99 -methylene-blue, 98 -thionin-blne, 98 Carbolic acid as antiseptic, 147 Carroll's method of making anaerobic cultures, 60 Carter on relapsing fever, 440 Cattle plague, 507 Cerebro- spinal fluid, examination by lumbar puncture, 68 Chamberland and Eoux, attenuation of b. anthracis, 460 Chamber-land's filter, 70 Chemiotaxis, 20, 495 Chlorine as antiseptic, 144 Cholera, 399 immunity against, 410 inoculation of man with, 410 methods of diagnosis of, 412 preventive inoculation against, 412 -red reaction, 404 Cholera spirillum, 400 distribution of, 402 inoculation with, 406 powers of resistance of, 405 relations to disease, 414 toxins of, 408 Cladothrices in soil, 132 Cladothrix, 15 asteroides, 295 Clubs in actinomyces, 289 Cocci, characters of, 11 Collodion capsules, preparation of, 123 Colonies, counting of, 65 Comma bacillus, 399 Commission on tuberculosis, 248 vaccination, 505 Complement, 478 deviation of, 488 Conjunctivitis, 191 Conradi-Drigalski medium, 42 596 INDEX Copeman on smallpox, 508 Cornet's forceps, 87 Corrosive films of blood, etc., 89 Corrosive sublimate, as antiseptic, 145 fixing by, 90 Councilman and Lafleur on dysentery, 537 Coimting of colonies, 65 dead bacteria in a culture, 67 living bacteria in a culture, 66 Cover-glasses, cleaning of, 87 Cowpox, relation to smallpox, 505 Cresceutic bodies in malaria, 523 Cultivation of anaerobes, 57 Culture media, preparation of : agar, 36 alkaline blood serum, 41 blood agar, 38 serum, 39 bouillon, 32 bread paste, 47, glucose agar, 37 broth, 32 gelatin, 35 glycerin agar, 37 broth, 35 litmus whey, 44 Loffler's serum medium, 40 Marmorek's serum media, 41 meat extract, 31 peptone gelatin, 35 solution, 38 potatoes, 44 serum agar, 38 Cultures, destruction ol, 83 filtration of, 69 from organs, 117, 124 hanging-drop, aerobic, 63 anaerobic, 64 incubation of, 79 microscopic examination of, 86 permanent preservation of, 82 plate, 52j pure, 48 "shake," 77 Cutting of sections, 92 Cystitis, 185, 224 Cytases, 478, 496 Cytolytic sera, 482 De Bary, definition of species, 23 Decolorising agents, 97 Deep cultures, 60 Delhi sore, 568 Dehydration of sections, 93 Delepine, 110 Deneke's spirillum, 419 Dextrose-free bouillon, 75 Diagnosis, bacteriological, 115, 118 Diphtheria, 352 diagnosis of, 368 immunity against, 467 origin and spread of, 365 paralysis in, 353, 361 results of treatment, 488 Diphtheria bacillus, action of, 358 bacilli allied to, 365 characters of, 353 distribution of, 354 inoculation with, 359 isolation of, 368 powers of resistance of, 359 staining of, 108, 359 toxins of, 163, 361 variations in virulence of, 364 Diplo-bacillus of conjunctivitis, 193 Diplococcus, 12 catarrhalis, 217 crassus, 217 endocarditidis encapsulatus, 188 intracellularis meningitidis, 213 pneumonise, 199 Disturbances of metabolism by bacteria, 159 Drigalski-Conradi medium, 42 Drying of sera, etc., in vacuo, 78 Ducrey's bacillus, 227 cultivation of, 228 Dum-Dum fever, 563 Durham's fermentation tubes, 76 Dysentery, amoebic, 537 bacteria in, 346 characters of amoeba of, 537 Dysentery, methods of examination in, 347, 542 East coast fever in cattle, 569 Eberth's bacillus, 319 Ehrlich on ricin and abrin, 464, 469 on toxins, 170 side-chain theory of antitoxin for- mation, 491 Eisner's medium, 46 Embedding in paraffin, 91 Empyema, 205, 423 Endocarditis, bacteria in, 188 Enhsemosphores (malaria), 523 Entamceba coli, 537 INDEX 597 Entamoeba histolytica, 537 cultivation of, 539 Enteric fever, 319 Enteritis, dysenteric, 347, 540 Epidemic cerebrospinal meningitis, 213 Eppinger's streptothrix, 295 Ermengem on botulism, 394 stain for flagella, 104 Erysipelas, 191 Escherich's bacillus, 319 Esmarch's roll-tubes, 55, 59 anaerobic, 60 Exaltation of virulence, 461 Examination of water, 135 Exhaust-pump, 70 Exotospores (malaria), 522 False membrane, 184, 353 Farcy, 276 Feeding, immunity by, 464 Fermentation by pneurno- bacillus, 204 by bacillus coli, 326 methods of observing, 74 of sugars by bacteria, 74 test of bacterial action, 74 tubes, 76 anaerobic, 60 Ferments formed by bacteria, 22, 168 in diphtheria, 364 in tetanus, 380 Fever, 159 Film preparations, dry, 86 wet, 88 staining of, 95 Filter, porcelain, gelatin ed, 166 Filtration of cultures, 69 Finkler and Prior's spirillum, 418 Fish, tuberculosis in, 251 Fixateurs, 497 Fixation of tissues, 89 Flagella, nature of, 8 staining of, 103 Flagellated organisms in malaria, 528 Fliigge, 14 Forceps for cover-glasses, 87 Formalin as antiseptic, 146 Foth's dry mallein, 283 Fraenkel's pneumococcus, 192, 198, 199 stain for tubercle, 101 Framboesia, spirochsetes in, 234 Frankland, on water bacteria, 137 Fraser, T. E., 462, 469, 475 Friedlander's pneumobacillus, 198, 203 Frisch on rhinoscleroma, 284 Fuchsin, carbol-, 99 Gamaleia on pneumonia, 206 Gaiuetocytes (malaria), 527 Gangrenous emphysema, 389, 392 pneumonia, 423 Gas formation, observation of, 44, 76 Gas -regulator, 80 Geissler's exhaust-pump, 70 Gelatin media, 35 phenolated, 345 separation by, 51 Gelatined porcelain filter, 166 Gentian-violet, 98 Germicides, 141 Geryk pump, 78 Giemsa's stain, 107 stain for spirochsetes in films, 107 Glanders, 275 diagnosis of, 283 in horses, 276 in man, 276 lesions in, 281 Glanders bacillus, 277 agglutination of, 282 inoculation with, 280 Glossina morsitans, 552 palpalis, 559 Glucose media, 35 et seq. Glucosides, fermentation of, 75 Glycerin media, 35 et seq. potato as culture medium, 46 Golgi on malaria, 521 Gonidia, 15 Gonococcus, characters of, 219 inoculation with, 222 toxin of, 223 Gonorrhoea, 219 Gonorrhoeal conjunctivitis, 225 endocarditis, 225 septicaemia, 226 Gram's method, 99^ Weigert's modification of, 100 Grease, 504 Greenfield on anthrax, 311, 446, 460 Griiber and Durham's phenomenon, 485 Guarnieri bodies in smallpox, 508 598 INDEX Gulland (methods), 89, 92 Haemamceba Danilewski, 530 malarise, 530 prsecox, 530 relicta, 530 * vivax, 530 Hsematozoon malaria?, 520 Hsemolytic sera, 479 Hsemolytic tests, methods of, 483 Haff kine on anti - cholera inocula- tion, 412 Haffkine's inoculation method against plague, 436 Halteridium, 528, 530 Hanging-drop cultures, 63 examination of, 8,5 Hankiu, 312 Hansen, leprosy bacilli, 269 Hesse's tube, 127 Hiss's serum water media, 41 Hofmann's bacillus, 366 Horsepox, 504 Houston on bacteriology of soil, 1 31 Hueppe, 7, 14 Hydrogen, supply of, 58 Hydrophobia, 510 diagnosis of, 519 Negri bodies in, 514 prophylactic treatment of, 516 the virus of, 513 Hypodermic syringes, 121 Immune-bodies, 478 origin of, 482 Immunity (v. also Special Diseases), 456 acquired, theories of, 490 active, 458, 459 artificial, 457 by feeding, 464 by toxins, 462 methods, 459 natural, 498 passive, 458, 464 unit of, 468 Impression preparations, 118 Incubators, 79 Indol, formation of, 77 Infection, conditions modifying, 149 nature of, 153 Inflammatory conditions due to bacteria, 157 Influenza, 420 bacilli, pseudo-, 423 Influenza, bacillus, cultivation of, 421 bacillus, inoculation, 424 lesions in, 422 sputum in, 422 Inoculation, methods of, 120 of animals, 120 separation by, 56 protective, 462 et seq. Intestinal changes in cholera, 402 amoebic dysentery, 539 bacterial dysentery, 347 typhoid fever, 329 Intestinal infection in cholera (ex- perimental), 407 Involution forms in bacteria, 4 Iodine solution, Gram's, 99 terchloride, 468 as antiseptic, 145 lodoform as antiseptic, 148 Issaeff, 464 Ivauoff s vibrio, 415 Japanese dysentery, 350 Jenner on vaccination, 503 Jeuner's stain, 106 Johne's bacillus, 254 Joints, gouococci in, 225 Kala-azar, 563 Kipp's apparatus, 58 Kitasato on bacillus of influenza, 420 of plague, 425 of tetanus, 372 et seq. Klebs-Loffler bacillus, 352 Klein, 345, 508 Klemperer on pneumonia, 210 Koch on avian tuberculosis, 250 bacillus of malignant oedema, 388 bovine tuberculosis, 248 cholera spirillum, 399 cultivation of b. anthracis, 301 leveller for plates, 53 tubercle bacillus, 235 tuberculin, 258 "tuberculin 0," and "R," 260 Koch-Weeks bacillus, 191 Korn's acid-fast bacillus, 253 Kruse and Pasquale on dysentery, 541 Kubel-Tiemann litmus solution, 42 Kiihne's methylene-blue, 98 modification of Gram's method, 100 Lamb on relapsing fever, 442 Laveran's malarial parasite, 521 Leishmau- Donovan bodies, 563 INDEX 599 Leisbman-Donovan bodies, cultivation of, 566 Irishman's opsonic technique, 111 serum method for staining try- panosomes, 545 stain, 106 Leishmauia donovaui, 567 tropica, 567, 568 Lenses, 85 Lepra cells, 269 Leprosy, 267 bacillus, 269 distribution of, 271 staining, 100, 270 diagnosis of, 274 etiology of, 272 Leprosy-like disease in rats, 273 Leptothrix, 15 Lesions produced by bacteria, 155 Leucocidin, 165 Leucocytosis, 156, 495 Leucomaines, 161 Levaditi's method for staining spiro- chsetes, 104 Litmus solution, Kubel-Tiemann's, 42 whey, 44 Liver abscess in dysentery, 540 Lockjaw, 371 Loffler's bacillus, 352 methylene-blue, 98 serum medium, 40 and Schutz' glanders bacillus, 275 Losch, amceba of, 537 Lumbar puncture, 68 Lustgarten's bacillus, 229 Lustig's anti-plague serum, 436 Lymph, vaccine, 506 Lymphangitis, 184 Lysogenic action of serum, 477 towards blood corpuscles, 479 MacConkey's bile-salt media, 43 medium, use of in dysentery, 347 in examining water, 136 in paratyphoid fever, 335 M'Fadyean on glanders, 282 Macrocytase, 497 Macrophages, 495 Madura disease, 297 Malaria, cycle in man, 522 in mosquito, 528 pathology of, 533 prevention of, 532 question of immunity against, 534 Malarial fever, examination of blood in, 535 malignant, 523, 531 mosquitoes in, 532 Malarial parasite, 52-1 inoculation of, 522 staining of, Leishman's method, 106 Romanowsky methods, 106 varieties of, 529 Malignant cedema, bacillus of, 388 diagnosis of, 393 immunity against, 393 Malignant pustule, 310 Mallein, 283 Malta fever, 446 methods of diagnosis, 450 spread of disease, 449 Mann's method of fixing sections, 92 Manson, 521 Maragliano's anti - tubercular serum 264 Marchiafava and Celli on malaria 521 Marmorek, on streptococci, 183 antistreptococcic serum, 476 Marmorek's serum media, 41 antitubercular serum, 265 Martin, Sidney, on albumoses, etc., 165 on anthrax, 312 on diphtheria, 363 Martin, C. J., on toxins, 166 on antitoxins, 475 Massowah vibrio, 416 Measuring bacteria, 119 Meat extract, 31 Meat-poisoning by bacillus botulinus, 393 by Gaertner's bacillus, 336 Mediterranean fever, 446 Meningitis, bacteria in, 217 epidemic cerebro-spinal, 174, 213 in influenza, 423 pneumococci in, 205 posterior basal, 216 Mercury perchloride as antiseptic, 145 Metabolism, disturbances of, by bacteria, 159 Metachromatic granules, 8 Metchnikoff on cholera in rabbits, 407 relapsing fever, 441 Metchnikoff's phagocytosis theory, 495 spirillum, 417 Methylene-blue, 95, 98 600 INDEX Methyl-violet, 94 Meyer and Ransom on tetanus toxin, 382 Micrococci of suppuration, 174 Micrococcus, 12 of gonorrhoea, 219 melitensis, 447 pyogenes tennis, 174 tetragenus, 181 lesions caused by, 185 urese, 20 """ Microcytase, 497 Microphages, 495 Microscope, use of, 85 Microtomes, 90 Migula, 12 Mikulicz, cells of, 284 Milk as culture medium, 46 Moller's stain for spores, 102 Moeller's Timothy-grass bacillus, 253 Morax, bacillus of, 192, 193 ' Mordants, 97 • Morgan's bacillus, No. 1, 351 Mosquitoes, in malaria, 528, 532 role in yellow fever, 453 Moulds, media for growing, 44 Muencke's filter, 72 Miiller's bacillus, 192 Mycetoma, 297 Myelocytes, neutrophile, 156 Nagana, 552 Natural immunity, 498 Neelsen's stain for tubercle, 101 Negative phase in immunisation, 262, 494 Negri bodies in rabies, 514 Neisser's gonococcus, 219 stain for b. diphtherise, 108 Neucki, 10 Neutral-red as indicator for media, 43 use of, 38 with b. typhosus, 327 Neutrophile leucocytes, 156 myelocytes, 156 Nicolaier, tetanus bacillus, 371 Nicolle's modification of Gram's method, 100 Nikati and Rietsch on cholera, 407 Nitrifying bacteria, 23 Nitroso-indol body, 78 Nordhafen vibrio, 418 Novy and MacNeal, medium for culture of trypanosomes, 546 Obermeier's spirillum, 438 (Edema, malignant, 388 Ogata's dysentery bacillus, 350 Ogston, 174 Oil, aniline, for dehydrating, etc., 93 Oil immersion lens, 85 Ookinete, 528, 547, 548 Opsonic action, nature of, 483 technique, 111 Opscnins, 112 absorption of, 484 in tuberculosis, 261 thermolabile, 484 thermostable, 484 Organisms lower than bacteria, 2, 452 Oriental plague, 425 Osteomyelitis, 190 Otitis, 205, 423 Oxygen, nascent, as antiseptic, 145 Ozoena bacillus, 285 Para-colon bacillus, 335 Paraffin embedding, 91 Paratyphoid bacillus, 335 Passage, 461 Passive immunity, 458, 464 Pasteur on exaltation of virulence of bacteria, 461 on hydrophobia, 516 on vaccination against anthrax, 314 septicemie de, 388 Pathogenicity of bacteria, 149 Peptone gelatin (v. Culture media), 35 solution, 38, 404 Periostitis, acute suppurative, 190 Peritonitis, 184, 224 Perlsucht, 236 Pestis major, 431 minor, 431 Petri's acid-fast bacillus, 253 capsules, 52 sand-filter for examining air, 128 Petruschky's litmus whey, 44 Pettenkofer on cholera, 410, 415 Pfeffer, 20 Pfeiffer on anti-serum, 477 cholera, 411 influenza, 420 typhoid, 333 Pfeiffer's phenomenon, 411, 477 Phagocytes, 156 Phagocytosis theory of Metchnikoff, 495 Phenol broth, 347 INDEX 601 Phenol-phthalein as indicator, 33 Phenomenon of Bordet, 477 Gru'ber and Durham, 485 Pfeiffer, 411, 477 Pigments, bacterial, 10 Pipettes, 66, 108, 110, 116 Piroplasmata as causes of disease, 569 Piroplasmosis, 568 Pitfield's flagella stain, 103 Plague, bacillus of, 426 et seq, Haffkiue's inoculation against, 436 immunity against, 435 infection in, 432 involution forms, 427 part played by rat fleas in the spread of, 433 preventive inoculation against, 436 serum diagnosis, 437 stalactite growths of, 429 varieties of, 431 Plasmolysis, 9 Plate cultures, agar, 55 gelatin, 51 gonococcus, 222 Platinum needles, 49 Piieumobacillus ( Fried! ander's), 199, 203 et seq. Pneumococcus (Fraenkel's), 199, 201 et seq. immunity against, 210 in endocarditis, 188 lesions caused by, 204 . toxins of, 209 Pneumonia, bacteria in, 197 gangrenous, 423 in influenza, 422 methods of examination of, 212 septic, 197 varieties of, 196 Polar granules, 8 Positive phase in immunisation, 262, 494 Potassium permanganate as antiseptic, 147 Potatoes as culture material, 44 Poynton and Payne on acute rheumatism, 193 Precipitius, 487 Preparations, impression, 118 Protective inoculation, 462 et seq. Proteosoma, 530 Protozoa described in hydrophobia, 513 smallpox, 508 Protozoou inalarise, 521 Pseudo- diphtheria bacillus, 365 -tuberculosis streptothricea, 296 Psittacosis bacillus, 337 Ptomaines, 161 Puerperal septicaemia, 184 Pus, examination of, 87, 195 Pustule, malignant, 310 Pyaemia, 184 et seq. nature of, 173 Quartan fever, 530 Quarter-evil, bacillus of, 396 Quotidian fever, 529 Rabies, 510 Rabinowitch's acid-fast bacillus, 253 Rauschbrand bacillus, 396 Ray-fungus (actinomyces), 286 Reaction of media, standardising of, oq OQ Receptors, 491 Recovery from disease, 457 Red stains, 95 Red- water fever in cattle, 569 Reichert's gas regulator, 80 Relapsing fever, agglutination of spirillum, 442 bactericidal serum in, 442 spirillum of, etc., 439 Reversibility of toxin-antitoxin reac- tion, 472 Rheumatism, acute, 193 Rhinoscleroma, bacillus of, 284 Ricin, 169 immunity against, 464, 469 Rivers, bacteria in, 137 Robin, 169 Rock fever, 446 Roll-tubes, Esmarch's, 55, 59 Romanowsky stains, 105 Roseubach (bacteria in suppuration), 174 Ross, on malaria, 521 thick film method for malarial parasite, 536 Roux on antitoxic sera, 469 and Yersin (diphtheria), 361 etseq. Sabouraud's medium, 44 Safraniu, 95 Salt-agar as medium for b. pestis, 427 Sanarelli (typhoid fever), 332 Sanderson, Burden, 460, 507 602 INDEX Saprophytes, 149 Sarciiia, 12 Sausage poisoning, bacillus botulinus in, 394 Schaudinn 011 biology of trypauosomes, 548 on amoebae of dysentery, 537 on morphology of spirilla, 550 on spirochsete pallida, 229 on spirillum Ziemanni, 550 Schizomycetes, 3 Schizophyceae, 3 Schizophyta, 3 Schiiffuer's dots, 106, 531 Sclavo's anti-anthrax serum, 315 Scorpion poison, 169 Section-cutting, 90 Sections, dehydration of, 93 Sedimentation methods, 109 test for typhoid, 338 Seiteuketten, 491 Septicaemia, nature of, 173 puerperal, 184 sputum, 197 Septicemie de Pasteur, 388 Septic pneumonia, 197 Sera, haemolytic, 479 Serum agar. 38 Serum, agglutinative action of, 485 anaphylaxis, 494 antibacterial, 476 anti- cholera, 411 antidiphtheritic, 467 anti-plague, 436 antipneumococcic, 210 antirabic, 519 antistreptococcic, 476 antitetanic, 384 antitoxic, preparation of, 467 et seq. antitubercular, 264 antityphoid, 344 bactericidal action of, 477 blood (v. Culture media), 39 diagnosis, 485 methods, 109 of typhoid, 337 inspissator, 39 lysogenic action of, 477 towards blood corpuscles, 479 Serum media, 39 Serum- water media, 41 Sewage, bacterial treatment of, 139 contamination of water by, 136 Shake cultures, 77 Sheep-pox, 507 Shiga's bacillus, 346 Side-chain theory, Ehrlich's, 491 Sleeping sickness, 555 Slides for hanging-drops, 63, 64 Sloped cultures, aerobic, 48 anaerobic, 62 Smallpox, 503 bacteria in, 507 Guarnieri bodies in, 508 Siuegma bacillus, 254 Smith's, Lorrain, serum medium, 41 Smith, Theobald, phenomenon of, 494 Snake poisons, 169 activating of, by serum, 170 constituents of, 169 immunity against, 462 Sobernheim's anti-anthrax serum, 316 Soft sore, 227 Soil, examination of, for bacteria, 131 Soudakewitch on relapsing fever, 442 Spinal cord, lesions by pyogenic organisms, 183 Spirilla, characters of (v. also Vibrio), 14, 550 like cholera spirillum, 417 Spirillosis in animals, 439 Spirillum Metchuikovi, 417 of cholera, 400 Deneke, 419 Fiukler and Prior, 418 Miller, 419 relapsing fever, inoculation with, etc., 438 Spirochsete, 14, 229, 550 pallida, 229 staining of, 104, 107 pertenuis, 234 refringeus, 230 Spirochsetes in syphilis, 229 in yaws, 234 staining of, in films, 107 staining of, in sections, 104 Spironema pallidum, 229 Splenic fever, 300 Spore formation, arthrosporous, 7 endogenous, 5 in b. anthracis, 304 Spores, staining of, 102 Sporoblasts, 529 Sporocyst (malaria), 529 Sporocytes, in malaria, 524 Sporozoites, 529 INDEX 603 Sporulation of malarial parasite, 522 Sputum, amoebae in, 541 influenza, 422, 425 in plague, 238 in pneumonia, 200 phthisical, 241, 255, 265 septicaemia, 197 Staining methods, 94 et seq. of capsules, Welch's method, 102 Richard Muir's method, 102 of flagella, 103 of leprosy bacilli, 270 of spores, 102 of tubercle bacilli, 100 principles, 94 Stains, basic aniline, 94 Standard of immunity, 468 Standardising reaction of media, 33 Staphylococci, lesions caused by, 184 Staphylococcus, 12 cereus albus, 176 flavus, 176 pyogenes albus, 176 — ' aureus, characters of, 174 inoculation with, 182 citreus, 174 Steam steriliser, Koch's, 27 Stegomyia fasciata, 453 Sterilisation by heat, 26 et seq. at low temperatures, 29 by steam at high pressure, 29 Streptococci in diphtheria, 356 in false membrane, 184 lesions caused by, 184 varieties of, 178 Streptococcus, 12 anginosus, 179 brevis, 178 conglomerate, 178, 179 equinus, 179 erysipelatis, 191 - faecalis, 179 longus, 178 mitis, 179 pneumoniae, 198 pyogenes, characters of, 176 inoculation with, 182 in air, 130 in soil, 133 salivarius, 179 Streptothrices allied to actinomyces, 294 Streptothrix, 15 actinomyces, 287 Streptothrix, anaerobic in actino- mycosis, 293 madurae, 297 Subcultures, 49 Sugars, classification of, 74 fermentation of, 74 Sulphurous acid as antiseptic, 147 Summer diarrhoea, bacteria in, 351 Suppuration, bacteria of, 174 gonococci in, 223 methods of examination of, 195 nature of, 172 origin of, 186 pneumococci in, 205 typhoid bacillus in, 330 Symptoms caused by bacteria, 161 Syphilis, bacillus of, 229 spirochaete pallida in, 229 transmission to animals, 233 Syringes for inoculation, 120, 121 Tabes mesenterica, 257 Taurocholate media, 43 Tertian fever, 530, 531 Test-tubes for cultures, 47 Tetanolysin, 380 Tetanospasmin, 380 Tetanus, 371 anti-serum of, 384, 467 et seq. intravenous injection of, 386 cerebral, 384 dolorosus, 383 immunity against, 384 methods of examination in, 388 treatment of, 386, 489 Tetanus bacillus, 372 inoculation with, 378 isolation of, 373 spores of, 373 toxins of, 163, 379 Tetrads, 12 Texas fever, 569 Theory of exhaustion, 490 of phagocytosis, 495 of retention, 490 humoral, 490 Thermophilic bacteria, 18 Thermostable opsonius, 484 Thionin-blue, 95, 98 Thiothrix, 15 Tick fever, African, 443 Timothy-grass bacillus, 153 Tissues, action of bacteria on, 155 fixation of, 89 604 INDEX Tizzoni and Cattani on tetanus, 385 Toxalbumius, 162 Toxic action, theory of, 170 Toxicity, estimation of, 467 Toxins, concentrated, method of ob- taining, 167 constitution of, 491 early work on, 161 effects of, 158 immunisation by, 467 intra- and extra- cellular, 162 nature of, 165 non-proteid, 166 of anthrax, cholera, etc. (vide Special Diseases) production, 154 susceptibility to, 491 vegetable, 169 Toxoids, 171, 472 Toxones, 171 Trachoma, bacteria in, 192, 424 Trichophyta, media for growing, 44 Tropical ulcer, 568 Trypanosoma gambiense, 558 Lewisi, 544, 551 noctuse, 548 of sleeping sickness, 555 • ugandense, 544, 558 ugandense, relation to Tr. Gam- biense, 561 Trypanosomata associated with various diseases, 544 culture of, 546 morphology of, 544 sexual cycle in, 548 Trypanosomiasis, 544 Tse-tse fly disease, 552 Tubercle bacillus, 237 action of dead, 255 avian, 250 cultivation of, 239 distribution of, 243 immunity against, 260 inoculation with, 246 powers of resistance of, 241 in sputum, etc., 255, 265 toxins of, 258 ' stains for, 100, 239 giant cells, 242 methods of examination of, 265 Tubercles, structure of, 242 Tubercular leprosy, 268 Tuberculin, 258 " Bazillenemulsion/' 260 Tuberculin, "0" and "R," 260 Tuberculosis, 235 aviau, 250 bovine, 248 its relation to human, 248 diagnosis by tuberculin, 259 Tuberculosis, in animals, 236 in fish, 251 modes of infection, 256 precautions in diagnosis of, 255 Tubes, cultures in, 47 Typhoid bacillus, 319 comparison with b. coli, 325 examination for, 344 immunity against, 332 inoculation with, 331 isolation from water supplies, 345 toxins of, 332 serum diagnosis, 337 suppurations in, 330 vaccination against, 343 Typhoid fever, 319 pathological changes in, 329 Ulcerative endocarditis, 188 experimental, 190 gouococci in, 225 Unit of immunity, 468 Urine, examination of, 69 staining of bacteria in, 88 tubercle bacilli in, 246, 265 typhoid bacilli in, 344 Vaccination against smallpox, 502 against hydrophobia, 516 against typhoid, 343 for infection by pyogenic bacteria, 194 nature of, 509 Variola, 505 et seq. Venins, 169 Vibrio (see also Spirillum), 14 berolinensis, 415 of cholera, 400 Danubicus, 415 Deneke's, 419 Finkler and Prior's, 418 Gindha, 416 Ivanoff, 415 Massowah, 408, 416 Metchnikovi, 417 Nordhafen, 418 of Pestana and Bettencourt, 416 Romanus, 416 Vibrion septique, 388 INDEX 605 Virulence, attenuation of, 459 exaltation of, 461 of bacteria, 150 Water, bacteria in, 135 contamination of by sewage, 138 examination of, 135 supplies, typhoid bacilli in, 345 Weichselbaum on pneumonia, 198 Weigert's method of dehydration, 93 modification of Gram's method, 100 Wertheim's medium, 220, 226 Whooping cough, bacteria in, 423 Widal on serum diagnosis, 485 Widal's reaction, synonym for agglu- tination of b. typhosus, q.v., 109, 337 Winogradski, 23 Winter-spring fevers, 529 Wolff and Israel's streptothrix, 295 Woodhead on tuberculosis, 257 Woody tongue, 292 Woolsorter's disease, 311 Wright's, A. E., calibrated pipette, 108 diluting pipette, 66 method of counting dead bacteria, 67 Wright's, A. 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